Amelioration of colorectal cancer-associated fibroblasts in immunosuppressive microenvironment by ferroptosis-based nanotherapy

Domain: neuroscience
Published: 2026-02-14
Editorial outcome: minor_revision

Abstract
Most colorectal cancers (CRCs) are insensitive to cancer immunotherapy due to the tumor-promoting activities and immunosuppressive effects of cancer-associated fibroblasts (CAFs), which profoundly drive cancer progression. Here, we provide a treatment strategy targeting CAFs to remodel the structural and immune tumor microenvironment of CRC based on magnetic nanoparticles. These nanoparticles not only induce the enhanced ferroptosis of CAFs through the synergistic effect of iron and copper ions but also ameliorate the immunosuppressive tumor microenvironment, simultaneously breaking down physical obstructions and activating the immune system. Mechanistically, these nanoparticles regulate the intrinsic signaling pathways of CAFs and alter chemokine secretion to improve the maturation of dendritic cells and the activation and function of CD8+ T cells, eventually resulting in boosting the immune response to inhibit CRC growth. Moreover, we verify the effectiveness of this strategy in the patient-derived xenograft and organoid models, further illustrating its universality and promising clinical translational potential. Collectively, we show that our magnetic nanoparticles targeting CAFs can reprogram the tumor microenvironment of CRC by breaking down physical barriers and alleviating immunosuppression, offering promising avenues for the therapeutic strategy in clinical CRC management.

Introduction
During the process of colorectal cancer (CRC) initiation, progression, and metastasis, cancer-associated fibroblasts (CAFs), as the most abundant stromal cells in the tumor microenvironment (TME), play a crucial role in providing a scaffold and nutrients for cancer cell growth1–3. Clinically, 85% of CRC identified as microsatellite stable CRC is resistant to cancer immunotherapy largely because of CAFs-mediated structural obstacles4 and immunosuppression5. On the one hand, CAFs typically induce immune-related multidrug resistance of CRC due to their endocytosis and permeation obstruction towards drugs4. Meanwhile, CAFs can assist tumor cells in evading immune surveillance by remodeling the extracellular matrix (ECM), secreting cytokines such as transforming growth factor (TGF)-β via an autocrine or paracrine manner6. Moreover, the ECM remodeling capacity of CAFs mediates T cell trapping through increased matrix rigidity and enhanced resistance to T cell movement7,8. On the other hand, CAFs induce immunosuppression through their biological functions5, including suppressing dendritic cell (DC) differentiation and inducing T cell exhaustion6,8–10. Hence, targeting CAFs is a very reasonable and promising therapeutic strategy in the treatment of CRC.

At present, the conventional strategies targeting CAFs in CRC mainly include drugs or antibodies directly targeting CAFs11, inhibitors targeting CAFs-secreted growth factors, cytokines, or exosomes12, and reprogramming CAFs to normal fibroblasts (NFs)13. However, the ineluctable bottlenecks of the aforementioned strategies still exist in the translational applications, thanks to inefficiency caused by CAFs-induced heterogeneity and cumulative side effects14–17. Hence, an advanced treatment needs to be urgently developed to breakthrough these obstacles.

Nanotherapy, an emerging treatment, has been extensively applied in cancer theranostics. However, most nanomaterials directly targeting cancer cells often have poor therapeutic efficacy, especially for advanced cancers, largely due to CAFs. By secreting high levels of ECM and increasing solid pressure and interstitial fluid pressure, CAFs limit the extravasation and diffusion of nanomaterials in tumor stroma18–20. Meanwhile, due to the preferential localization near blood vessels21, the phagocytosis of nanomaterials by CAFs is much greater than that by cancer cells, resulting in only a small amount of nanomaterials accumulated in cancer cells22,23. Therefore, nanomaterials designed to target CAFs can effectively overcome the above challenges. In the diverse range of nanomaterials, magnetic nanoparticles (NPs) represented by Fe3O4 NPs with excellent biocompatibility can act as photothermal therapy (PTT) and chemodynamic therapy (CDT) agents due to their good photothermal conversion properties and peroxidase-like activity24–28, which can be further optimized by the introduction of copper ions29.

In this work, we prepare Cu@Fe3O4 magnetic NPs consisting of human trace elements with the core-shell nanostructure to target CAFs of CRC for remodeling the structural and immune TME. Based on the intrinsic physicochemical properties of NPs, we provide a nanotherapy strategy targeting CAFs to significantly inhibit CRC growth by not only inducing ferroptosis of CAFs, thereby reducing pro-tumorigenic paracrine signaling, but also remodeling the TME, and alleviating CAF-mediated immunosuppression. Specifically, we find that NPs induce enhanced ferroptosis of CAFs through reactive oxygen species (ROS) generation caused by iron ions and glutathione peroxidase 4 (GPX4) downregulation caused by copper ions. Meanwhile, NPs modulate the secretion of chemokines, such as C-C motif chemokine ligand (CCL) 3 and C-X-C motif chemokine ligand (CXCL) 12, promoting DCs maturation and T cell activation, thereby enhancing T cell function and ultimately generating an immune-activated TME (Fig. 1a). Moreover, pronounced antitumor effects are observed in two kinds of subcutaneous tumor mouse models, a genetically engineered mouse model (GEMM), a patient-derived xenograft (PDX) model, and a patient-derived organoid (PDO) model, which more closely simulate the clinical tumor environment, further demonstrating the broad applicability and translational potential of our strategy as a next-generation CRC therapy.

Results
NPs induce synergistically-enhanced ferroptosis of CAFs
In our work, to evaluate the impact of CAFs on CRC patient survival, tumor samples from GSE17538 and GSE87211 were grouped by CAF scores using microenvironment cell populations-counter (MCPcounter), estimating the proportion of immune and cancer cells (EPIC), and estimation of stromal and immune cells in malignant tumor tissues using expression data (ESTIMATE)30–34. Higher CAF levels were consistently linked to poorer survival outcomes across all methods (Fig. 1b, c, and Supplementary Fig. 1). In addition, the impact of sex on CAF scores was evaluated in the GSE17538, GSE87211, and a single-cell RNA-sequencing (scRNA-seq) dataset E-MTAB-8017. The results consistently indicated that sex had no significant association with CAF abundance in the TME (Supplementary Fig. 2a–e). Based on these findings, sex was not included in the design of subsequent experiments. Firstly, in terms of NPs preparation, Cu@Fe3O4 NPs (simplified as NPs) were synthesized by thermal decomposition in the oil phase, consisting of a core of Cu NPs (~10 nm diameter) with a shell of Fe3O4 NPs (~15 nm thick) (Supplementary Fig. 3). Then, in terms of CRC patients-derived fibroblast lines preparation, NFs from three normal colonic tissues and CAFs from three CRC specimens were successfully isolated (Fig. 2a) and CAFs were identified by the higher expression of their characteristic markers35 (Fig. 2b). In vitro cytotoxicity assay revealed that CAFs were more sensitive to NPs compared to NFs (Fig. 2c) partly due to the higher NPs internalization by CAFs than that by NFs (Fig. 2d, and Supplementary Fig. 4a, b). Calcein-AM/PI double staining assay indicated that NPs combining 808 nm laser irradiation led to nearly complete cell death of CAFs, demonstrating the remarkable PTT effect of NPs (Fig. 2e), which was completely opposite in NFs (Supplementary Fig. 4c, d). Moreover, the most generation of ROS in CAFs was also observed in the NPs+Laser group (Supplementary Fig. 4e–h), and the better CDT effect of NPs combined with laser was also observed towards CAFs than NFs (Supplementary Fig. 4i–l). Similar results were also proved in the CRC cell line HCT116 and non-cancerous colon cell line NCM460 (Supplementary Fig. 5).

Given the significantly higher cytotoxicity of Cu@Fe3O4 NPs compared to Fe3O4 NPs alone (Supplementary Fig. 6a), we focused on the roles of Fe²⁺ and Cu²⁺ in exploring the mechanisms underlying NPs-induced cell death. NPs treatment led to a marked increase in Fe²⁺ levels in CAFs (Fig. 2f, and Supplementary Fig. 6b, c), accompanied by significant lipid peroxidation (LPO) (Fig. 2g, Supplementary Fig. 6d), suggesting that NPs elevated intracellular Fe²⁺ within the labile iron pool, enhancing iron metabolism to induce ferroptosis in CAFs36. Additionally, NPs treatment resulted in a dose-dependent reduction of GPX4 expression (Fig. 2h), consistent with previous reports that Cu²⁺ suppressed GPX4 expression, thereby promoting ferroptosis37. The cytotoxic effects of NPs were significantly attenuated by the Fe²⁺ chelator deferoxamine (DFO) and the Cu²⁺ chelator tetraethylenepentamine (TM), with combined treatment yielding an additive effect (Fig. 2i). DFO notably reduced NPs-induced Fe²⁺ elevation, while TM did not affect Fe²⁺ levels (Fig. 2j, Supplementary Fig. 6e, f). Both DFO and TM inhibited NPs-induced LPO, and their combination further reduced LPO levels in CAFs (Fig. 2k, and Supplementary Fig. 6g). Fe²⁺ modulation had minimal impact on GPX4 expression, whereas Cu²⁺ chelation significantly restored GPX4 levels (Fig. 2l). These findings indicated that NPs, following lysosomal uptake, elevated intracellular Fe²⁺ and Cu²⁺ levels, simultaneously regulating iron metabolism and GPX4 pathway to induce synergistically-enhanced ferroptosis.

Moreover, similar experiments were designed in CRC cells. Cu@Fe3O4 NPs exhibited a stronger cytotoxic effect on HCT116 cells compared to Fe3O4 NPs (Supplementary Fig. 7a). An increase in intracellular Fe²⁺ levels, a significant accumulation of lipid peroxides, and a progressive decrease in GPX4 expression were observed as NPs concentration increased, suggesting that CRC cell death was induced via iron metabolism and the GPX4 pathway (Supplementary Fig. 7b–g). Moreover, co-chelation of Fe²⁺ and Cu²⁺ restored cell viability more effectively than the chelation of Fe²⁺ or Cu²⁺ alone (Supplementary Fig. 7h). Further analysis of Fe²⁺ content, LPO, and GPX4 expression upon individual or combined chelation of Fe²⁺ and Cu²⁺ revealed that Fe primarily mediated ferroptosis through iron metabolism, whereas Cu exerted its effect predominantly via the GPX4 pathway, collectively inducing ferroptosis in tumor cells (Supplementary Fig. 7i-n).

NPs regulated multiple signaling pathways of CAFs in vitro
To evaluate the impact of NPs targeting CAFs on CRC cells, we collected conditioned media (CM) from NFs and CAFs without/with NPs treatment and assessed the effects of these CM on the proliferative and migratory potential of HCT116 cells (Fig. 3a). Compared to NFs-derived CM, CAFs-derived CM significantly enhanced the proliferation, migration, and expression of epithelial-mesenchymal transition (EMT) related markers of HCT116 cells. However, this pro-tumorigenic effect was markedly suppressed after NPs treatment in CAFs (Fig. 3b–d).

As the intrinsic molecular signaling pathways of CAFs can not only regulate their physiological proliferation and programmed cell death but also influence the interaction of CAFs with surrounding tumor cells, dysregulated pathways of CAFs treated with NPs were explored by RNA-sequencing (RNA-seq), with identification of 2380 significantly altered genes (1382 upregulated, 998 downregulated) (Supplementary Fig. 8a). Gene Ontology (GO) analysis of differentially expressed genes in NPs-treated CAFs revealed significant upregulation of chemokine receptor binding and chemokine activity, alongside reductions in cell growth, migration, and adhesion (Fig. 3e, Supplementary Fig. 8b). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis highlighted that ferroptosis and key signaling pathways such as tumor necrosis factor (TNF), nuclear factor ĸB (NF-ĸB), janus tyrosine kinase (JAK)-signal transducer and activator of transcription (STAT), and mitogen-activated protein kinase (MAPK) were upregulated, while TGF-β, wingless-related integration site (Wnt), and phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) pathways were downregulated (Fig. 3f, and Supplementary Fig. 8c). Pathways enriched by Gene Set Enrichment Analysis (GSEA) further showed high concordance with GO and KEGG results, emphasizing the critical roles of these pathways in reducing the pro-tumorigenic effects of CAFs upon NPs treatment (Fig. 3g, h, and Supplementary Fig. 8d–f). Given the well-established roles of TGF-β and Wnt pathways in CAFs17,38, we focused on three chemokine-related GO pathways. Pro-inflammatory chemokines (e.g., CCL2, CCL3, CCL7, CXCL8, CXCL10, and CXCL11) and C-C motif chemokine receptor (CCR) 7 were significantly upregulated, contributing to immune cell recruitment, antigen presentation, and antitumor responses38–43. Conversely, CXCL12, which supported CRC proliferation and immunosuppression6,42, was downregulated (Fig. 3i). According to RNA-seq findings, transcriptional alignment of CCL3, CXCL12, and CCR7 (Fig. 3j) and secreted protein levels of CCL3 and CXCL12 (Fig. 3k, l) were also further validated. Additionally, Western blot results substantiated the activation of MAPK and NF-κB pathways, along with attenuated AKT pathway, corroborating the transcriptional data (Fig. 3m).

The immunoregulatory effect of NPs towards CAFs in vivo
We established two kinds of CAFs-related cancer mouse models (CT26 and BALB/3T3 cells, MC38 and NIH3T3 cells) to explore the immunoregulatory effect of NPs towards CAFs (Fig. 4a). The growth rate of CRC cells co-inoculated with CAFs (as CT26+BALB/3T3 group) was much faster than that of only CRC cells (as CT26 group), and NPs treatment presented satisfactory suppression effects on both the CT26 group and the CT26+BALB/3T3 group. Considering the larger pre-treated tumor sizes of the CT26+BALB/3T3 group compared to the CT26 group, the tumor inhibition efficiency of NPs in the CT26+BALB/3T3 group was reasonably considered higher than that in the CT26 group (Fig. 4b–d). The consistent results were also observed in MC38-corresponding four groups (Supplementary Fig. 9a–c). Meanwhile, the good biosafety and biocompatibility of NPs in vivo were proved by metabolic performance assay29, coagulation and hemolysis assay (Supplementary Fig. 9d–e), and body weight curves (Fig. 4e, and Supplementary Fig. 9f). Furthermore, compared to the CT26 group, co-implantation with BALB/3T3 cells decreased CCL3 and increased CXCL12 expression in vivo and NPs treatment reversed these chemokine alterations through significantly elevating CCL3 and suppressing CXCL12, which resulted in transforming the immunosuppressive TME into a more active state (Fig. 4f). Hence, we subsequently made a comprehensive and systematic evaluation on the immunomodulatory effect of NPs. In the CT26+BALB/3T3 group, the costimulatory molecules CD80 and CD86 in DCs around tumor tissues or in the spleen were slightly down-regulated compared to the CT26 group, indicating CAFs’ inhibitory effect on DC activation. However, NPs treatment up-regulated CD80 and CD86 in both the CT26 group and the CT26+BALB/3T3 group, accelerating DC activation and leading to functional outcomes like DC maturation and enhanced antigen presentation (Fig. 4g, Supplementary Fig. 10). Although there was no significant change in the population of CD8+ T cells around tumor tissues or in the spleen in these four groups (Supplementary Fig. 11a–d), the activation and function of CD8+ T cells presented a surprising improvement after NPs administration, which were indicated by the up-regulated expression of T-bet/Eomesodermin (Eomes) (Fig. 4h, and Supplementary Fig. 11e–j) and interferon (IFN)-γ/TNF-α/CD107a (Fig. 4i, and Supplementary Fig. 12), respectively. All these results demonstrated the impairment of CD8+ T cell activation and function caused by CAFs, which were reversed visibly through the NPs treatment.

The targeting specificity of AS1411 in different cells
To improve the efficiency, specificity, and biosafety of NPs targeting CAFs in vivo, we integrated scRNA-seq and subcellular protein localization datasets to identify optimal cell surface targets. Approximately twenty reported nano-drug surface markers were examined, and nine of them were significantly upregulated in CAFs according to scRNA-seq data (Supplementary Fig. 13a). Among fibroblast subclusters, nucleolin (NCL) showed the broadest distribution and highest expression, followed by CD44. Conversely, CD44 was predominantly expressed in phagocytic myeloid cells (Fig. 5a-c), suggesting potential enhancement of clearance and reduced tumor accumulation of NPs. Nucleolin, although mainly localized to the nucleolus and nucleus, is known to translocate to the cell surface and is markedly upregulated in CRC44,45, representing a more specific and suitable target. In the further subdivided NF and CAF subpopulations, NCL mRNA expression was significantly higher in CAFs than in NFs (Supplementary Fig. 13b–d), and western blot analysis showed that nucleolin protein levels were markedly elevated in the three CAF lines compared with the three NF lines (Supplementary Fig. 13e). To determine nucleolin localization, immunofluorescence (IF) assays of living cells (for membrane nucleolin) and fixed cells (for nuclear nucleolin) were performed. Nucleolin membrane expression was strongest in CRC CAFs and tumor cells but minimal in macrophages and nearly undetectable in NFs, epithelial cells, and endothelial cells (Fig. 5d). To further validate the specific targeting of AS1411 binding to nucleolin and off-target effects evaluation of NPs, we isolated different types of cells from the in situ TME of the subcutaneous tumor model and detected their uptake of NPs. As shown in Fig. 5e–k and Supplementary Fig. 14a–i, B cells, T cells, and endothelial cells hardly engulfed NPs and NPs-AS1411, while DCs and macrophages had minimal phagocytosis of NPs and NPs-AS1411. As expected, CAFs had the highest uptake of NPs, followed by tumor cells. Moreover, due to the targeting specificity of AS1411, both CAFs and tumor cells had a significant increase in phagocytosis of NPs-AS1411. Moreover, the results of Supplementary Fig. 14j–k confirmed that both NPs and NPs-AS1411 were mainly distributed in the liver and kidney, and NPs-AS1411 were more enriched in the tumor site than NPs, further demonstrating the good metabolizability of NPs and the effectiveness of AS1411 targeting. All the results suggested the enhanced targeting specificity and biosafety of NPs, especially NPs-AS1411.

The synergistic treatment effect of NPs in vivo
Taking the excellent physicochemical properties, including photothermal conversion properties, peroxidase-like activity, and the immunoregulatory effect of NPs into account, a synergistic therapy strategy (i.e., PTT/CDT/immunotherapy) for CRC treatment was implemented based on the schematic illustration in Fig. 6a. Referring to previous results29, laser irradiation was applied to the mice with NPs and NPs-AS1411 treatment 24 h after injection, with the maximum enriched amounts of NPs at the tumor sites. As expected, compared to the other five groups, NPs-AS1411 treatment combined with the 808 nm laser irradiation presented the most satisfactory suppression effects on CT26 cells co-inoculated with BALB/3T3 cells and MC38 cells co-inoculated with NIH3T3 cells (Fig. 6b, c, and Supplementary Fig. 15a, b). Meanwhile, the body weight curves (Supplementary Fig. 15c, d), blood routine test (Supplementary Fig. 15e), liver and kidney function assay (Supplementary Fig. 15f), and hematoxylin and eosin (H&E) staining results (Supplementary Fig. 15g) demonstrated no differences in these six groups after different treatments, intensely revealing the good in vivo biosafety of this strategy again. Moreover, the NPs+Laser group showed the most CCL3 and the least CXCL12 expression in tumors, suggesting the strongest immune response (Fig. 6d, e). Among the six groups, the NPs-AS1411+Laser group exhibited the highest expression of CD80 and CD86 of DCs (Fig. 6f, g, and Supplementary Fig. 16), in accordance with the highest expression of T-bet and Eomes (Fig. 6h, and Supplementary Fig. 17) as well as IFN-γ, TNF-α, and CD107a in CD8+ T cells around tumor tissues or in the spleen (Fig. 6i, and Supplementary Fig. 18). All these results suggested NPs-mediated initiation of the strongest immune response and amelioration of CRC immunosuppression. To better simulate the process of CRC occurrence and development, as well as to more accurately substantiate the therapeutic efficacy of NPs and NPs-AS1411 against CRC, a kind of colorectal cancer genetically engineered mouse model (GEMM), namely the Apc-KO mouse model, was employed for further evaluation. Mice were injected with NPs or NPs-AS1411 intraperitoneally three times at 13, 14, and 15 weeks of age, with PBS-treated Apc-KO and wild-type (WT) C57BL/6J mice as controls. At 18 weeks, tumor progression in the colon and small intestine was evaluated (Fig. 6j). Both NPs and NPs-AS1411 markedly reduced intestinal tumor number and burden, increased colon length, and partially restored body weight, with stronger effects in the NPs-AS1411 group (Fig. 6k–l, Supplementary Fig. 19a–e). H&E staining confirmed significant alleviation of adenocarcinoma lesions (Fig. 6m, Supplementary Fig. 19d). ELISA assays revealed elevated CCL3 and reduced CXCL12 levels in peritoneal lavage fluid, particularly after NPs-AS1411 treatment (Fig. 6o). Immunohistochemistry (IHC) showed enhanced CD8⁺ T cells and CD86⁺ DCs infiltration, indicating robust immune activation mediated by NPs, especially NPs-AS1411 (Fig. 6n, and Supplementary Fig. 19e).

The tumor inhibitory effect of NPs on patient-derived CRC models
Adopting the same treatment strategy in the PDX model, the NPs-AS1411 group showed the most significant temperature rise at tumor sites after the laser irradiation, which proved the excellent photothermal conversion performance of NPs and the effective active targeting of AS1411 again (Fig. 7a). Meanwhile, the NPs-AS1411+Laser group also expectedly exhibited the strongest tumor-suppressive effect in the PDX model with tumors that almost disappeared (Fig. 7b, c). In addition, this strategy had no effect on the body weight of the PDX model (Supplementary Fig. 20a). The histochemical analysis of tumors indicated the more extensive necrosis appeared with severe cell shrinkage and loss of contact in the NPs-AS1411+Laser group compared to the other five groups (Supplementary Fig. 20c). The expression of carcinoembryonic antigen (CEA) and Ki67 as epithelial cells markers and α-SMA and Vimentin as CAFs markers were gradually decreased with the continuous enrichment of strategies, indicating the inhibition effect on both CRC cells and CAFs (Supplementary Fig. 20d). To evaluate the simultaneous effects of NPs on CAFs and cancer cells, co-cultured cells from the PDX model were treated with NPs and laser irradiation after confirming their cellular characteristics (Supplementary Fig. 20b). Primary CRC cells surrounded by CAFs were identified by CEA and Ki67 and CAFs were identified by α-SMA and Vimentin (Fig. 7d). In co-culture system, the dose of NPs that effectively eliminated CAFs had limited direct cytotoxicity against CRC cells. However, when active targeting capability and NPs-triggered PTT were introduced, the cytotoxic effects of NPs on both CAFs and tumor cells were markedly enhanced (Fig. 7e).

To better conform to the real pathological environment of clinical patients, organoids were derived from cancerous and adjacent normal colonic tissues. Normal colonic organoids exhibited thinner lumens, uniformed cell organization, and better differentiation compared to CRC organoids (Fig. 7f), which were confirmed by consistent marker expressions in these organoids with their respective source tissues (Fig. 7g, and Supplementary Fig. 20e). Additionally, the cytotoxic effects of NPs at CAF-eliminating doses on CRC organoids were further enhanced by the active targeting of aptamer AS1411 and laser irradiation, while these treatments caused nearly no damage to normal colonic organoids. It was worth noting that the targeting ability of AS1411 could increase the enrichment of NPs in tumor tissues, thus exhibiting stronger tumor inhibitory effects in CRC organoids, regardless of whether laser irradiation is present or not (Fig. 7h, i).

Methods
Ethics statement
All patient samples used in the present study were obtained from individuals undergoing surgical resection at the Department of Gastrointestinal Surgery IV, Peking University Cancer Hospital & Institute. None of the patients had received chemotherapy, radiotherapy, or immunotherapy prior to surgery. The study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the Ethics Committee of Peking University Cancer Hospital & Institute (approval no. 2020KT127). Written informed consent was obtained from all participants. All animal experiments were performed in strict accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Peking University, Beijing, China (MSE-HouYL-1). As approved by IACUC (MSE-HouYL-1), when measuring tumor size, the maximum diameter of no more than 20 mm was strictly as the upper limit for tumor measurement in all animal experiments and the final tumor volumes did not exceed 2000 mm³ or 10% of the animal’s body weight. The tumor volume calculation formula adopted the most widely accepted and used standard formula V=1/2 * L * W2 (V is the volume, L is the longest diameter, W is the width).

Synthesis of monodisperse Cu@Fe3O4 NPs
Cu@Fe3O4 NPs (simplified as NPs) were synthesized as follows: copper acetylacetonate (Cu(acac)2) (1 mmol, J & K), 1-octadecene (ODE) (46.875 mmol, Alfa Aesar), oleic acid (2 mmol, Alfa Aesar), and oleamine (OAm) (10 mmol, J & K) were mixed in a four necked flask. The solution was heated to 120°C and kept for 1 h to remove the organic impurities under the mild N2 flow. When the temperature reached 180°C, Fe(CO)5 (5 mmol) was injected into the reaction system and kept for 10 min. Then, the solution was heated to 280°C and kept for 2 h. At room temperature, the solution was washed with acetone and hexane three times, and the products were kept in trichloromethane.

Synthesis of Cu@Fe3O4-AS1411 NPs
As performed previously72, NPs were modified with DSPE-PEG-NH2. 1-Ethyl-3-(3-(dimethylamino)propyl)urea hydrochloride (EDC) (1.9 mg, Aldrich, 99%) and N-hydroxysuccinimide (NHS) (2.2 mg, Aldrich, 98%) were mixed with the aforementioned NPs (20 mg) in an aqueous solution for 4-8 h. AS1411 (60 μg) was added to the reaction system and mixed for 24 h. The products were washed three times and kept in an aqueous solution.

Characterization
Transmission electron microscopy (TEM) images were obtained from the FEI Tecnai T20 microscope. High-resolution TEM (HRTEM) images were obtained from the FEI Tecnai F30 microscope. X-ray photoelectron spectroscopy (XPS) measurements were performed on an imaging X-ray photoelectron spectrometer using Al Kα radiation (Axis Ultra DLD, Kratos Analytical Ltd.). All the collected spectra were calibrated with the contaminated C 1s peak at 284.8 eV and were analyzed using CasaXPS software (2.3.12 Dev7). X-ray diffraction (XRD) patterns were carried out using a Rigaku DMAX-2400 X-ray diffractometer equipped with Cu Kα (λ=0.15405 nm) radiation. The concentrations of Fe were quantified using an inductively coupled plasma-atomic emission spectrometer (ICP-AES, Prodigy 7, Leeman, USA).

Cell culture
Human colonic epithelial cell line NCM460, human CRC cell line HCT116, murine CRC cell lines CT26 and MC38, the human monocytic leukemia cell line THP-1, and murine fibroblast cell lines (BALB/3T3 and NIH3T3) were originally obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and China Infrastructure of Cell Line Resources, Institute of Basic Medical Sciences, and Chinese Academy of Medical Sciences. Immortalized human colonic microvascular endothelial cells (HCMEC, BL12-H) and human pulmonary microvascular endothelial cells (HPMEC, HTX2255) were obtained from Otwo Biotech (Shenzhen, China). All cells were cultured in either Dulbecco’s modified eagle medium (DMEM) or RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in a humidified environment at 37°C with 5% CO2. All the reagents for cell culture were purchased from Invitrogen.

Human subjects
Fresh CRC tissues and matched adjacent normal tissues used for the isolation and culture of fibroblasts and organoids were obtained from patients undergoing surgical resection at the Department of Gastrointestinal Surgery IV, Peking University Cancer Hospital & Institute. NF lines were established from the adjacent normal tissues of three patients (NF1, 58-year-old male; NF2, 63-year-old male; NF3, 54-year-old female), and a normal colonic organoid was derived from the adjacent normal tissue of a 66-year-old male patient. CAF lines were generated from primary CRC tissues of three patients (CAF1, 61-year-old male; CAF2, 55-year-old female; CAF3, 71-year-old male), and a CRC PDO as well as a PDX model were established from tumor tissues of two patients (PDO, 52-year-old female; PDX, 72-year-old male). Written informed consent was obtained from all participants. All human tissue samples were used exclusively for the isolation of fibroblast lines, organoids, and PDX models in this study, and no residual tissues were retained.

Isolation and culture of NFs and CAFs
CRC tissues and adjacent normal tissues (located at least 10 cm from the tumor margin) were surgically obtained and transferred to the laboratory under sterile conditions. After PBS washing and removal of necrotic areas, the tissues were minced into ~1 mm fragments. These fragments were explanted in 6 cm culture dishes containing DMEM/Nutrient Mixture F-12 (DMEM/F12) supplemented with 10% FBS and 1% penicillin-streptomycin. Migrating fibroblasts were allowed to expand until reaching 70–80% density, then harvested and enzymatically digested for further expansion. Three NFs from normal colonic tissues and three CAFs from CRC samples were successfully isolated, with NF1 and CAF1 validated and selected for further experiments.

Preparation of conditioned medium
Primary fibroblasts (NFs or CAFs) were seeded and cultured in complete growth medium until reaching approximately 80-90% confluence. Prior to collecting the CM, cells were gently washed twice with sterile PBS, and the culture medium was replaced with fresh medium. Fibroblasts were then incubated for 24-48 h at 37°C in a humidified atmosphere containing 5% CO2 to allow accumulation of secreted factors. The supernatant was collected and centrifuged at 300 g for 10 min to remove floating cells and debris, followed by filtration through a 0.22 µm sterile filter. For CAFs treated with NPs, the supernatant was additionally centrifuged at 12,000 g for 10 min to remove any residual nanoparticles. The resulting CM was aliquoted and stored at −80°C until use. Before application, CM was thawed on ice and mixed 1:1 with fresh complete medium for the subsequent treatment of CRC cells.

In vitro cytotoxicity assay
The cytotoxicity of NPs was evaluated using the Cell Counting Kit-8 (CCK-8; DOJINDO, Tokyo, Japan). Briefly, NFs, CAFs, NCM460, and HCT116 cells were seeded in a 96-well plate to reach 70–80% density. The cells were treated with NPs at varying Fe concentrations and incubated for 24 h. After washing, CCK-8 reagent was added to each well, and absorbance at 450 nm was measured using a microplate reader (Bio-Rad 680) after 2 h incubation. Each experiment was repeated in triplicate. Cytotoxicity was calculated as a percentage relative to untreated controls, and IC50 values were determined by fitting dose-response curves using nonlinear regression analysis in GraphPad Prism. All subsequent cell experiments were conducted at the following IC50 concentrations: 12.26 μM (95% CI: 11.35-13.90 μM) for NFs, 2.63 μM (95% CI: 2.36-2.89 μM) for CAFs, 7.08 μM (95% CI: 6.87–7.30 μM) for NCM460 cells, and 4.62 μM (95% CI: 4.25–5.00 μM) for HCT116 cells.

Bio-TEM characterization
Bio-TEM was utilized to determine the subcellular distribution of the NPs in NFs, CAFs, NCM460, and HCT116 cells. NFs, CAFs, NCM460, and HCT116 cells were seeded into a 6-well culture plate (105 cells per well). When the cell density reached 80%-90%, the cells were then incubated with NPs for 24 h. The treated cells were washed three times with PBS, fixed using paraformaldehyde and osmium tetroxide, and then dehydrated with ethanol. Eventually, the treated cells were embedded in Spurr resin and sectioned to 70 nm in thickness.

Double staining of living/dead cells assay
The viability of cultured cells and organoids was assessed using the Calcein-AM/PI Double Stain Kit (40747ES76, Yeasen, Shanghai, China). NFs, CAFs, NCM460, and HCT116 cells were seeded into a 24-well culture plate (2 × 104 cells per well), achieving approximately 70% density. Normal colonic and CRC organoids were seeded in 3D low-attachment cell culture plates (Thermo Scientific). Indicated cells or organoids were then incubated with NPs for 24 h. After washing out the free NPs with PBS, the fresh culture medium was added. Laser (808 nm, 0.25 W/cm2) was then used to irradiate the cells or organoids for 5 min. The staining method was directed by the instructions. Each experiment was repeated in triplicate. Stained cells and organoids were visualized using an inverted microscope (Olympus IX71) or a confocal microscope (Zeiss LSM880, Germany).

Intracellular ROS assay
Intracellular ROS production was determined by the ROS Assay Kit (S0033, Beyotime, China). NF, CAF, NCM460, and HCT116 cells were seeded into a 24-well culture plate (2 × 104 cells per well). When the cell density reached 70%, the cells were then incubated with NPs for 24 h. After washing out the free NPs with PBS, the fresh culture medium was added. Laser (808 nm, 0.25 W/cm2) was then used to irradiate the cells for 5 min. The staining method was directed by the instructions. Each experiment was repeated in triplicate. The cells were finally visualized using an inverted microscope (Olympus IX71) or detected through flow cytometry using a BD Fortessa flow cytometer (BD Biosciences).

Measurement of intracellular ferrous ions
The intracellular content of ferrous ions was assessed both qualitatively and quantitatively using the FerroOrange Kit (Dojindo, Japan) and the Ferrous Ion Content Assay Kit (BC5415, Solarbio, China), following the manufacturer protocols. Each experiment was repeated in triplicate.
For qualitative analysis, indicated cells were washed twice with PBS and incubated with 1 μM of the FerroOrange fluorescent probe at 37°C for 30 min. The cells were then visualized using a confocal laser scanning microscope (Zeiss LSM 880, Germany), and fluorescence intensity was quantified using ImageJ software.
For quantitative analysis of Fe²⁺ content, treated cells were ultrasonically lysed in an ice bath, and the lysates were centrifuged at 4°C for 10 min. The resulting supernatants were analyzed using the Ferrous Ion Content Assay Kit, with the absorbance measured at 593 nm using a microplate reader. A standard curve was generated based on standard samples, and the relative intracellular Fe2+ levels were calculated accordingly.

Liperfluo staining for LPO
Cells were assessed using Liperfluo staining (Dojindo, Japan). Cells were incubated with 10 μM Liperfluo in the dark at 37°C for 30 min. After incubation, cells were washed twice with PBS and visualized using a confocal laser scanning microscope (Zeiss LSM 880, Germany), and fluorescence intensity was quantified using ImageJ software. Each experiment was repeated in triplicate.

Immunofluorescence
To examine protein expression on the plasma membrane, living cell IF staining was performed. Cells were washed twice with PBS and incubated with a primary antibody at 4°C for 60 min without fixation, followed by an appropriate fluorophore-conjugated secondary antibody. After washing, nuclei were counterstained with Hoechest 33342 and imaged immediately under a confocal microscope. For conventional IF, cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.5% Triton-X100 for 10 min at 4°C, and blocked with 1% bovine serum albumin for 1 h at room temperature. Cells were then incubated overnight at 4°C with primary antibodies and with the corresponding secondary antibody for 1 h at room temperature the next day. Primary antibodies used were as follows Ki67 (Abcam, ab16667, 1:200), CEA (Huabio, ET1705-52, 1:500), α-SMA (STARTER, S0B2103, 1:200), Vimentin (Huabio, ET1610-39, 1:500), Nucleolin (Abcam, ab129200, 1:200), and CTNNB1 (Proteintech, 51067-2-AP, 1:200). Following primary antibody incubation, cells were washed three times with PBS for 5 min. Secondary antibody incubation was performed for 1 h with Goat Anti-Rabbit IgG (Alexa Fluor 488) (Abcam, ab150077, 1:200) and Goat Anti-Rabbit IgG (Alexa Fluor 594) (Abcam, ab150080, 1:200). Images were captured using an inverted microscope (Olympus IX71) or a laser scanning confocal microscope (Zeiss LSM 880, Germany).

ELISA
Supernatants from treated CAFs homogenized mouse tumor tissues, as well as the peritoneal lavage fluid of mice, were collected. CCL3 and CXCL12 levels in CAF supernatants and homogenized mouse tumor tissues were measured using the anti-human ELISA kits (CCL3 for SEKH-0246, and CXCL12 for SEKH-0310, Solaribio) and anti-mouse ELISA kits (CCL3 for SEKM-0112, and CXCL12 for SEKM-0050, Solaribio), respectively, following manufacturer protocols. All experiments were conducted in triplicate.

Animals and subcutaneous cancer mouse models
Mice were housed under standard specific pathogen-free (SPF) conditions with a 12-h light/12-h dark cycle, an ambient temperature of 22±1°C, and a relative humidity of 50±10%. BALB/c mice and C57BL/6J mice (female, 6-8 weeks) were purchased from Vital River Laboratory Animal Technology Co. Ltd (Beijing, China). For the subcutaneous cancer mouse model, CT26 cells (2×106 cells in 0.1 ml of saline) or CT26 cells (1.95×106 cells in 0.1 ml of saline) mixed with BALB/3T3 cells (0.05×106 cells) were injected subcutaneously into BALB/c mice at the root of the right hind legs. Similarly, MC38 cells (2×106 cells in 0.1 ml of saline) or MC38 cells (1.95×106 cells in 0.1 ml of saline) mixed with NIH3T3 cells (0.05×106 cells) were injected subcutaneously into C57BL/6J mice at the root of the right hind legs. All animal experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Peking University, Beijing, China.

Apc-KO mouse model
A genetically engineered colorectal cancer mouse model, the Apc-KO strain (C57BL/6JCya-Apcem2/Cya; C001511, Cyagen Biosciences, Saiye Biotechnology Co., Ltd., Guangzhou, China), was employed to evaluate the in vivo therapeutic efficacy of NPs. This model is established by deleting the mutation cluster region (MCR) of the Apc gene using CRISPR/Cas9-mediated gene editing. Homozygous Apc-KO mice are embryonically lethal, whereas heterozygous mice spontaneously develop multiple intestinal adenomas that progress to adenocarcinomas in both the small intestines and colon. For treatment, heterozygous Apc-KO male mice received intraperitoneal injections of either NPs or NPs-AS1411 at 13, 14, and 15 weeks of age, with WT mice and PBS-treated Apc-KO mice serving as controls. All mice were euthanized at 18 weeks of age, and tumor progression in the colon and small intestine was systematically evaluated.

Patient-derived xenograft (PDX) models
Surgically resected CRC tissues were implanted subcutaneously into the dorsal flanks of 6-8-week-old NOD/SCID mice (Hua-Fu-Kang Corporation, Beijing, China). Tumor xenografts were allowed to grow for 1-3 months, depending on the tumor growth rate. Successfully engrafted tumors were expanded and retransplanted into additional NOD/SCID mice for evaluation of the therapeutic effects.

Isolation, establishment, and maintenance of patient-derived organoids (PDOs)
PDOs were established as previously described73. Briefly, CRC tissues were minced and digested with collagenase and dispase. While adjacent normal tissues were treated with ethylenediaminetetraacetic acid (EDTA) to release crypts. Following filtration and washing, tumor cells or crypts were resuspended in ECM droplets for culture. Organoid passage and maintenance utilized the Human Colonic Organoid Kit (Biogenous, K2003-HC) for normal colonic organoids and the Colorectal Cancer Organoid Kit (Biogenous, K2103-CR) for CRC organoids.

Organoid viability assay
Normal colonic organoids and CRC organoids were treated with NPs or NPs-AS411 first. After 6–8 h, the supernatant was removed and replaced with fresh culture medium, followed by laser irradiation 4–6 h. After 24 h, CellTiter-Glo 3D reagent (G9681; Promega, Madison, WI) was added to each well at a volume equal to that of the culture medium. The organoids were lysed by vigorous mixing, and the plate was incubated at room temperature for 30 min to stabilize the luminescent signal. Luminescence was measured using a microplate reader (Bio-Rad 680), and organoid viability was calculated relative to untreated controls. All experiments were conducted in triplicate.

In vivo treatment experiments
For CT26 cells- and CT26+BALB/3T3 cells-bearing mouse models in four groups, as the schematic diagram showed (Fig. 4a), NPs (20 mg/kg, 200 μl) were intravenously injected into mice after 14, 17, and 20 days of cell inoculation, respectively. Similarly, for MC38 cells- and MC38+NIH3T3 cells-bearing mouse models in four groups, the same treatment was administrated, with the second and third injections repeated after 20 and 26 days of cell inoculation, respectively.
For CT26+BALB/3T3 cells-bearing mouse model in six groups, as the schematic diagram showed (Fig. 5c), NPs (20 mg/kg, 200 μl) and NPs-AS1411 (20 mg/kg, 200 μl) were intravenously injected into mice after 14 days of cell inoculation. After 24 h post injection, the tumors of mice were exposed to 808 nm laser irradiation (0.25 W/cm2) for 5 min. An infrared thermal imaging instrument (FLIR A325SC camera) was used to record the temperature detection and thermal image. The 808 nm high-power multimode pump laser (Shanghai Connect Fiber Optics Co.) was used for the NIR laser. The above injection and laser irradiation were repeated after 17 and 20 days of cell inoculation, respectively. Similarly, for MC38 cells- and MC38+NIH3T3 cells-bearing mouse models in six groups, the same treatment was administrated, with the second and third injections and laser irradiation repeated after 20 and 26 days of cell inoculation, respectively.
For the PDX mouse model in six groups, NPs (20 mg/kg, 200 μl) and NPs-AS1411 (20 mg/kg, 200 μl) were intravenously injected into mice after 22 days of tissue inoculation. After 24 h post injection, the tumors of mice were exposed to 808 nm laser irradiation (0.25 W/cm2) for 5 min. The above injection and laser irradiation were repeated after 34 and 46 days of cell inoculation, respectively.

Histological evaluation
PDX mice with different treatments were sacrificed at the end of treatment, and the tissues (heart, liver, spleen, lung, kidney, and tumor) were selected, followed by fixing with 4% paraformaldehyde. The tissues were embedded in paraffin and sectioned at 5 mm. H&E staining was performed for histological evaluation. The slides were observed under an optical microscope.

Immunohistochemistry
The paraffin sections of tumor tissues in mice were prepared at the end of treatment. Immunohistochemistry was performed as described previously74. Paraffin-embedded tissue sections were deparaffinized, rehydrated, and antigen-recovered. Sections were then blocked with 5% serum/PBS for 30 min, incubated with primary antibodies at 4°C overnight, and followed by a reaction using an immunohistochemistry staining kit (Yeasen, 36311ES50). Primary antibodies were used including Ki67 (Abcam, ab16667, 1:500), CEA (Huabio, ET1705-52, 1:200), CDX2 (Huabio, ET1605-4, 1:200), CK20 (Huabio, EM1901-97, 1:200), CK7 (Huabio, EM0702, 1:200), α-SMA (STARTER, S0B2103, 1:200), Vimentin (Huabio, ET1610-39, 1:200), CD8a (Servicebio, GB15068-100, 1:500), and CD86 (Servicebio, GB150054-100, 1:500).

Flow cytometry
Flow cytometry was performed using a BD Fortessa flow cytometer (BD Biosciences) and data were analyzed using FlowJo 10 software (TreeStar). All experiments were conducted in triplicate. Monoclonal antibodies against mouse were purchased from Biolegend as followed: CD45 (103111, 1:200), CD3 (100335, 1:200), CD4 (100405, 1:200), CD8a (100733, 1:200), T-bet (644809, 1:200), Eomes (157703, 1:200), CD107a (121613, 1:100), IFN-γ (113603, 1:200), TNF-ɑ (506323, 1:200), CD80 (104707, 1:200), CD86 (105027, 1:200), MHCII (107605, 1:200), MHCII (107607, 1:200), CD11c (117317, 1:200), CD11b (101215, 1:200), F4/80 (111603, 1:200), CD19 (152407, 1:200), CD31 (102409, 1:200), CD45 (103131, 1:200), Ep-CAM (118205, 1:200). Monoclonal antibodies against mouse were purchased from Absin as followed: α-SMA (abs172181, 1:200).

Uptake assay of NPs by different cells
According to the different color matching of antibodies marking the different cells, NPs and NPs-AS1411 were linked with the appropriate fluorescence molecules (FITC fluorescence molecules) for subsequent flow cytometry analysis. Splenocytes or cells around tumors were cocultured with NPs or NPs-AS1411. The specific experimental steps were performed as described previously75. To assess uptake into primary cells, single cell suspensions from the total spleen or tumors were used. Splenocytes or cells around tumors were cultured, and NPs or NPs-AS1411 were added and co-incubated with splenocytes or cells around tumors. Lastly, the uptake of NPs or NPs-AS1411 by different cells was analyzed by flow cytometry. All experiments were conducted in triplicate.

CD8+ T cell function assay
Splenocytes (2×106) or lymphocytes around tumors were collected. BD GolgiStop™ reagent (BD Biosciences) was used to inhibit intracellular protein transport, and meanwhile, APC-conjugated anti-CD107a antibody or respective control isotypes were added at the beginning of incubation. After co-culturing for 4 h, cells were harvested and stained with the indicated antibodies, fixed, permeabilized with Cytofix/Cytoperm Buffer (BD Biosciences), and then stained with CD107a, IFN-γ, and TNF-ɑ antibody. All experiments were conducted in triplicate.

RNA extraction and quantitative real-time PCR
Total RNA was extracted utilizing TRIzol reagent (Thermo Fisher Scientific, Carlsbad, USA) and subsequently reverse transcribed into cDNA using the GoScript™ Reverse Transcriptase (Promega, Madison, USA). The obtained cDNAs were then subjected to quantitative real-time PCR (qRT-PCR) analysis using SYBR Green PCR Master Mix (Toyobo Life Science, Osaka, Japan) on an ABI 7500 Real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.), with each reaction conducted in triplicate. GAPDH was used as the internal control gene for normalization, and the relative gene expression was analyzed using the 2−ΔΔCt method. The primers of CCL3, CXCL12, CCR7, and GAPDH were as follows:
CCL3 forward: AGTTCTCTGCATCACTTGCTG,
CCL3 Reverse: CGGCTTCGCTTGGTTAGGAA;
CXCL12 forward: ATTCTCAACACTCCAAACTGTGC,
CXCL12 Reverse: ACTTTAGCTTCGGGTCAATGC;
CCR7 forward: TGAGGTCACGGACGATTACAT,
CCR7 Reverse: GTAGGCCCACGAAACAAATGAT;
GAPDH forward: TGCACCACCACCTGCTTAGC,
GAPDH Reverse: GGCATGGACTGTGGTCATGAG.

Western blot
Cells (105 cells per well) were seeded in a 6-well plate and incubated with NPs for 24 h once reaching 80%-90% density, and then lysed in RIPA buffer (Cell Signaling Technology). Western blot analysis was performed using standard protocols76. Primary antibodies from Cell Signaling Technology were as followed: Akt (4691T, 1:1000), Pho-Akt (Ser473) (4060T, 1:1000), NF-κB (p65) (8242T, 1:1000), Pho-NF-κB (Ser536) (3033T, 1:1000), MAPK (p38) (8690T, 1:1000), Pho-MAPK (Thr180/Tyr182) (4511T, 1:1000), E-Cadherin (3195s, 1:1000), N-Cadherin (13116s, 1:500), and Vimentin (5741s, 1:500). Other primary antibodies used were Nucleolin (Abcam, ab129200, 1:1000), and β-actin (Sigma-Aldrich, A1978, 1:2000). All experiments were conducted in triplicate.

RNA-seq analysis
CAFs were seeded to 5×106 cells reaching 80%-90% density and then incubated with or without NPs for 24 h. The treated cells were washed three times with PBS and then harvested for total RNA extraction with a total RNA purification kit (GeneMarkbio). RNA purity and quantification were assessed using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). RNA integrity was confirmed with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Subsequently, libraries were constructed using the VAHTS Universal V6 RNA-seq Library Prep Kit, following the manufacturer’s instructions. Transcriptome sequencing and subsequent bioinformatic analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China).

Bioinformatic analysis
Whole human genome microarray data were obtained from GEO datasets GSE1753834 and GSE87211233 and analyzed by using the “GEOquery” R package77. The infiltration levels of CAFs were evaluated using the MCP-counter, EPIC, and ESTIMATE algorithms30–32, via the “IOBR” package78. The optimal cut-off values for CAF abundance were ascertained by the “surv_cutpoint” function in the “survminer” followed by Kaplan–Meier survival analysis with “survival” and “survminer” with log-rank test for p-values.
To further analyze the effects of NPs on CAFs, RNA-sequencing data of CAFs with different treatments were further explored. DEGs with log2 |fold change (FC)| ≥ 1 and adjusted P-value ≤ 0.05 were identified by the “limma” package79. and functional enrichment was performed through GO, KEGG, and Hallmark GSEA using clusterProfiler and enrichplot. Data visualization was conducted with ggplot2 and pheatmap80–82.
For single-cell analysis, the CRC dataset E-MTAB-8107 was downloaded and processed by using the “Seurat” R package83 (R software, version 4.3.3). Cells with >200 and <6000 detected genes and <15% mitochondrial reads were retained, followed by normalization, PCA, clustering (resolution 0.1–1), and UMAP visualization., Major cell types were annotated based on canonical markers including epithelial cells (EPCAM, KRT884), myeloid cells (CD14, CD6884), endothelial cells (PECAM1, VWF85), fibroblasts (COL1A1, COL1A286), enteric glial cells (S100B87, PLP184), B cells (CD79A, CD79B88), T cells (CD3D, CD3E89), and mast cells (KIT, CPA388). Fibroblast subsets were further re-clustered into CAFs and NFs using markers DCN, IGFBP6, and MFAP590.

Statistical analysis and reproducibility
All experiments were performed at least three times independently. Quantitative data were presented as means±SD. Statistical significance between two groups was assessed using a two-tailed unpaired Student’s t-test. Comparisons across more than two groups were performed using one-way ANOVA followed by appropriate post hoc tests to determine pairwise differences. For experiments involving two independent factors, such as treatment type and time, two-way ANOVA was applied to evaluate the main effects of each factor as well as their interaction. A two-tailed p<0.05 was considered statistically significant. Western blot, immunofluorescence, FerroOrange and Liperfluo staining, as well as H&E staining and IHC experiments were performed at least three times as independent experiments with similar results, and representative images are shown. Statistical analyses were performed using GraphPad Prism (version 10.0). All schematic diagrams in the article were generated by PowerPoint or Adobe Illustrator.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability
The RNA-seq data generated in this study have been deposited in the Gene Expression Omnibus database under accession number GSE301220. The microarray gene expression profiling data sets (GSE17538 and GSE87211) are downloaded from Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo/) database. The scRNA sequencing data were retrieved from the EMBL-EBI ArrayExpress database under accession number E-MTAB-8107 (https://www.ebi.ac.uk/biostudies/arrayexpress). All data supporting this work are available within the Article, Supplementary Information, or Source Data file. Source data are provided with this paper.

Code availability
The code used for the analysis of public GEO datasets, bulk RNA-seq data, and single-cell RNA-seq data in this study is available on GitHub (https://github.com/hoshino-tech/Nano-Shuren-Zaozao-2025) and has been archived in Zenodo at https://zenodo.org/records/1796043291.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Wang et al. investigated the impact of magnetic nanoparticles (NPs) on cancer-associated fibroblasts (CAFs) in colonic cancer, presenting extensive data that could potentially lead to new treatment options. However, several concerns need to be addressed, particularly regarding the robustness of the data. Additionally, the manuscript would benefit from a more concise and focused presentation.\nThe authors claim that \"NPs have good in vivo biosafety\" based on the observation that CAFs exhibited higher phagocytic efficiency for NPs compared to normal fibroblasts (NFs) and cancer cells. However, Fig. 2c shows that NFs were also killed by NPs, a result not further investigated in combination with laser treatment, which demonstrated improved efficiency throughout the rest of the study. In addition, the organoid model presented in Fig. 7 shows data from a single (representative?) experiment, where some killing of normal colonic organoids is suggested by PI staining and brightfield imaging. The manuscript lacks evidence demonstrating that NPs, in combination with laser treatment, do not produce harmful off-target effects. Increasing the number of replicates would strengthen the conclusions.\nSome data currently presented in the introduction should be moved to the results section.\nThe authors also tested whether NPs affect tumor cell lines (Fig. 3), as \"Previous studies indicated that directly targeting CAFs to disrupt the physical barrier within the tumor microenvironment (TME) might carry the risk of tumor stem cell dissemination.\" They observed some degree of cell death, but stronger evidence is needed to support the claim that \"NPs also directly killed tumor cells and prevented tumor cell escape.\"", "reviewer_2": "In this manuscript, Wang et al. report an innovative approach by incorporating copper ions into traditional magnetic nanoparticles (MNPs). These copper ions involved MNPs demonstrate selective targeting of cancer-associated fibroblasts (CAFs), inducing CAFs ferroptosis, remodeling the tumor microenvironment, reducing tumor volume, and promoting immune cell infiltration and activation in different tumor models. While this study presents novel and impactful findings, however, few concerns should be addressed to improve the overall quality of this manuscript.\nMajor Comments:\n1. One of the biggest challenges in MNP-based cancer therapy is the off-target effects, as MNPs may be taken up by immune cells such as macrophages and dendritic cells (DCs), which typically exhibit higher phagocytic activity than tumor cells or CAFs. The authors emphasize MNPs selectively targeting CAFs, it is crucial to address the potential accumulation of MNPs in macrophages, DCs, tumor cells and other cell types. To provide a comprehensive understanding of MNP distribution and specificity, the authors should include data on MNPs accumulation in various cell types (e.g., macrophages, DCs, tumor cells) and organs (e.g., spleen, tumor, draining lymph nodes, liver and kidney) in their in vivo model. This will offer a clearer landscape of copper-ion MNPs distribution post-administration and will help assess off-target toxicity effects.\n2. For RNA-seq analysis, it would be helpful if the authors could highlight marker genes for each cell type cluster in the UMAP shown in Figure 6a to improve clarity. In addition, can authors overlay NCL and another marker expression directly onto the UMAP plot in Figure 6a rather than summarizing it in a bubble plot? This would provide the expression pattern across the cell populations to show its specificity. The library preparation methods for both bulk RNA-seq and single-cell RNA-seq are missing and should be clearly described.\n3. The focus of this manuscript is on Cu@Fe₃O₄ nanoparticles, it remains unclear how these compare to traditional Fe₃O₄ NPs in toxicity and therapeutic efficacy. To strengthen the conclusions, the authors should provide comparative in vivo data on Fe₃O₄ NPs versus Cu@Fe₃O₄ NPs, particularly regarding their toxicity and antitumor effects.\nMinor Comments:\n1. The supplementary section contains a large number of figures, this valuable in demonstrating the study’s hypotheses, however, it may overwhelm the readers. To improve clarity and reduce redundancy, the authors should consider combine related figures. For example, Supplementary Figures 10–17, which all describe ROS production in HCT116 and NCM460 cells, could be combined into a single figure.\n2. The labeling of all cell infiltration data is unclear, for example, in Figure 5g “MHCII+ CD11c+ CD80+ cell (%)”, it is not clear whether the percentages refer to MHCII+ CD11c+ CD80+ cells in all the cells within tumor, in the total CD45+ population, in total CD11c+ cells, or in some other subset. The authors should specify the reference population to improve interpretability.\n3. The authors should clarify how many times each experiment was repeated, as many data with only 3 samples per group. Providing this information will enhance the rigor and reliability of the experimental results.\n4. In the Methods section, the authors list statistical analysis with only t-tests. However, the manuscript involves comparisons across more than two groups, which require one-way ANOVA (e.g., Figure 2e, figure 3 g, h and i). The tumor volume and mice body weight, is a time-dependent variable, need to be analyzed using two-way ANOVA. The authors should properly describe these statistical methods in both the Methods section and the relevant figure legends to improve the rigor of their analysis.\n5. the authors are encouraged to submit full, unedited images of all western blots as supplementary information.", "reviewer_3": "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.", "reviewer_4": "In the current study, the authors developed a nanoparticle (NP) consisting of a core of Cu with a shell of Fe3O4. Both cancer-associated fibroblasts (CAF) and cancer cells subjected to this NP undergo substantial ferroptosis caused by Fe-mediated oxidative stress and Cu-mediated GPX4 degradation. RNA seq analysis from CAF indicated that NP treatment upregulated the expression of pro-inflammatory chemokines such as CCL3 while downregulated CXCL12, which supports cancer proliferation and immunosuppression. They further tested this NP in two CAF-related cancer mice models as well as a PDX organoid model. Consistently, NP treatment improved the immune response and suppressed tumor growth. Overall, the data present in the manuscript are solid and support some of their conclusion. One major issue is that this NP has a similar impact on CAF and cancer cells to induce ferroptosis, making it fairly confusing why the authors brand it like a therapeutic strategy to target the immunosuppressive microenvironment.\n1. What does the Fe concentration in Fig 2C refer to? How is it determined?\n2. In Fig.2d, quantification of the NP internalization is required to support the claim.\n3. Authors claimed that “these NPs also directly killed tumor cells and prevented the escape of tumor cells, highlighting their potential for therapeutic intervention” (l175-176). While NP treatment effectively decreases the cell viability of CAF and cancer cells, no evidence was provided to indicate it prevents the escape of tumor cells, as cells under metastasis undergo substantial changes, making them distinct from the original cancer cells.\n4. Detailed methods of collecting contained media need to be provided. It is important to separate the effect caused by the conditioned media from a direct effect from NP remaining in the media. Since the NP treatment has almost the same impact on CAF and cancer cells (Fig 2 and 3)\n5. What is the expression level of NCL in cancer cells? Does NP-AS1411 impact cancer cell viability?\n6. As mentioned above, this NP similarly affects CAF and cancer cells. Being made from Cu and Fe, it is not surprising the NP will induce ferroptosis in cells. While inducing cell death in both CAF and cancer cells, from a therapeutic perspective, is not necessarily a bad thing, it is important to justify the focus on the tumor microenvironment. For instance, it will be beneficial to know the impact of an NP ONLY targeting the CAF (but not the cancer cells) on tumor growth.\n7. Considering the complex effect of ferroptosis on cancer immunity (pro vs. anti), it is important to include a colorectal cancer GEMM mice model to support all the claims related to the impact of NP on the tumor microenvironment.", "reviewer_5": "This manuscript details a study of nanoparticle-based therapy targeting CAFs to treat colorectal cancer. There is a good level of analysis presented, with correlating data in in vitro models and animal models, validated with patient-relevant organoids. The use of primary cells, mice and patient organoids is thorough and represents a large body of work and I can see how all of these orthogonal methods support the conclusions - there is a clear effect of the NPs demonstrated. That said, I feel like there are some gaps which I think the authors should address prior to considering for publication.\n1. The authors use 2 normal fibroblast lines and 3 CAF lines (line 128). I realise lots of things can happen in preparing primary cells but why only 2 NFs? It falls short of enabling statistical tests to be performed (although stats appear to be shown in eg figure 1c/1e - what are the experimental replicates here?). Having 3 paired patient-derived samples would surely have been preferable.\n2. The authors present treatments on HCT116, with the rationale that their approach might risk \"tumor stem cell dissemination\" (line 160). I can understand the purpose here, but I think the data on HCT116 raises more questions than answers. No data on stem cell populations or markers is shown, are there stem cell/quiescent cells within the cell population? Do these cells a) uptake the NPs or b) respond to the NPs? The authors show data on \"normal\" NCM460 cells in the supplementary data, my concern is that given that >30% of the cells remain viable after treatment (figure 3a), this will contain any stem-cell like cells which are notoriously resistant to therapy and likely have very different uptake through endocytosis (eg https://doi.org/10.1242/jcs.216804). There are some interesting experiments that could be done here, but at very least I think the authors need to revisit the rationale.\n3. The authors show increased immune response after treatment (figure 5). I was struggling to separate effects on CAFs from possible direct effects on immune cells however. Wouldn't macrophages be critical uptakers of the NPs? And then potentially drivers of chemokine release/activation of the immune response? To me, this isn't really considered in the experimental design but I am happy to be corrected. Since the authors have extensively analysed immune populations (supplementary data) it feels like there are some experiments that could clarify this.\n4. The authors show that cancer cells are not responsive to NPs alone in their PDX model (line 393). Why is this different from figure 3 in vitro? This links to point 2, how representative are the HCT116 experiments?\nSome minor points on manuscript preparation:\n5. results information and links to figures do not belong in the introduction. The introduction should provide context prior to discussing results, so I think links to results need removing from the introduction.\n6. there are some language issues, mainly minor but a proof-read is worthwhile eg in figure 1 \"cystine\", \"perxoidation\", line 279 \"presented the satisfactory suppression\", line 282 \"was reasonably considered\" - not sure what is meant.\n7. Figure 2m is a repeat of figure 1c, not sure why duplication of figure is needed."}, "author_response": "Dear Reviewers,\nWe appreciate your careful review and invaluable suggestions and comments,\nwhich are helpful for us to improve our work. According to your comments, we\ncarefully revised our manuscript accordingly. The comments from the reviewers\nand our responses are highlighted as follows.\nREVIEWER COMMENTS\nReviewer #1 (CAFs):\nWang et al. investigated the impact of magnetic nanoparticles (NPs) on cancer-\nassociated fibroblasts (CAFs) in colonic cancer, presenting extensive data that\ncould potentially lead to new treatment options. However, several concerns need\nto be addressed, particularly regarding the robustness of the data. Additionally,\nthe manuscript would benefit from a more concise and focused presentation.\n1. The authors claim that \"NPs have good in vivo biosafety\" based on the\nobservation that CAFs exhibited higher phagocytic efficiency for NPs compared\nto normal fibroblasts (NFs) and cancer cells. However, Fig. 2c shows that NFs were\nalso killed by NPs, a result not further investigated in combination with laser\ntreatment, which demonstrated improved efficiency throughout the rest of the\nstudy. In addition, the organoid model presented in Fig. 7 shows data from a single\n(representative?) experiment, where some killing of normal colonic organoids is\nsuggested by PI staining and brightfield imaging. The manuscript lacks evidence\ndemonstrating that NPs, in combination with laser treatment, do not produce\nharmful off-target effects. Increasing the number of replicates would strengthen\nthe conclusions.\nAuthor reply: We sincerely appreciate the reviewer’s valuable comments and\nsuggestions. Firstly, there is a significant difference in the sensitivity of CAFs and NFs\nto NPs. As shown in Fig. 2c, the results of the IC assay indicated that only 2.63 μM\n50\nof NPs could induce approximately 50% CAF death, whereas NFs maintained nearly\n100% viability at the same concentration. Based on these findings, we selected this\nconcentration of NPs for subsequent treatment experiments, as it could ensure the safety\n1\nof NFs while effectively eliminating CAFs. At this dosage, combining NPs with laser\nirradiation resulted in the near-complete eradication of CAFs, without detectable NF\ndamage (Fig. 2e and Supplementary Fig. 4c-d). Moreover, to further enhance the\nspecificity of NPs-mediated CAF killing effects and minimize the potential non-\nspecific toxicity to NFs, we conjugated the NPs with AS1411, an NCL-targeting\naptamer. NCL was highly expressed on the membranes of CAFs and CRC cells but was\nrarely detected on the membranes of NFs or normal colonic epithelial cells (Fig. 5d).\nThis active targeting modification further reduced the potential non-specific toxicity of\nour NPs and improved the safety in vivo.\nIn the former manuscript, the design of our normal colonic and CRC organoid\nexperiments is not optimal, and we are grateful to the reviewer for pointing out this\noversight. To kill tumor cells more reasonably within a safe concentration of NPs, we\nused lower doses of NPs to treat normal colonic and CRC organoids and supplemented\nthe results treated with NPs-AS1411 and NPs-AS1411 + Laser. The results\ndemonstrated that NPs-AS1411 exhibited a more effective tumor-killing function than\nNPs, and the combination of laser irradiation would further enhance this anti-tumor\neffect. On the contrary, regardless of laser irradiation or not, NPs or NPs-AS1411 with\nthe same dose had almost no killing effect on normal colonic organoids, which fully\ndemonstrated the biosafety of our NPs concentration and strategy and the effectiveness\nin avoiding off-target effects (Representative image shown in Fig. 7h-i; parallel\nreplicates are presented below).\nfluorescent images of Calcein-AM (live cells, green fluorescence) and PI (dead cells,\nred fluorescence) staining in (h) normal colonic organoids and (i) CRC organoids after\ntreatment with PBS, Laser, NPs, NPs-AS1411, NPs + Laser, and NPs-AS1411 + Laser.\n2. Some data currently presented in the introduction should be moved to the\nresults section.\nAuthor reply: We sincerely thank the reviewer for this helpful suggestion. In the\nrevised manuscript, we have reorganized the order of the Introduction and Results\nsections accordingly.\n3\n3. The authors also tested whether NPs affect tumor cell lines (Fig. 3), as \"Previous\nstudies indicated that directly targeting CAFs to disrupt the physical barrier\nwithin the tumor microenvironment (TME) might carry the risk of tumor stem\ncell dissemination.\" They observed some degree of cell death, but stronger\nevidence is needed to support the claim that \"NPs also directly killed tumor cells\nand prevented the escape of tumor cells.\"\nAuthor reply: We sincerely thank the reviewer for this insightful comment. Indeed,\nthe former manuscript might not present the conclusion accurately enough. The results\nof the former Fig. 3 primarily demonstrated that NPs could induce ferroptosis in CRC\ncells. The statement that NPs could “prevent tumor cell escape” was an interpretation\nderived from the integrated biological effects of NPs—namely, their ability to induce\nCAF death, activate antitumor immunity, suppress CAF-mediated epithelial-\nmesenchymal transition (EMT) in tumor cells, and directly kill CRC cells, along with\nestablished mechanisms associated with tumor cell dissemination and metastasis1. After\ncarefully reorganizing the logic of the manuscript, we believe that this interpretation\nwould be more appropriately discussed in the Discussion section.\nOzdemir et al. reported that genetic ablation of α-SMA⁺ myofibroblasts in a transgenic\nmouse model enhanced tumor cell EMT and impaired immune surveillance, suggesting\nthe direct depletion of CAFs by certain approaches may paradoxically convert the\ntumor microenvironment into a pro-tumorigenic state and even increase the risk of\ntumor dissemination2. In contrast, our study revealed that conditioned medium derived\nfrom CAFs significantly promoted CRC cell migration, whereas the supernatant from\nNPs-treated CAFs attenuated this effect (Fig. 3b). RNA-seq analysis further showed\nthat NPs markedly inhibited Wnt and TGF-β signaling pathways in CAFs, thereby\nsuppressing the pro-metastatic paracrine signals mediated by these pathways\n(Supplementary Fig. 8b-c). Notably, Wnt and TGF-β are well-established regulators\nthat promote EMT-driven metastasis and contribute to the establishment of an\nimmunosuppressive TME3-6. Hence, during the revision, we found that HCT116 cells\ncultured in conditioned medium from NPs-treated CAFs exhibited increased expression\nof E-cadherin and decreased expression of N-cadherin and Vimentin compared to those\ncultured in untreated CAF-conditioned medium (Fig. 3d). These findings further\nconfirmed that the cytotoxic effect of NPs on CAFs effectively reduced the metastatic\n4\npotential of CRC cells, without increasing the risk of tumor dissemination that might\nresult from CAF depletion.\nAnother key factor promoting tumor escape and metastasis is the formation of an\nimmunosuppressive TME1. The central mechanistic insight and therapeutic strategy of\nour study were that Cu@Fe O NPs induced the death of CAFs, attenuating their tumor-\n3 4\npromoting function, activating antitumor immunity in the TME, ultimately achieving\ntherapeutic efficacy against CRC (Fig.1a, schematic illustration). Furthermore, because\nCAFs exhibited high sensitivity to NPs, relatively low doses were sufficient to kill them\neffectively; at such doses, the direct cytotoxicity of NPs toward tumor cells remained\nlimited and acted only as an auxiliary antitumor mechanism.\nIn this manuscript, Wang et al. report an innovative approach by incorporating\ncopper ions into traditional magnetic nanoparticles (MNPs). These copper ions\ninvolved MNPs demonstrate selective targeting of cancer-associated fibroblasts\n(CAFs), inducing CAFs ferroptosis, remodeling the tumor microenvironment,\nreducing tumor volume, and promoting immune cell infiltration and activation in\ndifferent tumor models. While this study presents novel and impactful findings,\nhowever, few concerns should be addressed to improve the overall quality of this\nmanuscript.\nMajor Comments:\n1. One of the biggest challenges in MNP-based cancer therapy is the off-target\neffects, as MNPs may be taken up by immune cells such as macrophages and\ndendritic cells (DCs), which typically exhibit higher phagocytic activity than\ntumor cells or CAFs. The authors emphasize MNPs selectively targeting CAFs, it\nis crucial to address the potential accumulation of MNPs in macrophages, DCs,\ntumor cells and other cell types. To provide a comprehensive understanding of\nMNP distribution and specificity, the authors should include data on MNPs\naccumulation in various cell types (e.g., macrophages, DCs, tumor cells) and\norgans (e.g., spleen, tumor, draining lymph nodes, liver and kidney) in their in\nvivo model. This will offer a clearer landscape of copper-ion MNPs distribution\npost-administration and will help assess off-target toxicity effects.\nAuthor reply: We have made detailed revisions and provided an in-depth discussion\nin the revised manuscript. We fully agree that undesired uptake of NPs by non-target\ncells before reaching the intended site represents a major cause of poor targeting\nefficiency and limited therapeutic efficacy. The TME is extremely complex, especially\nthe mononuclear phagocyte system, mainly macrophages and immature DCs, is\ngenerally recognized as the principal cellular subgroup responsible for NP uptake and\nthe resulting off-target effects7-9. Strategies to minimize such off-target effects typically\nfocus on two aspects: (i) optimizing the physicochemical properties of the NPs\nthemselves, including the size and surface chemistry; and (ii) employing active\ntargeting delivery strategies.\nprone to phagocytosis by macrophages, whereas our Cu@Fe O NPs, with an average\n3 4\ndiameter of approximately 20 nm (Supplementary Fig. 3a-b), preferentially accumulate\nin tumor tissues via the EPR effect. Moreover, surface modification of Cu@Fe O with\n3 4\nDSPE-PEG-NH₂ forms a hydrated layer that reduces protein adsorption and\nconsequently decreases macrophage uptake significantly7-13.\nIn terms of the active targeting delivery strategy: we performed a comprehensive\nanalysis integrating scRNA-seq and subcellular protein localization data to identify\nsuitable targets. Approximately twenty previously reported nano-drug surface markers\nwere examined at the mRNA level. Based on the scRNA-seq data, nine surface markers\nwere found to be significantly upregulated in CAFs compared to NFs (Supplementary\nFig. 13a). Subsequently, following your comments, we further analyzed the distribution\nof these nine cell surface marker mRNAs across all cellular subpopulations using\nscRNA-seq data. The results showed that among fibroblast subclusters, NCL exhibited\nthe broadest distribution and highest expression, followed by CD44 whereas among\nmyeloid cells that possessed the strongest phagocytic capacity, CD44 displayed the\nwidest distribution and highest expression (Fig. 5a-c). Considering that CD44 is a\ntransmembrane glycoprotein and is highly expressed in myeloid cell populations such\nas DCs and macrophages, CD44-targeted nanocarriers would likely undergo enhanced\nphagocytosis by the mononuclear phagocyte system, thereby further reducing NP\naccumulation at tumor sites. In contrast, NCL is primarily localized in the nucleolus\nand nucleus; however, studies have demonstrated that in several cancers, including\nCRC, NCL can translocate to the cell surface, where its expression is markedly\nupregulated14, 15. Our further IF analysis revealed that NCL expression on the plasma\nmembrane was highest in CAFs and tumor cells, with only minimal membrane\nexpression observed in macrophages, and almost no detectable expression in normal\nintestinal epithelial cells, NFs, or endothelial cells (Fig. 5d). Therefore, we considered\nNCL, a multi-compartment-shuttling protein, to be a more specific and suitable\ntargeting molecule, which was highly expressed in both CAFs and tumor cells. Hence,\nAS1411 binding to NCL was selected for attachment to the surface of the NPs to\nenhance the specific targeting. Moreover, in vivo cellular uptake experiments in mice\ndemonstrated distinct phagocytic efficiencies among various cell types, further\nconfirming our conclusions. B cells, T cells, and endothelial cells hardly engulfed NPs\n7\nand NPs-AS1411, while DCs and macrophages had minimal phagocytosis of NPs and\nNPs-AS1411. As expected, CAFs had the highest uptake of NPs, followed by tumor\ncells, and due to the targeting specificity of AS1411, both CAFs and tumor cells had a\nsignificant increase in phagocytosis of NPs-AS1411 (Fig. 5e-k and Supplementary Fig.\n14a-i). Collectively, these findings indicated that the specific targeting property of\nAS1411 could significantly alleviate off-target effects, thereby achieving improved\nbiosafety in vivo.\nIn addition, our results validated that both NPs and NPs-AS1411 primarily accumulated\nin the liver and kidney, with NPs-AS1411 exhibiting greater tumor enrichment\ncompared to NPs, underscoring the favorable metabolic clearance of the NPs and\nconfirming the targeting efficacy conferred by AS1411 (Supplementary Fig. 14j-k).\n2. For RNA-seq analysis, it would be helpful if the authors could highlight marker\ngenes for each cell type cluster in the UMAP shown in Figure 6a to improve clarity.\nIn addition, can authors overlay NCL and another marker expression directly\nonto the UMAP plot in Figure 6a rather than summarizing it in a bubble plot?\nThis would provide the expression pattern across the cell populations to show its\nspecificity. The library preparation methods for both bulk RNA-seq and single-\ncell RNA-seq are missing and should be clearly described.\nAuthor reply: We sincerely thank the reviewer for this valuable suggestion. Indeed, in\nthe scRNA-seq dataset E-MTAB-8107 (derived from samples of seven colorectal\ncancer patients), displaying the cell type–specific marker genes for each cellular subset\ncould substantially improve the clarity of cell classification within the high-dimensional\nUMAP projection (Fig. 5a-b). We used CD14 and CD68 to identify myeloid cells16;\nCD3D and CD3E for T cells17; COL1A1 and COL1A2 for fibroblasts18; PECAM1 and\nVWF for endothelial cells19; KIT and CPA3 for mast cells20; EPCAM and KRT8 for\nepithelial cells16; CD79A and CD79B for B cells20; and S100B and PLP1 for enteric\nglial cells16, 21.\nBased on the scRNA-seq data, nine surface markers were identified as being\nsignificantly upregulated in CAFs (Supplementary Fig. 13a). We then evaluated the\nexpression level and abundance of these nine markers across different cellular subsets\n(Fig. 5c). The results showed that among fibroblast subclusters, NCL exhibited\nthe broadest distribution and highest expression, followed by CD44; whereas among\n8\nmyeloid cells that possessed the strongest phagocytic capacity, CD44 displayed the\nwidest distribution and highest expression. Considering that CD44 is a transmembrane\nglycoprotein and is highly expressed in myeloid cell populations such as DCs and\nmacrophages, CD44-targeted nanocarriers would likely undergo enhanced\nphagocytosis by the mononuclear phagocyte system, thereby further reducing NP\naccumulation at tumor sites. In contrast, NCL is primarily localized in the nucleolus\nand nucleus; however, studies have demonstrated that in several cancers, including\nCRC, NCL can translocate to the cell surface, where its expression is markedly\nupregulated14, 15. Therefore, we considered NCL, a multi-compartment-shuttling\nprotein, to be a more specific and suitable targeting molecule.\nAdditionally, following the reviewer’s suggestion, we have provided a detailed\ndescription of the bulk RNA-seq library preparation method and specified that the\nscRNA-seq data were obtained from the E-MTAB-8107 dataset. These details have\nbeen explicitly described and cited in the Methods section of the revised manuscript.\n3. The focus of this manuscript is on Cu@Fe₃O₄ nanoparticles, it remains unclear\nhow these compare to traditional Fe₃O₄ NPs in toxicity and therapeutic efficacy.\nTo strengthen the conclusions, the authors should provide comparative in vivo\ndata on Fe₃O₄ NPs versus Cu@Fe₃O₄ NPs, particularly regarding their toxicity\nand antitumor effects.\nAuthor reply: We sincerely thank the reviewer for this question. Compared to\ntraditional Fe O NPs, Cu@Fe O NPs exhibited a markedly enhanced cytotoxic effect\n3 4 3 4\nagainst CRC cells and CAFs (Supplementary Fig. 6a and Supplementary Fig. 7a). The\nfurther in vivo results indicated the more strengthened antitumor effect of Cu@Fe O\n3 4\nNPs than Fe O NPs (Supplementary Fig. 21a-b). Meanwhile, both Fe O and\n3 4 3 4\nCu@Fe O NPs exhibited excellent biocompatibility in vivo (Supplementary Fig. 21c).\n3 4\nMinor Comments:\n1. The supplementary section contains a large number of figures, this valuable in\ndemonstrating the study’s hypotheses, however, it may overwhelm the readers. To\nimprove clarity and reduce redundancy, the authors should consider combine\nrelated figures. For example, Supplementary Figures 10–17, which all describe\nfigure.\nAuthor reply: Thanks for the helpful suggestion. In the revised manuscript, we have\ncombined the related results into a single figure to improve clarity and reduce\nredundancy.\n2. The labeling of all cell infiltration data is unclear, for example, in Figure 5g\n“MHCII+ CD11c+ CD80+ cell (%)”, it is not clear whether the percentages refer\nto MHCII+ CD11c+ CD80+ cells in all the cells within tumor, in the total CD45+\npopulation, in total CD11c+ cells, or in some other subset. The authors should\nspecify the reference population to improve interpretability.\nAuthor reply: Thanks for your careful suggestion. We have specified the reference\npopulation to improve interpretability in the figure legends of the revised manuscript.\n3. The authors should clarify how many times each experiment was repeated, as\nmany data with only 3 samples per group. Providing this information will enhance\nthe rigor and reliability of the experimental results.\nAuthor reply: We sincerely thank the reviewer for the careful review and constructive\nsuggestions. In this study, each group included at least three samples, and all\nexperiments, including western blot analyses, were independently repeated at least\nthree times. We have provided this information in the revised manuscript to enhance\nthe rigor and reliability of our experimental results.\n4. In the Methods section, the authors list statistical analysis with only t-tests.\nHowever, the manuscript involves comparisons across more than two groups,\nwhich require one-way ANOVA (e.g., Figure 2e, figure 3 g, h and i). The tumor\nvolume and mice body weight, is a time-dependent variable, need to be analyzed\nusing two-way ANOVA. The authors should properly describe these statistical\nmethods in both the Methods section and the relevant figure legends to improve\nthe rigor of their analysis.\nAuthor reply: Thanks for your valuable suggestions. Statistical analyses involving\ncomparisons across more than two groups were performed using one-way analysis of\nvariance (ANOVA), followed by Tukey’s post hoc test to assess pairwise differences\n10\nbetween groups. When data did not meet the assumptions of normality or homogeneity\nof variance, the Kruskal–Wallis test was applied instead, followed by Dunn’s multiple-\ncomparison test. All statistical tests were two-tailed, and a P value < 0.05 was\nconsidered statistically significant.\nFor comparisons involving two independent factors, a two-way analysis of variance\n(two-way ANOVA) was performed to evaluate the main effects of each factor as well\nas their interaction. When a significant interaction or main effect was detected, Tukey’s\nmultiple-comparison post hoc test was applied to assess pairwise differences among\ngroups. Data were examined for normality and homogeneity of variance before analysis.\nAll statistical tests were two-tailed, and P values less than 0.05 were considered\nstatistically significant.\nTo improve the rigor of our analysis, we have described these statistical methods in the\nMethods section of the revised manuscript.\n5. the authors are encouraged to submit full, unedited images of all western blots\nas supplementary information.\nAuthor reply: The western blot data shown are derived from three independent\nexperiments. The original images with at least one marker above and below are\npresented below.\nFig. 2l\nSupplementary Fig. 7g\nSupplementary Fig. 13e\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports. This is part of the Nature Communications initiative to facilitate training\nin peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.\nReviewer #4 (ferroptosis induction, cell death):\nIn the current study, the authors developed a nanoparticle (NP) consisting of a\ncore of Cu with a shell of Fe3O4. Both cancer-associated fibroblasts (CAF) and\ncancer cells subjected to this NP undergo substantial ferroptosis caused by Fe-\nmediated oxidative stress and Cu-mediated GPX4 degradation. RNA seq analysis\nfrom CAF indicated that NP treatment upregulated the expression of pro-\ninflammatory chemokines such as CCL3 while downregulated CXCL12, which\nsupports cancer proliferation and immunosuppression. They further tested this\nNP in two CAF-related cancer mice models as well as a PDX organoid model.\nConsistently, NP treatment improved the immune response and suppressed tumor\ngrowth. Overall, the data present in the manuscript are solid and support some of\ntheir conclusion. One major issue is that this NP has a similar impact on CAF and\ncancer cells to induce ferroptosis, making it fairly confusing why the authors\nbrand it like a therapeutic strategy to target the immunosuppressive\nmicroenvironment.\nAuthor reply: We appreciate the reviewer’s careful evaluation and constructive\nfeedback, which have guided several important improvements to our work. We\napologize that the therapeutic strategy of NPs against CRC was not clearly described in\nthe previous manuscript. We have addressed this major issue in detail in our response\nto Question 6 and have carefully revised the manuscript to clarify previously ambiguous\ndescriptions, such that the rationale and logical framework of the Cu@Fe O -based\n3 4\ntherapeutic strategy for CRC are now clearly presented in the revised manuscript.\n1. What does the Fe concentration in Fig 2C refer to? How is it determined?\nAuthor reply: Thanks for the question. We determined the specific content of the Fe\n17\nelement via the Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-\nOES), and then calculated the molar concentration of the Fe element based on the molar\nmass.\n2. In Fig.2d, quantification of the NP internalization is required to support the\nclaim.\nAuthor reply: Thanks for the helpful suggestion. As shown in Supplementary Fig. 4a-\nb, the quantification of the NP internalization indicated again that CAFs engulfed more\nNPs than NFs.\n3. Authors claimed that “these NPs also directly killed tumor cells and prevented\nthe escape of tumor cells, highlighting their potential for therapeutic intervention”\n(l175-176). While NP treatment effectively decreases the cell viability of CAF and\ncancer cells, no evidence was provided to indicate it prevents the escape of tumor\ncells, as cells under metastasis undergo substantial changes, making them distinct\nfrom the original cancer cells.\nAuthor reply: We sincerely thank the reviewer for this valuable comment. Indeed, in\nthe former manuscript, our results primarily demonstrated that NPs induced ferroptosis\nin both CAFs and tumor cells (Fig. 2, Supplementary Fig. 5, and Supplementary Fig.\n7). However, the statement that NPs could “prevent tumor cell escape” lacked direct\nexperimental evidence. This interpretation was in fact an extrapolation based on the\nmultiple biological effects of NPs—namely, their ability to induce CAF death, activate\nantitumor immunity, suppress CAF-mediated tumor-promoting function, and directly\nkill CRC cells, along with established mechanisms associated with tumor cell\ndissemination and metastasis1. Therefore, it is more appropriate to include this\nstatement in the Discussion section.\nIn this study, we found that the treatment of NPs not only effectively reduced the\nviability of CAFs, but that the conditioned medium from NP-treated CAFs significantly\nsuppressed CRC cell proliferation, migration, and EMT (Fig. 3a-d). This phenomenon\nwas associated with the marked inhibition of Wnt and TGF-β paracrine signaling by\nNPs in CAFs, and these pathways are known to promote tumor proliferation, metastasis,\nand the formation of an immunosuppressive TME (Supplementary Fig. 8)3-6. Another\ncritical factor promoting tumor cell escape and metastasis is the presence of a local\n18\nimmunosuppressive microenvironment1. Interestingly, our results showed that NPs-\ntreated CAFs released abundant chemokines that activated antitumor immunity (Fig.\n3e-l, Fig. 4, Fig. 6, Supplementary Fig. 10-12, and Supplementary Fig. 16-19). Together\nwith the direct cytotoxicity of NPs against CRC cells (Supplementary Fig. 5 and\nSupplementary Fig. 7), these findings suggested that targeting CAFs with NPs could\nhelp prevent tumor cell escape.\n4. Detailed methods of collecting contained media need to be provided. It is\nimportant to separate the effect caused by the conditioned media from a direct\neffect from NP remaining in the media. Since the NP treatment has almost the\nsame impact on CAF and cancer cells (Fig 2 and 3)\nAuthor reply: Thanks for your constructive comments. We have added detailed\nmethods for the collection of conditioned media with different treatments in the\nMethods section of the revised manuscript. The description is as follows:\nPreparation of conditioned medium.\nPrimary fibroblasts (NFs or CAFs) were seeded and cultured in complete growth\nmedium until reaching approximately 80-90% confluence. Prior to collecting the\nconditioned medium, cells were gently washed twice with sterile PBS, and the culture\nmedium was replaced with fresh medium. Fibroblasts were then incubated for 24–48 h\nat 37°C in a humidified atmosphere containing 5% CO₂ to allow accumulation of\nsecreted factors. The supernatant was collected and centrifuged at 300 g for 10 min to\nremove floating cells and debris, followed by filtration through a 0.22 µm sterile filter.\nFor CAFs treated with NPs, the supernatant was additionally centrifuged at 12,000 g\nfor 10 min to remove any residual nanoparticles. The resulting conditioned medium\nwas aliquoted and stored at −80°C until use. Before application, conditioned medium\nwas thawed on ice and mixed 1:1 with fresh complete medium for the subsequent\ntreatment of CRC cells.\n5. What is the expression level of NCL in cancer cells? Does NP-AS1411 impact\ncancer cell viability?\nAuthor reply: Thanks for this insightful question. NCL is a multi-compartment–\nshuttling protein that is primarily localized in the nucleolus and nucleus but is markedly\nupregulated and translocated to the cell surface in CRC14, 15. In the revised manuscript,\n19\nwe performed IF staining to examine NCL expression on different cell surfaces, and the\nresults demonstrated that NCL is highly expressed on the membranes of both CAFs and\ntumor cells (Fig. 5d). Phagocytosis assays further confirmed that NPs-AS1411\ntreatment significantly enhanced NP uptake by both CAFs and CRC cells (Fig. 5e-k\nand Supplementary Fig. 14a-i). Moreover, Fig.7e and 7i show that NPs-AS1411\nmarkedly increased the cytotoxic effects of Cu@Fe O on CAFs, CRC cells, and\n3 4\npatient-derived organoids (PDOs).\n6. As mentioned above, this NP similarly affects CAF and cancer cells. Being made\nfrom Cu and Fe, it is not surprising the NP will induce ferroptosis in cells. While\ninducing cell death in both CAF and cancer cells, from a therapeutic perspective,\nis not necessarily a bad thing, it is important to justify the focus on the tumor\nmicroenvironment. For instance, it will be beneficial to know the impact of an NP\nONLY targeting the CAF (but not the cancer cells) on tumor growth.\nAuthor reply: We greatly appreciate the reviewer’s thoughtful suggestion, which has\nhelped us improve the clarity and rigor of the manuscript. We sincerely apologize for\nnot clearly describing the therapeutic strategy of NPs against CRC in the previous\nversion. Based on our repeated experimental validations, we found that the NPs\ntriggered ferroptosis in both CAFs and CRC cells through Fe-mediated oxidative stress\nand Cu-mediated GPX4 degradation. Interestingly, NPs-induced ferroptosis in CAFs\npromoted the release of numerous cytokines that enhance immune activation, whereas\ntheir major effect on CRC tumor cells was direct cytotoxicity.\nRegarding the reviewer’s question about the impact of targeting only CAFs with NPs\non tumor growth, our in vitro experiments showed that the conditioned medium from\nNPs-treated CAFs markedly inhibited the proliferation, migration, and EMT phenotype\nof HCT116 cells compared to the conditioned medium from untreated CAFs ( Fig. 3a-\nd). Further RNA-seq analysis revealed that this effect was possibly attributable to the\nmarked inhibition of Wnt and TGF-β signaling pathways in CAFs by NPs, thereby\nsuppressing the paracrine signals that promote tumor growth and metastasis\n(Supplementary Fig. 8). Wnt and TGF-β are well-established regulators involved in\ntumor proliferation, metastasis, and the formation of an immunosuppressive TME3-6.\nTherefore, even in the absence of immune components, targeting CAFs with NPs alone\ncan effectively suppress CRC growth.\nof CAFs led to significant changes in multiple immune-activating chemokines and\nsignaling pathways, revealing a crucial finding: NPs-treated CAFs could activate\nantitumor immunity, thereby contributing to CRC suppression. This immunoregulatory\neffect of NPs on CAFs was further validated in two CAFs-associated murine cancer\nmodels (CT26 with BALB/3T3 and MC38 with NIH3T3 cells), and a CRC genetically\nengineered mouse model (GEMM), as you suggested (Fig. 4, Fig. 6, Supplementary\nFig. 9-12, and Supplementary Fig. 16-19).\nIn addition, low-dose NPs treatment was sufficient to extensively eliminate CAFs,\ninhibit tumor-promoting paracrine signaling, and activate immune responses,\nmeanwhile maintaining low systemic toxicity and minimal side effects. In contrast,\ndirectly targeting CRC tumor cells required higher NPs doses, which significantly\nincreased off-target risks and treatment-related toxicity.\nTaken together, by integrating the observed effects of Cu@Fe O NPs on CAFs, tumor\n3 4\ncells, and the immune microenvironment, we established a therapeutic strategy centered\non targeting CAFs. Specifically, low-dose NPs treatment effectively suppressed CAFs’\ntumor-promoting function, and activated antitumor immunity, thereby achieving safe\nand efficient CRC therapy. Furthermore, the direct cytotoxic effect of NPs on CRC\ncells could synergistically enhance the overall therapeutic efficacy.\n7. Considering the complex effect of ferroptosis on cancer immunity (pro vs. anti),\nit is important to include a colorectal cancer GEMM mice model to support all the\nclaims related to the impact of NP on the tumor microenvironment.\nAuthor reply: We appreciate the reviewer’s careful consideration and valuable\nquestion. To further substantiate the therapeutic efficacy of NPs and NPs-AS1411\nagainst CRC, as well as to assess the impact of NPs treatment on the TME, we have\nemployed an additional CRC GEMM in the revised manuscript, specifically, the Apc-\nKO mouse model (full strain designation: C57BL/6JCya-Apcem2/Cya; Cyagen\nBiosciences, Saiye Biotechnology Co., Ltd., Guangzhou, China). The Apc-KO mice\nspontaneously develop intestinal adenomas that progress to adenocarcinomas\nthroughout both the small intestine and colon. This model is established using gene-\nediting technology to delete the mutation cluster region (MCR) within the Apc gene.\nmice spontaneously develop tumorigenic lesions in the gastrointestinal tract.\nSpecifically, Apc-KO mice were intraperitoneally injected with either NPs or NPs-\nAS1411 three times at 13, 14, and 15 weeks of age, with the wild-type (WT) C57BL/6J\nmice and PBS-treated Apc-KO mice as comparisons. At 18 weeks of age, all mice were\nsacrificed, and tumor progression in both the colon and small intestine was evaluated\n(schematic illustration shown in Fig. 6j). The results demonstrated that, compared to\nthe Apc-KO control group, Apc-KO mice treated with NPs or NPs-AS1411 exhibited a\nmarkedly reduced number and total burden of intestinal tumors, accompanied by\nincreased total colon length and partial recovery of body weight, which were more\npronounced in the NPs-AS1411 group (Fig. 6k-l and Supplementary Fig. 19a-c).\nHematoxylin-eosin (H&E) staining of colonic sections further confirmed that NPs,\nparticularly NPs-AS1411 treatment significantly alleviated the severity of\nadenocarcinoma lesions in both the colon and small intestine (Fig. 6m and\nSupplementary Fig. 19d). In addition, ELISA analyses revealed that NPs, especially\nNPs-AS1411 treatment led to a significant increase in CCL3 levels and a concomitant\ndecrease in CXCL12 levels in the peritoneal lavage fluid of treated mice (Fig. 6o).\nImmunohistochemical staining showed a substantial increase in CD8⁺ T cells and\nCD86⁺ DCs infiltration within tumor tissues, with the most robust immune activation\nobserved in the NPs-AS1411 group (Fig. 6n and Supplementary Fig. 19e).\nCollectively, these findings indicated that both NPs and NPs-AS1411 effectively\nactivated antitumor immunity and exerted potent therapeutic effects against CRC in\nvivo.\nReviewer #5 (CRC therapy):\nThis manuscript details a study of nanoparticle-based therapy targeting CAFs to\ntreat colorectal cancer. There is a good level of analysis presented, with correlating\ndata in in vitro models and animal models, validated with patient-relevant\norganoids. The use of primary cells, mice and patient organoids is thorough and\nrepresents a large body of work and I can see how all of these orthogonal methods\nsupport the conclusions - there is a clear effect of the NPs demonstrated. That said,\nconsidering for publication.\n1. The authors use 2 normal fibroblast lines and 3 CAF lines (line 128). I realise\nlots of things can happen in preparing primary cells but why only 2 NFs? It falls\nshort of enabling statistical tests to be performed (although stats appear to be\nshown in eg figure 1c/1e - what are the experimental replicates here?). Having 3\npaired patient-derived samples would surely have been preferable.\nAuthor reply: We sincerely appreciate the reviewer’s insightful comment, which has\nhelped us further improve the quality of our manuscript. Indeed, from the perspectives\nof experimental reproducibility and statistical reliability, it is preferable to include at\nleast three NFs and three CAFs. As the reviewer pointed out, having three matched\npairs of NFs and CAFs and conducting experiments using one representative pair would\nbe the ideal approach. However, due to the inherent uncertainty of primary cell culture,\nthe successful isolation of both CAFs and their corresponding NFs from the same\npatient is often not achievable. In some cases, only CAFs could be obtained from tumor\ntissues, whereas NFs from adjacent normal tissues failed to grow, and vice versa.\nAt the initial stage of this study, we successfully established two NF lines and three\nCAF lines. To evaluate whether NPs exhibited differential cytotoxicity between NFs\nand CAFs, we performed cytotoxicity assays across all available lines. The results\nrevealed that CAFs were generally more sensitive to NPs-induced cytotoxicity, whereas\nNFs showed noticeable damage only at relatively high NPs concentrations. Based on\nthese findings, we randomly selected NF1 and CAF1 for the subsequent mechanistic\nexperiments. As the study progressed, we continued culturing primary cells and\nsuccessfully established additional NF and CAF lines. Repeated cytotoxicity assays\nusing these new lines yielded results consistent with our earlier observations.\nUnfortunately, due to our oversight, we did not update the western blot data that\noriginally included only two NFs and three CAFs. We sincerely thank the reviewer for\ncarefully identifying this error. In the figures below and Fig. 2b, we have included\nphase-contrast images showing the isolation and culture process of three NFs and three\nCAFs. In addition, we have re-performed the experiments and replaced the previous\nresults with protein expression analyses of marker genes across the three NFs and three\nCAFs.\nper group. One representative set of results is shown in the figures. We apologize for\nnot including this information in the former manuscript and have now provided these\ndetails in the revised version.\nThree NFs and CAFs for revised Fig. 2b. Phase-contrast images illustrating the\nisolation and culture process of three NFs and three CAFs, which were subsequently\nused for the analysis of CAF marker protein expression.\n2. The authors present treatments on HCT116, with the rationale that their\napproach might risk \"tumor stem cell dissemination\" (line 160). I can understand\nthe purpose here, but I think the data on HCT116 raises more questions than\nanswers. No data on stem cell populations or markers is shown, are there stem\ncell/quiescent cells within the cell population? Do these cells a) uptake the NPs or\nb) respond to the NPs? The authors show data on \"normal\" NCM460 cells in the\n24\nsupplementary data, my concern is that given that >30% of the cells remain viable\nafter treatment (figure 3a), this will contain any stem-cell like cells which are\nnotoriously resistant to therapy and likely have very different uptake through\nendocytosis (eg https://doi.org/10.1242/jcs.216804). There are some interesting\nexperiments that could be done here, but at very least I think the authors need to\nrevisit the rationale.\nAuthor reply: We greatly appreciate this constructive suggestion, which has helped us\nfurther improve the logical structure and rigor of our manuscript. We apologize for not\nclearly presenting the rationale for our therapeutic design of NPs targeting CAFs in\nCRC in the former manuscript, and for placing the extended discussion of the function\nof NPs within the description of the former Fig. 3 results.\nAs the reviewer correctly pointed out, the results in the former Fig. 3 demonstrate that\nNPs can effectively induce ferroptosis in CRC cells, and only extremely high\nconcentrations of NPs are capable of completely eliminating tumor cells. However,\nsuch high doses inevitably cause damage to normal colonic epithelial cells (NCM460).\nAlthough combining laser irradiation can enhance the therapeutic efficacy due to the\nhigher sensitivity of tumor tissue to hyperthermia treatment22, using NPs alone to\ndirectly kill tumor cells cannot ensure the complete eradication of stem cell-like tumor\ncells and may lead to potential drug resistance and safety concerns associated with high\nNPs doses.\nOur subsequent findings revealed that, although NPs can induce ferroptosis in both\nCAFs and CRC cells, the dose required to kill CAFs is significantly lower than that\nneeded for tumor cells. Moreover, NPs treatment of CAFs not only suppresses the\nsecretion of paracrine signals that promote malignant tumor phenotypes but also\npromotes the release of cytokines that effectively activate antitumor immunity. These\nfindings prompted us to focus our therapeutic strategy on developing an NPs-based\napproach targeting CAFs to activate antitumor immunity for CRC treatment.\nThe discussion in the former manuscript regarding “direct targeting CAFs to disrupt the\nphysical barrier within the TME might carry the risk of tumor stem cell dissemination”\nwas primarily based on a previous work suggesting that the direct depletion of CAFs\nby genetic ablation might paradoxically convert the TME into a pro-tumorigenic state\nand even increase the risk of tumor dissemination2. In contrast, our experimental results\nshowed that NPs-treated CAFs markedly inhibited the proliferation, migration, and\nuntreated CAFs, due to inhibition of Wnt and TGF-β signaling (Fig. 3a-d;\nSupplementary Fig. 8b-c, and Supplementary Fig. 8e). Additionally, NPs-treated CAFs\nwere found to activate antitumor immunity by releasing multiple immune-activating\nchemokines. Together with the intrinsic tumor-killing capacity of Cu@Fe O NPs,\nour\n3 4\nfindings suggest that the risk of tumor cell escape may be substantially reduced in this\nCAF-targeting nanotherapeutic strategy.\nWe realize that our former manuscript not only failed to highlight the intended rationale\nbut also included discussion-related content in an inappropriate section, which made\nthe overall logic unclear. In the revised manuscript, we have reorganized the logic of\nthe study to clearly present the rationale and therapeutic concept of NPs targeting CAFs\nin CRC. In addition, since the NP concentration used in our study was a low dose\nspecifically designed for CAF targeting, NP-induced ferroptosis in CRC tumor cells\nplays only an auxiliary role in this therapeutic strategy. To maintain logical consistency\nthroughout the manuscript, we have moved these data to the Supplementary\nInformation.\n3. The authors show increased immune response after treatment (figure 5). I was\nstruggling to separate effects on CAFs from possible direct effects on immune cells\nhowever. Wouldn't macrophages be critical uptakers of the NPs? And then\npotentially drivers of chemokine release/activation of the immune response? To\nme, this isn't really considered in the experimental design but I am happy to be\ncorrected. Since the authors have extensively analysed immune populations\n(supplementary data) it feels like there are some experiments that could clarify\nthis.\nAuthor reply: Thank you for such a thoughtful comment. As you correctly noted, the\nmononuclear phagocyte system, particularly macrophages, is widely recognized as the\nprincipal cellular group responsible for NPs uptake7-9. Although iron oxide NPs have\nbeen reported to reprogram macrophages toward a proinflammatory phenotype and\nenhance antigen presentation in certain contexts23, excessive macrophage uptake may\ninduce undesirable off-target effects, reduce targeting efficiency, and even impair\nmacrophage viability due to the intrinsic catalytic activity of iron oxide NPs. Therefore,\n26\nthe design of our NPs focused primarily on maximizing specificity and biosafety while\nminimizing off-target accumulation, especially macrophage uptake.\nStrategies to mitigate such off-target effects generally rely on two key approaches: (i)\noptimizing the physicochemical properties of the NPs, including size and surface\nchemistry, and (ii) incorporating an active targeting strategy. NPs larger than 100 nm\nare more susceptible to macrophage phagocytosis, whereas our Cu@Fe O NPs (~20\n3 4\nnm) preferentially accumulate in tumor tissues via the EPR effect (Supplementary Fig.\n3a-b). In addition, surface modification with DSPE-PEG-NH₂ generates a hydrated\nsteric layer that reduces protein adsorption and markedly decreases macrophage\nuptake7-13.\nFor the active targeting design, we aimed to maximize the therapeutic efficacy of NPs\nby simultaneously targeting CAFs and CRC cells while ensuring safety through the use\nof well-characterized cell surface markers. To this end, we integrated scRNA-seq data\nwith subcellular protein localization analyses to identify optimal surface-binding\ntargets from approximately 20 reported nano-drug surface markers. Among these,\nNCL emerged as the most suitable candidate, exhibiting the broadest distribution and highest\nexpression in CAFs (Fig. 5a-c and Supplementary Fig. 13). IF analysis further\nconfirmed that NCL exhibited strong plasma membrane localization in CAFs and tumor\ncells, minimal expression in macrophages, and was nearly undetectable in normal\nintestinal epithelial cells, NFs, and endothelial cells (Fig. 5d). Hence, AS1411 binding\nto NCL was selected for attachment to the surface of the NPs to enhance the specific\ntargeting. Moreover, in vivo uptake studies demonstrated distinct phagocytic\nefficiencies among different cell populations, further validating these observations.\nB\ncells, T cells, and endothelial cells hardly engulfed NPs and NPs-AS1411, while DCs\nand macrophages had minimal phagocytosis of NPs and NPs-AS1411. As expected,\nCAFs had the highest uptake of NPs, followed by tumor cells, and due to the targeting\nspecificity of AS1411, both CAFs and tumor cells had a more significant increase in\nphagocytosis of NPs-AS1411 (Fig. 5e-k and Supplementary Fig. 3a-i). Collectively,\nthese findings indicate that AS1411-NPs targeting NCL exhibit the highest specificity\nfor CAFs, moderate affinity for tumor cells, and minimal effects on NFs, normal\nepithelial cells, and endothelial cells, while effectively reducing macrophage uptake to\nenhance targeting precision and biosafety.\nimmunity was mainly demonstrated in vivo, we believe that the immune activation\nobserved in the TME primarily results from NPs-induced CAF depletion. This effect\nsuppresses pro-tumor paracrine signaling and promotes the secretion of\nimmunostimulatory chemokines.\n4. The authors show that cancer cells are not responsive to NPs alone in their PDX\nmodel (line 393). Why is this different from figure 3 in vitro? This links to point 2,\nhow representative are the HCT116 experiments?\nAuthor reply: We sincerely thank the reviewer for this insightful comment. Indeed,\nthis issue is similar to the second comment and mainly arose because we did not clearly\npresent the rationale for our therapeutic design of CAFs-targeting NPs in CRC in the\nformer manuscript. Given the multifaceted biological effects of the NPs, they exert\ncytotoxicity toward both CRC cells and CAFs. Notably, low doses are sufficient to\ninduce ferroptosis in CAFs, thereby suppressing pro-tumor paracrine signaling and\nenhancing antitumor immunity, whereas higher doses are required to effectively kill\nCRC cells. Considering drug biosafety, we therefore selected a low, CAF-targeting\ndose for treatment.\nPDX-derived tumor cell and CAF coculture experiments confirmed that this dose\neffectively eliminated CAFs but had limited direct cytotoxicity against CRC cells.\nHowever, when active targeting capability and laser irradiation were introduced, the\ncytotoxic effects of NPs on both CAFs and tumor cells were markedly enhanced\n(Fig.7e). Since the induction of ferroptosis in CRC tumor cells serves only as an\nauxiliary mechanism in this therapeutic strategy, we have moved these data to the\nSupplementary Information (Supplementary Fig. 5 and Supplementary Fig. 7) to\nmaintain the logical coherence of the manuscript.\nSome minor points on manuscript preparation:\n5. results information and links to figures do not belong in the introduction. The\nintroduction should provide context prior to discussing results, so I think links to\nfigures need removing from the introduction.\nthe content in the Introduction section that was related to the results and relocated it to\nthe appropriate Results section.\n6. there are some language issues, mainly minor but a proof-read is worthwhile eg\nin figure 1 \"cystine\", \"perxoidation\", line 279 \"presented the satisfactory\nsuppression\", line 282 \"was reasonably considered\" - not sure what is meant.\nAuthor reply: Thanks so much for your careful review. In the revised manuscript, we\nhave thoroughly proofread the entire text to correct typographical and language errors.\nWe apologize for any confusion caused by the previous wording.\n7. Figure 2m is a repeat of figure 1c, not sure why duplication of figure is needed.\nAuthor reply: We are grateful for your thoughtful reading and valuable feedback. You\nare absolutely right that the schematic illustration in Fig. 2m overlapped with Fig. 1c.\nIn the revised manuscript, we have removed the redundant Fig. 2m accordingly.\nWe hope our revision would help to improve the quality of the manuscript and\nwe thank again the reviewers for his/her comments and suggestions.\nWith best regards\nSincerely yours\nYanglong Hou and co-authors\nReference\n1. Lambert, A.W., Pattabiraman, D.R. & Weinberg, R.A. Emerging Biological Principles\nof Metastasis. Cell 168, 670-691 (2017).\n2. Ozdemir, B.C. et al. 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Nat Nanotechnol 11, 986-\n994 (2016).\n30"}, {"round": 2, "reviewer_comments": {"reviewer_2": "The authors improved the manuscript and addressed all the concerns.", "reviewer_3": "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.", "reviewer_4": "The authors provided additional experimental data, data analyses, and text clarifications and have adequately addressed all my questions and concerns.", "reviewer_5": "As outlined in my original review, there is interesting work in this manuscript that I would like to see published. I noticed that other reviewers had concerns that overlapped with my own, and overall my feeling with this revision is that the authors have addressed my comments in a reasonable way.\n- there is more rigour, with experimental replicates.\n- they have provided explanations for the questions around the results and interpretation.\n- they have re-organised the manuscript to avoid issues such as results in introduction, and I think the structure does have more logic now.\nAs such, I think this manuscript warrants publication. I am interested to see comments of the other reviewers."}, "author_response": ""}]

Decision letter