Manganese-engineered Lactobacillus Reuteri with enhanced antitumor and immunomodulatory activities for colorectal cancer prevention and treatment

Domain: chemistry
Published: 2025-12-19
Editorial outcome: major_revision

Abstract
Probiotic-based biotherapy for colorectal cancer (CRC) faces significant challenges due to poor tumor-targeting and limited bioactivities within the complex tumor microenvironment. This study shows that Lactobacillus reuteri (L. reuteri)’s inherent bioactivities, including proliferation, metabolism, and colorectal colonization, can be enhanced by manganese, prompting the fabrication of a manganese-engineered strain (MnLR). Oral administration of MnLR with its metabolic substrate-glycerol (MnLR/Gly) promotes the enrichment of L. reuteri and its antitumor metabolites within colon tumors. Across multiple preclinical colon tumor models, MnLR/Gly alone achieves a 95.6% inhibition of orthotopic tumor growth, liver metastases reduction by 62.1%, and confers durable protection, with 75% of prophylactically treated mice remaining long-term tumor-free and 62.5% resisting tumor rechallenge. Mechanistically, MnLR/Gly induces both intratumoral and peripheral dendritic cell maturation, M1 macrophage polarization, and effector T-cell responses. In orthotopic CRC rabbit models, GlyMnLR enteric capsules demonstrate significant antitumor efficacy comparable to standard chemotherapy while exhibiting favorable safety. These findings highlight MnLR as an effective standalone probiotic therapy for CRC prevention and treatment.

Introduction
Colorectal cancer (CRC) ranks as the third most commonly diagnosed cancer and the second leading cause of cancer-related death worldwide, accounting for nearly 10% of the new cancer cases and deaths in 20201. Effective interventions are still lacking, especially for distant metastatic CRC that decreased the 5-year survival rate to only ~10%2. The principal treatment option for both primary and metastatic CRC remains chemotherapy, which is, however, challenged by inevitable drug resistance and adverse reactions. Moreover, there are currently no well-established pharmacological strategies capable of preventing or delaying the occurrence of CRC. While non-steroidal anti-inflammatory drugs, such as aspirin and celecoxib, have shown potential preventive effects against CRC, their clinical feasibility remains uncertain3,4. Long-term use of these drugs can lead to gastrointestinal bleeding and elevate the risk of cardiovascular disease4,5. The significant development of immunotherapy has now revolutionized the field of oncology. However, in CRC, only a minority of metastatic patients with DNA mismatch repair-deficient (dMMR)/ microsatellite instability-high (MSI-H) are potentially sensitive to immune checkpoint inhibitors. Most CRC patients are poorly responsive to immunotherapy due to low tumor neoantigen load, insufficient immune cell enrichment, and the inherent presence of immunosuppressive components within the tumor microenvironment, such as macrophages with an immunosuppressive phenotype6,7. Thus, novel preventive and therapeutic strategies are highly desired for CRC.

A growing body of evidence reveals that the host microbiota within the gastrointestinal (GI) tract and other niches highly implicates the development of CRC8, leading to new targets for medical intervention. Typically, the use of intestinal probiotics (e.g., Lactobacillus and Bifidobacterium) provides a safe and promising approach for CRC prevention and treatment due to their safety and potential to restore tumor-induced gut microbial dysbiosis, enhance the immune system, and directly inhibit tumor growth with their antitumor metabolic products9,10. However, current preclinical and clinical studies on probiotics in cancer prevention and treatment largely focus on their role as adjuvants to improve the efficacy and safety of chemotherapy and immunotherapy, rather than as standalone anticancer agents11–14, most likely due to the uncertain efficacy, mechanism, and safety in the complex human body. Significant advancements in interdisciplinary research among microbiology, bioengineering, chemistry, and material science have brought various genetically, chemically, and physically engineered bacteria with augmented functionalities15–17. Despite these strides, the continued development and clinical translation of these bacteria formulations for cancer therapy still face substantial impediments mainly arising from the complicated and low-yield preparation process, ethical concerns, and notably, the limited antitumor effects owing to the lack of tumor-targeting capability and the progressive decline in bacterial activity and quantity before reaching target sites, concomitantly with inadequate production of antitumor metabolites18–20. Therefore, realizing the full potential of probiotics in CRC management requires identifying and developing bacterial strains with enhanced antitumor efficacy through in-depth investigations of their biological functions and optimizing their dosage formulations to ensure the effective and safe usage of probiotics in the host.

Lactobacillus reuteri (L. reuteri) is a well-defined probiotic bacterium that naturally inhabits the GI tract of humans and many other animals20. L. reuteri is known to produce various bioactive metabolites, including organic acids, antimicrobial compounds, and nutrients, which can inhibit the growth of pathogenic bacteria, protect against infections, and contribute to overall health21–23. L. reuteri has shown promise in the fight against several common pathological conditions, such as infant colic, constipation, diarrhea, Helicobacter pylori infection, colitis, and autoimmune disorders24–26. Recent studies suggest that L. reuteri is significantly reduced in CRC-bearing mice and patients’ feces and tumor mucosa. Moreover, L. reuteri-derived metabolic products (e.g., reuterin and indole-3-aldehyde) presented efficacy in suppressing CRC growth by altering redox balance and improving immune checkpoint inhibitor response in melanoma27,28. Nevertheless, the multifaceted impacts of L. reuteri on complex cancer biological processes and its potential roles in cancer prevention and treatment remain poorly understood.

Metal ions (e.g., iron, copper, zinc, and manganese) are essential for supporting the survival, growth, and biochemical reactions of bacteria at a suitable level by maintaining bacterial structural stability, enhancing their catalytic activity, and regulating gene expression or signal transduction29–32. For instance, manganese (Mn) supports bacterial oxidative stress resistance through Mn-dependent antioxidant systems33,34, and modulates host innate immune signaling pathways to enhance antiviral and antitumor effects35,36. These properties make Mn modification especially suitable for customizing bacterial biological properties to withstand challenging environments and enhance functionality.

Here, we reveal that Mn enhances the proliferation, metabolic activity, and colorectal colonization of L. reuteri, enabling the development of a Mn-modified strain (MnLR) with improved bioactivity. MnLR combined with glycerol shows increased intratumoral accumulation and metabolite production, resulting in strong suppression of orthotopic colon tumors through cancer-selective cytotoxicity, restoration of tumor-induced dysbiosis, and activation of antitumor immunity. To facilitate translational application, we further formulate an enteric GlyMnLR capsule that achieves robust efficacy and favorable safety in a rabbit CRC model. Together, this study establishes MnLR as a highly efficient, safe, and cost-effective oral microbial agent with broad implications for CRC prevention, treatment, and future development of engineered probiotic therapeutics.

Results
L. reuteri-derived glycerol metabolites exhibit redox-dependent CRC inhibition and enrich multiple tumor-associated immune cells within colon tumors
The CRC-inhibitory effects of the metabolites produced by L. reuteri catabolizing glycerol were determined in vitro (colon cancer cell lines and organoids from CRC patients) and in vivo (subcutaneous colon cancer-bearing mice) (Fig. 1a). Reuterin (i.e., 3-hydroxypropionaldehyde (3-HPA)) is a bioactive intermediate in glycerol catabolism and has been determined to be capable of inhibiting CRC growth by altering redox balance in cancer cells27. Consistent with the previous findings27, the mixed metabolites from L. reuteri catabolizing glycerol exerted a potent growth-inhibitory effect preferentially on cancer cells over non-cancerous cells in a redox-dependent manner (Fig. 1b, c), as the metabolites stimulated the production of cytotoxic reactive oxygen species (ROS), reduced antioxidative glutathione level, and upregulated oxidative stress–related molecules at both the mRNA and protein levels in colon cancer cells (Supplementary Fig. 1); and their cytotoxic effect was significantly reduced in human normal colonic epithelial cells (NCM460), human umbilical vein endothelial cells (HUVEC), and normal human liver cells (L-02) (with the cell viability remaining above 80% at a reuterin concentration of 50 µg/mL), as well as in the presence of N-acetyl-cysteine (NAC, a ROS scavenger) (Fig. 1b, d). Notably, mass spectrometry analysis revealed the presence of various compounds from glycerol catabolism within the mixed metabolites in addition to reuterin, including reuterin’s downstream products and nearly 20 unidentified compounds (at a percentage of > 0.1%) (Supplementary Table 1). Furthermore, the growth-inhibitory efficacies of the pure reuterin and its downstream products (1,3-propanediol and 3-hydroxypropionic acid) on colon cancer cells were lower in comparison to the mixed metabolites (containing an equivalent amount of reuterin) (Fig. 1b and Supplementary Fig. 2), indicating the potential cytotoxic effects of those unidentified metabolites on cancer cells. Consistent with these findings, reuterin elevated intracellular ROS levels in colon cancer cells, but to a markedly lower extent than the mixed metabolites (Supplementary Fig. 3); whereas its downstream products only caused a slight increase in ROS (Supplementary Fig. 3), suggesting that these products may exert cytotoxicity through ROS-independent or partially ROS-independent mechanisms. Further, the more effective colon cancer suppression of the mixed metabolites than pure reuterin was confirmed in five independent CRC patient-derived organoid (PDO) models, including four microsatellite stable (MSS) and one MSI-H subtypes (Fig. 1e, f, Supplementary Fig. 4, and Supplementary Table 2), supporting the robustness and clinical relevance37.

Next, the in vivo antitumor efficacy of glycerol metabolites was examined using subcutaneous colon cancer-bearing mice that were treated with pure reuterin or glycerol metabolites either by intratumoral injection (i.t.) or oral gavage (o.g.). Consistent with the observations in vitro, the orally-delivered metabolites effectively suppressed tumor growth, with a tumor weight suppression rate of nearly 1.8-fold higher than oral reuterin, and 1.2-fold higher than intratumorally-injected metabolites (Fig. 1g and Supplementary Fig. 5). This finding suggests that the orally-delivered metabolites can be effectively distributed into tumors, and may even induce systemic impacts to promote antitumor effects. Thus, we analyzed the tumor-associated immune cells within the tumor tissues since the immune system plays a significant and complex role in CRC development and treatment38–40. Notably, the numbers of macrophages, DCs, and CD8+ T cells were increased in the tumor microenvironment of the metabolite-orally treated mice (Fig. 1h, i), indicating that apart from the direct cytotoxicity in tumor cells, the metabolites might exert immunological impacts, potentially suppressing tumor growth.

L. reuteri is chemically modified with Mn that facilitates the proliferation, glycerol metabolism, and bio-adhesion of L. reuteri
Based on the aforementioned antitumor effects of L. reuteri-derived glycerol metabolites, we proposed modifying L. reuteri to enhance its proliferation and glycerol metabolism activities, aiming to achieve a high local concentration of antitumor metabolites for effective tumor suppression. To this end, we incubated L. reuteri with diverse metal ions naturally present in the human body to identify specific metals capable of enhancing L. reuteri’s bioactivities. We found that the proliferation and glycerol metabolism (measured by the reuterin yield) of L. reuteri were simultaneously promoted by Mn in a concentration-dependent manner (Fig. 2a–d). Consistently, Mn enhanced the enzymatic activity of glycerol dehydratase (GDHt) that catalyzes the dehydration of glycerol into reuterin in L. reuteri (Supplementary Fig. 6a)41. The reduced L. reuteri proliferation and reuterin production in the presence of Mn ion chelating agents or oxidative phosphorylation uncouplers (for the inhibition of Mn ion uptake) further demonstrated the essential role of Mn in fostering L. reuteri (Supplementary Fig. 6b). Also, Mn2+ supplementation significantly promoted the proliferative and metabolic activities of L. reuteri under carbon or nitrogen deprivation (Supplementary Fig. 7), highlighting the specific biological role of Mn in sustaining and rescuing bacterial functionality under nutrient-limited conditions. Furthermore, to assess the long-term retention of Mn-mediated enhancements, L. reuteri strains were propagated up to the 50th generation, with no significant differences observing growth or glycerol metabolism compared with early passages (3rd generation) (Fig. 2e), demonstrating generational stability. In addition, Mn2+ supplementation sustained enhanced bacterial proliferation and glycerol metabolism in simulated intestinal fluid (SIF) (Fig. 2f), indicating that these effects are durable under conditions relevant to CRC therapy. To investigate the underlying biological mechanisms by which Mn promotes L. reuteri’s bioactivity, prokaryotic transcriptomics were employed. Mn-treated L. reuteri was grouped in a cluster (Fig. 2g) and exhibited 130 up-regulated genes and 143 down-regulated genes compared with untreated L. reuteri (Supplementary Fig. 8). Furthermore, the subsequent pathway enrichment analysis determined that Mn-treated L. reuteri exhibited significant upregulation of signaling pathways related to the ribosome, thiamine metabolism, and oxidative phosphorylation, with ribosome pathways being the most prominent (Fig. 2h). To functionally validate these transcriptomic findings, quantitative proteomic analysis confirmed elevated levels of ribosomal proteins (Fig. 2i). SDS-PAGE further demonstrated increased global protein synthesis in Mn-treated L. reuteri (Supplementary Fig. 9). These results collectively establish that the transcriptional upregulation of ribosome-related pathways is functionally translated into enhanced ribosomal activity and protein synthesis, suggesting that ribosomes may be a key target for the regulation of L. reuteri’s bioactivity by Mn ions42.

Subsequently, we set out to modify L. reuteri with Mn using tannic acid (TA) as a chemical linker to obtain a bioactivity-enhanced L. reuteri (MnLR) (Fig. 2j). The successful surface modification of Mn on L. reuteri was verified by the evident changes in morphological features and fluorescence labeling (Fig. 2k, left). The elemental mapping (Fig. 2k, right), X-ray photoelectron spectroscopy (XPS) (Supplementary Fig. 10a), and atomic absorption spectroscopy (AAS) (Supplementary Fig. 10b, c) analyses further confirmed the presence of Mn and its surface association, as well as its capability to release from MnLR. Notably, MnLR exhibited a faster Mn release profile in SIF compared with neutral PBS (Supplementary Fig. 10b, c), indicating a release behavior conducive to CRC targeting while limiting off-target distribution. As a result, compared to the raw L. reuteri, MnLR exhibited improved proliferation, reuterin production (Fig. 2l, m), as well as biofilm formation and adhesion to colon cancer cells (Supplementary Fig. 11), which would collectively enhance the antitumor efficacy within colon tumor sites. Furthermore, compared with PBS, both raw L. reuteri and MnLR exhibited reduced proliferative viability when exposed to simulated gastric fluid (SGF) and SIF (Fig. 2n, o). Nevertheless, MnLR maintained significantly higher growth activity than raw L. reuteri under both conditions (Fig. 2n, o), suggesting the protective role of Mn modification in enhancing resistance to gastrointestinal stresses.

MnLR delivered with glycerol inhibits colon cancer cell growth and promotes dendritic cell maturation in vitro
The tumor-inhibitory effect of MnLR delivered with glycerol (MnLR/Gly) was demonstrated in human colon cancer cell lines with diverse MMR statuses—including microsatellite instability-high (MSI-H; SW48, HCT116, RKO), and microsatellite stable (MSS; SW620, DLD-1), as well as in mouse colon cancer cell lines (MC38 and CT26) (Fig. 3a). This effect was attributed to the MnLR-derived glycerol metabolites, as neither L. reuteri nor glycerol alone exhibited cytotoxicity to colon cancer cells (Fig. 3a). Moreover, the inhibitory rates of MnLR/Gly were higher than L. reuteri/Gly (Fig. 3a) across all the cell types, indicating that Mn promotes L. reuteri proliferation and production of cytotoxic metabolites, thus enhancing the cancer cell-killing efficiency. Consistent with the effects observed for L. reuteri-derived mixed metabolites, MnLR/Gly exhibited preferential cytotoxicity toward colon cancer cells over non-cancerous cells (Fig. 3a and Supplementary Fig. 12), indicating its anticancer efficacy, alongside reduced toxicity to normal proliferating cells. Further, MnLR/Gly also induced the intracellular generation of cytotoxic ROS (Supplementary Fig. 13a), which has been thought to be a potent initiator of immunogenic cell death (ICD)43. Indeed, the cancer cells treated with either pure reuterin or MnLR/Gly underwent a series of biochemical events towards ICD, including the exposure of calreticulin (CRT) on the cellular membrane, and the release of high mobility group box 1 (HMGB1) and adenosine triphosphate (ATP) (Supplementary Fig. 13b–d)44. Moreover, Annexin V/PI flow cytometry revealed that MnLR/Gly treatment induced substantial cell death in colon cancer cells, with the apoptotic cell population reaching 42.6%, much higher than 2.0% in untreated cells and 19.2% in reuterin-treated cells (Supplementary Fig. 13e). Consistently, Live/Dead staining confirmed a significantly increased number of dead cells in the MnLR/Gly group (Supplementary Fig. 13f). Together, these results validate that the ICD-associated markers reflect bona fide immunogenic cell death rather than transient stress. These biochemical products generated by the cells experiencing ICD would be engulfed by DCs, stimulating DCs’ maturation and facilitating tumor antigen presentation to effector T cells, ultimately priming an antitumor immune response45. To investigate this immunological process, we subjected DCs derived from murine bone marrows or human peripheral blood mononuclear cells (mBMDCs or hPBMC-DCs) to the lysates from reuterin- or MnLR/Gly-treated colon cancer cells and observed a significant improvement in DC maturation, as evidenced by the increased proportion of mature DCs (CD11c+CD86+ mBMDCs and CD83+ hPBMC-DCs), and the production of immune functional cytokines (IL-12, TNF) (Fig. 3b, c and Supplementary Fig. 14). Notably, the lysates from the MnLR/Gly-treated cells exhibited a more pronounced immunostimulatory efficacy on DCs compared with those from the reuterin-treated cells (Fig. 3b, c). These findings collectively indicate the capability of MnLR/Gly to effectively induce ICD in cancer cells to stimulate the functional maturation of DCs, thereby potentially promoting the activation of antitumor immunity (Fig. 3n).

Next, we investigated whether MnLR/Gly had a direct impact on DCs’ functional phenotype. Compared with glycerol, TA, and L. reuteri, which did not affect the proportion of mature DCs, reuterin, Mn2+, and MnLR all significantly stimulated DCs maturation (Fig. 3d, e and Supplementary Fig. 15). Notably, the proportion of mature DCs was the highest in the MnLR/Gly-treated total DCs (1.7-fold higher than reuterin- or Mn2+-treated mBMDCs, 1.2-fold higher than reuterin- or Mn2+-treated hPBMC-DCs) (Fig. 3d, e and Supplementary Fig. 15). Inhibition of reuterin production with a GDHt inhibitor reduced DC maturation to the level induced by Mn2+ alone (Supplementary Fig. 16), suggesting that glycerol metabolites and Mn2+ released by MnLR contribute additively to DC maturation (Fig. 3n). In addition, MnLR/Gly-treated DCs secreted markedly higher levels of IL-12 and TNF than other groups (Fig. 3f, g), further supporting their enhanced functional maturation. To investigate how MnLR/Gly functionally regulates DCs mechanically, we performed RNA-sequencing (RNA-seq) and subsequent pathway enrichment analysis. The results showed that the DEGs in the MnLR/Gly-treated DCs were enriched in the tumor necrosis factor (TNF) signaling pathway and its downstream mitogen-activated protein kinase (MAPK) signaling pathway (Supplementary Fig. 17). Quantitative real-time PCR analysis further confirmed the upregulation of the genes related to these two pathways within MnLR/Gly-treated DCs (Supplementary Fig. 18). The MnLR/Gly-treated DCs also exhibited the increased protein levels of phosphorylated p38 (p-p38) (Fig. 3h, i); and the presence of AMG-548 (a p38 MAPK inhibitor) prevented the MnLR/Gly-induced p38 phosphorylation (Fig. 3h, i) and DCs maturation (Fig. 3j–m). These results suggest that the DCs’ maturation driven by MnLR/Gly in part depends on the p38 MAPK pathway.

Altogether, these findings demonstrate that MnLR/Gly can promote dendritic cell maturation, either indirectly by inducing immunogenic tumor cell death through glycerol metabolites or directly by using a combination of glycerol metabolites and Mn ions, thereby potentially enhancing the activation of antitumor immunity (Fig. 3n). This aligns with previous in vivo studies (Fig. 1g–i), indicating that L. reuteri-derived glycerol metabolites may regulate DCs in the tumor microenvironment. In addition, the concordance in observations between murine- and human-derived DCs underscore the conserved nature and translational potential of MnLR/Gly-mediated immunomodulation.

MnLR delivered with glycerol drives macrophage polarization towards antitumor phenotype in vitro
Next, we investigated how MnLR/Gly functionally regulated macrophage polarization in vitro, as the previous in vivo experiments (Fig. 1g–i) suggested the potential regulation of macrophage polarization by L. reuteri-derived glycerol metabolites. To test M1 (pro-inflammatory and tumor-killing) polarization, we stimulated resting macrophages (RAW264.7 cells, M0) with an M1 inducer (LPS/IFN-γ). MnLR/Gly significantly promoted the M1 polarization, as evidenced by the expanded M1 macrophage subpopulation among total LPS/IFN-γ-stimulated macrophages (Fig. 4a, b), accompanied by the upregulated expression of M1-related markers (Supplementary Fig. 19a), with the M1-promoting efficacy marginally higher than the pure reuterin. By contrast, the macrophage polarization into M2 phenotype (anti-inflammatory and tumor-promoting) was reversed by MnLR/Gly, since the percentage of M2 macrophages (Fig. 4c, d) and the level of M2-related genes (Supplementary Fig. 19b) were drastically decreased in the IL-4 (M2 inducer)-treated RAW264.7 cells with the presence of MnLR/Gly. We also established the 3D multicellular tumor spheroids (MCTs) by co-culturing colon cancer cells and macrophages under LPS/IFN-γ or IL-4 stimulation to investigate the MnLR’s antitumor efficacy in the tumor microenvironment containing macrophages. The results showed that the combined presence of MnLR/Gly and LPS/IFN-γ-stimulated macrophages most significantly inhibited MCT growth; meanwhile, MnLR/Gly effectively attenuated IL-4-treated macrophages’ stimulatory effects on MCT growth (Fig. 4e and Supplementary Fig. 20), suggesting the synergetic antitumor effects of MnLR/Gly with macrophages in the tumor microenvironment. Of note, glycerol, TA, Mn2+, L. reuteri, and MnLR did not affect or even suppress M1 polarization (Supplementary Fig. 21a); while glycerol, Mn2+, and L. reuteri also exhibited inhibitory effects on M2 polarization similar to reuterin and MnLR/Gly (Supplementary Fig. 21b). These observations indicate that MnLR-produced glycerol metabolites (especially reuterin) predominately stimulate M1 macrophage polarization, whereas the blockage of M2 macrophage polarization could also be attributed to MnLR-released Mn2+ and L. reuteri as well as the unmetabolized glycerol, in addition to glycerol metabolites. Human PBMC-macrophages exhibited consistent responses with RAW264.7 cells, wherein MnLR/Gly treatment induced M1 phenotypic polarization (Fig. 4f, g and Supplementary Fig. 22), accompanied by elevated IL-6 and TNF production and enhanced phagocytic activity against colon cancer cells (Fig. 4h–j), thereby reinforcing the robustness and translational potential of immunomodulatory effects mediated by MnLR/Gly.

Furthermore, the subsequent RNA-seq and pathway enrichment analysis revealed that the DEGs in both LPS/IFN-γ- and IL-4-stimulated macrophages with the supplement of MnLR/Gly were markedly enriched in hypoxia-inducible factor 1 (HIF-1) signaling pathway (Supplementary Fig. 23). Quantitative real-time PCR analysis (Supplementary Fig. 24) and immunoblot assay (Fig. 4k, l) further demonstrated the upregulation of HIF-1 signaling pathway-related genes and HIF-1 protein levels within MnLR/Gly-treated macrophages upon LPS/IFN-γ- or IL-4 stimulation. Moreover, the HIF-1 inhibition resulted in reduced M1 macrophages (Fig. 4m, n) but increased M2 macrophages (Fig. 4o, p) under MnLR/Gly treatment, suggesting that the macrophage polarization towards antitumor phenotype induced by MnLR/Gly partially relies on the HIF-1 signaling pathway (Fig. 4t). Consistently, this HIF-1–dependent regulation of macrophage polarization was also validated in human PBMC-macrophages (Fig. 4q–s). Additionally, treatment with the NAC partially reduced MnLR/Gly-induced HIF-1 upregulation, suggesting that this pathway activation is mediated, at least in part, by ROS production (Supplementary Fig. 25).

Together, MnLR/Gly promote the polarization of macrophages towards an antitumor phenotype, thereby enhancing tumor-killing effects.

Mn modification improves L. reuteri colonization and metabolite distribution at colon tumor sites without eliciting systemic adverse side effects in mice
According to the in vitro observations where MnLR presented an improved proliferative activity, biofilm formation, and adhesion to colon cancer cells, we then determined whether Mn promoted the L. reuteri colonization at the colon tumor site in vivo using the orthotopic colon tumor-bearing mice (Fig. 5a). Abundant MnLR arrived at the small intestine and colorectum (tumor site) 1 hour after oral administration, 1.7-fold higher than L. reuteri without Mn modification (Fig. 5a, b). The enhanced enrichment of MnLR at the colorectum was maintained for at least 24 hours and was also observed in healthy mice (Fig. 5c–e and Supplementary Fig. 26). Furthermore, L. reuteri quantity in the feces from MnLR-treated mice was higher than that from raw L. reuteri-treated mice (Fig. 5f), indicating an accelerated proliferation of MnLR in vivo. Of note, both MnLR and raw L. reuteri exhibited significantly higher 24-hour retention in the colorectal tissues of tumor-bearing mice compared with healthy mice (Fig. 5d, e and Supplementary Fig. 26). High-resolution ex vivo tissue fluorescence imaging further revealed that MnLR accumulated 1.4-fold more in colon tumor tissues than in adjacent normal tissues (Fig. 5g, h). These observations suggest a preferential localization and colonization of L. reuteri into tumor regions, likely influenced by distinct features of the tumor microenvironment (such as oxygen gradients), immunosuppressive states, and tumor-associated alterations of the gut microbiota. Additionally, MnLR preferred to be distributed to the liver, which is the most common metastatic site for CRC46, rather than other vital organs; and the enrichment of MnLR in the liver was also more significant than that of L. reuteri (Supplementary Fig. 27). Next, to determine whether the bioactivity-enhanced MnLR could promote glycerol metabolism and metabolic product accumulation within colon tumor sites in vivo, the orthotopic colon tumor-bearing mice received oral co-gavage with MnLR (or L. reuteri) and glycerol for 1 week followed by LC-MS/MS analysis on reuterin content within different tissues (Fig. 5i). Compared to L. reuteri/Gly treatment group, higher concentrations of reuterin were found within colon tumors, peripheral blood, and liver of the mice receiving MnLR/Gly treatment (Fig. 5j–l), suggesting an enhanced metabolic activity of MnLR in vivo. Of note, probably due to the poor bioavailability and rapid clearance of reuterin, significantly lower levels of reuterin were detected within colon tumors, peripheral blood, and liver of the mice receiving oral gavage of pure reuterin (Fig. 5j–l). Together, these observations demonstrate the critical role of Mn in facilitating the proliferation, colonization, and antitumor metabolite production of L. reuteri at colon tumor sites and in livers in vivo, which would be conducive to improved therapeutic efficacy in primary and even metastatic CRC.

The systemic biosafety of prolonged MnLR/Gly or reuterin treatments was assessed after 30 days of continuous oral administration (100 µl including 10 µl of glycerol and 5 × 10⁹ CFU of L. reuteri, once daily) followed by a 3-day withdrawal. Serum biochemical analyses (indicators for key organ functions, lipid profiles, and electrolytes) and histopathological examinations of major organs (heart, liver, spleen, lung, kidney, and intestine) showed no significant abnormalities compared with healthy controls (Supplementary Fig. 28a–f). Likewise, the levels of acute inflammatory cytokines in serum (IL-1β and TNF) returned to baseline after withdrawal, demonstrating the absence of persistent inflammatory responses (Supplementary Fig. 28g). Furthermore, manganese biodistribution analysis revealed that residual Mn levels in serum and major organs after one month of MnLR/Gly administration remained consistent with the physiological manganese concentrations in normal mice (Supplementary Fig. 28h), indicating efficient clearance and the absence of Mn accumulation. These findings demonstrate that MnLR/Gly and reuterin do not induce systemic toxicity and sustained inflammation, highlighting their potential value for clinical translation.

Oral delivery of MnLR with glycerol effectively inhibits orthotopic colon tumor growth and promotes antitumor macrophages, mature DCs, and effector T cells in mice
Next, we investigated the impacts of oral MnLR/Gly on primary tumor growth using the orthotopically transplanted colon tumor-bearing mice (Fig. 6a). Oral MnLR/Gly significantly suppressed the tumor growth by 95.6%, much higher than reuterin (74.6%), L. reuteri (58.4%), and L. reuteri/Gly (85%) (Fig. 6b, c and Supplementary Fig. 29). Additional dihydroethidium (DHE) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining revealed that MnLR/Gly or reuterin administration induced ROS generation and apoptosis in colon tumor tissues, whereas these signals were substantially attenuated in healthy colon tissues (Supplementary Fig. 30). These results further demonstrate in vivo that L. reuteri–derived metabolites exert ROS-mediated cytotoxicity selectively on colon tumor tissues, thereby contributing to the minimization of off-target damage to normal tissues consistent with the systemic biosafety confirmation (Supplementary Fig. 30).

To further evaluate the therapeutic potential of MnLR/Gly relative to established colon cancer therapies, we compared its efficacy with 5-fluorouracil (5-FU, a first-line chemotherapy drug), PD-1 antibody (a representative immune checkpoint inhibitor), and panitumumab (an EGFR monoclonal antibody) in the orthotopic colon tumor model. MnLR/Gly achieved a tumor growth inhibition rate 1.14-fold higher than PD-1 antibody, 1.25-fold higher than 5-FU, and 2.9-fold higher than panitumumab (Supplementary Fig. 31). Moreover, MnLR/Gly significantly extended overall survival, with 80% of mice surviving at least 60 days, in contrast to lower survival rates in the other groups (Fig. 6d). These advantages arise from MnLR/Gly’s potent antitumor activity, combined with its minimal systemic toxicity, and safe, convenient oral administration. In addition, MnLR/Gly holds promise for lower production costs compared to antibody- or chemotherapy-based regimens, highlighting its potential translational value.

Since MnLR/Gly has demonstrated functional regulation on macrophages and DCs in vitro, we hypothesized that it might similarly impact these tumor-associated immune cells and their downstream elements (such as effector T cells and their antitumor cytokines) in vivo, which could be a critical factor contributing to its enhanced antitumor efficacy. As expected, the activated M1 macrophages (F4/80+CD86+) and mature DCs (CD11c+CD86+) were significantly enriched in the tumor tissues of the mice treated with MnLR/Gly, concomitantly with an obvious reduction in M2 macrophages (F4/80+CD206+) (Fig. 6e, f). Furthermore, MnLR/Gly dramatically promoted the effector T cells activation and antitumor function, as evidenced by the substantially increased enrichment of IFN-γ-expressing CD4+ and CD8+ T cells within the tumor tissues of MnLR/Gly-treated mice, surpassing that observed in L. reuteri/Gly-treated mice (Fig. 6e, f). These observations suggest that MnLR/Gly actively remodels the tumor immune microenvironment into antitumor in vivo, thus potentiating inhibitory effects on primary colon tumors. We also analyzed the changes of immune cells in the peripheral immune organs, including the spleen and mesenteric lymph nodes (MLNs), which are critical for priming effector T cells and driving systemic antitumor immunity47,48. Only the spleens from the mice treated with MnLR/Gly exhibited a simultaneous increase in the ratio of M1 over M2 macrophages, mature DCs, and IFN-γ-expressing CD4+ and CD8+ T cells (Fig. 6g–j and Supplementary Fig. 32). Meanwhile, MnLR/Gly increased the presence of IFN-γ-expressing CD4+ and CD8+ T cells in MLNs (Fig. 6k, l and Supplementary Fig. 33). These data demonstrate the capability of oral MnLR/Gly to stimulate antitumor immune responses beyond local tumor microenvironment, which would be beneficial for preventing tumor occurrence and metastasis (see the following investigations)47,49. Of note, reuterin and L. reuteri elicited no significant effects on macrophages, DCs, and effector T cells within the tumor tissues (Fig. 6e, f), suggesting that their suppression of primary colon tumor growth is independent of the antitumor immunity mediated by these three immune cells. Additionally, depleting either CD8+ T cells or macrophages (Tdep. or Macdep.) just attenuated the tumor-inhibitory rate of MnLR/Gly to a level comparable to reuterin, rather than completely offsetting the antitumor effects (Fig. 6b, c and Supplementary Fig. 34). These observations collectively indicate a significant role of the direct cytotoxicity triggered by MnLR-produced metabolites (containing reuterin) in inhibiting primary tumors in vivo. Also, the antitumor immune responses elicited by MnLR/Gly are not confined to a single immune cell type and might even involve the participation of immune cells beyond T cells or macrophages. This might be particularly advantageous for treating tumor subtypes with deficiencies in specific immune cell populations.

We also analyzed the gut microbiota, an important factor influencing tumor growth and the immune microenvironment. Compared with stool samples from healthy mice, those from tumor-bearing mice displayed distinct microbial communities and reduced α-diversity (Fig. 6m and Supplementary Fig. 35a). MnLR/Gly treatment partially ameliorated the colon tumor-induced gut microbiota dysbiosis, concurrently enriching the bacterial genera with potential antitumor properties (including Lachnoclostridium50, Bacteroides51, and Lactobacillus52,53) (Supplementary Fig. 35b). Moreover, in orthotopic colon tumor-bearing mice pretreated with a cocktail of four broad-spectrum antibiotics to deplete gut microbiota, the inhibitory efficacy of MnLR/Gly on tumor growth was slightly attenuated though not completely abolished (Fig. 6n and Supplementary Fig. 36). These findings indicate that the antitumor effects of MnLR/Gly are mediated in part through gut microbiota modulation, in addition to its immunostimulatory and cytotoxic activities.

Oral delivery of MnLR with glycerol effectively prevents orthotopic primary colon tumor occurrence and liver metastasis in mice
Inspired by the aforementioned capability of MnLR/Gly to initiate systemic antitumor immunity, we subsequently investigated its potential effectiveness in preventing the occurrence of primary colon cancer and mitigating distant metastasis, which is crucial for substantially reducing cancer mortality rates. The mice were administered the given agents via oral gavage once daily for 10 consecutive days, followed by orthotopic inoculation of colon cancer cells and without further treatments (Fig. 7a). The pre-treatment with MnLR/Gly suppressed the formation of a primary colon tumor, resulting in the absence of visible tumor masses in the isolated colon tissues from MnLR/Gly-treated mice on day 14 post tumor inoculation, whereas tumors were developed in colons of the mice in the other treatment groups (Fig. 7b and Supplementary Fig. 37). When the observation period was extended, 75% (6/8) of MnLR/Gly-pretreated mice remained tumor-free for at least 8 weeks (Supplementary Fig. 38a). To further evaluate the durability of the preventive effect of MnLR/Gly, tumor rechallenge experiments were performed by re-inoculating colon cancer cells on day 56 after the initial inoculation in the mice that remained tumor-free following prophylactic treatment of MnLR/Gly (Fig. 7a). Strikingly, 62.5% (5/8) of MnLR/Gly-pretreated mice still exhibited no detectable tumors 3 weeks later (Fig. 7c and Supplementary Fig. 38b), indicating that MnLR/Gly confers a long-lasting protective effect against tumor recurrence. Additionally, the preventive efficacy of MnLR/Gly was validated in a genetic model of spontaneous intestinal tumorigenesis (Apcmin/+ mice) (Fig. 7d), where continuous oral administration of MnLR/Gly significantly reduced the intestinal tumor multiplicity and the Ki-67–positive proliferating epithelial cells compared with controls (Fig. 7e–h). Collectively, these findings indicate that MnLR/Gly provides durable protection against colorectal tumor development and recurrence, supporting its potential as a preventive strategy for CRC.

Next, we investigated whether the lasting antitumor effects of MnLR/Gly involved the enhanced systemic antitumor immunity. Indeed, MnLR/Gly promoted the splenic macrophage polarization towards M1 phenotype and DC maturation and concomitantly enriched the IFN-γ-expressing CD4+ and CD8+ T cells in MLNs (Fig. 7i–l and Supplementary Fig. 39). ELISA assays further revealed that MnLR/Gly treatment significantly elevated the levels of immune effector cytokines (IFN-γ, TNF, perforin, and granzyme) in serum and MLNs (Supplementary Fig. 40). Consistently, the depletion of CD8+ T cells or macrophages reduced the efficiency of MnLR/Gly in suppressing the formation of primary colon tumors (Fig. 7b and Supplementary Fig. 37). These findings collectively suggest that MnLR/Gly can simultaneously and functionally regulate multiple immune cells across the peripheral immune system in healthy individuals to generate a lasting antitumor effect for preventing colon tumor development.

Next, we determined the potential of MnLR/Gly for preventing colon tumor metastasis by developing a liver metastatic model via splenic injection of colon cancer cells (Fig. 7m). Compared to reuterin and L. reuteri/Gly that only reduced tumor burden in spleens, MnLR/Gly demonstrated high efficacy in preventing tumor metastasis into livers, as evidenced by a significant decrease in metastasized liver weight (decrease rate of 35.4%), numbers of metastatic nodules (decrease rate of 62.1%), and tumor invasion area (Fig. 7n–p and Supplementary Fig. 41). Depleting CD8+ T cells or macrophages resulted in reduced efficacy of MnLR/Gly in suppressing formation of liver metastatic nodules, highlighting the significance of antitumor immunity for preventing tumor metastasis (Fig. 7n–p).

Together, these results indicate that oral MnLR/Gly cannot only suppress primary colon tumor growth but also prevent primary colon tumor development and liver metastases by eliciting effective antitumor immunity, thus revealing MnLR’s clinical translation potential in preventing and treating CRC.

GlyMnLR capsule effectively inhibits primary colon tumor growth without eliciting systemic adverse side effects in rabbits
Since the viability of both raw L. reuteri and MnLR was lower in SGF than in SIF (Fig. 2n, o), and SEM imaging further revealed that Mn modification was less stable in SGF compared to SIF (Supplementary Fig. 42), we prepared a dimension-tunable enteric-soluble capsule for delivering MnLR encapsulated by glycerol (as one of the capsule materials) through 3D printing and casting technology to preserve MnLR bioactivity before reaching the therapeutic targets (Fig. 8a and Supplementary Fig. 43). In vitro and in vivo fluorescence imaging showed that the dissolution rate of the GlyMnLR capsule (MnLR labeled by Cy5.5) was significantly faster in simulated intestinal fluid (SIF) and real intestinal tract compared to simulated gastric fluid and real stomach (SGF) (Fig. 8b, d). This finding was corroborated by endoscopy examination (Supplementary Fig. 44) and X-ray imaging (Fig. 8c), which revealed that the capsule remained intact in the rabbit’s stomach for at least 4 hours after oral administration. After reaching the intestine, the barium signal of the capsule was weakened within 20 hours owing to the dissolution of the capsule in intestinal fluid (Fig. 8c). Consistently, in vivo quantitative analysis showed that only ~26.7% of MnLR was released within 4 hours in the stomach, whereas release markedly accelerated during gastrointestinal transit (4–10 hours) and intestinal residence (10–20 hours), with cumulative release reaching ~83.5% within 20 hours (Fig. 8d, e). These observations collectively demonstrated the capsule’s resistance to gastric juice and its solubility in the intestinal environment, ensuring sustained and targeted release of MnLR at the site of tumor growth. Next, the orthotopic colorectal tumor model was established in rabbits to investigate the GlyMnLR capsule’s antitumor efficacy (Fig. 8f). To optimize the dosing strategy of GlyMnLR capsules, we evaluated three regimens in the model: one capsule every other day, one capsule once daily, and two capsules once daily. Both once-daily regimens achieved stronger tumor suppression than one capsule every other day (approximately 3-fold higher), while the efficacy difference between one and two capsules daily was minimal (Supplementary Fig. 45), with the latter posing greater metabolic burden and safety concerns. The tumor-suppressive efficacy of once-daily oral administration of one GlyMnLR was further confirmed by in vivo CT imaging and photographs of the isolated tumors at the end of the experiments (Fig. 8g–i). Accordingly, we selected one capsule once daily as the optimal dosing regimen for subsequent therapeutic studies. GlyMnLR capsules exhibited a markedly higher tumor inhibition rate (67.9%) compared with free MnLR (25.4%) and reuterin capsules (8.0%), demonstrating the protective role of the enteric formulation and the advantage of sustained production of active metabolites by viable bacteria (Fig. 8j, k). Also, the antitumor efficacy of GlyMnLR capsules was slightly superior to that of the clinically used chemotherapeutic agent 5-FU (Fig. 8j, k), which is consistent with the previous findings in mouse colon tumor models. Immunofluorescence for Ki67 further confirmed the superior inhibitory effect of the GlyMnLR capsule on tumor cell proliferation (Fig. 8l). In addition, GlyMnLR capsules promoted the enrichment of CD8⁺ T cells in the tumor microenvironment (Fig. 8m, n), supporting the induction of antitumor immunity in the rabbit CRC model. Together with the favorable biosafety profile demonstrated in the rabbit model (Supplementary Fig. 46), these results further support the potential of MnLR to achieve robust antitumor activity while maintaining low toxicity.

Methods
Humansubjects
All procedures involving human participants were approved by the Ethics Committee of Wuhan Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (IORG No. IORG0003571, 2020-S197). Written informed consent was obtained from all participants prior to their inclusion in the study.

Bacterialandcellularculture
L.reuteri strain (ATCC 23272, GDMCC) was cultured in a sealed centrifuge tube containing de Man, Rogosa, and Sharpe (MRS) broth (HB0384, Hopebio, China) or in a carbon dioxide-generating bag containing MRS agar medium to maintain a microaerophilic environment at 37°C.
Murine colon cancer cells MC38 (CL-0972, Procell, China) and MC38-Luc (YC-A002-Luc-P, Ubigene, China), human colon cancer cells HCT116 (CL-0096), SW48 (CL-0513), RKO (CL-0196), SW620 (CL-0225), DLD-1 (CL-0074) and HT-29 (CL-0118) (Procell, China), human immortalized colonic epithelial cells (NCM460, H1-3701, OriCell, China), human umbilical vein endothelial cells (HUVEC, CP-H082, Procell), human liver cells (L-02, H1-2501, OriCell), and rabbit squamous carcinoma cells (VX2, BFN60700420, BLUEFBIO) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (C11995500BT, Gibco, USA) containing 10% (v/v) fetal bovine serum (FBS) (10099-141, Gibco, USA). Murine colon cancer cells (CT26, CL-0071, Procell) and macrophage-like cells (RAW264.7, CL-0190, Procell) were cultured in Rosewell Park Memorial Institute (RPMI) 1640 medium (A1049101, Gibco, USA) containing 10% (v/v) FBS. DCs generated from C57BL/6 mouse bone marrows according to our previous work44 were cultured in RPMI 1640 media containing 10% (v/v) FBS with the supplement of GM-CSF (20 ng/ml) (300-03, PeproTech, USA) and IL-4 (10 ng/ml) (200-04, PeproTech, USA). Peripheral blood mononuclear cells (PBMC), isolated from healthy donors according to the Human PBMC Isolation Kit (071A100.11, IPHASE, China), were cultured in RPMI 1640 media containing 10% (v/v) FBS. All cells were placed in a humidified incubator with 5% CO2 at 37°C.

AnalysisofL.reuteriproliferation,glycerolmetabolism,andbiofilmformation
L.reuteri was cultured in glycerol solution (500 mM in PBS) containing the given supplements under the microaerophilic environment at 37°C. After a 4-hour incubation, L. reuteri was collected by centrifugation (4000 g, 5 minutes) and resuspended in PBS. The concentration of reuterin in the supernatant was detected by a tryptophan colorimetric method according to previous studies69. The GDHt activity of L. reuteri was determined using 3-methyl-2-benzothiazoline hydrazone (H693567, Aladdin, China)70. For bacterial proliferation assessment, L. reuteri was centrifugally collected and resuspended by PBS after 18-hour incubation, following absorbance measurement at 600 nm by a multimode reader (RF-5301PC, Japan).
To investigate the bacterial biofilm formation and bio-adhesion, L. reuteri in MRS broth (OD600=0.8, 500 µl) was seeded in 48-well plates and incubated at 37°C for 24 hours. The broth was then removed followed by adding 0.5% (w/v) crystal violet (50 µl) to visualize the biofilm production. For bio-adhesion capability assessment, HT-29 cells were seeded in 6-well plates (5 × 105 in 1 ml culture media per well) and cultured for 24 hours. The cells were then washed with HANK’s buffer after the removal of culture media. Fresh culture media (2 ml) containing L. reuteri or MnLR (107 CFU/ml) was added to each well and incubated for 2 hours. After the removal of unattached L. reuteri or MnLR in culture media, the cells with attached L. reuteri or MnLR were washed with HANK’s buffer, digested with trypsin, suspended in PBS, and spread on MRS plates at 105 dilutions. L. reuteri colonies were counted on the next day. The adhesion rate was calculated as the percentage of L. reuteri attached to the cells in the initial total L. reuteri number.

FabricationandcharacterizationofMnLR
L.reuteri was amplified in MRS broth overnight, washed, and resuspended by PBS at a density of ~1010 CFU/ml. MnCl2 (100 µl, 1.25 mg/ml) was added to the bacterial suspension (500 µl) and swirled for 30 seconds. TA (100 µl, 5 mg/ml) and PBS (300 µl) were added to the mixture and vortexed for 30 seconds. After a 30-minute standing at 37°C, MnLR was obtained by centrifugation (4,000 g, 3 minutes). Morphology and element mapping were observed under a biological TEM (FEI Tecnai G2 F20, USA). The presence of Mn and TA within MnLR was determined by XPS (Thermo Scientific K-Alpha, USA) and fluorescence imaging (Nikon Ti-U, Japan).

EvaluationofthestabilityandretentionofMnLR
L.reuteri, cultured overnight in MRS broth, was resuspended in PBS to a density of OD600 of 1.0 and modified with Mn2+ to generate MnLR. MnLR or unmodified L. reuteri was incubated in simulated gastric fluid (SGF) or simulated intestinal fluid (SIF) at 37°C for 2 or 4 hours. The bacterial solution was then diluted 4 × 104 times, and 50 µl was evenly spread on an MRS agar medium. Colonies were counted after an anaerobic incubation at 37°C for 24 hours.

Invitrocytotoxicityassay
To investigate the cytotoxicity of metabolites, NCM460, HUVEC, L-02, HCT116, or MC38 were seeded in 96-well plates (5 × 103 cells in 100 µl culture media per well). After a 24-hour incubation, the media were replaced with fresh media containing the given agents. After a 24-hour treatment, the viabilities of these cells were detected by CCK8 assay according to the manufacturer’s instructions. To assess the cytotoxicity of MnLR/Gly, NCM460, HUVEC, L-02, CT26, MC38, HCT116, SW48, RKO, SW620, or DLD-1 cells were seeded in the lower chamber of transwell plates (aperture 0.4 µm) (2 × 105 cells 1 ml culture media per well) and cultured for 24 hours. Subsequently, fresh media (0.5 ml) containing L. reuteri (109 CFU), glycerol (5 µl), L. reuteri (109 CFU)/Gly (5 µl), or MnLR (109 CFU)/Gly (5 µl) were added to the upper chamber. The viabilities of colon cancer cells in the lower chamber were detected by CCK8 assay after a 24-hour incubation.

Colony-formingassay
HCT116 or MC38 cells were seeded in 12-well plates (500 cells in 1 ml culture media per well) and cultured for 48 hours. The cells were treated with the metabolites at the given concentrations for 24 hours, followed by culture medium renewal every 3 days. Colony-forming assays were performed on day 10 (HCT116) or day 15 (MC38 cells) after cell seeding by fixing with 4% paraformaldehyde for 10 minutes, staining with 0.1% crystal violet for 30 minutes, and imaging with an Olympus U-RFL-T microscope (Japan).

Organoidgrowthassay
Organoids derived from 5 independent CRC patients were prepared according to our previous work71. Organoids were embedded in 50 µl of Matrigel (CLS354263, Corning, USA) and cultured with 500 µl of modified medium (PRS-ICM-3D, PRECEDO, China) in 24-well plates. Reuterin (1.5 µg/ml; H916417, Macklin, China) or mixed glycerol metabolites (containing 1.5 µg/ml reuterin) were then added to the plates and incubated for 3 days. Organoids’ morphology and size were recorded daily. At the experimental endpoint, organoids were stained with a Live/Dead Cell Staining Kit (C1070, Beyotime Biotechnology, China) and imaged with an Olympus U-RFL-T microscope (Japan).

ROSgenerationandICDinductionincoloncancercells
MC38 cells were seeded in the lower chamber of transwell plates (2 × 105 cells in 1 ml culture media per well) and cultured for 24 hours. Fresh media (0.5 ml) containing pure reuterin (18.25 µg/ml) or MnLR (109 CFU)/Gly (5 µl) were added to the upper chamber. After a 12-hour incubation, cells were stained with DCFH-DA (Beyotime Biotechnology, China) and imaged with an Olympus U-RFL-T microscope (Japan) to determine intracellular ROS levels. For visualization and location of CRT, cells were stained with anti-CRT/FITC (bs-5913R-FITC, Bioss, China) for 30 minutes at 4 °C. The cells were subsequently stained with Dil membrane dye (S0033, Beyotime Biotechnology, China) for 15 minutes and Hoechst 33342 nuclear dye (14533, Sigma-Aldrich, USA) for another 15 minutes after fixation with 4% paraformaldehyde. Finally, the stained cells were observed under a Nikon Ti-U microscope (Japan) equipped with a CSU-X1 spinning-disk confocal unit (Yokogawa) and an EM-CCD camera (iXon+; Andor).

DCsmaturation
To investigate the DCs maturation stimulated by colon cancer cells undergoing ICD, MC38 or DLD-1 cells were treated with pure reuterin or MnLR/Gly as mentioned above. Subsequently, the cells together with the supernatants in each well were collected and repeatedly frozen-thawed for rapidly lysing cells. The obtained MC38 or DLD-1 cell lysates were then added to the 12-well plates, where murine bone marrow-derived DCs (mBMDCs) or human PBMC-DCs (hPBMC-DCs) were seeded. After a 24-hour incubation, the DCs in each well were collected for the following analysis. To determine the DCs maturation stimulated by pure reuterin or MnLR/Gly, mBMDCs or hPBMC-DCs were seeded in the lower chamber of transwell plates. Reuterin (6.25 µg/ml) or MnLR (109 CFU)/Gly (1 µl) was then added to the upper chamber to stimulate DCs for 24 hours following cell collection and analysis. The percentage of mature DCs in the total DCs collected was determined by flow cytometry analysis (Canto II, BD Company, USA) using the specific antibodies, including CD45-APC/CY 7 (30-F11, 103115), CD11c-BV421 (N418, 117329), and CD86-PE (A17199A, 159204) (BioLegend, USA) for mBMDCs, and CD45-APC/CY 7 (2D1, 368516), CD11c-APC (3.9, 301614), HLA-DR-PerCP/CY 5.5 (L243, 307629), and CD83-PE (HB15e, 305308) for hPBMC-DCs. The Gating strategies were shown in Supplementary Fig. 47. Functional cytokines IL-12 (ELK2507, ELK2263) and TNF (ELK1387, ELK1190) were measured using the ELISA Kits (ELK Biotechnology, China).

Macrophagepolarization
RAW264.7 cells were seeded in the lower chamber of transwell plates (2 × 105 cells in 1 ml culture media per well). M1 and M2 macrophage polarizations were stimulated by LPS (100 ng/ml)/IFN-γ (20 ng/ml) and IL-4 (20 ng/ml), respectively. The hPBMCs were stimulated by recombinant human M-CSF (20 ng/ml) for 6 days to obtain hPBMC-macrophages. After a 24-hour initial stimulation, reuterin (6.25 µg/ml) or MnLR (109 CFU)/Gly (1 µl) was added to the upper chamber to further stimulate the macrophages. After 24 hours, the macrophages in each well were collected for subsequent analyses. The percentages of M1 and M2 macrophages in the total RAW264.7 cells macrophages collected were determined by flow cytometry analysis using CD86-PE (A17199A, 159204) and CD206-PE (C068C2, 141706) antibodies (BioLegend, USA), respectively. The M1 (CD86+) to M2 (CD206+) ratio of hPBMC-macrophages was analyzed by flow cytometry using CD45-APC/CY 7 (2D1, 368516), CD68-BV421 (Y1/82A, 333827), CD86-PE (BU63, 374206), and CD206-APC (15-2, 321110) antibodies (BioLegend, USA). The Gating strategies were shown in Supplementary Fig. 47. The expression of M1-related markers (IL-1β, IL-6, TNF, and iNOS) and M2-related markers (Arg-1, CD206, and Mgl2) was detected by qRT-PCR according to our previous work44. The related primer sequences were summarized in the Supplementary Table 3. Functional cytokines (IL-6 and TNF) were measured using the ELISA Kits (ELK Biotechnology, China).

MCTsgrowthassay
MC38 cells, either alone or mixed with RAW264.7 cells at a 7:3 ratio, were seeded in ultralow-attachment 96-well plates (Corning, USA) (5 × 103 cells in 100 µl culture media per well) and cultured for 48 hours. The suspended MCTs were then harvested and added to the lower chamber of ultralow-attachment 24-transwell plates (Corning, USA). Cytokines (LPS/IFN-γ or IL-4) were supplemented to the culture media with or without the addition of MnLR/Gly to the upper chamber. The culture media were refreshed every two days. The MCTs were imaged at the given time points under an Olympus U-RFL-T microscope (Japan). Their volumes were calculated using the formula: major axis × (minor axis)2/2.

Phagocytosisassayoftumorcellsbymacrophages
Human PBMC-derived macrophages were stimulated by MnLR/Gly for 24 hours in the 12-transwell plates. DLD-1 cells were labeled with Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) using the CFDA-SE Cell Proliferation and Tracking Kit (C0051, Beyotime, China) according to the manufacturer’s instructions. Subsequently, stimulated or unstimulated macrophages were harvested and mixed with CFDA-SE–labeled DLD-1 cells at a 1:2 ratio, followed by co-culture in 24-well plates for 6 hours. After incubation, cells were gently resuspended, stained with CD11b-BV421 (LM2, 393114, BioLegend, USA) for 30 minutes, washed with PBS, and analyzed by flow cytometry to assess tumor cell phagocytosis. The Gating strategies were shown in Supplementary Fig. 47d.

RNA-seqandpathwayanalyses
Total RNAs were isolated from DCs or RAW264.7 cells with the given treatments using a TRIzol Reagent Kit (Invitrogen, USA). The RNA quality was evaluated by RNA integrity number (RIN) which above 6 was qualified. Genes with a |log2(fold change)| > 1 and an adjusted p value < 0.05 were determined to be DEGs. Pathway (e.g., environmental information processing, cellular processes, metabolism, and organismal systems pathway) enrichment analysis was performed with the Kyoto Encyclopedia of Genes and Genomes biological database. The impact of specific signaling pathways, including MAPK p38 and HIF-1, on DC maturation and macrophage polarization was further investigated through qRT-PCR, Western blot, and flow cytometry analyses. The expression of genes related to MAPK p38 and HIF-1 signaling pathways in DCs and macrophages was detected by qRT-PCR (the related primer sequences were summarized in Supplementary Table 3). The protein levels of p38, p-p38, and HIF-1α were determined by western blotting using SAPK4 (E-3) (1:1000, sc-271292, Santa Cruz Biotechnology, USA), Phospho-p38 MAPK-T180/Y182 pAb (1:2000, AP0526, ABclonal Biotechnology, China), and HIF-1α (28b) (1:1000, sc-13515, Santa Cruz Biotechnology, USA) antibodies, respectively. Scans of blots were supplied in the Source Data files. The percentages of mature DCs (mBMDCs and hPBMC-DCs) and M1/M2 macrophages (RAW264.7 and hPBMC-macrophages) were determined by flow cytometry analysis as mentioned above. AMG548 (4100 nM; 52952ES08, YEASEN, China) and YC-1 (10 µM; HY-14927, MedChem-Express, China) were applied to inhibit MAPK p38 and HIF-1 signaling pathways, respectively.

Animals
Specific pathogen-free (SPF) male C57BL/6J mice (stock #000664) (6-8 weeks old) and conventional male New Zealand white rabbits (NZW) (5-8 weeks old) were obtained from the Laboratory Animal Center at Huazhong University of Science and Technology in Wuhan, China. SPF female transgenic Apcmin/+ mice (B6/JGpt-Apcem1Cin(Min)/Gpt) (stock # 002020) (8 weeks old) were bought from Shulaibao (Wuhan) Biotechnology Co., Ltd. The experimental/control animals were bred separately with a 12-hour light/dark cycle, 22°C temperature, and 50% humidity. All animal experiments, including mice and rabbits, were approved by the ethics committee of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China (IACUC Number: 2846; 3706). For the subcutaneous tumor, the tumor volume does not exceed 2000 mm³. For the primary colon tumor, tumor volumes are not directly measured, as tumors are intra-abdominal. Animals are evaluated regularly, and the humane endpoints are defined by a loss of 20% body weight, signs of distress (facial grimace, hunch), difficult breathing, or lackluster hair.

Analysesofantitumoractivityandimmunecellenrichmentina subcutaneouscolontumor-bearingmousemodel
Male C57BL/6 mice were subcutaneously injected with MC38 cell suspension (106 cells in 100 µl PBS per mouse) in the right infra-axillary dermis. When the tumor volume reached approximately 50 mm3, the mice were randomly divided into five groups and intratumorally or orally gavage with PBS, pure reuterin (100 µg per mouse), or mixed metabolite (containing 100 µg reuterin per mouse) once daily. The tumor volumes (calculated by 1/2 × length × width2) were recorded every 2 days. At the experimental endpoint and the tumor volume of mice did not exceed 2000 mm³, the mice were euthanized by intraperitoneal injection of an overdose of pentobarbital sodium. Tumors in each mouse were isolated for weighing, photographing, and immunofluorescent staining with CD8-CY3.5 (1:500, ab209775, ABclonal Biotechnology, China), CD11c-CY 3.5 (1:200, 97585S, Cell Signaling Technology, USA), and F4/80-CY 3.5 (1:200, 70076, Cell Signaling Technology, USA). The immune cells in the tumor microenvironment were observed under a Pannoramic MIDI (3DHISTECH, Hungary).

DistributionofL.reuteriandreuterininprimarycolontumor-bearingmousemodel
The primary colon tumor-bearing mouse model was established by injecting MC38 or MC38-Luc cells (3 × 106 cells in 100 µl PBS per mouse) into the cecal cavity. CY5.5 dye was used for visualizing L. reuteri in vivo. Specifically, NHS-CY5.5 (50 µg; Q-0363099, Qiyue Biotechnology, China) was added to the L. reuteri suspension (1010 CFU/ml in 10 ml PBS) and incubated at 37 °C for 15 minutes. L. reuteri was then washed with PBS to remove the free dye. CY5.5-labeled L. reuteri was modified with Mn using the same method described in the “Fabrication and characterization of MnLR” section. One week after tumor inoculation, CY 5.5-labeled L. reuteri or MnLR (109 CFU in 100 µl PBS) was administered to both tumor-bearing and healthy mice. After 1, 6, or 24 hours, the entire GI tracts and other internal organs were isolated and imaged with an in vivo imaging system (IVIS) (BRUKER In-Vivo FX PRO, USA). Subsequent fluorescence quantitative analysis was conducted with the supporting software Bruker MI SE. To investigate the spatial distribution of bacteria within colon tumor tissues and adjacent intestinal tissues, mice were euthanized 24 hours after oral gavage. The isolated tissues were stained with mouse anti-MUC2-FITC (1:100, sc-7314, Santa Cruz Biotechnology, USA) for visualizing the intestinal mucin layer. Quantification of stool L. reuteri was achieved through the utilization of qPCR to identify the pdu cluster encoding GDHt72. Total bacterial DNA was extracted from the stool samples using a FastDNA Stool Mini kit (51604, QIAGEN, USA). The qPCR amplification was performed by One Step SYBR PrimeScript PLUS Kit (Takara, Japan) on Applied Biosystem StepOne Plus (USA). The pdu primer sequences were forward 5’-CCTGAAGTAAAYCGCATCTT-3’ and reverse 5’-GAAACYATTTCAGTTTACGG-3’. The copy number of crude DNA templates in the gut was determined by the correlation of the copy number of serially diluted plasmid DNA standards with cycle threshold run on the same procedure. Quantitative results were expressed as log10 gene copies in 1 ng stool DNA (log10 copies/ng).
To determine the reuterin production and distribution, primary colon tumor-bearing mice were orally gavage with PBS (100 µl per mouse), pure reuterin (100 µg per mouse), L. reuteri/Gly, or MnLR/Gly (5 × 109 CFU and 10 µl glycerol per mouse) once daily. One week later, tumor tissues, blood serum, and livers were collected and examined by LC-MS/MS (UltiMate 3000 UHPLC UHPLC/Q Exactive, Thermo Fisher Scientific, USA) for quantifying reuterin.

Thebiosafetyevaluationinmice
For systemic toxicity and inflammatory response assessments, the normal mice received continuous oral administration of MnLR/Gly (100 µl, including 10 µl of glycerol and 5 × 10⁹ CFU of L. reuteri, once daily) or reuterin (100 µg, once daily) for 30 days. After a 3-day withdrawal, orbital blood and major organs (heart, liver, spleen, lung, kidney, and intestine) were isolated. Blood samples were collected for biochemical analysis of liver, heart, and kidney function, electrolytes, and lipids with a fully automatic biochemical analyzer (Catalyst One, IDEXX, China), as well as the detection of inflammatory cytokines by ELISA Kits (ELK Biotechnology, China). The isolated organs were fixed with 4% paraformaldehyde, embedded in paraffin, and sliced for haematoxylin and eosin (HE) staining. Residual Mn levels in organs and blood were measured by ICP-OES (Agilent 5800 ICP-OES, Malaysia).

Antitumoractivity,gutmicrobiota,andantitumorimmunityassessmentsinprimarycolontumor-bearingmousemodel
The primary colon tumor-bearing model was established as described above. On the second day after tumor inoculation, mice were orally gavage with the given agents (dosage per mouse: PBS, 100 µl; Glycerol, 10 µl; reuterin, 100 µg; L. reuteri/MnLR, 5 × 109 CFU) once daily. To deplete CD8T cells or macrophages, the tumor-bearing mice were intraperitoneally injected with anti-CD8 (200 µg per mouse; clone 2.43; A2102, Selleck, China) or clodronate-encapsulated liposomes (200 µl per mouse; LIPOSOMA, Netherlands) on days 6, 2, and 1 before tumor inoculation, and on days 4, 8, and 12 following tumor inoculation. For monitoring tumor growth by bioluminescence imaging, the mice (inoculated with MC38-Luc cells) in each group were intraperitoneally injected with D-Luciferin potassium salt (150 mg/kg mouse weight; Y023738, Beyotime Biotechnology, China) and imaged by IVIS. At the experimental endpoint (day 14 after tumor inoculation) and the weight loss did not exceed 20% according to humane endpoint criteria, the mice were euthanized and intestines were isolated from the mice in each group for observing and photographing tumors. Additionally, stool samples were collected for 16S rRNA sequencing analysis to reveal gut microorganisms (OE Biotech, China). Gut microbiota depletion was induced by administering an antibiotic cocktail (consisting of ampicillin (A433389), neomycin (N109017), and metronidazole (M109874) (0.2 g/L each) together with vancomycin (0.1 g/L, V301569) (Aladdin, China)) in the drinking water for two weeks prior to tumor cell inoculation.
To compare MnLR/Gly with current standard-of-care treatments, the orthotopic colon tumor-bearing mice were treated with MnLR/Gly daily as mentioned above or (1) PD-1 antibody (RMP1-14; A2122, Selleck, China; 100 µg once every three days, i.p.), (2) 5-FU (F476739, Aladdin, China; 400 µg every other day, i.p.), and (3) Panitumumab (A2018, Selleck, China; 200 µg once every three days, i.p.). The tumor growth was recorded weekly, and mouse survival was evaluated using Kaplan-Meier analysis until spontaneous death occurred or the end of the 2-month observation period.
To assess the antitumor immunity, colon tumor tissues, spleens, and MLNs were isolated from the mice in each group on day 14 after tumor inoculation. The tumor tissues were fixed with 4% paraformaldehyde and stained with the specific antibodies, including F4/80-FITC (1:1000, 70076), CD86-CY 5 (19589), CD11c-FITC (1:1000, 97585S), CD206-CY 5 (1:1000, 24595) (Cell Signaling Technology, USA), IFN-γ-FITC (1:400, Df6045, Affinity Biosciences, China), CD4-CY 5 (1:400, Ab183685, Abcam, China), and CD8-CY 5 (1:4000, Ab209775, Abcam, China). Spleens and MLNs were dissociated into single-cell suspensions, stained with antibodies, and then washed and resuspended with PBS. Collected cell samples were analyzed by flow cytometry. The following antibodies were used: CD45-BV510 (30-F11), CD11c-PE/CY 7 (N418), CD86-APC (GL-1), CD11b-FITC (M1/70), F480-BV421 (BM8), CD206-PE (C068C2), CD3-APC (17A2), CD4-FITC (RM4-5), CD8-Percp/CY5.5 (53-6.7), and IFN-γ-PE (XMG1.2) (BioLegend, USA). The Gating strategies were shown in Supplementary Fig. 47.

Evaluationofthepreventiveeffectsonprimarycolontumoroccurrenceandlivermetastasisinmousemodel
To evaluate the preventive effects on primary colon tumor occurrence, mice were orally administered the given agents once daily. 10 days later, the mice ceased oral medication and received the injection of MC38-Luc cells (3 × 106 cells in 100 µl PBS per mouse) into the cecal cavity, followed by tumor growth monitoring. On day 14 after tumor inoculation, the intestines were isolated from the mice for observing and photographing tumors. The agent dosage, methods for depleting CD8T cells or macrophages, tumor growth monitoring approach with IVIS, and the procedures for analyzing immune cells in spleens and MLNs using flow cytometry have been described in detail in the previous section. The levels of perforin (ELK3068), granzyme (ELK1660), TNF, and IFN-γ (ELK1132) in both serum and MLNs were measured by ELISA Kits (ELK Biotechnology, China).
For the tumor rechallenge study, healthy mice were first pre-treated with MnLR/Gly by oral gavage for 10 consecutive days, followed by orthotopic inoculation with MC38 cells. MnLR/Gly administration was discontinued thereafter. At week 8 post-initial inoculation, tumor growth was evaluated, and mice that remained tumor-free were subjected to a second orthotopic MC38 cell inoculation. Age-matched mice receiving tumor inoculation at the same time point served as controls. Tumor growth was visualized and quantified by IVIS after 3 weeks. For the Apcmin/+ transgenic mouse model, 8-week-old mice were randomly divided into 4 groups and orally administered with the given agents daily. After 9 weeks of treatment, mice were euthanized, and intestinal tissues were harvested for HE staining and Ki67 (1:200, 9129, Cell Signaling Technology, USA) immunohistochemical analysis.
To investigate the inhibitory effects on liver metastasis of colon cancer, mice received a splenic injection of MC38 cells (5 × 105 cells in 100 µl PBS per mouse). Swabs were employed to massage the spleen for 5 minutes to accelerate cell flow into the hepatic portal vein. On the second day after tumor inoculation, mice were orally gavaged with the given agents. The agent dosage and methods for depleting CD8T cells or macrophages were the same as mentioned above. On day 14 after tumor inoculation, spleens and livers were harvested from the mice in each group for weighing, photographing, counting of tumor nodules, and histological analysis.

FabricationofGlyMnLRcapsules
The rod-shaped master molds (with a length of 10 mm) for the capsule’s outer (4 mm in diameter) and inner (3 mm in diameter) shells were designed with the Solidworks 2021 software and printed in an X190 3D printer (JGAURORA, China) using polylactic acid (PLA) (Jiguang Erwo Technology, China) as the raw materials. The negative molds were fabricated from the master molds using polydimethylsiloxane (PDMS) (PHR1518, Sigma-Aldrich, USA). The precursor solution for capsule shells was prepared by mixing glycerol (2 ml) and gelatin (2 g) in ddH2O (4 ml). The mixture was placed in an oven at 75°C for approximately 30 minutes until the gelatin was fully dissolved. Genipin (2% w/v; G101204 Aladdin, China) was added to the mixture that was subsequently filled into the negative PDMS molds. The rod-shaped PLA molds with diameters of 2 mm (for outer shell) or 1 mm (for inner shell) produced by 3D printing were vertically inserted into the mixture and left for 5 minutes to form the capsule cavity. The gel-like capsule shell precursors fully solidified after being placed at 37°C for 2 days. The negative molds were then removed to obtain the outer capsule shell with a diameter of 3.5 mm and the inner capsule shell with a diameter of 2.5 mm. The outer and inner shells were cut to lengths of 5 mm, and following joined together to form the complete capsule with a length of 8 mm. The enteric-coated polymer was prepared by dissolving 200 mg of Eudragit L-100 (P756765, Macklin, China) in an organic solvent mixture containing 1 ml of dichloromethane (Sinopharm, China) and 1 ml of anhydrous ethanol (Sinopharm, China), followed by stirring until the solution became transparent. The polymer solution was then cast to the capsule’s surface for 3 times. The coated capsules were left to dry at 37°C for 24 hours, resulting in the formation of enteric-coated capsules. MnLR (1011 CFU in 20 µl PBS) was slowly injected into the capsule using a syringe to obtain the final MnLR. This specific size of GlyMnLR capsules was used in subsequent in vitro and in vivo experiments. Capsule shells of other sizes presented in Fig. 8b were prepared using the same method.

MonitoringofGlyMnLRcapsuledissolutionandL.reuteri releaseintheGItract
To observe the capsule dissolution and L. reuteri release in vitro, capsules loaded with CY5.5-labeled MnLR were immersed into the SGF (pH 2.0, containing 0.01 M HCl and 3.2 g/l pepsin) or SIF (pH 6.8, containing 0.01 M PBS, 0.2 M NaOH, and 10 g/l trypsin) and imaged by IVIS. To further determine GlyMnLR capsules’ resistance against gastric fluid in vivo, they were administered to the throats of rabbits that had been fasted for 2 days to assist with active swallowing, followed by endoscopic observation (Endoscope, Yuyanbio, China). Finally, to demonstrate that the capsules can dissolve in intestinal fluids, the rabbits were orally administered capsules containing barium sulfate for abdominal radiographic imaging using an X-ray photographic system (Mindray, China). For Quantitative analysis of capsule dissolution kinetics and MnLR release in vivo, capsules loaded with Cy5.5-labeled MnLR were administered to rabbits by oral gavage. Four experimental groups were euthanized at designated time points: immediately (0 h), 4 h, 10 h, and 20 h post-gavage. Rabbits were euthanized by first inducing deep anesthesia with isoflurane inhalation, followed by an intravenous injection of an overdose of pentobarbital sodium through the auricular vein. In the first group (0 h), the capsule was immediately retrieved from the stomach and imaged to determine the initial fluorescence intensity (Intensity0). In the second group (4 h), the capsule was recovered from the stomach, and the remaining fluorescence (Intensity4) was measured; the cumulative release at 4 h was calculated as (Intensity0 – Intensity4)/Intensity0 × 100%. In the third and fourth groups, capsules retrieved from the intestinal tract at 10 h and 20 h were similarly imaged, and cumulative release fractions were computed. Fluorescent imaging was performed using IVIS to quantify residual capsule signal in situ.

AntitumoractivityandbiosafetyassessmentsintheCRC rabbitmodel
To establish the primary colon cancer model in rabbits, VX2 cell suspension (2 × 108 cells in 200 µl PBS) was inoculated into the thigh muscle of 5-week-old rabbits. After 4 weeks, the resulting solid tumors in the muscles were excised and minced with surgical scissors in a petri dish. The tumor tissues were then transferred to a homogenizer and processed by grinding no more than five times. The homogenized tissues were suspended in sterile PBS, centrifuged at 4°C (2000 g, 5 minutes), washed twice, resuspended by PBS, and injected into the colon wall (approximately 5 cm below the cecum along the median abdominal line) of 6-8-week-old rabbits (500 µl tumor suspension per rabbit). Two days later, the tumor-bearing rabbits were administered with the given agents for one month: (1) PBS, once daily (o.g.), (2) GlyMnLR, one capsule every other day, one capsule once daily, or two capsules once daily (o.g.), (3) free MnLR/Gly, once daily (o.g.), (4) one reuterin (2 mg) capsule, once daily (o.g.), and (5) 5-FU, 20 mg/kg every three days (i.p.). Tumor progression was monitored at designated time points by CT imaging (SOMATOM, Siemens, Germany) and endoscopic examination (YAN-E30, YUYAN INSTRUMENTS, China). On day 31 and the weight loss did not exceed 20% according to humane endpoint criteria, the rabbits were euthanized and tumors in each group were finally isolated for ex vivo imaging, Ki67 staining, and immunofluorescence staining with rabbit-specific antibodies against CD45 (1:1000, L12/201, LS-C187600) and CD8 (1:200, 12.C7, LS-C187471) (LSBio, USA).
To assess the systemic toxicity, tumor-free rabbits (6-8 weeks old) received the GlyMnLR capsule once daily for one month. Blood samples were then collected and biochemically analyzed in the same way as in the previous section.

Statisticalinformation
All experiments were performed at least 3 times. Data were presented as mean ± standard deviation (SD). Statistical analyses were conducted using Student’s t-test for comparisons between two independent groups and one-way ANOVA with Tukey’s post-hoc tests for comparisons among three or more independent groups. Statistical significance was indicated by the p-value.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The concept of preventing and treat cancer with probiotics is, of course, extremely fascinating and in case of CRC prevention also obvious. Thus, the current manuscript is of high potnetial relevance for the field. Also the concept of chemically modify/improve known probiotics instead of genetically modify them is pretty clever.\nHowever, in its current form, the study of Cao and colleauges has some major points that need to be adressed to improve the scientific quality and, most of all, the potential relevance for patients and society.\nThe major problem for this reviewer is a lack of data obtained from human cells. Since the target species will be neither mouse nor rabbit, the absolute majority of in vitro data should be obtained by using human cells. Thus, more CRC cell models must be tested in vitro. The presented data are based on measurements by SW48 and HCT116. Both are highly-passaged CRC models - and both are microsatellite-instable and this fact could be a potential bias since no further molecular subtypes have been tested. It would be superior to add further data from patient-derived models in low passages and from different molecular background.\nSome additional data were presented from PDO - but it became not absolutely clear, if this was just one single model or more. Here, more PDO models from different patients are necessary.\nIn line with this argumentation, the in vitro immune cell tests presented should be done with immune cells from healthy donors and - at best - from patients!\nAnothe major issue is the choice of animal model(s) and the conclusions drawn from the experiments done. With none of the mouse models chosen, the prevention of primary CRC can be examined. For that kind of conclusion, a genetic model would be the right choice. Albeit this might not be transferrable to the human situation. Similarly, the splenic injection model is no longer acceptable as a model of liver metastasis in biological terms of the metastatatic procedure. All effects presented in the manuscript can be explained by direct or indirect effect on the CRC cells used - maybe in combination of such effects on immune cells. Finally, the rabbit model might be fine for the stability measurements of the GlyMnLR capsules, but the CRC part adds no relevant information.\nMinor comments:\n- English has to be improved in the neglcted parts of the manuscript: materials and methods, as well as results.\n- some data are presented in different styles (for example flow cytometry in Fig. 3) - should be the same.\n- some of the schemes used to explain experimental procedures are not really helping and can be omitted (again Fig. 3)\n- whenever a certain cell line is used, it should be named in the results part.", "reviewer_2": "This manuscript presents an innovative approach to probiotic-based biotherapy for colorectal cancer (CRC), demonstrating that manganese-engineered Lactobacillus reuteri (MnLR), administered with glycerol, can achieve potent antitumor effects through enhanced colon-specific colonization, metabolic activity, and immune modulation. The authors provide robust preclinical data, including in orthotopic tumor models, and address both mechanistic and translational aspects of this novel therapy. I find this work to be of high scientific interest and potentially significant impact. The strategy of using manganese to enhance probiotic function offers a promising and accessible alternative to gene editing, with potential implications for broader applications in microbial therapeutics. The demonstration of systemic and local immune modulation, combined with a practical encapsulation strategy for oral delivery, positions this research as both scientifically novel and clinically relevant.\nI consider this manuscript worthy of publication in Nature Communications upon the authors’ satisfactory resolution of the following issues and clarifications, which are important for validating the robustness and generalizability of the approach:\n1. The study lacks comprehensive investigation into the effects of L. reuteri metabolites on non-cancerous rapidly dividing cells throughout the body. The redox-dependent mechanism of L. reuteri metabolites, such as reuterin, might affect other fast-proliferating cells in the body, which could lead to unintended toxicity in a therapeutic context. This is crucial for understanding the true specificity of the metabolites and potential side effects beyond the gut environment. Further studies are needed to examine the effects of L. reuteri metabolites on a broader range of rapidly dividing non-cancerous cell types to ensure safety and specificity in potential therapeutic applications.\n2. The experiment lacks characterization of the specific metabolites responsible for the enhanced antitumor effect. While Figures 1e and 1f demonstrate the superior efficacy of mixed metabolites, they do not identify which specific components beyond reuterin contribute to this effect. Understanding the composition and relative contributions of different metabolites is crucial for optimizing the therapeutic potential and ensuring reproducibility. The identification of specific bioactive metabolites could not only elucidate the mechanism of action but also potentially serve as diagnostic biomarkers. Furthermore, comprehensive metabolite characterization could aid in optimizing the efficacy of natural product-derived therapies and reducing side effects by pinpointing cancer-specific metabolic vulnerabilities. Addressing this gap would align the study with current best practices in metabolomics-based cancer research and significantly enhance its impact on therapeutic development.\n3. The study lacks a direct comparison to current standard-of-care treatments for colon cancer, which is crucial for assessing the true clinical potential of GlyMnLR. While the efficacy of GlyMnLR against a PBS control is impressive, its performance relative to existing colon cancer treatments remains unknown. Current literature demonstrates extensive comparative effectiveness studies among established treatments such as bevacizumab, cetuximab, and panitumumab. These studies have shown varying impacts on overall survival and progression-free survival. Additionally, emerging therapies like immune checkpoint inhibitors and RNA-based treatments are being actively researched, mainly in MSI-high CRC. Without direct comparison to these established and emerging treatments, it's challenging to determine whether GlyMnLR offers advantages in terms of efficacy or safety. Future studies should include head-to-head comparisons with current standard-of-care treatments, considering factors such as overall survival, progression-free survival, and safety profiles, as demonstrated in existing comparative studies.\n4. The study lacks quantitative analysis of capsule dissolution kinetics and MnLR release profiles in vivo. While Figures 8c and 8d provide qualitative evidence of capsule behavior, they do not offer precise measurements of dissolution rates or MnLR release over time in the gastrointestinal tract. This quantitative data is crucial for understanding the exact timing and extent of MnLR delivery, which directly impacts its therapeutic efficacy. A more detailed kinetic analysis would strengthen the claim about the capsule's targeted delivery capabilities.\n5. The study lacks a dose-response analysis for GlyMnLR capsule treatment, which is critical for understanding its optimal efficacy and potential toxicity threshold. While the current data in Figures 8f-j demonstrate significant tumor suppression at a single dose, the absence of a dose-response curve limits our understanding of the treatment's full potential. This gap is particularly significant given the established importance of dose-response relationships in drug development and the potential risks associated with high-dose antioxidant supplementation. A comprehensive dose-response study would help determine the therapeutic window and guide dosing strategies for future studies or potential clinical trials, aligning with best practices in drug development and antioxidant research.\n6. The long-term stability and retention of Mn2+ induced enhancements in L. reuteri have not been assessed. While Figures 2b-e show clear short-term enhancements, the durability of these effects over multiple generations or in varying environmental conditions is crucial for practical applications. Long-term stability would impact the efficacy of Mn2+ modified L. reuteri in therapeutic settings, especially considering potential variations in gut conditions.\n7. The stability and retention of manganese modification in MnLR under simulated gastrointestinal conditions have not been adequately assessed. The stability of the Mn modification in acidic stomach conditions and the presence of digestive enzymes is crucial for its effectiveness as an oral probiotic treatment\n8. The study lacks validation of the functional consequences of the observed gene expression changes in L. reuteri. While the RNA-seq data in Figures 2g and 2h demonstrate clear changes in gene expression, and Figure 2i shows enrichment of ribosome-related pathways, there is no direct evidence that these transcriptional changes translate to increased ribosomal activity or protein synthesis. This gap leaves open the possibility that the observed gene expression changes may not have the assumed functional impact on L. reuteri's bioactivity.\n9. The study lacks a direct comparison of MnLR/Gly cytotoxicity on normal colon epithelial cells versus cancer cells. While the study demonstrates enhanced cytotoxicity of MnLR/Gly on various colon cancer cell lines (Figures 3a and 3b), it does not address potential effects on healthy colon cells. This comparison is crucial to establish the specificity of MnLR/Gly's action and assess potential side effects on normal tissue. However, without specific data on MnLR/Gly's effects on normal colon epithelial cells, it remains challenging to fully evaluate its therapeutic potential and safety profile. This gap is particularly important given the observed preferential activation of manganese-based nanomaterials in cancer cells due to higher GSH levels, which may or may not apply to MnLR/Gly. Additional experiments comparing MnLR/Gly's effects on both cancerous and normal colon epithelial cells are necessary to address this concern.\n10. The study does not provide direct measurement of cell death and long-term cell fate following MnLR/Gly treatment. While the measured markers are associated with ICD, they do not definitively prove that cells undergo immunogenic death. As shown in Figures 3c-f, these markers could potentially indicate cellular stress without necessarily leading to cell death. Direct assessment of cell viability and analysis of long-term cell fate are crucial to confirm the occurrence of ICD and rule out potential adaptive responses.\n11. The research does not include in vivo validation of the combined effect of MnLR/Gly and M1 macrophages on tumor growth. While the 3D MCT model provides valuable insights, it may not fully recapitulate the complex tumor microenvironment found in vivo. In vivo studies are crucial to confirm the translational potential of these findings, assess any potential systemic effects or unexpected interactions, and validate the efficacy of the combination therapy in a physiologically relevant context.\n12. The study lacks direct visualization of bacterial localization within tumor tissues at a cellular level. High-resolution imaging of tumor sections, possibly using optimized intravital microscopy techniques, could reveal the precise distribution of bacteria within the tumor microenvironment and healthy tissues, addressing this critical gap in the current study.\n13. The study does not provide a comprehensive mechanistic explanation for how the enhanced systemic immunity prevents tumor initiation and early growth. While the study demonstrates changes in immune cell populations and shows that CD8+ T cells and macrophages are important for the preventive effect, it doesn't elucidate the specific molecular mechanisms by which these immune cells prevent tumor formation. However, the precise mechanisms by which enhanced systemic immunity leads to prevention of tumor initiation and early growth remain unclear. Understanding these mechanisms, including potential metabolic interactions and immune evasion prevention, is crucial for fully explaining the preventive effect and potentially identifying new therapeutic targets.\n14. The study lacks functional assays to confirm the enhanced antitumor activity of the observed immune cell populations. While the flow cytometry data in Figures 7f-i show increased presence of immune cells associated with antitumor responses, it does not directly demonstrate their functional capacity. Without assessing cytokine production, cytotoxicity, or antigen-specific responses, it's unclear whether these cells are truly more effective at combating tumors.", "reviewer_3": "Review Title\nManganese-Engineered Lactobacillus reuteri with Enhanced Antitumor and Immunomodulatory Activities for CRC Prevention and Treatment\nSummary\nThe authors present a novel approach by engineering Lactobacillus reuteri with manganese (MnLR) to enhance its antitumor and immunomodulatory effects in colorectal cancer (CRC). The study is ambitious, integrating microbial engineering, immunology, and cancer biology, and provides compelling data that MnLR promotes increased bacterial proliferation, metabolite production, biofilm formation, and adhesion to CRC cells. These enhanced traits appear to improve tumor targeting and therapeutic efficacy. Additionally, the observed increase in tumor-infiltrating macrophages, dendritic cells (DCs), and CD8⁺ T cells suggests dual mechanisms of action direct cytotoxicity and immune modulation, which are particularly relevant in cancer therapy. Despite these strengths, several key aspects require clarification and additional experimentation to solidify the impact and translational potential of the study.\nMajor Comments and Suggestions\nIntroduction\n• While the general role of metal ions is introduced, the rationale for selecting manganese over other biologically relevant ions (e.g., iron, zinc) is not fully developed. The authors should better articulate manganese’s unique benefits in microbial physiology and immune regulation.\nResults\nFigure 1\n1. Include histopathology or biochemical assays (ALT, AST, creatinine) to assess systemic toxicity following oral administration of MnLR and metabolites.\n2. Investigate whether ROS-mediated cytotoxicity affects non-tumor tissues. This is critical for assessing off-target toxicity.\n3. Perform antioxidant or redox pathway inhibitor co-treatment to confirm the cytotoxic effects are redox-dependent.\n4. Provide Western blot analyses for redox-responsive proteins (e.g., NRF2, HO-1, GPx) to support the proposed mechanism.\n5. Conduct metabolomic profiling to identify and quantify redox-active metabolites, especially since reuterin only accounts for a minor portion of the glycerol metabolite pool.\n6. While increases in immune cells are reported, phenotypic and functional characterization is missing (e.g., M1 vs. M2 macrophages, DC activation markers, cytotoxic assays for CD8⁺ T cells).\n7. Clarify why additional controls (e.g., glycerol alone, unmodified L. reuteri) were not used consistently across models to isolate individual effects.\nFigure 2\n1. Describe how Mn localization (surface-bound or intracellular) was confirmed after treatment.\n2. Evaluate Mn release profiles in simulated gastrointestinal environments to ensure colon delivery.\n3. Provide dose comparisons to physiological or dietary manganese levels. The risk of Mn-induced toxicity (e.g., neurotoxicity) is not addressed. Include or reference data on biodistribution and clearance.\n4. Test bacterial growth in nutrient-limited media, and perform rescue experiments with Mn to validate its specific effects.\n5. Clarify the enrichment of “photosynthesis pathways” in transcriptomics, as L. reuteri is non-photosynthetic—this may suggest annotation artifacts. Key findings should be validated by qRT-PCR.\nFigure 3\n1. Differentiate synergistic vs. additive effects of reuterin and Mn²⁺ on DC maturation using MnLR with metabolite inhibition or depletion.\n2. Include cytokine profiling (e.g., IL-12, TNF-α) to confirm functional DC maturation.\nFigure 4\n1. Confirm results in primary macrophages (e.g., BMDMs or human MDMs).\n2. Investigate the engagement of HIF-1 by MnLR/Gly using ROS inhibitors or reporters.\n3. Assess macrophage function, including cytokine production (IL-6, TNF-α) and phagocytosis.\nFigure 5\n1. Quantify tumor burden, growth kinetics, and survival to evaluate efficacy.\n2. Assess whether hypoxia attracts MnLR passively or whether the strain has active tumor-homing capabilities.\n3. Compare MnLR with encapsulated reuterin to evaluate the added benefit of using live probiotics.\n4. Investigate how the tumor microenvironment, immune state, or gut microbiota influence MnLR colonization.\nFigure 6\n1. Clarify how reuterin exerts in vivo cytotoxicity—via ROS, DNA damage, or metabolic interference.\n2. Use fecal transfer or microbiota depletion experiments to establish causal roles of gut microbial changes.\nFigure 7\n1. Immune cell findings are largely phenotypic; functional assays (e.g., cytotoxicity, antigen specificity) are needed.\n2. Test protective memory by re-challenging cured mice with tumor cells.\n3. Include histological analysis of major organs and inflammatory markers following prolonged MnLR/Gly treatment.\nFigure 8\n1. Provide immune profiling in rabbits (e.g., T cells, DCs, macrophages) to support mechanistic parallels to mice.\n2. Compare GlyMnLR capsules vs. free MnLR/Gly vs. standard CRC therapies (e.g., 5-FU) to strengthen translational value.\nGeneral Recommendations and suggestions\n1. The precise role of Mn²⁺ in enhancing immune responses remains unclear and should be dissected further.\n2. The study lacks comparison with standard-of-care therapies. Benchmarking would clarify clinical competitiveness.\n3. Long-term studies are missing: colonization persistence, immune memory, and chronic safety are all key for translation.\n4. It might be worthwhile to consider in the future a germ-free or antibiotic- treated mice and to validate the findings in human monocytes derived DC.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Responses to Reviewers’ Comments\nWe express our sincere appreciation to the reviewers for their valuable comments and suggestions,\nwhich have significantly contributed to enhancing the quality of our manuscript.\n(1) Highlight: The changes to the text in the revised manuscript have been highlighted in red,\nincluding newly added descriptions and corrections of errors.\n(2) Figures: Fig. 2e,f,i,n,o, Fig. 3c,e-g,i,k,m, Fig. 4f-j,q-s, Fig. 5g,h, Fig. 6d,n, Fig. 7c-h, and Fig.\n8d,e,j-n are the newly added main data.\n(3) Supporting Information: Supplementary Fig. 3,4,6-9,12-16,22,25,28,30,31,36,37,40,42,43,45,46,\nand Supplementary Table 2 are the newly added supplementary data.\nThe concept of preventing and treat cancer with probiotics is, of course, extremely fascinating and in\ncase of CRC prevention also obvious. Thus, the current manuscript is of high potnetial relevance for\nthe field. Also the concept of chemically modify/improve known probiotics instead of genetically\nmodify them is pretty clever.\nHowever, in its current form, the study of Cao and colleauges has some major points that need to be\nadressed to improve the scientific quality and, most of all, the potential relevance for patients and\nsociety.\n1. The major problem for this reviewer is a lack of data obtained from human cells. Since the target\nspecies will be neither mouse nor rabbit, the absolute majority of in vitro data should be obtained by\nusing human cells. Thus, more CRC cell models must be tested in vitro. The presented data are based\non measurements by SW48 and HCT116. Both are highly-passaged CRC models - and both are\nmicrosatellite-instable and this fact could be a potential bias since no further molecular subtypes have\nbeen tested.\nResponse: We sincerely thank the reviewer for this important comment. To address the concern\nregarding human relevance and molecular subtype diversity, we expanded our in vitro studies to\ninclude additional human colorectal cancer cell lines: RKO (microsatellite instability-High, MSI-H)\nand SW620 and DLD-1 (microsatellite stable, MSS). Across these models, MnLR/Gly consistently\nexhibited strong inhibitory effects, with cell viability reduced by 87.9%-93.7%, higher than that of L.\nreuteri/Gly (Fig. 3a). These results indicate that the Mn-enhanced antitumor activity of L. reuteri is\nnot limited to MSI-H CRC models but extends to MMR-proficient tumors, thereby strengthening the\ngeneralizability and translational relevance of our findings.\nCytotoxicity of L. reuteri, glycerol, L. reuteri/Gly, and MnLR/Gly in multiple human colon cancer cell\nlines (SW48, HCT116, RKO, SW620, DLD-1) and mouse colon cancer cell lines (MC38, CT26) after\na 24-hour incubation (n = 3 or 4). Data are shown as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001,\n****p < 0.0001; one-way ANOVA.\nThe relevant description on page 11, lines 260-267：\n“The tumor-inhibitory effect of MnLR delivered with glycerol (MnLR/Gly) was demonstrated in\nhuman colon cancer cell lines with diverse MMR statuses—including microsatellite instability-high\n(MSI-H; SW48, HCT116, RKO), and microsatellite stable (MSS; SW620, DLD-1), as well as in mouse\ncolon cancer cell lines (MC38 and CT26) (Fig. 3a). This effect was attributed to the MnLR-derived\nglycerol metabolites, as neither L. reuteri nor glycerol alone exhibited cytotoxicity to colon cancer\ncells (Fig. 3a). Moreover, the inhibitory rates of MnLR/Gly were higher thanL. reuteri/Gly (Fig. 3a)\nacross all the cell types, indicating that Mn promotes L. reuteri proliferation and production of\ncytotoxic metabolites, thus enhancing the cancer cell-killing efficiency.”\n2. It would be superior to add further data from patient-derived models in low passages and from\ndifferent molecular background. Some additional data were presented from PDO - but it became not\nabsolutely clear, if this was just one single model or more. Here, more PDO models from different\npatients are necessary.\nResponse: We sincerely thank the reviewer for this valuable suggestion, which has indeed\nstrengthened the translational relevance of our study. In response, we expanded our analysis to include\nfive independent patient-derived organoid (PDO) models established from colorectal cancer (CRC)\n3\npatients with distinct molecular backgrounds, whose clinical characteristics are summarized in\nSupplementary Table 2. These models comprised four microsatellite stable (MSS) PDOs and one\nmicrosatellite instability- High (MSI-H) PDO, thereby covering molecular heterogeneity. Consistent\nwith our earlier results, morphological evaluation and live/dead cell staining demonstrated that L.\nreuteri-derived glycerol metabolites exerted robust cytotoxic effects across all five PDOs, with the\nefficacy consistently superior to that of pure reuterin (Fig. 1e,f, and Supplementary Fig. 4). These\nfindings confirm that the potent antitumor activity of the mixed metabolites is preserved in patient-\nderived CRC models of diverse molecular subtypes, enhancing the clinical relevance of our work.\nFig. 1: Glycerol metabolites from L. reuteri inhibit CRC growth in vitro and in vivo, and increase\nimmune cells in colon tumors. e, The representative microscopic and live/dead staining images of\nthe PDOs derived from MSI-H and MSS CRC patients treated with pure reuterin (1.5 μg/ml) or mixed\nmetabolites (containing 1.5 μg/mlof reuterin) for 3 days. Scale bar, 50 μm. f, The organoid diameter\nafter a 3-day treatment with the given agents (n = 10). Data are shown as mean ± SD; *p < 0.05, **p\n< 0.01, ***p < 0.001, ****p < 0.0001 (compared to Ctrl); one-way ANOVA.\nSupplementary Fig. 4 | The cytotoxic effects of reuterin and mixed metabolites on 3 different\npure reuterin (1.5 μg/ml) or mixed metabolites (containing 1.5 μg/mlof reuterin) for 3 days. Scale bar,\n50 μm. b, The organoid diameter after a 3-day treatment with the given agents (n = 10). Data are shown\nas mean ± SD; **p < 0.01, ***p < 0.001 (compared to Ctrl); one-way ANOVA.\nSupplementary Table 2 | The clinical information of patients providing PDO models.\nSample ID 1 2 3 4 5\nGender Female Female Male Male Male\nRace Asian Asian Asian Asian Asian\nDiagnosis CRC CRC CRC CRC CRC\nSize 3*2.5 cm 4.5*4 cm 2*1 cm 5.5*3.5 cm 4*2.5 cm\nTNM stage T4aN1bM0 IIIb T3N0M0 IIa T3N2bM0 IIIc T3N0M0 IIa T3aN1aM0 IIIb\nAnatomic region Descending colon Ascending colon Rectum Descending colon Sigmoid colon\nPathological type Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma\nMMR status MSI-H MSS MSS MSS MSS\nThe relevant description on page 6, lines 151-154：\n“Further, the more effective colon cancer suppression of the mixed metabolites than pure reuterin was\nconfirmed in five independent CRC patient-derived organoid (PDO) models, including four\nmicrosatellite stable (MSS) and one MSI-H subtypes (Fig. 1e,f, Supplementary Fig. 4, and\nSupplementary Table 2), supporting the robustness and clinical relevance37.”\n3. In line with this argumentation, the in vitro immune cell tests presented should be done with immune\ncells from healthy donors and - at best - from patients!\nResponse: We sincerely thank the reviewer for this constructive suggestion. We fully agree that\nvalidating the immune-modulatory effects in human immune cells is of high importance. To address\nthis, we performed additional experiments using peripheral blood mononuclear cells (PBMCs) from\nhealthy donors. Specifically, MnLR/Gly was found to promote human PBMC-derived dendritic cell\nmaturation, both indirectly through induction of immunogenic tumor cell death and directly via the\n5\np38 MAPK pathway (Fig. 3c,e,g,i,k,m,n, and Supplementary Fig. 14c,d,15b). In parallel, MnLR/Gly\nskewed PBMC-derived macrophages toward an antitumor phenotype, as evidenced by an increased\nM1/M2 ratio and enhanced phagocytic activity, at least partly dependent on HIF-1 signaling (Fig. 4f-\nj,4q-s, and Supplementary Fig.22). These human PBMC-based findings are consistent with the results\nobtained from murine immune cells, collectively providing direct evidence that MnLR/Gly can\nmodulate immune function across species and further strengthening the translational relevance of our\nstudy.\nCytotoxicity of L. reuteri, glycerol, L. reuteri/Gly, and MnLR/Gly in multiple human colon cancer cell\nlines (SW48, HCT116, RKO, SW620, DLD-1) and mouse colon cancer cell lines (MC38, CT26) after\na 24-hour incubation (n = 3 or 4). b, Quantification of mature DCs (CD11c+CD86+) in the total CD45+\nmBMDCs after being stimulated by the cell lysates from MC38 cells treated with reuterin or\nMnLR/Gly (n = 3). c, Quantification of mature DCs (CD83+) in the total CD11c+HLADR+ hPBMC-\n= 3).d,e, The representative flow cytometric images and corresponding quantification of mature DCs\n(CD11c+CD86+ or CD83+) in the total CD45+or CD11c+HLADR+ DCs after direct stimulation by the\ngiven agents (n = 3). f, g, The levels of immune functional cytokines (IL-12, TNF-α) produced by\nmBMDCs or hPBMC-DCs after direct stimulation by the given agents (n = 3). h,i, Immunoblotting\nassays of p-p38 in the mBMDCs or hPBMC-DCsafter direct stimulation by the given agents. iMAPK,\nMAPK inhibitor. j-m, The representative flow cytometric images (j,k) and corresponding\nquantification (l,m) of mature DCs (CD11c+CD86+or CD83+) in the total CD45+ or CD11c+HLADR+\nDCs after the given treatments (n = 3). n, The schematic showing that MnLR/Gly indirectly (via ICD)\nor directly promotes DC maturation. Data are shown as mean ± SD; *p < 0.05, **p < 0.01, ***p <\n0.001, ****p < 0.0001 (compared to Ctrl in f,g); one-way ANOVA.\nSupplementary Fig. 14 | DCs maturation stimulated by reuterin- or MnLR/Gly-treated colon\ncancer cells. a,c, The representative flow cytometric images of mature mouse BMDCs (CD11c+\nCD86+) in the total CD45+ BMDCs (a) and mature human PBMC-DCs (CD83+) in the total CD11c+\nHLADR+ PBMC-DCs (c) after stimulation by the lysates from reuterin- or MnLR/Gly-treated colon\ncancer cells (MC38 or DLD-1). b,d, The levels of immune functional cytokines (IL-12, TNF-α) in\nmature mBMDCs (b) and hPBMC-DCs (d) detected by ELISA Kit (ELK Biotechnology, China) (n =\n3). Data are shown as mean ± SD; **p < 0.01, ***p < 0.001, ****p < 0.0001 (compare to Ctrl); one-\nway ANOVA.\nquantification of the proportion of mature mouse BMDCs (CD11c+CD86+) in the total CD45+ BMDCs\n(a) and mature human PBMC-DCs (CD83+) in the total CD11c+ HLADR+ PBMC-DCs (b) after direct\nstimulation by the given agents for 24 hours (n = 3). Data are shown as mean ± SD; *p < 0.05, **p <\n0.01, ****p < 0.0001 (compare to Ctrl); one-way ANOVA.\nThe relevant description on pages 12, 13, lines 283-321：\n“To investigate this immunological process, we subjected DCs derived from murine bone marrows or\nhuman peripheral blood mononuclear cells (mBMDCs or hPBMC-DCs) to the lysates from reuterin-\nor MnLR/Gly-treated colon cancer cells and observed a significant improvement in DC maturation, as\nevidenced by the increased proportion of mature DCs (CD11c+CD86+ mBMDCs and CD83+ hPBMC-\nDCs), and the production of immune functional cytokines (IL-12, TNF-α) (Fig. 3b,c, and\nSupplementary Fig. 14) ...... Notably, the proportion of mature DCs was the highest in the MnLR/Gly-\ntreated total DCs (1.7-fold higher than reuterin- or Mn2+-treated mBMDCs, 1.2-fold higher than\nreuterin- or Mn2+-treated hPBMC-DCs) (Fig. 3d,e, and Supplementary Fig. 15). Inhibition of reuterin\nproduction with a GDHt inhibitor reduced DC maturation to the level induced by Mn2+ alone\n(Supplementary Fig. 16), suggesting that glycerol metabolites and Mn2+ released by MnLR contribute\nadditively to DC maturation (Fig. 3n). In addition, MnLR/Gly-treated DCs secreted markedly higher\nlevels of IL-12 and TNF-α than other groups (Fig. 3f,g), further supporting their enhanced functional\nmaturation ...... In addition, the concordance in observations between murine- and human-derived DCs\nunderscores the conserved nature and translational potential of MnLR/Gly-mediated\nimmunomodulation.”\ncytometry analysis of M1 (CD86+) polarization in total murine macrophages (RAW264.7 cells)\nwithout any treatments (Blank), with only LPS/IFN-γ stimulation (Ctrl), with LPS/IFN-γ stimulation coupled by\npure reuterin (Reuterin) or MnL/Gly treatments (n = 3). c,d, The representative image and\ncorresponding quantification of flow cytometry analysis of M2 (CD206+) polarization in total\nmacrophages (RAW264.7 cells) without any treatments (Blank), with only IL-4 stimulation (Ctrl), with\nIL-4 stimulation coupled by reuterin or MnLR/Gly treatments (n = 3). e, Images of the 3D MCTs with\ndifferent treatments at the given time points. Scale bar, 25 μm. f,g, The representative image and\ncorresponding quantification of flow cytometry analysis of the ratio of M1 (CD86+) to M2 (CD206+)\nmacrophages in CD45+CD68+ hPBMC-macrophages after reuterin or MnLR/Gly treatments (n = 3).\nh, The levels of immune functional cytokines (IL-6, TNF-α) produced by hPBMC-macrophages after\nthe given treatments (n = 3). i,j, The representative images and corresponding quantification of flow\ncytometry of the proportion of CFDA-SE+ cells (DLD-1) in CD11b+ hPBMC-macrophages after being\nstimulated by the given agents (phagocytic function assessment) (n = 4). k,l, Immunoblotting assays\nof HIF-1α protein within the RAW264.7 cells with the given treatments. m-p, The representative image\nand corresponding quantification of flow cytometry analysis of M1 (m,n) or M2 (o,p) polarization in\nthe total LPS/IFN-γ-stimulated or IL-4-stimulated RAW264.7 cells with the given treatments (n = 3).\nq, Immunoblotting assays of HIF-1α protein within the hPBMC-macrophages with the given\ntreatments.r,s, The representative image and corresponding quantification of flow cytometry analysis\nof the ratio of M1 (CD86+) to M2 (CD206+) macrophages in CD45+CD68+ hPBMC-macrophages with\nthe given treatments (n = 3). iHIF-1α, HIF-1α inhibitor (YC-1). t, The schematic illustration of the\nimpact of MnLR/Gly on macrophage polarization. Data are shown as mean ± SD; *p < 0.05, **p <\n0.01, ***p < 0.001, ****p < 0.0001 (compared to Ctrl in g,h,j); one-way ANOVA.\nSupplementary Fig. 22 | Flow cytometry analysis of macrophage polarization in human\nPBMC-macrophages. The flow cytometric quantification of the proportion of the ratio of M1 (CD86+) to M2\n(CD206+) macrophages in CD45+CD68+ PBMC-macrophages after direct stimulation by the given\n11\nagents for 24 hours (n = 3). Data are shown as mean ± SD; ****p < 0.0001 (compare to Ctrl); one-\nway ANOVA.\nThe relevant description on page 16, lines 367-384：\n“Human PBMC-macrophages exhibited consistent responses with RAW264.7 cells, wherein\nMnLR/Gly treatment induced M1 phenotypic polarization (Fig. 4f,g, and Supplementary Fig. 22),\naccompanied by elevated IL-6 and TNF-α production and enhanced phagocytic activity against colon\ncancer cells (Fig. 4h-j), thereby reinforcing the robustness and translational potential of\nimmunomodulatory effects mediated by MnLR/Gly ...... Consistently,this HIF-1–dependent regulation\nof macrophage polarization was also validated in human PBMC-macrophages (Fig. 4q-s). Additionally,\ntreatment with the NAC partially reduced MnLR/Gly-induced HIF-1 upregulation, suggesting that this\npathway activation is mediated, at least in part, by ROS production (Supplementary Fig. 25).”\n4. Another major issue is the choice of animal model(s) and the conclusions drawn from the\nexperiments done. With none of the mouse models chosen, the prevention of primary CRC can be\nexamined. For that kind of conclusion, a genetic model would be the right choice. Albeit this might\nnot be transferrable to the human situation. Similarly, the splenic injection model is no longer\nacceptable as a model of liver metastasis in biological terms of the metastatic procedure. All effects\npresented in the manuscript can be explained by direct or indirect effect on the CRC cells used - maybe\nin combination of such effects on immune cells.\nResponse:We sincerely thank the reviewer for this thoughtful and critical assessment, which helps us\nto clarify the scope and strengthen the interpretation of our in vivo findings. We fully acknowledge the\nlimitations of the models used and have carefully considered the reviewer’s concerns.\n(1) Prevention of primary CRC: To explore preventive efficacy, we first employed a pretreatment\nprotocol in which mice received MnLR/Gly for 10 days before orthotopic tumor inoculation. This\nregimen markedly suppressed primary tumor formation, with 75% (6/8) of mice remaining tumor-free\nfor at least 8 weeks (Supplementary Fig. 38a), and 62.5% (5/8) continuing to resist tumor growth upon\nrechallenge (Fig. 7c, and Supplementary Fig. 38b). To further substantiate these findings, we\n12\nconducted additional studies in the Apcmin/+ transgenic model, a well-established system for\nspontaneous intestinal tumorigenesis. MnLR/Gly treatment significantly reduced intestinal tumor\nburden and proliferative activity (Fig. 7e-h), reinforcing its potential role in CRC prevention.\n(2) Liver metastasis model: We recognize the reviewer’s valid concern that the splenic injection model\ndoes not fully recapitulate the complex biology of metastasis. Nevertheless, as it reflects portal venous\ndissemination, it remains widely applied in CRC research (Cancer Cell. 2021 May 10;39(5):708-\n724.e11; Nat Commun. 2019 Oct 16;10(1):4695.2). In this setting, MnLR/Gly treatment markedly\nreduced metastatic liver burden, both in terms of liver weight and nodule counts (Fig. 7n-p). Moreover,\ndepletion of CD8⁺ T cells or macrophages diminished these antimetastatic effects, indicating that\nMnLR/Gly acts not only directly on tumor cells but also through engagement of host antitumor\nimmunity (Fig. 7n-p).\nTaken together, while each model has intrinsic limitations, the complementary use of orthotopic,\ntransgenic, and splenic injection approaches provides convergent evidence that MnLR/Gly can\nprevent primary CRC development and suppress liver metastasis. We are grateful to the reviewer for these\nconstructive comments, which have allowed us to refine both the experimental strategy and the\ninterpretation of our findings.\n13\nillustration of the pre-treatments with the given agents (once-daily oral gavage for 10 days),\nbiochemical and immunological analyses on blood, spleen, and MLN samples, initial inoculation and\nrechallenge of colon cancer cells, and macrophage or CD8+ T cell (Mac/CD8 T) depletion. b,\nBioluminescence imaging of the colon tumors in the mice receiving different treatments at the given\ntime points after initial orthotopic tumor inoculation (n = 5). c, Bioluminescence imaging and\nrepresentative images of orthotopic tumor development in mice after the rechallenge of colon cancer\ncells. Scale bar, 1 cm. d, Schematic illustration of the prevention in transgenic APCmin/+ mice receiving\nthe given treatments for 9 weeks. e,f, Representative images and the corresponding quantification of\ntumors in the intestine of APCmin/+ mice after different treatments (n = 5). Red triangular arrows\nindicate the tumor nodules. Scale bar, 1 cm. g,h, Representative images of H&E staining (g) and\nimmunohistochemistry staining of Ki67 (h) of the intestinal segments. Blue triangular arrows indicate\nthe tumor nodules. Scale bar, 2.5 mm. i,j,Quantification of the ratio of M1 (CD11b+F4/80+CD86+) to\nM2 (CD11b+F4/80+CD206+) macrophages, and the percentages of mature DCs (CD11c+CD86+) in\ntotal CD45+ cells within the spleen from the mice receiving the given treatments for 10 days (n = 5).\nk,l, Quantification of the percentages of activated CD4+ and CD8+ T cells (IFN-γ+CD4+ and IFN-\nγ+CD8+) in total CD3+ cells within the MLNs from the mice receiving the given treatments once daily\nfor 10 days (n = 5). m, Schematic illustration of the establishment of the liver metastasis model by\ninjecting MC38 cells into the spleen of mice, once-daily oral gavage of the given agents, and\nmacrophage or CD8+ T cell (Mac/CD8 T) depletion. n, Representative images of the liver and spleen\ntissues isolated from the mice 14 days after tumor inoculation. Blue triangular arrows indicate the liver\ntumor nodules.o,The weight of livers and spleens isolated from the mice in each group 14 days after\ntumor implantation (n = 5). p, The quantification of liver tumor nodules in mice in each group 14 days\nafter tumor inoculation (n = 5). Data are shown as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001,\n****p < 0.0001 (compared to PBS group); one-way ANOVA.\nSupplementary Fig. 38 | The long-term preventive effect of pre-oral MnLR/Gly on the\n15\noccurrence of primary colon tumor. a, Bioluminescence imaging of colon tumors in the mice\n(receiving pre-oral MnLR/Gly for 10 days) at the given time points post orthotopic tumor implantation\n(n = 8). b, The fluorescence intensity quantification of the colon tumors in rechallenged mice on day\n77 after the initial inoculation (n = 8). Data are shown as mean ± SD; ***p < 0.001; Student’s t-test.\nThe relevant description on pages 26, 27, lines 580-592：\n“When the observation period was extended, 75% (6/8) of MnLR/Gly-pretreated mice remained\ntumor-free for at least 8 weeks (Supplementary Fig. 38a). To further evaluate the durability of the\npreventive effect of MnLR/Gly, tumor rechallenge experiments were performed by re-inoculating\ncolon cancer cells on day 56 after the initial inoculation in the mice that remained tumor-free following\nprophylactic treatment of MnLR/Gly (Fig. 7a). Strikingly, 62.5% (5/8) of MnLR/Gly-pretreated mice\nstill exhibited no detectable tumors 3 weeks later (Fig. 7c, and Supplementary Fig. 38b), indicating\nthat MnLR/Gly confers a long-lasting protective effect against tumor recurrence. Additionally, the\npreventive efficacy of MnLR/Gly was validated in a genetic model of spontaneous intestinal\ntumorigenesis (Apcmin/+ mice) (Fig. 7d), where continuous oral administration of MnLR/Gly\nsignificantly reduced the intestinal tumor multiplicity and the Ki-67–positive proliferating epithelial\ncells compared with controls (Fig. 7e-h). Collectively, these findings indicate that MnLR/Gly provides\ndurable protection against colorectal tumor development and recurrence, supporting its potential as a\npreventive strategy for CRC.”\n5. Finally, the rabbit model might be fine for the stability measurements of the GlyMnLR capsules,\nbut the CRC part adds no relevant information.\nResponse:We sincerely thank the reviewer for this important comment. In line with the suggestion,\nwe focused on using the rabbit model to evaluate the in vivo stability and release kinetics of the\nGlyMnLR capsules. As shown by in vitro and in vivo fluorescence imaging, endoscopy, and X-ray\nanalyses, the capsules remained intact in the stomach and subsequently dissolved during\ngastrointestinal transit and in the intestine, resulting in a concentrated release of MnLR within\ntherapeutic targets (Fig. 8b-e, and Supplementary Fig. 44).\nTo further assess the translational potential, we also compared the antitumor efficacy of GlyMnLR\n16\ncapsules with free MnLR and reuterin capsules in the rabbit orthotopic CRC model. Although we\nrecognize the limitations of this model in reflecting the full complexity of CRC, the results\ndemonstrated significantly higher tumor inhibition by GlyMnLR capsules (Fig. 8f-l), supporting the\nprotective role of the enteric coating and the advantage of sustained active metabolite production.\nTaken together, these data confirm the capsule’s functional stability and therapeutic relevance in vivo,\nwhile we acknowledge that more sophisticated models will be needed to fully explore clinical\ntranslatability.\nrabbit models. a, The images of the serial glycerol capsules with different external diameters. Scale bar, 2\nmm. b, Fluorescence images showing the capsule dissolving with the release of CY 5.5-labeled MnLR\nin SGF and SIF. c, X-ray images showing the GlyMnLR capsule containing barium sulfate during\n18\ngastrointestinal transportation after being orally taken by rabbits. Scale bar, 5 mm (left); 2 cm (right).\nd,e, Fluorescence images of GlyMnLR capsule dissolution and corresponding quantification of MnLR\n(labeled with CY5.5) release at the given time points after oral administration (n = 5). f, Schematic\nillustration of the therapeutic regime, endoscopic/CT imaging, and biochemical and immunological\nanalyses on blood and tumor tissues in the primary CRC-bearing rabbit model.g, CT images obtained\n28 days after the treatment initiation showing the tumor occupation at different locations highlighted\nby red, yellow, and green colors. Scale bar, 5 cm. h,Quantification of the tumor occupation relative to\nthe PBS group (n = 5). i, Representative images showing the tumors (highlighted with yellow dashed\nbox) isolated from the primary colon tumor-bearing rabbits 31 days after the treatment initiation.\nScale bar, 5 mm. j, Representative in vivo (endoscopic, the top and middle panels) and ex vivo\n(the bottom panel) images of tumors (highlighted with yellow dashed boxes) on days 10, 28, and 31 post oral\nadministration of the indicated agents. Scale bar, 1 cm. k, The tumor volumes obtained from the\ncorresponding groups at the end of the experiment (n = 5). l, The representative immunofluorescent\nimages and corresponding statistical analysis of the green Ki67-stained proliferating cells within the\nisolated tumor tissues from rabbits receiving the given treatments at the experimental end (n = 5 fields).\nScale bar, 200 μm. m,n, Representative immunofluorescence staining images and corresponding\nquantification of CD45+CD8+ T cells within the tumor tissues isolated from the rabbits with the given\ntreatments (n = 9 fields).Scale bar, 50 μm. Data are shown as mean ± SD; *p < 0.05, **p < 0.01, ***p <\n0.001, **** p < 0.0001 (compared to PBS group); Student’s t-test or one-way ANOVA.\nSupplementary Fig. 44 | The morphological change of the GlyMnLR capsule within a rabbit’s\nstomach observed by endoscopy. The capsule remained intact in the gastric environment for at least\n4 hours without disintegration following oral administration.\nThe relevant description on pages 30, 31, lines 651-675：\n“In vitro and in vivo fluorescence imaging showed that the dissolution rate of the GlyMnLR capsule\n(MnLR labeled by Cy5.5) was significantly faster in simulated intestinal fluid (SIF) and real intestinal\ntract compared to simulated gastric fluid and real stomach (SGF) (Fig. 8b,d). This finding was\ncorroborated by endoscopy examination (Supplementary Fig. 44) and X-ray imaging (Fig. 8c), which\nrevealed that the capsule remained intact in the rabbit’s stomach for at least 4 hours after oral\nadministration. After reaching the intestine, the barium signal of the capsule was weakened within 20\nhours owing to the dissolution of the capsule in intestinal fluid (Fig. 8c). Consistently, in vivo\nquantitative analysis showed that only ~26.7% of MnLR was released within 4 hours in the stomach,\nwhereas release markedly accelerated during gastrointestinal transit (4–10 hours) and intestinal\nresidence (10–20 hours), with cumulative release reaching ~83.5% within 20 hours (Fig. 8d,e). These\nobservations collectively demonstrated the capsule’s resistance to gastric juice and its solubility in the\nintestinal environment, ensuring sustained and targeted release of MnLR at the site of tumor growth ......\nAccordingly, we selected one capsule once daily as the optimal dosing regimen for subsequent\ntherapeutic studies. GlyMnLR capsules exhibited a markedly higher tumor inhibition rate (67.9%)\ncompared with free MnLR (25.4%) and reuterin capsules (8.0%), demonstrating the protective role of\nthe enteric formulation and the advantage of sustained production of active metabolites by viable\nbacteria (Fig. 8j,k).”\n6. Minor comments:\n1) English has to be improved in the neglected parts of the manuscript: materials and methods, as well\nas results.\nResponse: We sincerely thank the reviewer for pointing this out. We have carefully revised the\nlanguage throughout the entire manuscript to improve clarity, accuracy, and readability.\n2) some data are presented in different styles (for example flow cytometry in Fig. 3) - should be the\nsame.\nResponse: We appreciate the reviewer’s suggestion. The presentation style of all flow cytometry and\nother related data has been standardized for consistency across the figures.\n3) some of the schemes used to explain experimental procedures are not really helping and can be\nomitted (again Fig. 3)\nResponse: We thank the reviewer for the constructive comment. We have removed the redundant\nschemes in Fig. 3 to improve the overall conciseness and focus of the figure.\n20\n4) whenever a certain cell line is used, it should be named in the results part.\nResponse: We agree with the reviewer’s suggestion and have added the information on the cell lines\nused in each experiment to the Results section and/or figure legends.\nThis manuscript presents an innovative approach to probiotic-based biotherapy for colorectal\ncancer (CRC), demonstrating that manganese-engineered Lactobacillus reuteri (MnLR),\nadministered with glycerol, can achieve potent antitumor effects through enhanced colon-\nspecific colonization, metabolic activity, and immune modulation. The authors provide robust\npreclinical data, including in orthotopic tumor models, and address both mechanistic and\ntranslational aspects of this novel therapy. I find this work to be of high scientific interest and\npotentially significant impact. The strategy of using manganese to enhance probiotic function\noffers a promising and accessible alternative to gene editing, with potential implications for\nbroader applications in microbial therapeutics. The demonstration of systemic and local\nimmune modulation, combined with a practical encapsulation strategy for oral delivery,\npositions this research as both scientifically novel and clinically relevant.\nI consider this manuscript worthy of publication in Nature Communications upon the authors’\nsatisfactory resolution of the following issues and clarifications, which are important for\nvalidating the robustness and generalizability of the approach:\n1. The study lacks comprehensive investigation into the effects of L. reuteri metabolites on\nnon-cancerous rapidly dividing cells throughout the body. The redox-dependent mechanism of\nL. reuteri metabolites, such as reuterin, might affect other fast-proliferating cells in the body,\nwhich could lead to unintended toxicity in a therapeutic context. This is crucial for\nunderstanding the true specificity of the metabolites and potential side effects beyond the gut\nenvironment. Further studies are needed to examine the effects of L. reuteri metabolites on a\nbroader range of rapidly dividing non-cancerous cell types to ensure safety and specificity in\npotential therapeutic applications.\nResponse: We sincerely thank the reviewer for raising this important concern regarding the\npotential effects of L. reuteri metabolites on non-cancerous proliferating cells. In response, we\nperformed additional experiments using several representative non-cancerous cell types.\nSpecifically, we included (i) normal human colonic epithelial cells (NCM460) as counterparts\nto colon cancer cells; (ii) human umbilical vein endothelial cells (HUVEC), which are widely\nused as a model of proliferating endothelial cells involved in angiogenesis and vascular\nhomeostasis; and (iii) normal human liver cells (L-02), since both L. reuteri and MnLR were\nfound to preferentially localize in the liver compared with other major organs. Our results\ndemonstrated that, compared with colon cancer cells, L. reuteri metabolites and MnLR/Gly\n22\nexhibited markedly reduced cytotoxicity toward these non-cancerous cells (Fig. 1b, 3a, and\nSupplementary Fig. 12). Specifically, at a reuterin concentration of 50 µg/mL, the viability of\nNCM460, HUVECs, and L-02 cells remained above 80%, whereas the viability of colon cancer\ncells decreased to approximately 14.5% (Fig. 1b). Moreover, hematological and\nhistopathological analyses further confirmed that continuous oral administration of MnLR/Gly\nor reuterin did not induce pathological alterations in major organs or abnormalities in blood\nbiochemical parameters (Supplementary Fig.28). Collectively, these findings indicate that both\nL. reuteri metabolites and MnLR/Gly exhibit preferential cytotoxicity toward cancer cells\nwhile sparing non-cancerous proliferating cells, thereby supporting their safety and therapeutic\nspecificity. These new results and the corresponding description have been incorporated into\nthe revised manuscript.\nFig. 1: Glycerol metabolites from L. reuteri inhibit CRC growth in vitro and in vivo, and\nincrease immune cells in colon tumors. b, Cell viabilities of NCM460, HUVEC, L-02,\nHCT116, and MC38 cells after a 24-hour culture with the collected metabolites at different\nreuterin concentrations (n = 5).\nThe relevant description on pages 5, 6, lines 131-140：\n“Consistent with the previous findings 27, the mixed metabolites from L. reuteri catabolizing\nglycerol exerted a potent growth-inhibitory effect preferentially on cancer cells over non-\ncancerous cells in a redox-dependent manner (Fig. 1b,c), as the metabolites stimulated the\nproduction of cytotoxic reactive oxygen species (ROS), reduced antioxidative glutathione level,\nand upregulated oxidative stress–related molecules at both the mRNA and protein levels in\ncolon cancer cells (Supplementary Fig. 1); and their cytotoxic effect was significantly reduced\nin human normal colonic epithelial cells (NCM460), human umbilical vein endothelial cells\n(HUVEC), and normal human liver cells (L-02) (with the cell viability remaining above 80%\nat a reuterin concentration of 50 µg/mL),as well asin the presence of N-acetyl-cysteine (NAC,\na ROS scavenger) (Fig. 1b,d).”\nFig. 3: In vitro colon cancer cell inhibition and DCs maturation mediated by MnLR/Gly.\na, Cytotoxicity of L. reuteri, glycerol, L. reuteri/Gly, and MnLR/Gly in multiple human colon\ncancer cell lines (SW48, HCT116, RKO, SW620, DLD-1) and mouse colon cancer cell lines\n(MC38, CT26) after a 24-hour incubation (n = 3 or 4). Data are shown as mean ± SD; *p <\n0.05, **p < 0.01, ****p < 0.0001; one-way ANOVA.\nSupplementary Fig. 12 | The cytotoxicity on normal non-cancerous rapidly dividing cell\nlines. Cell viabilities of NCM460, HUVEC, and L-02 cells after being treated with L. reuteri\n(109 CFU), glycerol (5 μl), L. reuteri/Gly, or MnLR (109 CFU)/Gly in 500 μl of culture media\nfor 24 hours (n = 4). Data are shown as mean ± SD.\nThe relevant description on page 11, lines 267-270：\n“Consistent with the effects observed for L. reuteri-derived mixed metabolites, MnLR/Gly\nexhibited preferential cytotoxicity toward colon cancer cells over non-cancerous cells (Fig. 3a\nand Supplementary Fig. 12), indicating its anticancer efficacy, alongside reduced toxicity to\n24\nnormal proliferating cells.”\nSupplementary Fig. 28 | Biosafety of oral MnLR/Gly and reuterin in vivo. Healthy\nC57BL/6 mice were orally gavaged with PBS, reuterin or MnLR/Gly daily for 30 days,\nfollowed by a 3-day withdrawal. a, The histopathological analysis of the vital organs. Scale\nbar, 250 μm. b-g, The biochemical assays and inflammation assessment on the sera (n = 5).\nTP (g/l), ALT (u/l), AST (u/l), ALP (u/l), TBIL-Z (umol/l), TC (mmol/l), HDL-C (mmol/l), LDL-\nC (mmol/l), LP(a) (mg/l). h, Mn residual contents within the organs and serum of mice with or\nwithout MnLR treatment detected by ICP-OES (Agilent 5800 ICP-OES, Malaysia) (n = 4).\nData are shown as mean ± SD;there was no significant difference among the groups; one-way\nThe relevant description on pages 19, 20, lines 445-457：\n“The systemic biosafety of prolonged MnLR/Gly or reuterin treatments was assessed after 30\ndays of continuous oral administration (100 μl including 10 μl of glycerol and 5×10⁹ CFU of\nL. reuteri, once daily) followed by a 3-day withdrawal. Serum biochemical analyses (indicators\nfor key organ functions, lipid profiles, and electrolytes) and histopathological examinations of\nmajor organs (heart, liver, spleen, lung, kidney, and intestine) showed no significant\nabnormalities compared with healthy controls (Supplementary Fig. 28a-f). Likewise, the levels\nof acute inflammatory cytokines in serum (IL-1β and TNF-α) returned to baseline after\nwithdrawal, demonstrating the absence of persistent inflammatory responses (Supplementary\nFig. 28g). Furthermore, manganese biodistribution analysis revealed that residual Mn levels in\nserum and major organs after one month of MnLR/Gly administration remained consistent with\nthe physiological manganese concentrations in normal mice (Supplementary Fig. 28h),\nindicating efficient clearance and the absence of Mn accumulation. These findings demonstrate\nthat MnLR/Gly and reuterin do not induce systemic toxicity and sustained inflammation,\nhighlighting their potential value for clinical translation.”\n2. The experiment lacks characterization of the specific metabolites responsible for the\nenhanced antitumor effect. While Figures 1e and 1f demonstrate the superior efficacy of mixed\nmetabolites, they do not identify which specific components beyond reuterin contribute to this\neffect. Understanding the composition and relative contributions of different metabolites is\ncrucial for optimizing the therapeutic potential and ensuring reproducibility. The identification\nof specific bioactive metabolites could not only elucidate the mechanism of action but also\npotentially serve as diagnostic biomarkers. Furthermore, comprehensive metabolite\ncharacterization could aid in optimizing the efficacy of natural product-derived therapies and\nreducing side effects by pinpointing cancer-specific metabolic vulnerabilities. Addressing this\ngap would align the study with current best practices in metabolomics-based cancer research\nand significantly enhance its impact on therapeutic development.\nResponse: We sincerely thank the reviewer for emphasizing the importance of identifying\nbioactive metabolites beyond reuterin. We fully acknowledge that pinpointing additional\nmetabolites is critical for mechanistic understanding, therapeutic optimization, and potential\n26\nbiomarker development. In our original manuscript, mass spectrometry analysis revealed that,\nin addition to reuterin (constituting only 2.48% of the total metabolites), the mixed metabolites\ncontained reuterin’s downstream products and nearly 20 additional compounds with relative\nabundances above 0.1% (Supplementary Table 1). We demonstrated that pure reuterin and its\nchemically defined downstream products exerted weaker growth-inhibitory effects on colon\ncancer cells than the complete metabolite mixture (Fig. 1b and Supplementary Fig. 2),\nindicating that unidentified metabolites likely contribute to the overall cytotoxicity. We further\nanalyzed intracellular ROS levels and found that reuterin significantly increased ROS, but to a\nsubstantially lower extent than the mixed metabolites, while its downstream products only\nslightly elevated ROS (Supplementary Fig. 3), suggesting additional or alternative mechanisms\nbeyond ROS induction. Although we detected over 20 metabolites with significant relative\nabundance, the majority lack well-defined chemical identities and are not commercially\navailable, which limits direct functional testing. We agree that further systematic\ncharacterization of the unidentified metabolites and their individual or combined contributions\nwill be necessary. These findings and corresponding descriptions have been provided in the\nrevised manuscript.\nSupplementary Fig. 2 | The growth-inhibitory effects of pure reuterin, 1,3-propanediol\n(1,3-PD), and 3-hydroxypropionic acid (3-HP) (two downstream products of reuterin) on\ndifferent cells. a,c,d, Cell viability of the NCM460, HCT116, and MC38 cell lines after 24-\nhour incubation with the pure reuterin, 1,3-PD, and 3-HP at different concentrations (n = 5 per\ngroup).\npropanediol (1,3-PD), and 3-hydroxypropionic acid (3-HP). a,b, The flow cytometric\nimages and corresponding quantification of ROS (labeled by DCFH-DA) in MC38 cells with\nthe indicated treatments (n = 3). Data are shown as mean ± SD; **p < 0.01, ****p < 0.0001\n(compared to Ctrl); one-way ANOVA.\nThe relevant description on page 6, lines 140-151：\n“Notably, mass spectrometry analysis revealed the presence of various compounds from\nglycerol catabolism within the mixed metabolites in addition to reuterin, including reuterin’s\ndownstream products and nearly 20 unidentified compounds (at a percentage of > 0.1%)\n(Supplementary Table 1). Furthermore, the growth-inhibitory efficacies of the pure reuterin and\nits downstream products (1,3-propanediol and 3-hydroxypropionic acid) on colon cancer cells\nwere lower in comparison to the mixed metabolites (containing an equivalent amount of\nreuterin) (Fig. 1b and Supplementary Fig. 2), indicating the potential cytotoxic effects of those\nunidentified metabolites on cancer cells. Consistent with these findings, reuterin elevated\nintracellular ROS levels in colon cancer cells, but to a markedly lower extent than the mixed\nmetabolites (Supplementary Fig. 3); whereas its downstream products only caused a slight\nincrease in ROS (Supplementary Fig. 3), suggesting that these products may exert cytotoxicity\nthrough ROS-independent or partially ROS-independent mechanisms.”\nThe relevant description in the Discussion section on page 34, lines 729-733：\n“According to our compositional analysis of glycerol metabolites, reuterin constituted only\n2.48%, with its known downstream metabolites (1,3-PD and 3-HP) accounting for 13.45%.\nTherefore, it is necessary to further explore and identify other metabolites with tumor-\n28\ninhibitory effects, which would be critical for mechanistic understanding, therapeutic\noptimization, and potential biomarker development.”\n3. The study lacks a direct comparison to current standard-of-care treatments for colon cancer,\nwhich is crucial for assessing the true clinical potential of GlyMnLR. While the efficacy of\nGlyMnLR against a PBS control is impressive, its performance relative to existing colon cancer\ntreatments remains unknown. Current literature demonstrates extensive comparative\neffectiveness studies among established treatments such as bevacizumab, cetuximab, and\npanitumumab. These studies have shown varying impacts on overall survival and progression-\nfree survival. Additionally, emerging therapies like immune checkpoint inhibitors and RNA-\nbased treatments are being actively researched, mainly in MSI-high CRC. Without direct\ncomparison to these established and emerging treatments, it's challenging to determine whether\nGlyMnLR offers advantages in terms of efficacy or safety. Future studies should include head-\nto-head comparisons with current standard-of-care treatments, considering factors such as\noverall survival, progression-free survival, and safety profiles, as demonstrated in existing\ncomparative studies.\nResponse: We sincerely thank the reviewer for raising this important point regarding the need\nto compare MnLR/Gly with current standard-of-care treatments for colon cancer to better\nassess its true clinical potential. Following this suggestion, we performed additional\ncomparative experiments in the MC38 orthotopic colon cancer model (MSI-H),\ncomparing\nMnLR/Gly (100 μl including 10 μl of glycerol and 5×10⁹ CFU of L. reuteri, orally once daily)\nwith 5-fluorouracil (5-FU; one of the first-line chemotherapeutic agents for CRC; 400 μg,\nintraperitoneally every other day; Biomed Pharmacother. 2017 Nov;95:387-393), PD-1\nantibody (the most widely used immune checkpoint inhibitor; 100 μg, intraperitoneally every\nthree days; Nat Nanotechnol. 2025 Jul;20(7):935-946), and panitumumab (EGFR monoclonal\nantibody; 200 μg, intraperitoneally every three days; MAbs. 2015;7(2):440-50.).\nThe results showed that MnLR/Gly achieved a tumor inhibition rate 1.14-fold higher than\nPD-1 antibody, 1.25-fold higher than 5-FU, and 2.9-fold higher than panitumumab\n(Supplementary Fig. 31). Moreover, MnLR/Gly significantly prolonged survival, with 80% of\nmice surviving at least 60 days, compared with lower survival in the other groups (Fig. 6d).\nThese advantages arise from MnLR/Gly’s potent antitumor efficacy, combined with its\nminimal systemic toxicity (Supplementary Fig. 28), and safe, convenient oral administration.\nproduced more economically via scalable bacterial culture followed by straightforward\nmanganese modification, further underscoring its translational potential.\nWe appreciate the reviewer’s suggestion, which significantly strengthens the translational\nrelevance of our work. The new data and corresponding descriptions have been incorporated\ninto the revised manuscript.\nFig. 6: In vivo orthotopic primary colon tumor growth inhibition, gut microbiota\nmodulation, and antitumor immunity activation by oral MnLR/Gly. d, Survival\npercentages of colon tumor-bearing mice receiving different treatments (n = 10).\nSupplementary Fig. 31 | Comparative antitumor efficacy of MnLR/Gly and standard-of-\ncare treatments in the orthotopic colon tumor model. a, In vivo bioluminescence and in vitro\nimages of the colon tumors in mice receiving the indicated treatments at the given time points\n30\nafter orthotopic tumor inoculation. Scale bar, 1 cm. b,c The corresponding fluorescence\nquantification of colon tumors (n = 5). Data are shown as mean ± SD; *p < 0.05 (compared to\nPBS group), one-way ANOVA.\nThe relevant description on pages 22, 23, lines 491-501：\n“To further evaluate the therapeutic potential of MnLR/Gly relative to established colon cancer\ntherapies, we compared its efficacy with 5-fluorouracil (5-FU, a first-line chemotherapy drug),\nPD-1 antibody (a representative immune checkpoint inhibitor), and panitumumab (an EGFR\nmonoclonal antibody) in the orthotopic colon tumor model. MnLR/Gly achieved a tumor\ngrowth inhibition rate 1.14-fold higher than PD-1 antibody, 1.25-fold higher than 5-FU, and\n2.9-fold higher than panitumumab (Supplementary Fig. 31). Moreover, MnLR/Gly\nsignificantly extended overall survival, with 80% of mice surviving at least 60 days, in contrast\nto lower survival rates in the other groups (Fig. 6d). These advantages arise from MnLR/Gly’s\npotent antitumor activity, combined with its minimal systemic toxicity, and safe, convenient\noral administration. In addition, MnLR/Gly holds promise for lower production costs compared\nto antibody- or chemotherapy-based regimens, highlighting its potential translational value.”\n4. The study lacks quantitative analysis of capsule dissolution kinetics and MnLR release\nprofiles in vivo. While Figures 8c and 8d provide qualitative evidence of capsule behavior, they\ndo not offer precise measurements of dissolution rates or MnLR release over time in the\ngastrointestinal tract. This quantitative data is crucial for understanding the exact timing and\nextent of MnLR delivery, which directly impacts its therapeutic efficacy. A more detailed\nkinetic analysis would strengthen the claim about the capsule's targeted delivery capabilities.\nResponse:We sincerely thank the reviewer for this insightful comment regarding the need for\nquantitative analysis of capsule dissolution kinetics and MnLR release in vivo. To address this,\nwe performed quantitative in vivo experiments in rabbits using glycerol capsules loaded with\nCy5.5-labeled MnLR. Fluorescence analysis revealed that only ~26.7% of MnLR was released\nwithin 4 hours in the stomach, consistent with endoscopic and X-ray imaging that exhibited\ncapsule integrity at this stage (Fig. 8b-e and Supplementary Fig. 44). During the subsequent\ngastrointestinal transit (4–10 hours) and intestinal residence (10–20 hours), the capsules\nprogressively disintegrated, leading to marked MnLR release, with cumulative release reaching\n~83.5% within 20 hours (Fig. 8d,e). These results demonstrate that GlyMnLR capsules remain\n31\nstructurally stable in the gastric environment and achieve sustained, intestine-targeted release\nof MnLR over time, thereby ensuring efficient delivery at the site of tumor growth. The\ncorresponding quantitative data and descriptions have been incorporated into the revised\nmanuscript.\nFig. 8: Characterization of GlyMnLR capsule and its antitumor efficacy and biosafety in\nrabbit models. a, The images of the serial glycerol capsules with different external diameters.\nScale bar, 2 mm. b, Fluorescence images showing the capsule dissolving with the release of\nCY 5.5-labeled MnLR in SGF and SIF. c, X-ray images showing the GlyMnLR capsule\ncontaining barium sulfate during gastrointestinal transportation after being orally taken by\nrabbits. Scale bar, 5 mm (left); 2 cm (right). d,e, Fluorescence images of GlyMnLR capsule\ndissolution and corresponding quantification of MnLR (labeled with CY5.5) release at the\ngiven time points after oral administration (n = 5).\nSupplementary Fig. 44 | The morphological change of the GlyMnLR capsule within a rabbit’s\nstomach observed by endoscopy. The capsule remained intact in the gastric environment for at least\n4 hours without disintegration following oral administration.\n“In vitro and in vivo fluorescence imaging showed that the dissolution rate of the GlyMnLR\ncapsule (MnLR labeled by Cy5.5) was significantly faster in simulated intestinal fluid (SIF)\nand real intestinal tract compared to simulated gastric fluid and real stomach (SGF) (Fig. 8b,d).\nThis finding was corroborated by endoscopy examination (Supplementary Fig. 44) and X-ray\nimaging (Fig. 8c), which revealed that the capsule remained intact in the rabbit’s stomach for\nat least 4 hours after oral administration. After reaching the intestine, the barium signal of the\ncapsule was weakened within 20 hours owing to the dissolution of the capsule in intestinal fluid\n(Fig. 8c). Consistently, in vivo quantitative analysis showed that only ~26.7% of MnLR was\nreleased within 4 hours in the stomach, whereas release markedly accelerated during\ngastrointestinal transit (4–10 hours) and intestinal residence (10–20 hours), with cumulative\nrelease reaching ~83.5% within 20 hours (Fig. 8d,e). These observations collectively\ndemonstrated the capsule’s resistance to gastric juice and its solubility in the\nintestinal environment, ensuring sustained and targeted release of MnLR at the site of tumor growth.”\n5. The study lacks a dose-response analysis for GlyMnLR capsule treatment, which is critical\nfor understanding its optimal efficacy and potential toxicity threshold. While the current data\nin Figures 8f-j demonstrate significant tumor suppression at a single dose, the absence of a\ndose-response curve limits our understanding of the treatment's full potential. This gap is\nparticularly significant given the established importance of dose-response relationships in drug\ndevelopment and the potential risks associated with high-dose antioxidant supplementation. A\ncomprehensive dose-response study would help determine the therapeutic window and guide\ndosing strategies for future studies or potential clinical trials, aligning with best practices in\ndrug development and antioxidant research.\nResponse:We thank the reviewer for raising this important point regarding the lack of dose–\nresponse analysis. We have in fact performed GlyMnLR capsule dose-optimization\nexperiments by comparing three dosing regimens in the orthotopic colon tumor model: (1) one\ncapsule every other day, (2) one capsule once daily, and (3) two capsules once daily (each\nGlyMnLR capsule containing 1011 CFU of MnLR). The results showed that both regimens (2)\nand (3) achieved significantly stronger tumor suppression than regimen (1), with inhibition\nrates approximately 2.99-fold higher (Supplementary Fig. 45). Importantly, the difference in\nefficacy between regimens (2) and (3) was marginal (Supplementary Fig. 45), while the latter\n33\nmight raised concerns of increased metabolic burden and potential toxicity. Therefore, we\nselected “one capsule once daily” as the optimal regimen for subsequent in vivo studies,\nbalancing therapeutic efficacy and safety.\nSupplementary Fig. 45 | Dose optimization of GlyMnLR capsules for rabbit CRC models.\na, Representative in vivo (endoscopic, the top and middle panels) and ex vivo (the bottom panel)\nimages of tumors (highlighted with yellow dashed boxes) on days 10, 28, and 31 post oral\nadministration of the indicated agents. b, The tumor volumes obtained from the corresponding\ngroups at the end of the experiment (n = 5). Scale bar, 1 cm. Data are shown as mean ± SD;\n**p < 0.01, ***p < 0.001 (compared to PBS group); one-way ANOVA.\nThe relevant description on page 31, lines 664-672：\n“To optimize the dosing strategy of GlyMnLR capsules, we evaluated three regimens in the\nmodel: one capsule every other day, one capsule once daily, and two capsules once daily. Both\nonce-daily regimens achieved stronger tumor suppression than one capsule every other day\n(approximately 3-fold higher), while the efficacy difference between one and two capsules\ndaily was minimal (Supplementary Fig. 45), with the latter posing greater metabolic burden\nand safety concerns. The tumor-suppressive efficacy of once-daily oral administration of one\nGlyMnLR was further confirmed by in vivo CT imaging and photographs of the isolated tumors\nat the end of the experiments (Fig. 8g-i). Accordingly, we selected one capsule once daily as\nthe optimal dosing regimen for subsequent therapeutic studies.”\n6. The long-term stability and retention of Mn2+ induced enhancements in L. reuteri have not\nbeen assessed. While Figures 2b-e show clear short-term enhancements, the durability of these\neffects over multiple generations or in varying environmental conditions is crucial for practical\napplications. Long-term stability would impact the efficacy of Mn2+ modified L. reuteri in\n34\ntherapeutic settings, especially considering potential variations in gut conditions.\nResponse:We appreciate the reviewer’s insightful comment regarding the long-term stability\nof Mn²⁺-induced enhancements in L. reuteri. To address this, we evaluated both generational\npersistence and stability under simulated gut conditions: (1) L. reuteri strains were propagated\nto the 3rd and 50th generations on MRS agar medium, and comparative analyses revealed no\nsignificant differences in Mn²⁺-mediated promotion of bacterial growth or glycerol metabolism\nbetween early (3rd generation) and late (50th generation) passages (Fig. 2e), indicating that the\nenhancements are retained over extended cultivation. (2) In simulated intestinal fluid (SIF)\nsupplemented with 500 µM Mn²⁺, the bacteria continued to exhibit enhanced proliferation and\nglycerol metabolism (Fig. 2f), suggesting that these effects are durable under conditions\nrelevant to CRC therapy. These results and corresponding descriptions have been incorporated\ninto the revised manuscript.\nFig. 2: Screening, preparation, and characterization of MnLR. e, The proliferation and\nreuterin yield of L. reuteri of 3rd or 50th generations cultured with or without Mn2+ (n = 5). f,\nThe proliferation and reuterin yield of L. reuteri cultured in SIF with or without Mn2+ (n = 5).\nData are shown as mean ± SD; ****p < 0.0001; one-way ANOVA.\nThe relevant description on pages 8, 9, lines 202-207：\n“Furthermore, to assess the long-term retention of Mn-mediated enhancements, L. reuteri\nstrains were propagated up to the 50th generation, with no significant differences observed in\ngrowth or glycerol metabolism compared with early passages (3rd generation) (Fig. 2e),\ndemonstrating generational stability. In addition, Mn²⁺ supplementation sustained enhanced\nbacterial proliferation and glycerol metabolism in simulated intestinal fluid (SIF) (Fig. 2f),\nindicating that these effects are durable under conditions relevant to CRC therapy.”\n7. The stability and retention of manganese modification in MnLR under simulated\n35\ngastrointestinal conditions have not been adequately assessed. The stability of the Mn\nmodification in acidic stomach conditions and the presence of digestive enzymes is crucial for\nits effectiveness as an oral probiotic treatment\nResponse:We sincerely thank the reviewer for highlighting the importance of evaluating the\nstability and retention of manganese modification under gastrointestinal conditions. To address\nthis concern, we conducted comparative assays evaluating the proliferative viability of raw L.\nreuteri and MnLR in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF),\nrelative\nto PBS. Both raw L. reuteri and MnLR exhibited reduced proliferative activity in SGF and SIF\ncompared with PBS; however, MnLR consistently maintained significantly higher viability\nthan raw L. reuteri (Fig. 2n,o), demonstrating the advantage of manganese modification in\nenhancing bacterial robustness under gastrointestinal stress.\nNotably, the proliferative activity of both raw L. reuteri and MnLR was lower in SGF than\nin SIF (Fig. 2n,o). The scanning electron microscopy also revealed that manganese\nmodification exhibited relatively lower stability in SGF compared to SIF (Supplementary Fig.\n42). These observations indicate that the acidic gastric environment and digestive enzymes\nexert intensive destabilizing effects. To preserve the bioactivity of MnLR prior to reaching the\ntherapeutic site, we therefore encapsulated MnLR together with glycerol into an enteric-soluble\ncapsule (GlyMnLR capsule). Both in vitro and in vivo experiments demonstrated that this\ncapsule design ensured relative stability in the gastric environment and efficient MnLR release\nin the intestine (Fig. 8b-e). Furthermore, in a rabbit orthotopic colon tumor model, the\nGlyMnLR capsule achieved significantly higher antitumor efficacy compared with free\nMnLR/Gly (Fig. 8j,k). Collectively, these results validate the necessity and effectiveness of the\ncapsule formulation in maintaining the therapeutic potential of MnLR for oral delivery.\nWe have incorporated these new experimental data and corresponding descriptions into the\nrevised manuscript to provide a more comprehensive assessment of the gastrointestinal\nstability of MnLR and the rationale for the GlyMnLR capsule formulation.\nFig. 2: Screening, preparation, and characterization of MnLR. n,o, The representative\nimages and corresponding colony counts of raw L. reuteri and MnLR cultured in SGF or SIF\n36\nfor 2 or 4 hours (n = 3). Data are shown as mean ± SD; *p < 0.05; one-way ANOVA.\nThe relevant description on pages 9,10, lines 231-235：\n“Furthermore, compared with PBS, both raw L. reuteri and MnLR exhibited reduced\nproliferative viability when exposed to simulated gastric fluid (SGF) and SIF (Fig. 2n,o).\nNevertheless, MnLR maintained significantly higher growth activity than raw L. reuteri under\nboth conditions (Fig. 2n,o), suggesting the protective role of Mn modification in enhancing\nresistance to gastrointestinal stresses.”\nSupplementary Fig. 42 | The stability and retention of manganese modification on L.\nreuteri under simulated gastrointestinal conditions.SEM imaging (the left panel) shows the\nsurface morphology of MnLR after a 4-hour incubation in SGF or SIF. Elemental mapping (the\nmiddle panel) indicates the presence of manganese on the bacterial surface, visualized as\nyellow signals. Scale bar, 2 μm.\nrabbit models. a, The images of the serial glycerol capsules with different external diameters.\nScale bar, 2 mm. b, Fluorescence images showing the capsule dissolving with the release of\nCY 5.5-labeled MnLR in SGF and SIF. c, X-ray images showing the GlyMnLR capsule\n38\ncontaining barium sulfate during gastrointestinal transportation after being orally taken by\nrabbits. Scale bar, 5 mm (left); 2 cm (right). d,e, Fluorescence images of GlyMnLR capsule\ndissolution and corresponding quantification of MnLR (labeled with CY5.5) release at the\ngiven time points after oral administration (n = 5). f, Schematic illustration of the therapeutic\nregime, endoscopic/CT imaging, and biochemical and immunological analyses on blood and\ntumor tissues in the primary CRC-bearing rabbit model. g, CT images obtained 28 days after\nthe treatment initiation showing the tumor occupation at different locations highlighted by red,\nyellow, and green colors. Scale bar, 5 cm. h,Quantification of the tumor occupation relative to\nthe PBS group (n = 5). i, Representative images showing the tumors (highlighted with yellow\ndashed box) isolated from the primary colon tumor-bearing rabbits 31 days after the treatment\ninitiation. Scale bar, 5 mm. j, Representative in vivo (endoscopic, the top and middle panels)\nand ex vivo (the bottom panel) images of tumors (highlighted with yellow dashed boxes) on\ndays 10, 28, and 31 post oral administration of the indicated agents. Scale bar, 1 cm. k, The\ntumor volumes obtained from the corresponding groups at the end of the experiment (n = 5). l,\nThe representative immunofluorescent images and corresponding statistical analysis of the\ngreen Ki67-stained proliferating cells within the isolated tumor tissues from rabbits receiving\nthe given treatments at the experimental end (n = 5 fields). Scale bar, 200 μm. m,n,\nRepresentative immunofluorescence staining images and corresponding quantification of\nCD45+CD8+ T cells within the tumor tissues isolated from the rabbits with the given treatments\n(n = 9 fields).Scale bar, 50 μm. Data are shown as mean ± SD; *p < 0.05, **p < 0.01, ***p <\n0.001, **** p < 0.0001 (compared to PBS group); Student’s t-test or one-way ANOVA.\nThe relevant description on pages 30, 31, lines 646-675：\n“Since the viability of both raw L. reuteri and MnLR was lower in SGF than in SIF (Fig. 2n,o),\nand SEM imaging further revealed that Mn modification was less stable in SGF compared to\nSIF (Supplementary Fig. 42), we prepared a dimension-tunable enteric-soluble capsule for\ndelivering MnLR encapsulated by glycerol (as one of the capsule materials) through 3D\nprinting and casting technology to preserve MnLR bioactivity before reaching the therapeutic\ntargets (Fig. 8a, and Supplementary Fig. 43) …… GlyMnLR capsules exhibited a markedly\nhigher tumor inhibition rate (67.9%) compared with free MnLR (25.4%) and reuterin capsules\n(8.0%), demonstrating the protective role of the enteric formulation and the advantage of\nsustained production of active metabolites by viable bacteria (Fig. 8j,k).”\n8. The study lacks validation of the functional consequences of the observed gene expression\nchanges in L. reuteri. While the RNA-seq data in Figures 2g and 2h demonstrate clear changes\nin gene expression, and Figure 2i shows enrichment of ribosome-related pathways, there is no\ndirect evidence that these transcriptional changes translate to increased ribosomal activity or\n39\nprotein synthesis. This gap leaves open the possibility that the observed gene expression\nchanges may not have the assumed functional impact on L. reuteri's bioactivity.\nResponse:We thank the reviewer for raising this important point. To functionally validate the\ntranscriptional changes observed in our RNA-seq analysis, we performed quantitative\nproteomic analysis (LC-MS/MS), which confirmed elevated levels of ribosomal proteins,\nincluding both small and large subunit components, in Mn²⁺-treated L. reuteri (Fig. 2i). In\nparallel, SDS-PAGE analysis revealed a clear increase in total protein expression compared\nwith the untreated control (Supplementary Fig. 9), further supporting enhanced global\ntranslational activity. Together, these findings provide direct functional evidence that the\ntranscriptional upregulation of ribosome-related pathways translates into increased ribosomal\nactivity and protein synthesis, thereby reinforcing our conclusion that Mn²⁺ modification\nenhances the bioactivity of L. reuteri by promoting its translational capacity. The\ncorresponding data and descriptions have now been incorporated into the revised manuscript.\nFig. 2: Screening, preparation, and characterization of MnLR. i, The quantification of\nribosomal proteins of L. reuteri with or without Mn2+ treatment by LC-MS/MS (n = 3). Data\nare shown as mean ± SD; *p < 0.05, **p < 0.01 (compared to Ctrl group); Student’s t-test.\nuntreated control. Whole-cell lysates were prepared from L. reuteri cultures treated with 500\nμM Mn2+ for 4 hours or from untreated control cultures. Prior to lysis, cultures were adjusted\nto identical optical density (OD600) so that sample input was normalized by cell density; equal\nvolumes of lysate were then resolved by SDS–PAGE and visualized with Coomassie Brilliant\nBlue staining. The gel shows relative changes in total protein banding patterns and overall\nprotein abundance between Mn-treated and control samples.\nThe relevant description on page 9, lines 213-219：\n“To functionally validate these transcriptomic findings, quantitative proteomic analysis\nconfirmed elevated levels of ribosomal proteins (Fig. 2i). SDS-PAGE further demonstrated\nincreased global protein synthesis in Mn-treated L. reuteri (Supplementary Fig. 9). These\nresults collectively establish that the transcriptional upregulation of ribosome-related pathways\nis functionally translated into enhanced ribosomal activity and protein synthesis, suggesting\nthat ribosomes may be a key target for the regulation of L. reuteri’s bioactivity by Mn ions42.”\n9. The study lacks a direct comparison of MnLR/Gly cytotoxicity on normal colon epithelial\ncells versus cancer cells. While the study demonstrates enhanced cytotoxicity of MnLR/Gly on\nvarious colon cancer cell lines (Figures 3a and 3b), it does not address potential effects on\nhealthy colon cells. This comparison is crucial to establish the specificity of MnLR/Gly's action\nand assess potential side effects on normal tissue. However, without specific data on\nMnLR/Gly's effects on normal colon epithelial cells, it remains challenging to fully evaluate\nits therapeutic potential and safety profile. This gap is particularly important given the observed\npreferential activation of manganese-based nanomaterials in cancer cells due to higher GSH\n41\nlevels, which may or may not apply to MnLR/Gly. Additional experiments comparing\nMnLR/Gly's effects on both cancerous and normal colon epithelial cells are necessary to\naddress this concern.\nResponse:We thank the reviewer for this important comment regarding the need to evaluate\nMnLR/Gly cytotoxicity in normal cells. To address this, we performed additional experiments\nand found that MnLR/Gly exhibited markedly lower cytotoxicity in normal intestinal epithelial\ncells (NCM460), human umbilical vein endothelial cells (HUVEC), and norma human liver\ncells (L-02), with cell viability remaining above 80%, in contrast to the pronounced inhibitory\neffects on multiple CRC cells (Fig. 3a and Supplementary Fig. 12). This result is consistent\nwith previous reports that L. reuteri-derived glycerol metabolites (including reuterin) induces\noxidative stress in a GSH-dependent manner, which may explain its preferential toxicity toward\ncancer cells (Fig. 1b,d and Supplementary Fig.1; Cancer Cell. 2022 Feb 14;40(2):185-200.e6).\nAdditionally, serum biochemical analyses and histopathological examinations demonstrated\nthat continuous oral administration of MnLR/Gly did not cause discernible functional\nimpairments or structural damage to major organs (heart, liver, spleen, lung, kidney, and\nintestine) (Supplementary Fig. 28). We believe these results address the reviewer’s concern\nand strengthen the evidence for its therapeutic specificity.\nFig. 3: In vitro colon cancer cell inhibition and DCs maturation mediated by MnLR/Gly.\na, Cytotoxicity of L. reuteri, glycerol, L. reuteri/Gly, and MnLR/Gly in multiple human colon\ncancer cell lines (SW48, HCT116, RKO, SW620, DLD-1) and mouse colon cancer cell lines\n(MC38, CT26) after a 24-hour incubation (n = 3 or 4). Data are shown as mean ± SD; *p <\n0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; one-way ANOVA.\nlines. Cell viabilities of NCM460, HUVEC, and L-02 cells after being treated with L. reuteri\n(109 CFU), glycerol (5 μl), L. reuteri/Gly, or MnLR (109 CFU)/Gly in 500 μl of culture media\nfor 24 hours (n = 4). Data are shown as mean ± SD.\nThe relevant description on page 11, lines 267-270：\n“Consistent with the effects observed for L. reuteri-derived mixed metabolites, MnLR/Gly\nexhibited preferential cytotoxicity toward colon cancer cells over non-cancerous cells (Fig. 3a\nand Supplementary Fig. 12), indicating its anticancer efficacy, alongside reduced toxicity to\nnormal proliferating cells.”\nC57BL/6 mice were orally gavaged with PBS, reuterin or MnLR/Gly daily for 30 days,\nfollowed by a 3-day withdrawal. a, The histopathological analysis of the vital organs. Scale\nbar, 250 μm. b-g, The biochemical assays and inflammation assessment on the sera (n = 5).\nTP (g/l), ALT (u/l), AST (u/l), ALP (u/l), TBIL-Z (umol/l), TC (mmol/l), HDL-C (mmol/l), LDL-\nC (mmol/l), LP(a) (mg/l). h, Mn residual contents within the organs and serum of mice with or\nwithout MnLR treatment detected by ICP-OES (Agilent 5800 ICP-OES, Malaysia) (n = 4).\nData are shown as mean ± SD;there was no significant difference among the groups; one-way\nANOVA (b-g) or Student’s t-test (h).\nThe relevant description on pages 19, 20, lines 445-457：\n“The systemic biosafety of prolonged MnLR/Gly or reuterin treatments was assessed after 30\ndays of continuous oral administration (100 μl including 10 μl of glycerol and 5×10⁹ CFU of\nL. reuteri, once daily) followed by a 3-day withdrawal. Serum biochemical analyses (indicators\nfor key organ functions, lipid profiles, and electrolytes) and histopathological examinations of\nmajor organs (heart, liver, spleen, lung, kidney, and intestine) showed no significant\nabnormalities compared with healthy controls (Supplementary Fig. 28a-f). Likewise, the levels\nof acute inflammatory cytokines in serum (IL-1β and TNF-α) returned to baseline after\nwithdrawal, demonstrating the absence of persistent inflammatory responses (Supplementary\nFig. 28g). Furthermore, manganese biodistribution analysis revealed that residual Mn levels in\n44\nserum and major organs after one month of MnLR/Gly administration remained consistent with\nthe physiological manganese concentrations in normal mice (Supplementary Fig. 28h),\nindicating efficient clearance and the absence of Mn accumulation. These findings demonstrate\nthat MnLR/Gly and reuterin do not induce systemic toxicity and sustained inflammation,\nhighlighting their potential value for clinical translation.”\n10. The study does not provide direct measurement of cell death and long-term cell fate\nfollowing MnLR/Gly treatment. While the measured markers are associated with ICD, they do\nnot definitively prove that cells undergo immunogenic death. As shown in Figures 3c-f, these\nmarkers could potentially indicate cellular stress without necessarily leading to cell death.\nDirect assessment of cell viability and analysis of long-term cell fate are crucial to confirm the\noccurrence of ICD and rule out potential adaptive responses.\nResponse: We thank the reviewer for this important suggestion. In accordance with the\ncomment, we performed Annexin V/PI flow cytometry and Live/Dead staining assays to\ndirectly evaluate cell death and long-term cell fate following MnLR/Gly treatment. Flow\ncytometry results revealed that MnLR/Gly induced substantial cell death in colon cancer cells,\nwith the apoptotic population reaching 42.6%, which was significantly higher than 2.0% in\nuntreated cells and 19.2% in reuterin-treated cells (Supplementary Fig. 13e). Consistently,\nLive/Dead staining demonstrated a significantly increased number of dead colon cancer cells\nin the MnLR/Gly group compared with the other groups (Supplementary Fig. 13f). These\nfindings confirm that the ICD-associated markers indeed reflect bona fide immunogenic cell\ndeath rather than transient stress, thereby strengthening our conclusion regarding the ICD-\ninducing capacity of MnLR/Gly.\nMnLR/Gly. a. The intracellular ROS (stained by green 2’,7’-dichlorofluorescein diacetate\n(DCFH-DA)) in MC38 cells after a 12-hour incubation with MnLR/Gly. Scale bar, 200 μm. b,\nThe representative immunofluorescence images showing the expression and subcellular\n46\nlocalization of CRT (green, stained with anti-CRT/FITC) in MC38 cells after being treated with\nreuterin or MnLR/Gly for 12 hours. Red, cell membrane stained with Dil; blue, nuclei stained\nwith Hoechst 33342. Scale bar, 10 μm. c,d, The HMGB1 and ATP levels in MC38 cells after a\n24-hour incubation with reuterin or MnLR/Gly (n = 4 or 5). e. The proportion of apoptotic\nMC38 cells treated with reuterin or MnLR/Gly for 24 hours was analyzed by flow cytometry\nusing Annexin V-Fluor488/PI Apoptosis Detection Kit (Beyotime Biotechnology, China) (n =\n3). f. The live/dead staining images and statistical analysis of the dead cell counts after a 24-\nhour treatment with the given agents (n = 3). Scale bar, 50 μm. Data are shown as mean ± SD;\n**p < 0.01, ***p < 0.001, ****p < 0.0001 (compared to Ctrl); one-way ANOVA.\nThe relevant description on pages 11, 12, lines 275-281：\n“Moreover, Annexin V/PI flow cytometry revealed that MnLR/Gly treatment induced\nsubstantial cell death in colon cancer cells, with the apoptotic cell population reaching 42.6%,\nmuch higher than 2.0% in untreated cells and 19.2% in reuterin-treated cells (Supplementary\nFig. 13e). Consistently, Live/Dead staining confirmed a significantly increased number of dead\ncells in the MnLR/Gly group (Supplementary Fig. 13f). Together, these results validate that the\nICD-associated markers reflect bona fide immunogenic cell death rather than transient stress. ”\n11. The research does not include in vivo validation of the combined effect of MnLR/Gly and\nM1 macrophages on tumor growth. While the 3D MCT model provides valuable insights, it\nmay not fully recapitulate the complex tumor microenvironment found in vivo. In vivo studies\nare crucial to confirm the translational potential of these findings, assess any potential systemic\neffects or unexpected interactions, and validate the efficacy of the combination therapy in a\nphysiologically relevant context.\nResponse: Wesincerely thank the reviewer for raising this important point. In our study, we\nhave demonstrated that MnLR/Gly treatment promotes macrophage polarization toward the\nM1 phenotype in vivo (Fig. 6e-g and Fig. 7i). To further assess the functional relevance of\nmacrophages, we performed macrophage depletion experiments using clodronate-encapsulated\nliposomes. The results showed that depletion of macrophages attenuated the therapeutic and\npreventive effects of MnLR/Gly in both orthotopic and metastatic colon tumor models,\nalthough these effects were not completely abolished (Fig. 6b,c, Fig. 7b, and Supplementary\nFig. 37).These findings indicate that the antitumor efficacy of MnLR/Gly is at least partially\ndependent on its immunomodulatory regulation of macrophages.\nthe role of macrophages in mediating the therapeutic effects of MnLR/Gly, they may not fully\nrecapitulate the synergistic interplay between MnLR/Gly and adoptively transferred M1\nmacrophages as modeled in the 3D MCT system. Future studies involving co-administration\nof MnLR/Gly with exogenous M1 macrophages in vivo would be valuable to better validate\nthis combination strategy.\nIn addition, we would like to emphasize with caution that the focus of our study is to explore\nthe potential of MnLR/Gly not only as an adjuvant, but also as a candidate with independent\ntherapeutic and preventive activity. Unlike prior approaches that typically employ probiotics\nas supportive supplements, our findings highlight that MnLR/Gly exerts multifaceted\nantitumor activities—including specific cytotoxicity against tumor cells, activation of\nantitumor immunity, and modulation of the gut microbiota. We recognize that further\nvalidation is required, but we believe these results provide a meaningful step toward\nunderstanding probiotics as potential stand-alone therapeutic options.\nantitumor immunity activation by oral MnLR/Gly. a, Schematic illustration of the\nestablishment of primary colon tumor model by orthotopic inoculation of MC38 cells into\ncolon cavity of mice, once-daily oral gavage of the given agents, macrophage or CD8+ T cell\n(Mac/CD8 T) depletion by clodronate liposomes or anti-CD8 antibody, and analyses on the\nbiological samples (stool, tumor, spleen, and MLNs) isolated from the mice at the endpoint.\n49\nb,c, Bioluminescence imaging and the corresponding quantification of the colon tumors in the\nmice receiving different treatments at the given time points afterorthotopic tumor inoculation\n(n = 5). d, Survival percentages of colon tumor-bearing mice receiving different treatments (n\n= 10). e,f, Representative immunofluorescence staining images and the corresponding\nquantification of M1 macrophages (F4/80+CD86+), M2 macrophages (F4/80+CD206+), mature\nDCs (CD11c+CD86+), activated CD4+ and CD8+ T cells (IFN-γ+CD4+ and IFN-γ+CD8+) within\nthe tumor tissues from the mice receiving the given treatments 14 days after tumor inoculation\n(n = 15 fields from 3 mice per group). Scale bar, 50 μm. g-j, Quantification of the ratio of M1\nto M2 macrophages, and the percentages of mature DCs in total CD45+ cells, activated CD4+\nand CD8+ T cells in total CD3+ cells within the spleen from the colon tumor-bearing mice\nreceiving the given treatments 14 days after tumor inoculation (n = 5). k,l, Quantification of\nthe percentages of activated CD4+ and CD8+ T cells in total CD3+ cells within the MLNs from\nthe colon tumor-bearing mice receiving the given treatments 14 days after tumor inoculation\n(n = 5). m,Principal component analysis illustrating the β-diversity from 16S rRNA sequencing\nof stool samples collected from the healthy or primary colon tumor-bearing mice receiving the\ngiven treatments on day 14 after tumor inoculation (n = 6 to 8). n, Quantification of colon\ntumors in mice at the experimental endpoint under the indicated treatments. Mice were\npretreated with antibiotics (ABX) for two weeks prior to tumor cell inoculation to deplete gut\nmicrobiota (n = 5).Data are shown as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p\n< 0.0001 (compared to PBS group); one-way ANOVA.\nThe relevant description on pages 23, 24, lines 505-532：\n“As expected, the activated M1 macrophages (F4/80+CD86+) and mature DCs (CD11c+CD86+)\nwere significantly enriched in the tumor tissues of the mice treated with MnLR/Gly,\nconcomitantly with an obvious reduction in M2 macrophages (F4/80+CD206+) (Fig. 6e,f) ......\nWe also analyzed the changes of immune cells in the peripheral immune organs, including the\nspleen and mesenteric lymph nodes (MLNs), which are critical for priming effector T cells and\ndriving systemic antitumor immunity 47,48. Only the spleens from the mice treated with\nMnLR/Gly exhibited a simultaneous increase in the ratio of M1 over M2 macrophages, mature\nDCs, and IFN-γ-expressing CD4+ and CD8+ T cells (Fig. 6g-j and Supplementary Fig. 32) ......\nAdditionally, depleting either CD8+ T cells or macrophages (T dep. or Mac dep.) just attenuated\nthe tumor-inhibitory rate of MnLR/Gly to a level comparable to reuterin, rather than completely\noffsetting the antitumor effects (Fig. 6b,c, and Supplementary Fig. 34). These observations\ncollectively indicate a significant role of the direct cytotoxicity triggered by MnLR-produced\nmetabolites (containing reuterin) in inhibiting primary tumors in vivo. Also, the antitumor\nimmune responses elicited by MnLR/Gly are not confined to a single immune cell type and\nmight even involve the participation of immune cells beyond T cells or macrophages. This\n50\nmight be particularly advantageous for treating tumor subtypes with deficiencies in specific\nimmune cell populations.”\nFig. 7: In vivo prevention of primary colon tumor occurrence and metastasis. b,\nBioluminescence imaging of the colon tumors in the mice receiving different treatments at the\ngiven time points after initial orthotopic tumor inoculation (n = 5). i, Quantification of the ratio\nof M1 (CD11b+F4/80+CD86+) to M2 (CD11b+F4/80+CD206+) macrophages within the spleen\nfrom the mice receiving the given treatments for 10 days (n = 5). Data are shown as mean ±\nSD; ***p < 0.001 (compared to PBS group); one-way ANOVA.\nSupplementary Fig. 37 | Prevention of primary colon tumor occurrence by oral\nMnLR/Gly. a, The fluorescence intensity quantification of the colon tumors in the mice\nreceiving different treatments at the given time points after orthotopic tumor inoculation (n =\n5). b, Representative images showing the colon tumors (highlighted with blue dashed boxes)\nisolated from the mice 14 days after orthotopic tumor inoculation. Scale bar, 1 cm. G1, PBS;\nG2, Gly; G3, Reuterin; G4, L. reuteri; G5, L. reuteri/Gly; G6, MnLR/Gly; G7, MnLR/Gly +\nCD8 T dep; G8, MnLR/Gly + Mac dep. Data are shown as mean ± SD; *p < 0.05, **p < 0.01\n(compared to PBS); one-way ANOVA.\n“Next, we investigated whether the lasting antitumor effects of MnLR/Gly involved the\nenhanced systemic antitumor immunity. Indeed, MnLR/Gly promoted the splenic macrophage\npolarization towards M1 phenotype and DC maturation and concomitantly enriched the IFN-\nγ-expressing CD4+ and CD8+ T cells in MLNs (Fig. 7i-l and Supplementary Fig. 39). ELISA\nassays further revealed that MnLR/Gly treatment significantly elevated the levels of immune\neffector cytokines (IFN-γ, TNF-α, perforin, and granzyme) in serum and MLNs\n(Supplementary Fig. 40). Consistently, the depletion of CD8+ T cells or macrophages reduced\nthe efficiency of MnLR/Gly in suppressing the formation of primary colon tumors (Fig. 7b,\nand Supplementary Fig. 37). These findings collectively suggest that MnLR/Gly can\nsimultaneously and functionally regulate multiple immune cells across the peripheral immune\nsystem in healthy individuals to generate a lasting antitumor effect for preventing colon tumor\ndevelopment.”\n12. The study lacks direct visualization of bacterial localization within tumor tissues at a\ncellular level. High-resolution imaging of tumor sections, possibly using optimized intravital\nmicroscopy techniques, could reveal the precise distribution of bacteria within the tumor\nmicroenvironment and healthy tissues, addressing this critical gap in the current study.\nResponse: We sincerely thank the reviewer for highlighting the importance of directly\nvisualizing bacterial localization within tumor tissues. To address this, we performed ex vivo\ntissue immunofluorescence staining 24 hours after oral gavage of MnLR, with L. reuteri\nlabeled using Cy5.5. The analysis revealed bacterial distribution in the intestinal lumen,\nintestinal wall, and tumor tissues. Quantitative fluorescence analysis further indicated that\nbacterial signals in tumors were approximately 1.4-fold higher than those in adjacent normal\nintestinal tissues (Fig. 5g,h). These findings provide supportive evidence that MnLR\npreferentially accumulates in tumor tissues, which may contribute to its therapeutic advantage\nby enhancing local efficacy while potentially minimizing off-target effects in healthy tissues.\nWe also acknowledge that the mechanisms underlying this tumor tropism remain to be fully\nelucidated. Future studies employing higher-resolution imaging approaches, such as intravital\nmicroscopy or advanced spatial mapping techniques, will be important to reveal the precise\n52\nintratumoral distribution of MnLR at the cellular level and to better understand the biological\nbasis of its selective localization.\nFig. 5: Dynamic distribution of oral MnLR and L. reuteri within the GI tract. g,h, The\nrepresentative fluorescence images and corresponding quantification of the MnLR distribution in tumor\nor adjacent normal intestinal tissues (n = 3). Scale bar, 500 μm. Data are shown as mean ± SD; *p <\n0.05; Student’s t-test.\nThe relevant description on page 19, lines 425-430：\n“High-resolution ex vivo tissue fluorescence imaging further revealed that MnLR accumulated\n1.4-fold more in colon tumor tissues than in adjacent normal tissues (Fig. 5g,h). These\nobservations suggest a preferential localization and colonization of L. reuteri in tumor regions,\nlikely influenced by distinct features of the tumor microenvironment (such as oxygen\ngradients), immunosuppressive states, and tumor-associated alterations of the gut\nmicrobiota.”\n13. The study does not provide a comprehensive mechanistic explanation for how the enhanced\nsystemic immunity prevents tumor initiation and early growth. While the study demonstrates\nchanges in immune cell populations and shows that CD8+ T cells and macrophages are\nimportant for the preventive effect, it doesn't elucidate the specific molecular mechanisms by\nwhich these immune cells prevent tumor formation. However, the precise mechanisms by\nwhich enhanced systemic immunity leads to prevention of tumor initiation and early growth\nremain unclear. Understanding these mechanisms, including potential metabolic interactions\nand immune evasion prevention, is crucial for fully explaining the preventive effect and\npotentially identifying new therapeutic targets.\nResponse:We sincerely thank the reviewer for this insightful comment. Based on our current\nfindings, the preventive effect of MnLR/Gly against colorectal tumor initiation and early\ngrowth likely arises from the combined contributions of multiple biological processes:\n(1) Direct, tumor-selective cytotoxicity of microbial metabolites: L. reuteri–derived\n53\nmetabolites (including reuterin) induce ROS-mediated apoptosis in colon tumor cells while\nsparing normal tissues, thereby contributing to selective antitumor activity.\n(2) Modulation of tumor-associated immune cells: MnLR/Gly treatment promotes maturation\nand functional activation of DCs, induces the M1 macrophage polarization, and increases\ncytotoxic T cell activity. Depletion of CD8⁺ T cells or macrophages substantially attenuates the\npreventive effect, indicating that these immune populations play important functional roles.\n(3) Regulation of gut microbiota: MnLR/Gly partially restores gut microbial diversity disrupted\nby tumor development and enriches bacterial genera with potential antitumor properties,\nsuggesting that gut microbiota modulation may contribute to systemic immune activation and\ntumor suppression.\nTogether, these processes may create an immune-supportive environment that facilitates\nthe suppression of early tumor establishment. Nevertheless, we acknowledge that the precise\nmolecular mechanisms, including potential metabolic interactions and the prevention of\nimmune evasion, remain to be fully elucidated. Future studies will aim to dissect these\npathways in greater detail to deepen our understanding and potentially identify novel\ntherapeutic targets.\nThe relevant description in the Discussion section on pages 34, lines 733-744：\n“More importantly, in addition to the direct cytotoxicity to tumor cells, L. reuteri (MnLR) with\nits glycerol metabolites (including reuterin) was revealed to functionally regulate DCs and\nmacrophages towards antitumor property, and promote the enrichment of effector T cells. This\nmultifaceted immune modulation affected both the tumor microenvironment and key\nperipheral immune organs (spleen and MLNs). Consequently, oral administration of MnLR\nand glycerol not only significantly inhibited the growth of the primary colon tumor but also\neffectively prevented its development and liver metastases. Additionally, oral MnLR with\nglycerol improved gut dysbiosis induced by colon tumors, which may also be advantageous to\nthe activation of antitumor immunity and improved tumor-inhibitory effects. Collectively,\nMnLR exerts antitumor effects through multiple biological pathways and holds promise as an\nindependent strategy for CRC treatment and prevention. Nevertheless, the precise molecular\nand cellular mechanisms underlying these processes warrant further in-depth investigation.”\n54\n14. The study lacks functional assays to confirm the enhanced antitumor activity of the\nobserved immune cell populations. While the flow cytometry data in Figures 7f-i show\nincreased presence of immune cells associated with antitumor responses, it does not directly\ndemonstrate their functional capacity. Without assessing cytokine production, cytotoxicity, or\nantigen-specific responses, it's unclear whether these cells are truly more effective at combating\ntumors.\nResponse:We sincerely thank the reviewer for this constructive comment regarding the need\nto functionally validate the antitumor activity of the immune cell populations observed. We\nhave showed in the original analysis that MnLR/Gly significantly increased (1) the M1/M2\n(F4/80⁺CD86⁺/F4/80⁺CD206⁺) ratio in the spleen, (2) the proportion of mature DCs\n(CD11c⁺CD86⁺) in the spleen, and (3) the proportions of IFN-γ⁺ CD4⁺ and CD8⁺ T cells in\nmesenteric lymph nodes (MLNs). Moreover, depletion of either macrophages or T cells\nimpaired the preventive effect of MnLR/Gly, suggesting their functional relevance (Fig. 7b,i-\nl, and Supplementary Fig. 37).To further strengthen this point, we have now conducted ELISA\nassays to evaluate the production of immune effector cytokines. The results revealed\nsignificantly elevated levels of granzyme, IFN-γ, TNF-α, and perforin in serum or MLNs in\nthe MnLR/Gly-treated group (Supplementary Fig. 40). These findings provide additional\nfunctional evidence that the expanded immune populations are associated with enhanced\nantitumor activity.\nFig. 7: In vivo prevention of primary colon tumor occurrence and metastasis. b,\ngiven time points after initial orthotopic tumor inoculation (n = 5). i,j, Quantification of the\nratio of M1 (CD11b+F4/80+CD86+) to M2 (CD11b+F4/80+CD206+) macrophages, and the\npercentages of mature DCs (CD11c+CD86+) in total CD45+ cells within the spleen from the\nmice receiving the given treatments for 10 days (n = 5). k,l, Quantification of the percentages\nof activated CD4+ and CD8+ T cells (IFN-γ+CD4+ and IFN-γ+CD8+) in total CD3+ cells within\nthe MLNs from the mice receiving the given treatments once daily for 10 days (n = 5). Data\nare shown as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (compared to\nPBS group); one-way ANOVA.\nSupplementary Fig. 37 | Prevention of primary colon tumor occurrence by oral\nMnLR/Gly. a, The fluorescence intensity quantification of the colon tumors in the mice\nreceiving different treatments at the given time points after orthotopic tumor inoculation (n =\n5). b, Representative images showing the colon tumors (highlighted with blue dashed boxes)\nisolated from the mice 14 days after orthotopic tumor inoculation. Scale bar, 1 cm. G1, PBS;\nG2, Gly; G3, Reuterin; G4, L. reuteri; G5, L. reuteri/Gly; G6, MnLR/Gly; G7, MnLR/Gly +\nCD8 T dep; G8, MnLR/Gly + Mac dep. Data are shown as mean ± SD; *p < 0.05, **p < 0.01\n(compared to PBS); one-way ANOVA.\nSupplementary Fig. 40 | Immune effector cytokine production following different\ntreatments. ELISA Kits (ELK Biotechnology, China) were used to detect TNF-α, IFN-γ,\n56\nperforin, and GZMB concentrations in sera (a) and MLNs (b) isolated from the mice receiving\nthe given treatments for 10 days (n = 3). G1, PBS; G3, Reuterin; G5, L. reuteri/Gly; G6,\nMnLR/Gly. Data are shown as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001 (compared to\nPBS group);one-way ANOVA.\nThe relevant description on page 27, lines 593-603：\n“Next, we investigated whether the lasting antitumor effects of MnLR/Gly involved the\nenhanced systemic antitumor immunity. Indeed, MnLR/Gly promoted the splenic macrophage\npolarization towards M1 phenotype and DC maturation and concomitantly enriched the IFN-\nγ-expressing CD4+ and CD8+ T cells in MLNs (Fig. 7i-l and Supplementary Fig. 39). ELISA\nassays further revealed that MnLR/Gly treatment significantly elevated the levels of immune\neffector cytokines (IFN-γ, TNF-α, perforin, and granzyme) in serum and MLNs\n(Supplementary Fig. 40). Consistently, the depletion of CD8+ T cells or macrophages reduced\nthe efficiency of MnLR/Gly in suppressing the formation of primary colon tumors (Fig. 7b,\nand Supplementary Fig. 37). These findings collectively suggest that MnLR/Gly can\nsimultaneously and functionally regulate multiple immune cells across the peripheral immune\nsystem in healthy individuals to generate a lasting antitumor effect for preventing colon tumor\ndevelopment.”\nReview Title\nManganese-Engineered Lactobacillus reuteri with Enhanced Antitumor and\nImmunomodulatory Activities for CRC Prevention and Treatment\nSummary\nThe authors present a novel approach by engineering Lactobacillus reuteri with manganese\n(MnLR) to enhance its antitumor and immunomodulatory effects in colorectal cancer (CRC).\nThe study is ambitious, integrating microbial engineering, immunology, and cancer biology,\nand provides compelling data that MnLR promotes increased bacterial proliferation, metabolite\nproduction, biofilm formation, and adhesion to CRC cells. These enhanced traits appear to\nimprove tumor targeting and therapeutic efficacy. Additionally, the observed increase in tumor-\ninfiltrating macrophages, dendritic cells (DCs), and CD8⁺ T cells suggests dual mechanisms of\naction direct cytotoxicity and immune modulation, which are particularly relevant in cancer\ntherapy. Despite these strengths, several key aspects require clarification and additional\nexperimentation to solidify the impact and translational potential of the study.\nMajor Comments and Suggestions\nIntroduction\n• While the general role of metal ions is introduced, the rationale for selecting manganese over\nother biologically relevant ions (e.g., iron, zinc) is not fully developed. The authors should\nbetter articulate manganese’s unique benefits in microbial physiology and immune regulation.\nResponse: We sincerely appreciate the reviewer’s insightful comment. We agree that our\noriginal introduction provided only a general overview of metal ions without sufficiently\narticulating the unique advantages of manganese. To address this, we have revised the\nIntroduction to highlight manganese’s distinct biological roles, as reported in previous studies.\nSpecifically, manganese has been shown to act as a contributor to oxidative stress resistance in\nbacteria (Microbiology. 2002 Oct;148(Pt 10):3255-3263; Antioxid Redox Signal. 2013 Sep\n20;19(9):933-44), and modulate host immune responses to enhance antiviral and antitumor\neffects (Int Immunopharmacol. 2022 Dec;113(Pt B):109471; Cell Res. 2020 Nov;30(11):966-\n58\n979). We also emphasize that, consistent with these properties, our screening experiments\nuniquely identified Mn²⁺—but not other biologically relevant ions (e.g., Na⁺, Mg²⁺, K⁺, Ca²⁺)—\nas capable of simultaneously enhancing L. reuteri proliferation and metabolic activity, which\nwe further linked mechanistically to ribosomal function. These revisions provide a stronger\nrationale for our selection of manganese. The corresponding changes are now included in the\nIntroduction\nThe relevant description in the Introduction section on page 4, lines 100-103：\n“For instance, manganese (Mn) supports bacterial oxidative stress resistance through Mn-\ndependent antioxidant systems 33,34, and modulates host innate immune signaling pathways to\nenhance antiviral and antitumor effects 35,36.These properties make Mn modification especially\nsuitable for customizing bacterial biological properties towithstand challenging environments\nand enhance functionality.”\nFigure 1\n1. Include histopathology or biochemical assays (ALT, AST, creatinine) to assess systemic\ntoxicity following oral administration of MnLR and metabolites.\nResponse: We sincerely thank the reviewer for this valuable suggestion regarding systemic\ntoxicity assessment. In our original manuscript, we demonstrated that continuous oral\nadministration of MnLR/Gly for 10 days did not cause abnormal changes in serum biochemical\nparameters, indicating good systemic safety. Following the reviewer’s advice, we have now\nextended our evaluation by (1) assessing the biosafety of the MnLR/Gly and metabolite reuterin\nvia oral administration for a continuous 30 days, and (2) performing histopathological\nexaminations of the heart, liver, spleen, lung, kidney, and intestine in addition to serum\nbiochemical analysis. These supplementary experiments consistently showed no\nhistopathological alterations in major organs and no abnormal changes in blood biochemical\nindices, confirming that continuous oral administration of MnLR/Gly or reuterin does not\ninduce systemic toxicity. Relevant data and description have been added to the revised\nmanuscript.\nC57BL/6 mice were orally gavaged with PBS, reuterin or MnLR/Gly daily for 30 days,\nfollowed by a 3-day withdrawal. a, The histopathological analysis of the vital organs. Scale\nbar, 250 μm. b-g, The biochemical assays and inflammation assessment on the sera (n = 5).\nTP (g/l), ALT (u/l), AST (u/l), ALP (u/l), TBIL-Z (umol/l), TC (mmol/l), HDL-C (mmol/l), LDL-\nC (mmol/l), LP(a) (mg/l). h, Mn residual contents within the organs and serum of mice with or\nwithout MnLR treatment detected by ICP-OES (Agilent 5800 ICP-OES, Malaysia) (n = 4).\nData are shown as mean ± SD;there was no significant difference among the groups; one-way\nANOVA (b-g) or Student’s t-test (h).\n“The systemic biosafety of prolonged MnLR/Gly or reuterin treatments was assessed after 30\ndays of continuous oral administration (100 μl including 10 μl of glycerol and 5×10⁹ CFU of\nL. reuteri, once daily) followed by a 3-day withdrawal. Serum biochemical analyses (indicators\nfor key organ functions, lipid profiles, and electrolytes) and histopathological examinations of\nmajor organs (heart, liver, spleen, lung, kidney, and intestine) showed no significant\nabnormalities compared with healthy controls (Supplementary Fig. 28a-f). Likewise, the levels\nof acute inflammatory cytokines in serum (IL-1β and TNF-α) returned to baseline after\nwithdrawal, demonstrating the absence of persistent inflammatory responses (Supplementary\nFig. 28g). Furthermore, manganese biodistribution analysis revealed that residual Mn levels in\nserum and major organs after one month of MnLR/Gly administration remained consistent with\nthe physiological manganese concentrations in normal mice (Supplementary Fig. 28h),\nindicating efficient clearance and the absence of Mn accumulation. These findings demonstrate\nthat MnLR/Gly and reuterin do not induce systemic toxicity and sustained inflammation,\nhighlighting their potential value for clinical translation.”\n2. Investigate whether ROS-mediated cytotoxicity affects non-tumor tissues. This is critical for\nassessing off-target toxicity.\nResponse: We thank the reviewer for highlighting the importance of evaluating whether ROS-\nmediated cytotoxicity affects non-tumor tissues. In our original manuscript, we demonstrated\nin vitro that the mixed metabolites produced by L. reuteri from glycerol inhibited colon cancer\ncell proliferation while exerting significantly reduced cytotoxicity toward noncancerous cells,\nwhich aligns with previous reports showing that reuterin-mediated ROS-dependent\ncytotoxicity preferentially affects colon cancer cells rather than normal colonic epithelial cells\n(Cancer Cell. 2022 Feb 14;40(2):185-200.e6). In response to the reviewer’s comment, we\nfurther performed in vivo analyses, showing that ROS accumulation and apoptotic signals of ROS accumulation and apoptotic signals\ninduced by MnLR/Gly or reuterin were markedly weaker in healthy colon tissues than in colon\ntumor tissues (Supplementary Fig. 30), consistent with our systemic toxicity assessment\nshowing no histopathological abnormalities of intestinal tissues in MnLR/Gly- or reuterin-\ntreated healthy mice (Supplementary Fig. 28), presented in the response to the previous\nquestion. These findings demonstrate that L. reuteri–derived metabolites exert colon tumor-\nselective ROS-mediated cytotoxicity while minimizing off-target toxicity to normal tissues.\ntissues following different treatments. Colon tumor tissues (the upper two panels) and\nhealthy colon tissues (the lower two panels) were stained with dihydroethidium (DHE, red) for\nROS and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL, green) for\napoptosis. Representative images (left) and fluorescence quantification (right) are shown for\neach treatment group (n = 3 or 5 fields). Scale bar, 200 μm.Data are shown as mean ± SD; *p\n< 0.05, ****p < 0.0001 (compared to PBS group);one-way ANOVA.\nThe relevant description on page 22, lines 484-490：\n“Additional dihydroethidium (DHE) and terminal deoxynucleotidyl transferase dUTP nick end\nlabeling (TUNEL) staining revealed that MnLR/Gly or reuterin administration induced ROS\ngeneration and apoptosis in colon tumor tissues, whereas these signals were substantially\nattenuated in healthy colon tissues (Supplementary Fig. 30). These results further demonstrate\nin vivo that L. reuteri–derived metabolites exert ROS-mediated cytotoxicity selectively on\ncolon tumor tissues, thereby contributing to the minimization of off-target damage to normal\ntissues consistent with the systemic biosafety confirmation (Supplementary Fig. 30).”\n3. Perform antioxidant or redox pathway inhibitor co-treatment to confirm the cytotoxic effects\nare redox-dependent.\nwhether the cytotoxic effects are redox-dependent. As presented in our original manuscript,\nco-treatment with the ROS scavenger N-acetyl-cysteine (NAC) markedly attenuated the\ninhibitory effect of L. reuteri–derived metabolites on colon cancer cell proliferation, thereby\nsupporting that the observed cytotoxicity is indeed redox-dependent.\nFig. 1: Glycerol metabolites from L. reuteri inhibit CRC growth in vitro and in vivo, and\nincrease immune cells in colon tumors. d, Viabilities of MC38 cells after treatment with\nmixed metabolites (containing 6.25 μg/mlof reuterin) or pretreatment with 1.5 mg/ml NAC for\n24 h (n = 5). Data are shown as mean ± SD; ****p < 0.0001; one-way ANOVA.\nThe relevant description on pages 5, 6, lines 131-140：\n“Consistent with the previous findings 27, the mixed metabolites from L. reuteri catabolizing\nglycerol exerted a potent growth-inhibitory effect preferentially on cancer cells over non-\ncancerous cells in a redox-dependent manner (Fig. 1b,c), as the metabolites stimulated the\nproduction of cytotoxic reactive oxygen species (ROS), reduced antioxidative glutathione level,\nand upregulated oxidative stress–related molecules at both the mRNA and protein levels in\ncolon cancer cells (Supplementary Fig. 1); and their cytotoxic effect was significantly reduced\nin human normal colonic epithelial cells (NCM460), human umbilical vein endothelial cells\n(HUVEC), and normal human liver cells (L-02) (with the cell viability remaining above 80%\nat a reuterin concentration of 50 µg/mL),as well asin the presence of N-acetyl-cysteine (NAC,\na ROS scavenger) (Fig. 1b,d).”\n4. Provide Western blot analyses for redox-responsive proteins (e.g., NRF2, HO-1, GPx) to\nsupport the proposed mechanism.\nadditional Western blot analyses of representative redox-responsive proteins, and the results\nwere consistent with our previous mRNA expression data (Supplementary Fig. 1). These\nfindings demonstrate that the mixed metabolites upregulate oxidative stress–related molecules\nin colon cancer cells at both the mRNA and protein levels, further supporting the proposed\nmechanism.\nSupplementary Fig. 1 | The alteration of redox balance in colon cancer cells after a 24-\nhour treatment with the mixed metabolites. c,d, Expression of oxidative stress–related\nmolecules at mRNA level (measured by qPCR) (c) and protein level (measured by Western\nblotting) (d).Data are shown as mean ± SD; ****p < 0.0001; Student’s t-test.\nThe relevant description on pages 5, 6, lines 131-140：\n“Consistent with the previous findings 27, the mixed metabolites from L. reuteri catabolizing\nglycerol exerted a potent growth-inhibitory effect preferentially on cancer cells over non-\ncancerous cells in a redox-dependent manner (Fig. 1b,c), as the metabolites stimulated the\nproduction of cytotoxic reactive oxygen species (ROS), reduced antioxidative glutathione level,\nand upregulated oxidative stress–related molecules at both the mRNA and protein levels in\ncolon cancer cells (Supplementary Fig. 1); and their cytotoxic effect was significantly reduced\nin human normal colonic epithelial cells (NCM460), human umbilical vein endothelial cells\n(HUVEC), and normal human liver cells (L-02) (with the cell viability remaining above 80%\nat a reuterin concentration of 50 µg/mL),as well asin the presence of N-acetyl-cysteine (NAC,\na ROS scavenger) (Fig. 1b,d).”\n5. Conduct metabolomic profiling to identify and quantify redox-active metabolites, especially\nsince reuterin only accounts for a minor portion of the glycerol metabolite pool.\nand the need to assess redox-active metabolites. In our original manuscript, mass spectrometry\nanalysis revealed that, in addition to reuterin, the mixed metabolites contained reuterin’s\ndownstream products and nearly 20 unidentified compounds (>0.1%) (Supplementary Table\n1). We also demonstrated that pure reuterin and its downstream products exerted weaker\ngrowth-inhibitory effects on colon cancer cells than the complete metabolite mixture (Fig. 1b\nand Supplementary Fig. 2), suggesting potential cytotoxic contributions from the unidentified\nmetabolites.\nIn response to the reviewer’s suggestion, we further analyzed intracellular ROS levels in\ncolon cancer cells treated with pure reuterin or its downstream products (1,3-propanediol and\n3-hydroxypropionic acid). The results showed that reuterin significantly increased intracellular\nROS levels compared to the blank control, but to a substantially lower extent than the mixed\nmetabolites (Supplementary Fig. 3); whereas its downstream products only caused a slight\nelevation of ROS (Supplementary Fig. 3), suggesting that their cytotoxic effects may involve\nadditional or alternative mechanisms beyond ROS induction. These new findings and\ncorresponding descriptions have been incorporated into the revised manuscript.\nSupplementary Fig. 3 | ROS production induced by mixed metabolites, pure reuterin, 1,3-\npropanediol (1,3-PD), and 3-hydroxypropionic acid (3-HP). a,b, The flow cytometric\nimages and corresponding quantification of ROS (labeled by DCFH-DA) in MC38 cells with\nthe indicated treatments (n = 3). Data are shown as mean ± SD; **p < 0.01, ****p < 0.0001\n(compared to Ctrl); one-way ANOVA.\nThe relevant description on page 6, lines 143-151：\n“Furthermore, the growth-inhibitory efficacies of the pure reuterin and its downstream products\n65\n(1,3-propanediol and 3-hydroxypropionic acid) on colon cancer cells were lower in comparison\nto the mixed metabolites (containing an equivalent amount of reuterin) (Fig. 1b and\nSupplementary Fig. 2), indicating the potential cytotoxic effects of those unidentified\nmetabolites on cancer cells. Consistent with these findings, reuterin elevated intracellular ROS\nlevels in colon cancer cells, but to a markedly lower extent than the mixed metabolites\n(Supplementary Fig. 3); whereas its downstream products only caused a slight increase in ROS\n(Supplementary Fig. 3), suggesting that these products may exert cytotoxicity through ROS-\nindependent or partially ROS-independent mechanisms.”\n6. While increases in immune cells are reported, phenotypic and functional characterization is\nmissing (e.g., M1 vs. M2 macrophages, DC activation markers, cytotoxic assays for CD8⁺ T\ncells).\nResponse: Wethank the reviewer for pointing out the importance of phenotypic and functional\ncharacterization of the immune cells beyond quantification. In Figure 1, we reported that the\nnumbers of macrophages, DCs, and CD8⁺ T cells were increased in the tumor\nmicroenvironment of metabolite-treated mice (Fig.1h,i), suggesting that the metabolites might\nexert immunological effects in addition to their direct cytotoxicity. Our intent at that stage was\nto introduce the possibility that L. reuteri–derived glycerol metabolites suppress tumor growth\nnot only through reuterin-mediated cytotoxicity, as previously reported, but also through\nimmune regulation.To further substantiate this, we have performed detailed phenotypic and\nfunctional analyses of these immune populations in vitro and in vivo to determine whether\nMnLR/Gly (Mn-modified L. reuteri/Glycerol) and other critical controls modulate polarization\nof macrophages (M1 vs. M2), activation of DCs, and effector functions of CD4⁺ and CD8⁺ T\ncells (Figs. 3, 4, 6, 7, and the corresponding Supplementary Figs). The results presented in\nthese figures collectively demonstrated that MnLR/Gly not only exerts direct cytotoxic effects\non colon tumor cells but also reshapes the immune microenvironment to favor antitumor\nimmunity.\na, Cytotoxicity of L. reuteri, glycerol, L. reuteri/Gly, and MnLR/Gly in multiple human colon\ncancer cell lines (SW48, HCT116, RKO, SW620, DLD-1) and mouse colon cancer cell lines\n(MC38, CT26) after a 24-hour incubation (n = 3 or 4). b, Quantification of mature DCs\n(CD11c+CD86+) in the total CD45+ mBMDCs after being stimulated by the cell lysates from\nMC38 cells treated with reuterin or MnLR/Gly (n = 3).c, Quantification of mature DCs (CD83+)\nin the total CD11c+HLADR+ hPBMC-DCs after being stimulated by the cell lysates from DLD-\n1 cells treated with reuterin or MnLR/Gly (n = 3). d,e, The representative flow cytometric\nimages and corresponding quantification of mature DCs (CD11c+CD86+ or CD83+) in the total\nCD45+or CD11c+HLADR+ DCs after direct stimulation by the given agents (n = 3). f, g, The\nlevels of immune functional cytokines (IL-12, TNF-α) produced by mBMDCs or hPBMC-DCs\nafter direct stimulation by the given agents (n = 3). Data are shown as mean ± SD; *p < 0.05,\n**p < 0.01, ***p < 0.001, ****p < 0.0001 (compared to Ctrl in f,g); one-way ANOVA.\nM2 polarization. a,b, The representative image and corresponding quantification of flow\ncytometry analysis of M1 (CD86+) polarization in total murine macrophages (RAW264.7 cells)\nwithout any treatments (Blank), with only LPS/IFN-γ stimulation (Ctrl), with LPS/IFN-γ\nstimulation coupled by pure reuterin (Reuterin) or MnL/Gly treatments (n = 3). c,d, The\nrepresentative image and corresponding quantification of flow cytometry analysis of M2\n(CD206+) polarization in total macrophages (RAW264.7 cells) without any treatments (Blank),\nwith only IL-4 stimulation (Ctrl), with IL-4 stimulation coupled by reuterin or MnLR/Gly\ntreatments (n = 3). e, Images of the 3D MCTs with different treatments at the given time points.\nScale bar, 25 μm. f,g, The representative image and corresponding quantification of flow\ncytometry analysis of the ratio of M1 (CD86+) to M2 (CD206+) macrophages in CD45+CD68+\nhPBMC-macrophages after reuterin or MnLR/Gly treatments (n = 3). h, The levels of immune\nfunctional cytokines (IL-6, TNF-α) produced by hPBMC-macrophages after the given\ntreatments (n = 3). i,j, The representative images and corresponding quantification of flow\ncytometry of the proportion of CFDA-SE+ cells (DLD-1) in CD11b+ hPBMC-macrophages\nafter being stimulated by the given agents (phagocytic function assessment) (n = 4). Data are\nshown as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (compared to Ctrl\nin g,h,j); one-way ANOVA.\nantitumor immunity activation by oral MnLR/Gly. a, Schematic illustration of the\nestablishment of primary colon tumor model by orthotopic inoculation of MC38 cells into\ncolon cavity of mice, once-daily oral gavage of the given agents, macrophage or CD8+ T cell\n(Mac/CD8 T) depletion by clodronate liposomes or anti-CD8 antibody, and analyses on the\nbiological samples (stool, tumor, spleen, and MLNs) isolated from the mice at the endpoint.\n69\nb,c, Bioluminescence imaging and the corresponding quantification of the colon tumors in the\nmice receiving different treatments at the given time points afterorthotopic tumor inoculation\n(n = 5). d, Survival percentages of colon tumor-bearing mice receiving different treatments (n\n= 10). e,f, Representative immunofluorescence staining images and the corresponding\nquantification of M1 macrophages (F4/80+CD86+), M2 macrophages (F4/80+CD206+), mature\nDCs (CD11c+CD86+), activated CD4+ and CD8+ T cells (IFN-γ+CD4+ and IFN-γ+CD8+) within\nthe tumor tissues from the mice receiving the given treatments 14 days after tumor inoculation\n(n = 15 fields from 3 mice per group). Scale bar, 50 μm. g-j, Quantification of the ratio of M1\nto M2 macrophages, and the percentages of mature DCs in total CD45+ cells, activated CD4+\nand CD8+ T cells in total CD3+ cells within the spleen from the colon tumor-bearing mice\nreceiving the given treatments 14 days after tumor inoculation (n = 5). k,l, Quantification of\nthe percentages of activated CD4+ and CD8+ T cells in total CD3+ cells within the MLNs from\nthe colon tumor-bearing mice receiving the given treatments 14 days after tumor inoculation\n(n = 5). m,Principal component analysis illustrating the β-diversity from 16S rRNA sequencing\nof stool samples collected from the healthy or primary colon tumor-bearing mice receiving the\ngiven treatments on day 14 after tumor inoculation (n = 6 to 8). n, Quantification of colon\ntumors in mice at the experimental endpoint under the indicated treatments. Mice were\npretreated with antibiotics (ABX) for two weeks prior to tumor cell inoculation to deplete gut\nmicrobiota (n = 5).Data are shown as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p\n< 0.0001 (compared to PBS group); one-way ANOVA.\n70\nillustration of the pre-treatments with the given agents (once-daily oral gavage for 10 days),\nbiochemical and immunological analyses on blood, spleen, and MLN samples, initial\ninoculation and rechallenge of colon cancer cells, and macrophage or CD8+ T cell (Mac/CD8\nT) depletion. b, Bioluminescence imaging of the colon tumors in the mice receiving different\ntreatments at the given time points after initial orthotopic tumor inoculation (n = 5). c,\nBioluminescence imaging and representative images of orthotopic tumor development in mice\nafter the rechallenge of colon cancer cells. Scale bar, 1 cm. d, Schematic illustration of the\nprevention in transgenic APCmin/+ mice receiving the given treatments for 9 weeks. e,f,\nRepresentative images and the corresponding quantification of tumors in the intestine of APC\nmin/+ mice after different treatments (n = 5). Red triangular arrows indicate the tumor nodules.\nScale bar, 1 cm. g,h, Representative images of H&E staining (g) and immunohistochemistry\nstaining of Ki67 (h) of the intestinal segments. Blue triangular arrows indicate the tumor\nnodules. Scale bar, 2.5 mm. i,j,Quantification of the ratio of M1 (CD11b+F4/80+CD86+) to M2\n(CD11b+F4/80+CD206+) macrophages, and the percentages of mature DCs (CD11c+CD86+) in\ntotal CD45+ cells within the spleen from the mice receiving the given treatments for 10 days (n\n= 5). k,l, Quantification of the percentages of activated CD4+ and CD8+ T cells (IFN-γ+CD4+\nand IFN-γ+CD8+) in total CD3+ cells within the MLNs from the mice receiving the given\ntreatments once daily for 10 days (n = 5). m, Schematic illustration of the establishment of the\nliver metastasis model by injecting MC38 cells into the spleen of mice, once-daily oral gavage\nof the given agents, and macrophage or CD8+ T cell (Mac/CD8 T) depletion. n,Representative\nimages of the liver and spleen tissues isolated from the mice 14 days after tumor inoculation.\nBlue triangular arrows indicate the liver tumor nodules. o, The weight of livers and spleens\nisolated from the mice in each group 14 days after tumor implantation (n = 5). p, The\nquantification of liver tumor nodules in mice in each group 14 days after tumor inoculation (n\n= 5). Data are shown as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001\n(compared to PBS group); one-way ANOVA.\n7. Clarify why additional controls (e.g., glycerol alone, unmodified L. reuteri) were not used\nconsistently across models to isolate individual effects.\nResponse: Weappreciate the reviewer’s concern regarding the use of consistent controls across\ndifferent models. In Figure 1, our primary objective was to verify that L. reuteri–derived\nglycerol metabolites exert inhibitory effects on CRC, and to introduce the possibility that these\neffects may involve not only direct cytotoxicity but also immune regulation. Therefore, the\ndesign at this stage did not include all controls systematically. In subsequent in vitro and in\nvivo experiments (Figs. 3, 4, 6, 7, and the corresponding Supplementary Figs; as presented in\nthe response to the previous question), we carefully compared MnLR/Gly with the key controls,\nincluding glycerol alone, unmodified L. reuteri, reuterin, L. reuteri/Gly, etc. These\ncomprehensive analyses demonstrated the superior antitumor and immunomodulatory\n72\nactivities of MnLR/Gly, indicating that the observed effects cannot be ascribed to glycerol,\nunmodified L. reuteri, reuterin, or L. reuteri/Gly alone, but are specifically associated with the\nenhanced functional properties endowed by manganese engineering.\nFigure 2\n1. Describe how Mn localization (surface-bound or intracellular) was confirmed after treatment.\nResponse: We thank the reviewer for raising this important question regarding the localization\nof Mn after the modification of L. reuteri. Our data consistently indicate that Mn is\npredominantly surface-bound. During preparation of MnLR (Fig. 2j), intact bacteria were\ntreated exogenously with MnCl₂ and tannic acid (without permeabilization) for 30 minutes,\nconditions that favor coordination of Mn²⁺/tannic acid complexes with surface functional\ngroups (e.g., hydroxyl and carboxyl groups) rather than intracellular incorporation.SEM and\nelemental mapping revealed clear Mn signals restricted to the bacterial surface (Fig. 2k).\nMoreover, Mn was readily released from MnLR in PBS, SIF, and SGF, further supporting that\nMn is not intracellularly incorporated but instead coordinated at the bacterial surface\n(Supplementary Fig. 10 and 42).Consistently, XPS, which detects only a few nanometers in\ndepth, showed strong Mn peaks, providing direct evidence of surface modification\n(Supplementary Fig. 10). Taken together, these findings demonstrate that Mn is localized\nmainly on the bacterial surface rather than within the intracellular compartment.\nFig. 2: Screening, preparation, and characterization of MnLR. j, The schematic presenting\nthe fabrication of MnLR. k, The representative scanning electronic microscopy (SEM) (Scale bar 1 μm),\nfluorescence (Scale bar 5 μm), and elemental mapping images of MnLR. CY 3 red fluorescence labels\nTA; and SYTO 9 green fluorescence labels nucleus.\nMnLR under the given conditions. a, The XPS spectrum of MnLR. b,c, MnLR solution (1\nml, 109 CFU/ml) was shaken at 200 rpm. At the given time intervals, Mn ion concentration in\nthe supernatant of PBS (b) or SIF (c) was detected by AAS (Thermo iCETM3500, USA) after\ncentrifugation (12,000 rpm, 20 minutes), n = 3 per time point. Data are shown as mean ± SD.\nThe relevant description on page 9, lines 220-228：\n“Subsequently, we set out to modify L. reuteri with Mn using tannic acid (TA) as a chemical\nlinker to obtain a bioactivity-enhanced L. reuteri (MnLR) (Fig. 2j). The successful surface\nmodification of Mn on L. reuteri was verified by the evident changes in morphological features\nand fluorescence labeling (Fig. 2k, left). The elemental mapping (Fig. 2k, right), X-ray\nphotoelectron spectroscopy (XPS) (Supplementary Fig.10a), and atomic absorption\nspectroscopy (AAS) (Supplementary Fig.10b,c) analyses further confirmed the presence of Mn\nand its surface association, as well as its capability to release from MnLR. Notably, MnLR\nexhibited a faster Mn release profile in SIF compared with neutral PBS (Supplementary Fig.\n10b,c), indicating a release behavior conducive to CRC targeting while limiting off-target\ndistribution.”\nreuteri under simulated gastrointestinal conditions.SEM imaging (the left panel) shows the\nsurface morphology of MnLR after a 4-hour incubation in SGF or SIF. Elemental mapping (the\nmiddle panel) indicates the presence of manganese on the bacterial surface, visualized as\nyellow signals. Scale bar, 2 μm.\nThe relevant description on page 30, lines 646-651：\n“Since the viability of both raw L. reuteri and MnLR was lower in SGF than in SIF (Fig. 2n,o),\nand SEM imaging further revealed that Mn modification was less stable in SGF compared to\nSIF (Supplementary Fig. 42), we prepared a dimension-tunable enteric-soluble capsule for\ndelivering MnLR encapsulated by glycerol (as one of the capsule materials) through 3D\nprinting and casting technology to preserve MnLR bioactivity before reaching the therapeutic\ntargets (Fig. 8a, and Supplementary Fig. 43). ”\n2. Evaluate Mn release profiles in simulated gastrointestinal environments to ensure colon\ndelivery.\nResponse: We thank the reviewer for raising the important point regarding the Mn release\nbehavior under gastrointestinal conditions. In the original manuscript, we only reported the\nrelease profile of Mn from MnLR in neutral PBS. In response to the reviewer’s suggestion, we\nadditionally examined Mn release in simulated intestinal fluid (SIF) (Supplementary Fig.\n10b,c). The results showed that MnLR released Mn more rapidly in SIF than in PBS, which is\nexpected to facilitate functional activity at the intestinal tract, the primary site of tumor\npresence, while reducing the risk of off-target Mn release and systemic toxicity. These new\nresults and descriptions have been incorporated into the revised manuscript.\nSupplementary Fig. 10 | Mn element characterization and release profiles of Mn from\n200 rpm. At the given time intervals, Mn ion concentration in the supernatant of PBS (b) or\nSIF (c) was detected by AAS (Thermo iCETM3500, USA) after centrifugation (12,000 rpm,\n20 minutes), n = 3 per time point. Data are shown as mean ± SD.\nThe relevant description on page 9, lines 226-228：\n“Notably, MnLR exhibited a faster Mn release profile in SIF compared with neutral PBS\n(Supplementary Fig. 10b,c), indicating a release behavior conducive to CRC targeting while\nlimiting off-target distribution.”\n3. Provide dose comparisons to physiological or dietary manganese levels. The risk of Mn-\ninduced toxicity (e.g., neurotoxicity) is not addressed. Include or reference data on\nbiodistribution and clearance.\nResponse: We thank the reviewer for this valuable comment. To address safety concerns, we\nhave assessed systemic biosafety after 30 days of continuous oral administration of MnLR/Gly\nor reuterin, followed by a 3-day withdrawal period. Serum biochemical analyses and\nhistopathological examinations of major organs showed no significant abnormalities compared\nwith controls (Supplementary Fig. 28a,b). Likewise, IL-1β and TNF-α levels returned to\nbaseline after withdrawal, indicating the absence of persistent systemic inflammation\n(Supplementary Fig. 28c). Moreover, manganese biodistribution and clearance were evaluated\nafter one month of MnLR/Gly treatment. The residual Mn levels in serum and major organs\nafter one month of MnLR/Gly administration remained consistent with the physiological\nmanganese concentrations in normal mice (Supplementary Fig. 28d; J Biol Chem. 2020 May\n8;295(19):6312-6329.), demonstrating effective clearance and ruling out manganese\naccumulation beyond normal dietary or physiological ranges. Taken together, these results\nconfirm that MnLR/Gly treatment does not induce systemic toxicity or Mn overload, thereby\nsupporting its translational potential.\nC57BL/6 mice were orally gavaged with PBS, reuterin or MnLR/Gly daily for 30 days,\nfollowed by a 3-day withdrawal. a, The histopathological analysis of the vital organs. Scale\nbar, 250 μm. b-g, The biochemical assays and inflammation assessment on the sera (n = 5).\nTP (g/l), ALT (u/l), AST (u/l), ALP (u/l), TBIL-Z (umol/l), TC (mmol/l), HDL-C (mmol/l), LDL-\nC (mmol/l), LP(a) (mg/l). h, Mn residual contents within the organs and serum of mice with or\nwithout MnLR treatment detected by ICP-OES (Agilent 5800 ICP-OES, Malaysia) (n = 4).\nData are shown as mean ± SD;there was no significant difference among the groups; one-way\nANOVA (b-g) or Student’s t-test (h).\n“The systemic biosafety of prolonged MnLR/Gly or reuterin treatments was assessed after 30\ndays of continuous oral administration (100 μl including 10 μl of glycerol and 5×10⁹ CFU of\nL. reuteri, once daily) followed by a 3-day withdrawal. Serum biochemical analyses (indicators\nfor key organ functions, lipid profiles, and electrolytes) and histopathological examinations of\nmajor organs (heart, liver, spleen, lung, kidney, and intestine) showed no significant\nabnormalities compared with healthy controls (Supplementary Fig. 28a-f). Likewise, the levels\nof acute inflammatory cytokines in serum (IL-1β and TNF-α) returned to baseline after\nwithdrawal, demonstrating the absence of persistent inflammatory responses (Supplementary\nFig. 28g). Furthermore, manganese biodistribution analysis revealed that residual Mn levels in\nserum and major organs after one month of MnLR/Gly administration remained consistent with\nthe physiological manganese concentrations in normal mice (Supplementary Fig. 28h),\nindicating efficient clearance and the absence of Mn accumulation. These findings demonstrate\nthat MnLR/Gly and reuterin do not induce systemic toxicity and sustained inflammation,\nhighlighting their potential value for clinical translation.”\n4. Test bacterial growth in nutrient-limited media, and perform rescue experiments with Mn to\nvalidate its specific effects.\nResponse: We appreciate the reviewer’s insightful suggestion. In the revised study, we\nassessed the proliferation and glycerol metabolism of L. reuteri under nutrient-limited\nconditions, and evaluated whether Mn exerts specific rescue effects. The results showed that L.\nreuteri maintained proliferative capacity under carbon or nitrogen deprivation but exhibited\nmarkedly reduced metabolic activity. Mn supplementation significantly enhanced both\nproliferative and metabolic activities of L. reuteri under single-carbon or single-nitrogen\ndeprivation, indicating that Mn plays a critical role in sustaining and rescuing L. reuteri\nfunctionality under nutrient stress (Supplementary Fig. 7c-f). However, under combined\ncarbon and nitrogen deprivation, the enhancement and rescue effect of Mn was attenuated\n(Supplementary Fig. 7g,h), likely because the simultaneous deficiency of two essential\nnutrients imposes a profound restriction of L. reuteri functionality, thereby limiting the ability\nof Mn to restore bacterial activity. The new results and descriptions have been incorporated\ninto the revised manuscript.\nstress. L. reuteri proliferation (OD600) (a,c,e, and g) and reuterin yield (b,d,f, and h) with or\nwithout Mn2+ (500 μM) treatment were assessed in nutrient-limited media (C +, 20 mM\nglycerol; C -, 6 mM glycerol; N +, 20 mM ammonium chloride; N -, 3 mM ammonium chloride)\n(n = 3).Data are shown as mean ± SD; *p < 0.05, ***p < 0.001; Student’s t-test.\nThe relevant description on page 8, lines 199-202：\n“Also, Mn2+ supplementation significantly promoted the proliferative and metabolic activities\nof L. reuteri under carbon or nitrogen deprivation (Supplementary Fig. 7), highlighting the\nspecific biological role of Mn in sustaining and rescuing bacterial functionality under nutrient-\nlimited conditions.”\n5. Clarify the enrichment of “photosynthesis pathways” in transcriptomics, as L. reuteri is non-photosynthetic—this may suggest annotation artifacts. Key findings should be validated by\nqRT-PCR.\nResponse: We sincerely thank the reviewer for pointing out this important issue. We fully\nagree that L. reuteri is a non-photosynthetic bacterium and that the observed enrichment of the\n“photosynthesis” pathway does not reflect a biologically relevant activation. Upon careful re-\nexamination, we found that this enrichment likely arose from annotation artifacts in cross-\nspecies databases. Specifically, inspection of the enriched gene IDs revealed that the\n79\n“photosynthesis” pathway (KEGG ko00195) shares exactly the same gene set with the\n“oxidative phosphorylation” pathway (ko00190), including LREU_RS02420,\nLREU_RS02440, LREU_RS02450, and LREU_RS02455. This redundancy in annotation led\nto a false-positive enrichment signal. To avoid confusion, we have now removed the\n“photosynthesis” pathway from the transcriptomic results (Fig. 2h).\nTo further validate the transcriptomic findings, quantitative proteomics confirmed elevated\nexpression of ribosomal proteins (Fig. 2i), and SDS-PAGE demonstrated increased global\nprotein synthesis in Mn-treated L. reuteri (Supplementary Fig. 9). These results indicate that\nthe observed transcriptional upregulation of ribosome-related pathways is functionally\ntranslated into enhanced ribosomal activity and protein synthesis, suggesting the role of\nribosomes as a key target of Mn regulation.\nFig. 2: Screening, preparation, and characterization of MnLR. h, The top nine enriched\npathways in Mn-treated L. reuteri versus untreated control from Kyoto Encyclopedia of Genes and\nGenomes (KEGG) enrichment analysis. i, The quantification of ribosomal proteins of L. reuteri with or\nwithout Mn2+ treatmentby LC-MS/MS (n = 3).\nSupplementary Fig. 9 | The total protein abundance in Mn-treated L. reuteri versus\nuntreated control. Whole-cell lysates were prepared from L. reuteri cultures treated with 500\nμM Mn2+ for 4 hours or from untreated control cultures. Prior to lysis, cultures were adjusted\nto identical optical density (OD600) so that sample input was normalized by cell density; equal\n80\nvolumes of lysate were then resolved by SDS–PAGE and visualized with Coomassie Brilliant\nBlue staining. The gel shows relative changes in total protein banding patterns and overall\nprotein abundance between Mn-treated and control samples.\nThe relevant description on page 9, lines 211-219：\n“Furthermore, the subsequent pathway enrichment analysis determined that Mn-treated L.\nreuteri exhibited significant upregulation of signaling pathways related to the ribosome,\nthiamine metabolism, and oxidative phosphorylation, with ribosome pathways being the most\nprominent (Fig. 2h). To functionally validate these transcriptomic findings, quantitative\nproteomic analysis confirmed elevated levels of ribosomal proteins (Fig. 2i). SDS-PAGE\nfurther demonstrated increased global protein synthesis in Mn-treated L. reuteri\n(Supplementary Fig. 9). These results collectively establish that the transcriptional\nupregulation of ribosome-related pathways is functionally translated into enhanced ribosomal\nactivity and protein synthesis, suggesting that ribosomes may be a key target for the regulation\nof L. reuteri’s bioactivity by Mn ions42.”\nFigure 3\n1. Differentiate synergistic vs. additive effects of reuterin and Mn²⁺ on DC maturation using\nMnLR with metabolite inhibition or depletion.\nResponse: We thank the reviewer for this important suggestion regarding the interaction\nbetween Mn²⁺ and reuterin in driving DC maturation. To investigate this, we employed but-3-\nene-1,2-diol, a reported glycerol dehydratase inhibitor, to block reuterin production\n(Chembiochem. 2008 Sep 22;9(14):2268-75.).We confirmed that this compound effectively\ninhibited glycerol dehydratase activity and reuterin generation in vitro, even in the presence of\nMn²⁺ (Supplementary Fig. 16a,b). When DCs were stimulated with MnLR/Gly in the presence\nof the inhibitor (iGDHt), the proportion of mature DCs (CD11c⁺CD86⁺) decreased from 29.3%\nto 19.3%, closely resembling the Mn²⁺ group (19.1%) (Supplementary Fig. 16c,d). These\nfindings indicate that glycerol metabolites and Mn²⁺ independently contribute to DC maturation,\nand their combined effect in MnLR/Gly-treated DCs approximates the sum of their individual\neffects. Thus, the evidence supports an additive, rather than synergistic, relationship.\neffects of Mn2+ and glycerol metabolites on DC maturation. a,b, Validation of but-3-ene-\n1,2-diol, a glycerol dehydratase inhibitor (iGDHt), in suppressing glycerol dehydratase activity\nand reuterin production in L. reuteri cultured with glycerol (n = 3 or 5). c,d, The representative\nflow cytometric images and corresponding quantification of mature DCs (CD11c+CD86+ in the\ntotal CD45+ DCs) after the given treatments (n = 3). Data are shown as mean ± SD; **p < 0.01,\n***p < 0.001, ****p < 0.0001 (compared to Ctrl in d); one-way ANOVA.\nThe relevant description on page 12, lines 299-302：\n“Inhibition of reuterin production with a GDHt inhibitor reduced DC maturation to the level\ninduced by Mn2+ alone (Supplementary Fig. 16), suggesting that glycerol metabolites and Mn2+\nreleased by MnLR contribute additively to DC maturation (Fig. 3n).”\n2. Include cytokine profiling (e.g., IL-12, TNF-α) to confirm functional DC maturation.\nResponse: We thank the reviewer for highlighting the importance of functional validation of\nDC maturation. In response, we performed cytokine profiling and found that MnLR/Gly-\ntreated colon cancer cells and the direct treatment of MnLR/Gly significantly promoted the\nmBMDCs or hPBMC-DCs secretion of functional cytokines, including IL-12 and TNF-α, at\n82\nlevels higher than those induced by other groups (Fig. 3f,g, and Supplementary Fig. 14). These\nfindings further confirm the functional maturation of DCs and have been incorporated into the\nrevised manuscript.\nSupplementary Fig. 14 | DCs maturation stimulated by reuterin- or MnLR/Gly-treated\ncolon cancer cells. a,c, The representative flow cytometric images of mature mouse BMDCs\n(CD11c+ CD86+) in the total CD45+ BMDCs (a) and mature human PBMC-DCs (CD83+) in\nthe total CD11c+ HLADR+ PBMC-DCs (c) after stimulation by the lysates from reuterin- or\nMnLR/Gly-treated colon cancer cells (MC38 or DLD-1). b,d, The levels of immune functional\ncytokines (IL-12, TNF-α) in mature mBMDCs (b) and hPBMC-DCs (d) detected by ELISA\nKit (ELK Biotechnology, China) (n = 3). Data are shown as mean ± SD; **p < 0.01, ***p <\n0.001, ****p < 0.0001 (compare to Ctrl); one-way ANOVA.\nThe relevant description on page 12, lines 283-288：\n“To investigate this immunological process, we subjected DCs derived from murine bone\nmarrows or human peripheral blood mononuclear cells (mBMDCs or hPBMC-DCs) to the\nlysates from reuterin- or MnLR/Gly-treated colon cancer cells and observed a significant\nimprovement in DC maturation, as evidenced by the increased proportion of mature DCs\n(CD11c+CD86+ mBMDCs and CD83+ hPBMC-DCs), and the production of immune functional\ncytokines (IL-12, TNF-α) (Fig. 3b,c, and Supplementary Fig. 14).”\nf, g, The levels of immune functional cytokines (IL-12, TNF-α) produced by mBMDCs or\nhPBMC-DCs after direct stimulation by the given agents (n = 3). Data are shown as mean ±\nSD; *p < 0.05, **p < 0.01, ****p < 0.0001 (compared to Ctrl in f,g); one-way ANOVA.\nThe relevant description on page 12, lines 302-303：\n“In addition, MnLR/Gly-treated DCs secreted markedly higher levels of IL-12 and TNF-α than\nother groups (Fig. 3f,g), further supporting their enhanced functional maturation.”\nFigure 4\n1. Confirm results in primary macrophages (e.g., BMDMs or human MDMs).\nResponse: We appreciate the reviewer’s valuable suggestion to validate our findings in\nprimary macrophages. In response, we conducted additional experiments using human\nperipheral blood mononuclear cell (PBMC)-derived macrophages. The results were consistent\nwith those obtained in RAW264.7 cells, demonstrating that MnLR/Gly preferentially\nstimulated macrophage polarization toward the M1 phenotype, promoted the secretion of\nfunctional cytokines IL-6 and TNF-α, and strengthened tumor cell phagocytosis (Fig. 4f-j).\nMoreover, we verified that, similar to RAW264.7 cells, the MnLR/Gly-induced macrophage\npolarization in human PBMC-derived macrophages was also partially dependent on the HIF-1\nsignaling pathway (Fig. 4q-s). These data further substantiate the translational relevance of our\nfindings and have been incorporated into the revised manuscript.\nM2 polarization. f,g, The representative image and corresponding quantification of flow\ncytometry analysis of the ratio of M1 (CD86+) to M2 (CD206+) macrophages in CD45+CD68+\nhPBMC-macrophages after reuterin or MnLR/Gly treatments (n = 3). h, The levels of immune\nfunctional cytokines (IL-6, TNF-α) produced by hPBMC-macrophages after the given\ntreatments (n = 3). i,j, The representative images and corresponding quantification of flow\ncytometry of the proportion of CFDA-SE+ cells (DLD-1) in CD11b+ hPBMC-macrophages\nafter being stimulated by the given agents (phagocytic function assessment) (n = 4). q,\nImmunoblotting assays of HIF-1α protein within the hPBMC-macrophages with the given\ntreatments. r,s, The representative image and corresponding quantification of flow cytometry\nanalysis of the ratio of M1 (CD86+) to M2 (CD206+) macrophages in CD45+CD68+ hPBMC-\nmacrophages with the given treatments (n = 3). iHIF-1α, HIF-1α inhibitor (YC-1). Data are\nshown as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (compared to Ctrl\nin g,h,j); one-way ANOVA.\nPBMC-macrophages. The flow cytometric quantification of the proportion of the ratio of M1\n(CD86+) to M2 (CD206+) macrophages in CD45+CD68+ PBMC-macrophages after direct\nstimulation by the given agents for 24 hours (n = 3). Data are shown as mean ± SD; ****p <\n0.0001 (compare to Ctrl); one-way ANOVA.\nThe relevant description on page 16, lines 367-371, and lines 380-382：\n“Human PBMC-macrophages exhibited consistent responses with RAW264.7 cells, wherein\nMnLR/Gly treatment induced M1 phenotypic polarization (Fig. 4f,g, and Supplementary Fig.\n22), accompanied by elevated IL-6 and TNF-α production and enhanced phagocytic activity\nagainst colon cancer cells (Fig. 4h-j), thereby reinforcing the robustness and translational\npotential of immunomodulatory effects mediated by MnLR/Gly.”\n“Consistently,this HIF-1–dependent regulation of macrophage polarization was also validated\nin human PBMC-macrophages (Fig. 4q-s).”\n2. Investigate the engagement of HIF-1 by MnLR/Gly using ROS inhibitors or reporters.\nResponse: We thank the reviewer for raising this insightful suggestion. To further assess the\nengagement of HIF-1 by MnLR/Gly, we performed experiments with the ROS scavenger N-\nacetyl-cysteine (NAC). The results showed that NAC treatment partially attenuated\nMnLR/Gly-induced HIF-1 upregulation (Supplementary Fig. 25), indicating that the activation\nof the HIF-1 pathway by MnLR/Gly is at least partly dependent on ROS generation. These\nfindings have been incorporated into the revised manuscript.\nmacrophages. Immunoblot analysis of HIF-1α in human PBMC-derived macrophages\nfollowing a 24-hour treatment with MnLR/Gly, with or without a 4-hour pretreatment of N-\nacetyl-cysteine (NAC, 1.5 mg/ml).\nThe relevant description on page 16, lines 382-384：\n“Additionally, treatment with the NAC partially reduced MnLR/Gly-induced HIF-1\nupregulation, suggesting that this pathway activation is mediated, at least in part, by ROS\nproduction (Supplementary Fig. 25).”\n3. Assess macrophage function, including cytokine production (IL-6, TNF-α) and phagocytosis.\nResponse: We sincerely thank the reviewer for this valuable suggestion. In accordance with\nthe reviewer’s comments, we performed additional experiments to evaluate macrophage\nfunction. The results demonstrated that MnLR/Gly not only promoted the polarization of\nmacrophages toward the M1 phenotype but also increased the production of functional\ncytokines (IL-6, TNF-α) and enhanced their phagocytic activity against colon cancer cells (Fig.\n4f-j). These findings further support the functional relevance of MnLR/Gly–induced\nmacrophage polarization.\nFig. 4: MnLR/Gly promotes the polarization of M1 macrophages, meanwhile inhibiting\ncytometry analysis of the ratio of M1 (CD86+) to M2 (CD206+) macrophages in CD45+CD68+\nhPBMC-macrophages after reuterin or MnLR/Gly treatments (n = 3). h, The levels of immune\nfunctional cytokines (IL-6, TNF-α) produced by hPBMC-macrophages after the given\ntreatments (n = 3). i,j, The representative images and corresponding quantification of flow\ncytometry of the proportion of CFDA-SE+ cells (DLD-1) in CD11b+ hPBMC-macrophages\nafter being stimulated by the given agents (phagocytic function assessment) (n = 4). Data are\nshown as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (compared to Ctrl\nin g,h,j); one-way ANOVA.\nThe relevant description on page 16, lines 367-371：\n“Human PBMC-macrophages exhibited consistent responses with RAW264.7 cells, wherein\nMnLR/Gly treatment induced M1 phenotypic polarization (Fig. 4f,g, and Supplementary Fig.\n22), accompanied by elevated IL-6 and TNF-α production and enhanced phagocytic activity\nagainst colon cancer cells (Fig. 4h-j), thereby reinforcing the robustness and translational\npotential of immunomodulatory effects mediated by MnLR/Gly.”\nFigure 5\n1. Quantify tumor burden, growth kinetics, and survival to evaluate efficacy.\nResponse: We appreciate the reviewer’s suggestion. As clarified, the primary purpose of\nFigure 5 was to illustrate the in vivo distribution of L. reuteri and its metabolite (reuterin),\nand\nto examine whether Mn modification enhances the proliferative and metabolic activity of L.\nreuteri in vivo. The evaluation of tumor burden, growth kinetics, and survival was addressed\nin subsequent figures: therapeutic and preventive efficacy against orthotopic colon cancer (Figs.\n6-8), and inhibition of metastatic colon cancer (Fig. 7). These data were included in the original\nmanuscript, and we have added a transitional statement at the end of the Figure 5 results to\nguide readers more clearly toward the subsequent efficacy evaluations.\nThe relevant description on page 20, lines 458-460：\n“Given the enhanced proliferative and metabolic activity as well as the favorable biosafety of\nMnLR/Gly observed in vivo, we next investigated its therapeutic and preventive efficacy\nagainst CRC in orthotopic and metastatic models.”\n88\n2. Assess whether hypoxia attracts MnLR passively or whether the strain has active tumor-homing capabilities.\nResponse: We appreciate the reviewer’s insightful comment regarding the tumor-homing\ncapabilities of MnLR. In the revised study, fluorescence imaging revealed that MnLR\ndisplayed 1.4-fold higher distribution in colon tumor tissues compared to adjacent normal colon\ntissues (Fig. 5g,h). While this observation seems to be consistent with hypoxia-induced passive\nrecruitment, given the well-established lower oxygen content in tumors relative to normal\ntissues, further studies are warranted to determine whether MnLR also possesses active tumor-\nhoming mechanisms beyond passive attraction.\nFig. 5: Dynamic distribution of oral MnLR and L. reuteri within the GI tract. g,h, The\nrepresentative fluorescence images and corresponding quantification of the MnLR distribution in tumor\nor adjacent normal intestinal tissues (n = 3). Scale bar, 500 μm. Data are shown as mean ± SD; *p <\n0.05; Student’s t-test.\nThe relevant description on page 18, 19, lines 423-430：\n“Of note, both MnLR and raw L. reuteri exhibited significantly higher 24-hour retention in the\ncolorectal tissues of tumor-bearing mice compared with healthy mice (Fig. 5d,e, and\nSupplementary Fig. 26). High-resolution ex vivo tissue fluorescence imaging further revealed\nthat MnLR accumulated 1.4-fold more in colon tumor tissues than in adjacent normal tissues\n(Fig. 5g,h). These observations suggest a preferential localization and colonization of L. reuteri\nin tumor regions, likely influenced by distinct features of the tumor microenvironment (such\nas oxygen gradients), immunosuppressive states, and tumor-associated alterations of the gut\nmicrobiota.”\n3. Compare MnLR with encapsulated reuterin to evaluate the added benefit of using live\nprobiotics.\nResponse: We thank the reviewer for this insightful comment. To directly evaluate the added\n89\nbenefit of using live probiotics, we compared GlyMnLR capsules with reuterin-containing\ncapsules in the rabbit orthotopic colorectal tumor model. GlyMnLR consistently exhibited a\nmarkedly higher tumor inhibition rate (67.9%) relative to reuterin capsules (8.0%) (Fig. 8j,k),\nindicating that the presence of viable Mn-modified L. reuteri enables sustained in situ\nproduction of bioactive metabolites. These results highlight the functional advantage of live\nprobiotic delivery over administration of metabolites alone.\nFig. 8: Characterization of GlyMnLR capsule and its antitumor efficacy and biosafety in\nrabbit models. j, Representative in vivo (endoscopic, the top and middle panels) and ex vivo\n(the bottom panel) images of tumors (highlighted with yellow dashed boxes) on days 10, 28,\nand 31 post oral administration of the indicated agents. Scale bar, 1 cm. k, The tumor volumes\nobtained from the corresponding groups at the end of the experiment (n = 5). l, The\nrepresentative immunofluorescent images and corresponding statistical analysis of the green\nKi67-stained proliferating cells within the isolated tumor tissues from rabbits receiving the\ngiven treatments at the experimental end (n = 5 fields).Scale bar, 200 μm.m,n, Representative\n90\nimmunofluorescence staining images and corresponding quantification of CD45+CD8+ T cells\nwithin the tumor tissues isolated from the rabbits with the given treatments (n = 9 fields).Scale\nbar, 50 μm. Data are shown as mean ± SD; **p < 0.01, ***p < 0.001, **** p < 0.0001\n(compared to PBS group); one-way ANOVA.\nThe relevant description on page 31, lines 672-675：\n“GlyMnLR capsules exhibited a markedly higher tumor inhibition rate (67.9%) compared with\nfree MnLR (25.4%) and reuterin capsules (8.0%), demonstrating the protective role of the\nenteric formulation and the advantage of sustained production of active metabolites by viable\nbacteria (Fig. 8j,k).”\n4. Investigate how the tumor microenvironment, immune state, or gut microbiota influence\nMnLR colonization.\nResponse: We thank the reviewer for raising this insightful point. We have compared dynamic\nL. reuteri profiles in the gastrointestinal tract between healthy and colon tumor-bearing mice\nfollowing oral administration of MnLR or raw L. reuteri. We observed that the 24-hour\nretention of L. reuteri (including MnLR and raw L. reuteri) in the colorectum of tumor-bearing\nmice was significantly higher than that in healthy mice (Fig. 5d,e, and Supplementary Fig. 26),\nin line with the above-mentioned preferential accumulation observed in colon tumor tissues\nrelative to adjacent normal tissues (Fig. 5g,h). These observations suggest that MnLR shows a\npropensity for localization and colonization in tumor regions. While the precise mechanisms\nremain to be fully elucidated, differences in tumor versus normal tissues—including hypoxia\nand metabolic alterations in the tumor microenvironment (Cancer Cell Int. 2021 Jan\n20;21(1):62), immunosuppressive states (Science. 2015 Apr 3;348(6230):74-80), and altered\ngut microbiota composition (Cell Mol Immunol. 2025 Sep;22(9):1012-1031)—may contribute\nto the enhanced bacterial colonization and proliferation of in tumors. We have discussed this\npoint in the revised Results section accordingly.\nillustration and the representative fluorescence images of the L. reuteridistribution within the\nGI tract of orthotopic colon tumor-bearing mice 1, 6, and 24 hours after receiving oral gavage\nwith CY 5.5-labeled L. reuteri or MnLR. In the schematic illustration, the stomach, small\nintestine, and colorectum are highlighted in pink, blue, and yellow, respectively; the tumor\nlocated at the cecal site is shown in dark red. b-d, The corresponding statistical analysis of\nfluorescence signals in the stomach, small intestine, and colorectum at the given time points\nafter oral gavage. e, Proportions of fluorescence signals within the colorectum of healthy and\ncolon tumor-bearing mice 24 hours after oral gavage with L. reuterior MnLR. f, The quantities\nof L. reuteriin the feces from healthy or colon tumor-bearing mice 24 hours after oral gavage\nwith L. reuteri or MnLR. g,h, The representative fluorescence images and corresponding\nquantification of the MnLR distribution in tumor or adjacent normal cecum tissues (n = 3).\nScale bar, 500 μm. Data are shown as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001,****p\n< 0.0001; Student’s t-test or one-way ANOVA.\nof healthy mice.a-c, The representative images (left) and corresponding statistical analysis of\nfluorescence signals in the stomach, small intestine, and colorectum (right) at the given time\npoints after oral gavage with CY 5.5-labeled L. reuteri or MnLR. Data are shown as mean ±\nSD; *p < 0.05, **p < 0.01, ***p < 0.001; Student’s t-test.\nThe relevant description on page 18, 19, lines 423-430：\n“Of note, both MnLR and raw L. reuteri exhibited significantly higher 24-hour retention in the\ncolorectal tissues of tumor-bearing mice compared with healthy mice (Fig. 5d,e, and\nSupplementary Fig. 26). High-resolution ex vivo tissue fluorescence imaging further revealed\nthat MnLR accumulated 1.4-fold more in colon tumor tissues than in adjacent normal tissues\n(Fig. 5g,h). These observations suggest a preferential localization and colonization of L. reuteri\nin tumor regions, likely influenced by distinct features of the tumor microenvironment (such\nas oxygen gradients), immunosuppressive states, and tumor-associated alterations of the gut\nmicrobiota.”\nFigure 6\n93\n1. Clarify how reuterin exerts in vivo cytotoxicity—via ROS, DNA damage, or metabolic\ninterference.\nResponse: We thank the reviewer for this insightful comment. Our additional analyses provide\nin vivo evidence that MnLR/Gly and reuterin exert cytotoxicity primarily through ROS-\nmediated mechanisms. Specifically, DHE and TUNEL staining revealed marked ROS\naccumulation and apoptosis in colon tumor tissues following MnLR/Gly or reuterin\nadministration, whereas these effects were substantially attenuated in normal colon tissues\n(Supplementary Fig. 30). These findings suggest that ROS generation is a key driver of tumor-\nselective cytotoxicity, consistent with both our systemic toxicity assessment (Supplementary\nFig. 28) and in vitro results (Fig. 1b). While we cannot completely exclude additional\nmechanisms such as metabolic interference, the current evidence supports ROS-mediated\ncytotoxicity as the important pathway in vivo, although further studies will be needed to fully\ndelineate the underlying mechanisms.\nSupplementary Fig. 30 | ROS and apoptosis in colon tumor tissues and normal colon\ntissues following different treatments. Colon tumor tissues (the upper two panels) and\nhealthy colon tissues (the lower two panels) were stained with dihydroethidium (DHE, red) for\nROS and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL, green) for\napoptosis. Representative images (left) and fluorescence quantification (right) are shown for\neach treatment group (n = 3 or 5 fields). Scale bar, 200 μm.Data are shown as mean ± SD; *p\n< 0.05, ****p < 0.0001 (compared to PBS group);one-way ANOVA.\n“Additional dihydroethidium (DHE) and terminal deoxynucleotidyl transferase dUTP nick end\nlabeling (TUNEL) staining revealed that MnLR/Gly or reuterin administration induced ROS\ngeneration and apoptosis in colon tumor tissues, whereas these signals were substantially\nattenuated in healthy colon tissues (Supplementary Fig. 30). These results further demonstrate\nin vivo that L. reuteri–derived metabolites exert ROS-mediated cytotoxicity selectively on\ncolon tumor tissues, thereby contributing to the minimization of off-target damage to normal\ntissues consistent with the systemic biosafety confirmation (Supplementary Fig. 30).”\n2. Use fecal transfer or microbiota depletion experiments to establish causal roles of gut\nmicrobial changes.\nResponse: We thank the reviewer for this valuable suggestion. Following the comment, we\nperformed additional experiments in which orthotopically colon tumor-bearing mice were\npretreated with a broad-spectrum antibiotic cocktail (ampicillin, neomycin, metronidazole, and\nvancomycin) for two weeks to deplete the gut microbiota.We found that the inhibitory effect\nof MnLR/Gly on colon tumors was slightly attenuated in antibiotic-treated mice (Fig. 6n and\nSupplementary Fig. 36). These findings indicate that the antitumor efficacy of MnLR/Gly\ndepends, at least in part, on modulation of the gut microbiota, in addition to its\nimmunomodulatory effects and cytotoxicity. The new data and corresponding descriptions\nhave been incorporated into the revised manuscript.\nFig. 6: In vivo orthotopic primary colon tumor growth inhibition, gut microbiota\nmodulation, and antitumor immunity activation by oral MnLR/Gly. n, Quantification of\ncolon tumors in mice at the experimental endpoint under the indicated treatments. Mice were\npretreated with antibiotics (ABX) for two weeks prior to tumor cell inoculation to deplete gut\n95\nmicrobiota (n = 5). Data are shown as mean ± SD; **p < 0.01, ***p < 0.001 (compared to\nPBS group); one-way ANOVA.\nSupplementary Fig. 36 | Impact of microbiota depletion on the antitumor efficacy of\nMnLR/Gly. a, Bioluminescence imaging of colon tumors in the mice receiving different\ntreatments at the given time points afterorthotopic tumor inoculation (n = 5). b, Representative\nimages of colon tumors harvested on day 21 post-orthotopic tumor inoculation. Scale bar, 1 cm.\nThe relevant description on page 24, lines 533-543：\n“We also analyzed the gut microbiota, an important factor influencing tumor growth and the\nimmune microenvironment. Compared with stool samples from healthy mice, those from\ntumor-bearing mice displayed distinct microbial communities and reduced α-diversity (Fig. 6m\nand Supplementary Fig. 35a). MnLR/Gly treatment partially ameliorated the colon tumor-\ninduced gut microbiota dysbiosis, concurrently enriching the bacterial genera with potential\nantitumor properties (including Lachnoclostridium 50, Bacteroides 51, and Lactobacillus 52,53)\n(Supplementary Fig. 35b). Moreover, in orthotopic colon tumor-bearing mice pretreated with\na cocktail of four broad-spectrum antibiotics to deplete gut microbiota, the inhibitory efficacy\nof MnLR/Gly on tumor growth was slightly attenuated though not completely abolished (Fig.\n6n, and Supplementary Fig. 36). These findings indicate that the antitumor effects of\nMnLR/Gly are mediated in part through gut microbiota modulation, in addition to its\nimmunostimulatory and cytotoxic activities.”\n1. Immune cell findings are largely phenotypic; functional assays (e.g., cytotoxicity, antigen\nspecificity) are needed.\nResponse:We sincerely thank the reviewer for this insightful comment. In our original analysis,\nMnLR/Gly treatment was shown to increase the M1/M2 macrophage ratio, promote DC\nmaturation, and expand IFN-γ⁺ CD4⁺ and CD8⁺ T cells. Furthermore, depletion of macrophages\nor T cells abrogated the preventive effect of MnLR/Gly, indicating their functional involvement.\nTo provide additional evidence beyond phenotypic characterization, we have now performed\nELISA assays, which demonstrated significantly elevated levels of granzyme, IFN-γ, TNF-α,\nand perforin in serum or MLNs of MnLR/Gly-treated mice (Supplementary Fig. 40). These\nresults support that the observed immune cell populations are functionally linked to enhanced\nantitumor activity.\nSupplementary Fig. 40 | Immune effector cytokine production following different\ntreatments. ELISA Kits (ELK Biotechnology, China) were used to detect TNF-α, IFN-γ,\nperforin, and GZMB concentrations in sera (a) and MLNs (b) isolated from the mice receiving\nthe given treatments for 10 days (n = 3). G1, PBS; G3, Reuterin; G5, L. reuteri/Gly; G6,\nMnLR/Gly. Data are shown as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001 (compared to\nPBS group);one-way ANOVA.\nThe relevant description on page 27, lines 593-603：\n“Next, we investigated whether the lasting antitumor effects of MnLR/Gly involved the\nenhanced systemic antitumor immunity. Indeed, MnLR/Gly promoted the splenic macrophage\npolarization towards M1 phenotype and DC maturation and concomitantly enriched the IFN-\nγ-expressing CD4+ and CD8+ T cells in MLNs (Fig. 7i-l and Supplementary Fig. 39). ELISA\nassays further revealed that MnLR/Gly treatment significantly elevated the levels of immune\neffector cytokines (IFN-γ, TNF-α, perforin, and granzyme) in serum and MLNs\n(Supplementary Fig. 40). Consistently, the depletion of CD8+ T cells or macrophages reduced\nthe efficiency of MnLR/Gly in suppressing the formation of primary colon tumors (Fig. 7b,\nand Supplementary Fig. 37). These findings collectively suggest that MnLR/Gly can\nsimultaneously and functionally regulate multiple immune cells across the peripheral immune\nsystem in healthy individuals to generate a lasting antitumor effect for preventing colon tumor\ndevelopment.”\n97\n2. Test protective memory by rechallenging cured mice with tumor cells.\nResponse: We sincerely thank the reviewer for this valuable suggestion. Following the\nreviewer’s advice, we performed additional re-challenge experiments to assess protective\nmemory. Mice were pretreated with MnLR/Gly via daily oral gavage for 10 days, followed by\ninitial tumor inoculation (Fig. 7a). At day 56 (week 8), the tumor-free mice were re-challenged\nwith colon cancer cells (Fig. 7a). After three additional weeks of monitoring, we found that 5\nout of 8 mice (62.5%) remained free of detectable tumors (Fig. 7c, and Supplementary Fig.\n38b). These results provide supportive evidence that MnLR/Gly treatment can induce a durable\nprotective effect against tumor rechallenge.\nFig. 7: In vivo prevention of primary colon tumor occurrence and metastasis. a, Schematic\nillustration of the pre-treatments with the given agents (once-daily oral gavage for 10 days),\n98\nbiochemical and immunological analyses on blood, spleen, and MLN samples, initial\ninoculation and rechallenge of colon cancer cells, and macrophage or CD8+ T cell (Mac/CD8\nT) depletion. b, Bioluminescence imaging of the colon tumors in the mice receiving different\ntreatments at the given time points after initial orthotopic tumor inoculation (n = 5). c,\nBioluminescence imaging and representative images of orthotopic tumor development in mice\nafter the rechallenge of colon cancer cells. Scale bar, 1 cm.\nSupplementary Fig. 38 | The long-term preventive effect of pre-oral MnLR/Gly on the\noccurrence of primary colon tumor. a, Bioluminescence imaging of colon tumors in the mice\n(receiving pre-oral MnLR/Gly for 10 days) at the given time points post orthotopic tumor\nimplantation (n = 8). b, The fluorescence intensity quantification of the colon tumors in\nrechallenged mice on day 77 after the initial inoculation (n = 8). Data are shown as mean ± SD;\n***p < 0.001; Student’s t-test.\nThe relevant description on pages 26, 27, lines 576-587：\n“The pre-treatment with MnLR/Gly suppressed the formation of a primary colon tumor,\nresulting in the absence of visible tumor masses in the isolated colon tissues from MnLR/Gly-\ntreated mice on day 14 post tumor inoculation, whereas tumors were developed in colons of\nthe mice in the other treatment groups (Fig. 7b and Supplementary Fig. 37). When the\nobservation period was extended, 75% (6/8) of MnLR/Gly-pretreated mice remained tumor-\nfree for at least 8 weeks (Supplementary Fig. 38a). To further evaluate the durability of the\npreventive effect of MnLR/Gly, tumor rechallenge experiments were performed by re-\ninoculating colon cancer cells on day 56 after the initial inoculation in the mice that remained\ntumor-free following prophylactic treatment of MnLR/Gly (Fig. 7a). Strikingly, 62.5% (5/8) of\nMnLR/Gly-pretreated mice still exhibited no detectable tumors 3 weeks later (Fig. 7c, and\nagainst tumor recurrence.”\n3. Include histological analysis of major organs and inflammatory markers following prolonged\nMnLR/Gly treatment.\nResponse: We sincerely thank the reviewer for this important suggestion. In response, we\nperformed histological analyses of major organs (heart, liver, spleen, lung, kidney, and intestine)\nafter 30 days of continuous oral MnLR/Gly administration, which revealed no detectable\nabnormalities compared with healthy controls (Supplementary Fig. 28a,b). In addition, serum\nlevels of representative inflammatory cytokines (IL-1β and TNF-α) returned to baseline after\ntreatment, 3 days of withdrawal (Supplementary Fig. 28c), indicating the absence of sustained\ninflammatory responses.These results confirm that prolonged MnLR/Gly administration does\nnot induce systemic toxicity and persistent inflammation.\nC57BL/6 mice were orally gavaged with PBS, reuterin or MnLR/Gly daily for 30 days,\nfollowed by a 3-day withdrawal. a, The histopathological analysis of the vital organs. Scale\nbar, 250 μm. b-g, The biochemical assays and inflammation assessment on the sera (n = 5).\nTP (g/l), ALT (u/l), AST (u/l), ALP (u/l), TBIL-Z (umol/l), TC (mmol/l), HDL-C (mmol/l), LDL-\nC (mmol/l), LP(a) (mg/l). Data are shown as mean ± SD;there was no significant difference\namong the groups; one-way ANOVA (b-g) or Student’s t-test (h).\nThe relevant description on pages 19, 20, lines 445-457：\n“The systemic biosafety of prolonged MnLR/Gly or reuterin treatments was assessed after 30\ndays of continuous oral administration (100 μl including 10 μl of glycerol and 5×10⁹ CFU of\nL. reuteri, once daily) followed by a 3-day withdrawal. Serum biochemical analyses (indicators\nfor key organ functions, lipid profiles, and electrolytes) and histopathological examinations of\nmajor organs (heart, liver, spleen, lung, kidney, and intestine) showed no significant\nabnormalities compared with healthy controls (Supplementary Fig. 28a-f). Likewise, the levels\nof acute inflammatory cytokines in serum (IL-1β and TNF-α) returned to baseline after\nwithdrawal, demonstrating the absence of persistent inflammatory responses (Supplementary\nFig. 28g). Furthermore, manganese biodistribution analysis revealed that residual Mn levels in\nserum and major organs after one month of MnLR/Gly administration remained consistent with\nthe physiological manganese concentrations in normal mice (Supplementary Fig. 28h),\n101\nindicating efficient clearance and the absence of Mn accumulation. These findings demonstrate\nthat MnLR/Gly and reuterin do not induce systemic toxicity and sustained inflammation,\nhighlighting their potential value for clinical translation.”\nFigure 8\n1. Provide immune profiling in rabbits (e.g., T cells, DCs, macrophages) to support mechanistic\nparallels to mice.\nResponse: We sincerely thank the reviewer for this insightful suggestion regarding immune\nprofiling in rabbits. To address this point, we conducted immunofluorescence analysis of the\ntumor microenvironment using rabbit-specific antibodies, Rabbit Anti-CD45 (L12/201) and\nRabbit Anti-CD8 (12.C7) (LSBio, USA). The results revealed a marked increase in CD8⁺ T\ncell infiltration in GlyMnLR capsule–treated rabbits compared with controls (Fig. 8l,m),\nsuggesting that GlyMnLR treatment can elicit antitumor immune responses in rabbits, in line\nwith our findings in mice.We acknowledge, however, that due to the limited availability of\nrabbit-specific antibodies, comprehensive profiling of DCs and macrophages could not be\nachieved at this stage. Future work will therefore require broader immunological toolkits to\nenable deeper mechanistic dissection in this species.\nFig. 8: Characterization of GlyMnLR capsule and its antitumor efficacy and biosafety in\nrabbit models. j, Representative in vivo (endoscopic, the top and middle panels) and ex vivo\n(the bottom panel) images of tumors (highlighted with yellow dashed boxes) on days 10, 28,\nand 31 post oral administration of the indicated agents. Scale bar, 1 cm. k, The tumor volumes\nobtained from the corresponding groups at the end of the experiment (n = 5). l, The\nrepresentative immunofluorescent images and corresponding statistical analysis of the green\nKi67-stained proliferating cells within the isolated tumor tissues from rabbits receiving the\ngiven treatments at the experimental end (n = 5 fields).Scale bar, 200 μm.m,n, Representative\n102\nimmunofluorescence staining images and corresponding quantification of CD45+CD8+ T cells\nwithin the tumor tissues isolated from the rabbits with the given treatments (n = 9 fields).Scale\nbar, 50 μm. Data are shown as mean ± SD; **p < 0.01, ***p < 0.001, **** p < 0.0001\n(compared to PBS group); one-way ANOVA.\nThe relevant description on pages 31, lines 679-681：\n“In addition, GlyMnLR capsules promoted the enrichment of CD8⁺ T cells in the tumor\nmicroenvironment (Fig. 8m,n), supporting the induction of antitumor immunity in the rabbit\nCRC model.”\n2. Compare GlyMnLR capsules vs. free MnLR/Gly vs. standard CRC therapies (e.g., 5-FU) to\nstrengthen translational value.\nResponse: We sincerely appreciate the reviewer’s valuable suggestion to benchmark\nGlyMnLR capsules against free MnLR/Gly and standard CRC therapy. To address this, we\nconducted comparative studies showing that GlyMnLR capsules achieved significantly higher\ntumor inhibition than both free MnLR/Gly and reuterin capsules (Fig. 8j,k),\nunderscoring the\nprotective effect of the enteric formulation and the advantage of sustained metabolite\nproduction by viable bacteria. Notably, their antitumor efficacy was slightly superior to that of\n5-FU (Fig. 8j-m), a standard chemotherapeutic agent, which is consistent with our earlier\nfindings in mouse colon tumor models. In addition, immune profiling of tumor tissue\ndemonstrated that numbers of CD8+ T cells were obviously increased in the GlyMnLR capsule-\ntreated rabbits compared to PBS and 5-FU groups (Fig. 8n,o). These results further support the\ntranslational relevance of GlyMnLR capsules.\nrabbit models. j, Representative in vivo (endoscopic, the top and middle panels) and ex vivo\n(the bottom panel) images of tumors (highlighted with yellow dashed boxes) on days 10, 28,\nand 31 post oral administration of the indicated agents. Scale bar, 1 cm. k, The tumor volumes\nobtained from the corresponding groups at the end of the experiment (n = 5). l, The\nrepresentative immunofluorescent images and corresponding statistical analysis of the green\nKi67-stained proliferating cells within the isolated tumor tissues from rabbits receiving the\ngiven treatments at the experimental end (n = 5 fields).Scale bar, 200 μm.m,n, Representative\nimmunofluorescence staining images and corresponding quantification of CD45+CD8+ T cells\nwithin the tumor tissues isolated from the rabbits with the given treatments (n = 9 fields).Scale\nbar, 50 μm. Data are shown as mean ± SD; **p < 0.01, ***p < 0.001, **** p < 0.0001\n(compared to PBS group); one-way ANOVA.\nThe relevant description on pages 31, 32, lines 675-683：\n“Also, the antitumor efficacy of GlyMnLR capsules was slightly superior to that of the\nclinically used chemotherapeutic agent 5-FU (Fig. 8j,k), which is consistent with the previous\nfindings in mouse colon tumor models. Immunofluorescence for Ki67 further confirmed the\nsuperior inhibitory effect of the GlyMnLR capsule on tumor cell proliferation (Fig. 8l). In\naddition, GlyMnLR capsules promoted the enrichment of CD8⁺ T cells in the tumor\nmicroenvironment (Fig. 8m,n), supporting the induction of antitumor immunity in the rabbit\nCRC model. Together with the favorable biosafety profile demonstrated in the rabbit model\n(Supplementary Fig. 46), these results further support the potential of MnLR to achieve robust\nantitumor activity while maintaining low toxicity.”\nGeneral Recommendations and suggestions\n1. The precise role of Mn²⁺ in enhancing immune responses remains unclear and should be\ndissected further.\nResponse: We sincerely appreciate the reviewer’s insightful comment regarding the role of\nMn²⁺ in immune regulation. Previous studies have reported that Mn²⁺ can directly promote\nantitumor immune responses by activating the cGAS–STING pathway. In our study, we\nobserved that both Mn²⁺ and MnLR individually promoted dendritic cell maturation, but their\neffects were substantially weaker compared to MnLR/Gly stimulation, suggesting that Mn²⁺\nexerts a dual regulatory role—both as a direct immunomodulator and as an enhancer of L.\nreuteri bioactivity. Notably, our transcriptomic analysis revealed that the STING pathway was\n104\nnot significantly upregulated in MnLR/Gly-treated dendritic cells, indicating that alternative\nmechanisms beyond canonical cGAS–STING signaling may underlie the observed immune\nactivation. Taken together, while the precise contribution of Mn²⁺ to the overall immune\nresponse remains to be fully dissected, our results suggest that Mn modification ultimately\nbenefits antitumor immunity through complementary mechanisms. These considerations have\nbeen elaborated in the Discussion section.\n2. The study lacks comparison with standard-of-care therapies. Benchmarking would clarify\nclinical competitiveness.\nResponse: We thank the reviewer for this important suggestion. To benchmark the therapeutic\npotential of MnLR/Gly against standard-of-care treatments, we conducted comparative studies\nin the orthotopic colon tumor model. MnLR/Gly demonstrated superior efficacy, achieving a\ntumor growth inhibition rate 1.14-fold higher than PD-1 antibody, 1.25-fold higher than 5-\nfluorouracil (5-FU), and 2.9-fold higher than panitumumab (Supplementary Fig. 30).\nFurthermore, MnLR/Gly significantly prolonged overall survival, with a higher proportion of\nmice remaining alive beyond 60 days compared to other groups (Fig. 6d). These benefits are\nattributable to the combined effects of potent antitumor activity, minimal systemic toxicity,\nand the practicality of oral administration. Together, these findings highlight the translational\npotential of MnLR/Gly relative to conventional chemotherapy and immunotherapy. The\ncorresponding data and descriptions have been incorporated into the revised manuscript.\n3. Long-term studies are missing: colonization persistence, immune memory, and chronic\nsafety are all key for translation.\nResponse: We appreciate the reviewer’s insightful comment. To address this, we performed\nlong-term evaluations. In preventive settings, MnLR/Gly not only suppressed primary colon\ntumor formation but also conferred durable protection, as 75% of pretreated mice remained\ntumor-free for at least 8 weeks and 62.5% still resisted tumor growth upon rechallenge (Fig.\n7c, and Supplementary Fig. 37). Consistently, continuous administration of MnLR/Gly in\nApcmin/+ mice significantly reduced spontaneous intestinal tumor multiplicity and epithelial\nproliferation (Fig. 7d-h), further supporting a lasting preventive effect. In parallel, systemic\nsafety studies showed no significant abnormalities in organ histology, serum biochemical\nindices, or inflammatory cytokines following 30 days of MnLR/Gly administration\n105\n(Supplementary Fig. 27a-c). Moreover, Mn levels in serum and vital organs remain well below\nphysiological thresholds (Supplementary Fig. 27d), excluding risk of Mn accumulation. These\nfindings collectively demonstrate that MnLR/Gly confers durable antitumor protection while\nmaintaining favorable long-term biosafety. The corresponding data have been incorporated\ninto the revised manuscript.\n4. It might be worthwhile to consider in the future a germ-free or antibiotic- treated mice and\nto validate the findings in human monocytes derived DC.\nResponse: We thank the reviewer for raising this important point. To address the role of gut\nmicrobiota, we profiled microbial composition and diversity. Tumor-bearing mice exhibited\ndistinct dysbiosis compared with healthy controls, while MnLR/Gly treatment partially\nrestored microbial balance and enriched bacterial genera with reported antitumor potential (Fig.\n6m, and Supplementary Fig. 34). Furthermore, antibiotic pretreatment slightly attenuated\nindicating that gut microbiota contributes to, but does not fully account for, its antitumor effects\n(Fig. 6n and Supplementary Fig. 35). In addition, to extend the immune relevance to humans,\nwe tested human PBMC-derived dendritic cells and found that MnLR/Gly promoted DCs\nmaturation both indirectly through induction of immunogenic tumor cell death and directly via\np38 MAPK signaling, consistent with the findings in murine-derived DCs (Fig. 3c,e, and i-m).\nThese results collectively support that the antitumor efficacy of MnLR/Gly is mediated through\nmicrobiota modulation and conserved immunostimulatory mechanisms.\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who coreview\nmanuscripts.\nResponse: We sincerely thank the reviewer for their efforts and contribution to the peer-review\nprocess, and value the opportunity provided by the Nature Communications initiative to\nsupport Early Career Researchers in co-reviewing. We greatly appreciate the efforts and\nconstructive feedback provided by both the primary reviewers and the co-reviewer, which have\nbeen important in improving the quality and clarity of our manuscript. We have carefully\nconsidered all of the reviewers’ comments and have made our best effort to address each of the\nconcerns raised in detail in our revised manuscript and responses.\nWe express our sincere appreciation to the reviewers for their valuable comments and\nkind recognition, which have been invaluable in strengthening our manuscript.\nReviewer #1:\nMy concerns have all been appropriately addressed. It would be helpful to add the\npatients' age into the PDO clinical data Table.\nResponse: We sincerely thank the reviewer for their positive evaluation and for the\nhelpful suggestion. As recommended, we have added the patients’ age information to\nthe PDO clinical data table in the revised Supplementary Table 2.\nSupplementary Table 2 | The clinical information of patients providing PDO models.\nSample ID 1 2 3 4 5\nGender Female Female Male Male Male\nAge 70 79 77 53 69\nRace Asian Asian Asian Asian Asian\nDiagnosis CRC CRC CRC CRC CRC\nSize 3*2.5 cm 4.5*4 cm 2*1 cm 5.5*3.5 cm 4*2.5 cm\nTNM stage T4aN1bM0 IIIb T3N0M0 IIa T3N2bM0 IIIc T3N0M0 IIa T3aN1aM0 IIIb\nAnatomic region Descending colon Ascending colon Rectum Descending colon Sigmoid colon\nPathological type Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma\nMMR status MSI-H MSS MSS MSS MSS\nReviewer #2:\nThe authors fully addressed all my comments. Congratulations for the work thoroughly\nperformed!\nResponse: We sincerely thank the reviewer for the positive feedback and kind\nrecognition of our work. We truly appreciate the reviewer’s time and thoughtful\ncomments, which have been invaluable in improving the quality of our manuscript.\nReviewer #3:\nThe authors have satisfactorily addressed the questions we have raised. I also looked\nup the other reviewers' comments and believe the revised manuscript has much\nimproved.\nResponse: We sincerely thank the reviewer for the encouraging feedback and\nthoughtful assessment. We truly appreciate the reviewer’s recognition of our efforts\nand are glad that the revisions have improved the overall quality of the manuscript.\nReviewer #4:\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who coreview\nmanuscripts.\nResponse: We sincerely thank the reviewer and the co-reviewer for their efforts and\nvaluable contributions to the peer-review process. We greatly appreciate the Nature\nCommunications initiative supporting Early Career Researchers in co-reviewing and\nare grateful for the constructive feedback that has helped us improve the quality of our\nmanuscript."}, {"round": 2, "reviewer_comments": {"reviewer_1": "My concerns have all been appropriately adressed.\nIt would be helpful to add the patients' age into the PDO clinical data Table.", "reviewer_2": "The authors fully addressed all my comments. Congratulations for the work thoroughly performed!", "reviewer_3": "The authors have satisfactorily addressed the questions we have raised. I also looked up the other reviewers' comments and believe the revised manuscript has much improved.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Responses to Reviewers’ Comments\nWe express our sincere appreciation to the reviewers for their valuable comments and\nkind recognition, which have been invaluable in strengthening our manuscript.\nReviewer #1:\nMy concerns have all been appropriately addressed. It would be helpful to add the\npatients' age into the PDO clinical data Table.\nResponse: We sincerely thank the reviewer for their positive evaluation and for the\nhelpful suggestion. As recommended, we have added the patients’ age information to\nthe PDO clinical data table in the revised Supplementary Table 2.\nSupplementary Table 2 | The clinical information of patients providing PDO models.\nSample ID 1 2 3 4 5\nGender Female Female Male Male Male\nAge 70 79 77 53 69\nRace Asian Asian Asian Asian Asian\nDiagnosis CRC CRC CRC CRC CRC\nSize 3*2.5 cm 4.5*4 cm 2*1 cm 5.5*3.5 cm 4*2.5 cm\nTNM stage T4aN1bM0 IIIb T3N0M0 IIa T3N2bM0 IIIc T3N0M0 IIa T3aN1aM0 IIIb\nAnatomic region Descending colon Ascending colon Rectum Descending colon Sigmoid colon\nPathological type Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma\nMMR status MSI-H MSS MSS MSS MSS\nReviewer #2:\nThe authors fully addressed all my comments. Congratulations for the work thoroughly\nperformed!\nResponse: We sincerely thank the reviewer for the positive feedback and kind\nrecognition of our work. We truly appreciate the reviewer’s time and thoughtful\ncomments, which have been invaluable in improving the quality of our manuscript.\nReviewer #3:\nThe authors have satisfactorily addressed the questions we have raised. I also looked\nup the other reviewers' comments and believe the revised manuscript has much\nimproved.\nResponse: We sincerely thank the reviewer for the encouraging feedback and\nthoughtful assessment. We truly appreciate the reviewer’s recognition of our efforts\nand are glad that the revisions have improved the overall quality of the manuscript.\nReviewer #4:\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who coreview\nmanuscripts.\nResponse: We sincerely thank the reviewer and the co-reviewer for their efforts and\nvaluable contributions to the peer-review process. We greatly appreciate the Nature\nCommunications initiative supporting Early Career Researchers in co-reviewing and\nare grateful for the constructive feedback that has helped us improve the quality of our\nmanuscript."}]

Decision letter