THBS1-producing tumor-infiltrating monocyte-like cells contribute to immunosuppression and metastasis in colorectal cancer

Domain: earth_science
Published: 2023-09-25
Editorial outcome: minor_revision

Abstract
Mesenchymal activation, characterized by dense stromal infiltration of immune and mesenchymal cells, fuels the aggressiveness of colorectal cancers (CRC), driving progression and metastasis. Targetable molecules in the tumor microenvironment (TME) need to be identified to improve the outcome in CRC patients with this aggressive phenotype. This study reports a positive link between high thrombospondin-1 (THBS1) expression and mesenchymal characteristics, immunosuppression, and unfavorable CRC prognosis. Bone marrow-derived monocyte-like cells recruited by CXCL12 are the primary source of THBS1, which contributes to the development of metastasis by inducing cytotoxic T-cell exhaustion and impairing vascularization. Furthermore, in orthotopically generated CRC models in male mice, THBS1 loss in the TME renders tumors partially sensitive to immune checkpoint inhibitors and anti-cancer drugs. Our study establishes THBS1 as a potential biomarker for identifying mesenchymal CRC and as a critical suppressor of antitumor immunity that contributes to the progression of this malignancy with a poor prognosis.

Introduction
Accumulating evidence has determined the critical role of stroma-infiltrating cells in the tumor microenvironment (TME) of colorectal cancer (CRC)1–4. CRC subtypes with mesenchymal phenotypes, consensus molecular subtype 4 (CMS4), exhibit aggressive behavior and poor prognosis with an immunosuppressive TME5–7. Although tumor-infiltrating myeloid cells constitute the primary population of immune components in the TME, targeting myeloid cells remains challenging because of the functional heterogeneity of their subsets8. Therefore, a deeper understanding of how myeloid cells contribute to the creation of a tumor-promoting microenvironment is essential for identifying rational combinatorial strategies against mesenchymal CRC.

Recent studies have revealed that CMS4 CRC have a higher expression of inflammation-related pathways, suggesting a strong association between inflammation and stromal activation5,6,9,10. Thrombospondin-1 (THBS1) is a matricellular protein that is highly expressed in inflammatory processes and has various functions, such as restraining angiogenesis and immune activity11,12. The anti-angiogenic effect of THBS1 is believed to contribute to tumor growth inhibition; however, the roles of THBS1 in carcinogenesis are multifaceted and contradictory13–15. To date, cell type-specific analyses of THBS1 function in intestinal tumorigenesis are lacking, and the contribution of THBS1 to CRC progression and metastasis remains unclear. Furthermore, the importance of THBS1 in regulating the immune landscape of TME remains to be uncovered.

In this work, we elucidate the cellular sources and functions of THBS1, particularly during the progression and metastasis of mesenchymal CRC, with a focus on the differences between the primary and metastatic sites. The combined effects of THBS1 loss and currently available chemotherapy and immune checkpoint inhibitors (ICI) are also determined. This study highlights the critical importance of bone marrow (BM)-derived monocyte lineages in enhancing tumor progression and provides evidence for the potential application of THBS1-targeted therapy as an effective treatment for aggressive CRC.

Results
Increased infiltration of THBS1-expressing cells in stroma correlates with aggressiveness of human CRC
Immunostaining of human CRC revealed that THBS1 was highly expressed in the stromal areas of both primary and metastatic lesions compared to the paracancerous tissues (Fig. 1a). The RNAscope for THBS1 transcript and co-immunofluorescence of THBS1 with epithelial marker EPCAM confirmed the localization of THBS1 in the stroma, which was supported by transcriptomic analysis showing higher THBS1 expression in the tumor stroma (Fig. 1b, c and Supplementary Fig. 1a). Analysis of the CRC dataset from the cancer genome atlas (TCGA) demonstrated a positive correlation between THBS1 and mesenchyme-related genes (VIM and FN1) and pan-myeloid markers (CD14 and CD33) (Supplementary Fig. 1b–e). THBS1 expression was highly enriched in mesenchymal CMS4 CRC and correlated with altered genes in this subtype (Fig. 1d, e). Recent advances in single-cell technology have identified distinct intrinsic epithelial signatures and defined the most aggressive fibrotic i3_MSS CRC (i3_MSS_F)16, in which THBS1 expression was highly enriched (Fig. 1d). THBS1-high CRC exhibited an advanced stage, increased metastases to distant organs and lymph nodes, and a poorer prognosis, indicating the aggressive nature of these CRC (Fig. 1f and Supplementary Fig. 1f–i).

To further determine its clinical relevance, we applied tissue microarrays (TMAs) of surgically resected CRC4. High THBS1 expression was positively correlated with extensive stromal reaction, as indicated by dense collagen deposition, enrichment of CMS4 subtype, and accumulation of myeloid marker (CD14, CD11b, and CD68)-positive cells (Fig. 1g–j and Supplementary Fig. 1j, k). We found that the THBS1-high group correlated with a more advanced stage and increased lymph node metastases compared to the THBS1-low group (Fig. 1k, l and Supplementary Fig. 1l). Furthermore, high THBS1 expression was independently associated with recurrence, lymph node metastasis positivity, undifferentiated pathology, and microsatellite stable (MSS) status in the multivariate analyses (Supplementary Fig. 1m and Supplementary Tables 1–4). The THBS1-high group exhibited the lowest overall, disease-specific, and recurrence-free survival rates (Fig. 1m, n). These results highlighted the stromal localization of THBS1 and demonstrated a strong association between high THBS1 levels, aggressive phenotypes, and poor prognosis in human CRC.

TME-derived THBS1 suppresses inflammation and immunosurveillance in aggressive murine CRC
To investigate the role of TME-derived THBS1 in aggressive CRC, we performed the orthotopic implantation of mouse tumor organoids (MTO) that had a CMS4-like mesenchymal phenotype, harboring mutations in Apc, Trp53, Kras, and Tgfbr2 in the mouse rectum17. The implanted MTO led to the development of primary rectal lesions, followed by lymph node and liver metastases in immunocompetent mice, thereby successfully mimicking human CRC progression (Supplementary Fig. 2a). THBS1 expression was upregulated in primary and metastatic lesions in MTO-inoculated mice, like that in human CRC, and its stroma-specific localization was confirmed by RNAscope and immunofluorescence analyses (Supplementary Fig. 2b–d). MTO-derived tumors exhibited enhanced stromal activation, as indicated by abundant collagen deposition, accompanied by increased THBS1 expression, in contrast to tumors derived from MC38, a widely used mouse colon tumor cell line resembling immunogenic CRC (Supplementary Fig. 2e–g). This suggests that the high expression of THBS1 can be attributed to the mesenchymal phenotype of MTO-derived tumors.

We next analyzed orthotopic MTO tumors in Thbs1−/− mice (Fig. 2a). This system enabled selective evaluation of the effect of THBS1 loss in non-epithelial cells of the TME. We observed a sharp reduction in THBS1 expression in tumors in Thbs1−/− mice (Fig. 2b and Supplementary Fig. 2h, i), consistent with the stroma-specific enrichment of THBS1. Although there was no significant difference in the size of primary lesions between the genotypes, tumors in Thbs1−/− mice displayed extensive necrotic areas (Fig. 2b and Supplementary Fig. 2i, j). Consistent with this, immunostaining revealed a substantial increase in cleaved caspase-3 (C-Cas3)+ apoptotic cells in tumors in Thbs1−/− mice, which was reinforced by RNA sequencing (RNA-seq) analyses of these tumors, showing increased apoptotic pathways (Fig. 2b, c and Supplementary Fig. 2i, k). Consistent with the immunosuppressive function of THBS112, RNA-seq analyses of tumors in Thbs1−/− mice also showed an upregulation of inflammatory and active immune responses (Fig. 2c). In addition, consistent with the anti-angiogenic properties of THBS1, tumors in Thbs1−/− mice demonstrated increased CD31+ vascularity and angiogenesis-related signatures (Fig. 2d and Supplementary Fig. 2l, m). Collectively, these results highlight that THBS1 contributes to the development of an immunosuppressive and hypovascular TME.

Loss of THBS1 blocks T cell exhaustion and increases stem-like CD8+ T cells
To characterize the immune landscape governed by THBS1, we performed fluorescence-activated cell sorting (FACS) and immunohistochemical analyses of orthotopic MTO tumors. We observed increased infiltration of CD8+ T cells in tumors in Thbs1-/- mice, although there was no significant change in CD4+ T cells or FOXP3+ regulatory T cells (Treg) (Fig. 2e, f and Supplementary Fig. 2n, o). Because THBS1 has been shown to have a potential regulatory effect on myeloid lineages18, we examined myeloid cell populations including dendritic cells (DC), monocytes, neutrophils, and tumor-associated macrophages (TAMs) (Supplementary Fig. 2p); however, we observed no change in these populations. Myeloid-derived suppressor cells (MDSCs), including monocytic MDSCs (MO-MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs), also did not show any changes (Supplementary Fig. 2q). Consistent with this, qRT-PCR confirmed that there was no significant change in the expression of myeloid-derived immunosuppressive genes such as Il10, Arg1, and Mrc1 (Supplementary Fig. 2r). These results suggest the existence of a direct mechanism by which THBS1 regulates CD8+ T cell activity. To test this, we performed single-cell RNA-seq (scRNA-seq) on orthotopic MTO tumors. There were no major changes in the myeloid cell subsets and myeloid-related immunosuppressive gene expression such as M2 macrophage-related signature (Supplementary Fig. 3a–f). We further characterized the CD8+ T cell population in these tumors (Fig. 2g and Supplementary Fig. 3g). Analysis of orthotopic tumors in wild-type (WT) mice revealed that mT2_Exhausted CD8 cells were the most abundant population (Fig. 2g, h). In contrast, mT1_Stem-like CD8 cells were the most enriched in tumors in Thbs1-/- mice, comprising more than 60% of all the infiltrating CD8+ T cells (Fig. 2g, h). Consistent with previous reports, mT1_Stem-like CD8 cells displayed an upregulation in genes related to active T cell functions such as stemness, memory, migration, and anti-apoptosis and a downregulation of the dysfunctional signature (Fig. 2i–k)19–21. In contrast, mT2_Exhausted CD8 cells were enriched in genes related to the dysfunctional state, including apoptosis, T cell receptor (TCR) signaling, and exhaustion (Fig. 2j, k). Furthermore, FACS analysis demonstrated decreased CD8+ T cells expressing the dysfunctional marker CTLA4 in tumors in Thbs1-/- mice (Supplementary Fig. 3h). These data highlighted the role of TME-derived THBS1 in inducing the transition from an active stem-like state to dysfunction of CD8+ T cells. This is confirmed by the downregulation of TCF7, a stem-like T cell marker, in THBS1-high human CRC (Fig. 2l).

To examine the underlying mechanism by which THBS1 regulates T cell activity, we stimulated T cells isolated from the spleen with anti-CD3/CD28 antibodies in the presence of recombinant THBS1 (Fig. 2m). Consistent with bulk RNA-seq data, showing enhanced IFNγ signaling in tumors in Thbs1-/- mice (Fig. 2c and Supplementary Fig. 3i), THBS1 treatment suppressed anti-CD3/CD28-mediated T cell activation in a concentration-dependent manner as indicated by decreased proportions of CD69+, IFNγ+, and TCF7+ CD8+ T cells (Fig. 2n). Furthermore, THBS1 treatment increased the number of CTLA4+ CD8+ T cells, although it did not significantly alter PDCD1+ CD8+ T cells (Fig. 2o), which was consistent with the FACS results (Supplementary Fig. 3h). Immunostaining revealed an increase in TCF7+ CD8+ T cells and a decrease in CTLA4+ CD8+ T cells in tumors in Thbs1-/- mice (Fig. 2p). These findings are consistent with a previous study that found suppressive activity of THBS1 on TCR-mediated T cell activation22. The strong suppression of ex vivo T cell activation by THBS1 seems at odds with the enrichment for exhausted/dysfunctional CD8+ T cells, which is generally associated with chronic TCR activation, in THBS1-high orthotopic MTO tumors. This discrepancy is probably attributed to the strong and acute activation of the ex vivo protocol compared to the more long-term and likely lower-level TCR triggering by endogenous antigens in vivo. To test whether THBS1 functionally attenuates T cell cytotoxicity, we performed a killing assay by co-culturing CFSE-labeled OVA-expressing MTO and OVA53 T cells in the presence of THBS123. Notably, THBS1 treatment suppressed the cytotoxic activity of T cells under all conditions tested (Fig. 2q). The TMA analyses demonstrated a negative correlation between THBS1 expression and CD8+ T cell infiltration in human CRC (Supplementary Fig. 3j). These results demonstrate that TME-derived THBS1 contributes to immune evasion by directly suppressing T-cell activation and inducing dysfunction.

Loss of TME-derived THBS1 suppresses metastasis
Given the role of cytotoxic T cells in preventing tumor progression, we investigated whether THBS1-deficiency in the TME suppresses the metastasis of mesenchymal CRC. MTO-bearing WT mice developed multiple metastases in the liver and lymph nodes (CK19+) (Fig. 3a, b and Supplementary Fig. 4a). In contrast, Thbs1-/- mice inoculated with MTO demonstrated a sharp decrease in metastatic burden and prolonged overall survival (Fig. 3a–d, Supplementary Fig. 4a). We hypothesized that the increased infiltration of active CD8+ T cells may account for the reduction in metastasis in Thbs1-/- mice. Anti-CD8 antibody treatment effectively depleted CD8+ T cells and concomitantly decreased the number of apoptotic cells in primary tumors (Fig. 3e, f), confirming the cytotoxic activity of infiltrating CD8+ T cells. Notably, CD8+ T cell depletion restored metastases in MTO-bearing Thbs1-/- mice (Fig. 3g, h). To further clarify the impact of THBS1 loss on metastasis, we injected MTO into the spleen of WT and Thbs1-/- mice and evaluated liver metastasis (Fig. 3i). Similar to the results of orthotopic implantation, Thbs1-/- mice demonstrated decreased metastasis, which reverted to WT levels when CD8+ T cells were depleted (Fig. 3j, k). These data suggest that TME-derived THBS1 plays a pivotal role in enhancing the metastasis of intestinal tumors with a mesenchymal phenotype. Although immunostaining and transcriptomic analyses demonstrated the localization of THBS1 in the tumor mesenchyme, as described above, orthotopic tumors in Thbs1-/- mice showed low levels of THBS1 (Supplementary Fig. 2h). However, we observed that Thbs1-knockdown MTO showed no significant difference in growth and metastatic formation in the splenic injection model (Supplementary Fig. 4b–g), suggesting no obvious contribution of tumor epithelium-derived THBS1 to tumor progression. Taken together, these findings indicate that TME-derived THBS1 plays a critical role in promoting CRC metastasis by restraining antitumor immunity.

Higher antitumor immune activity in metastasis of Thbs1-/- mice
Metastasis was strongly suppressed in Thbs1-/- mice, whereas the primary tumor was not affected despite increased cell death in these mice, prompting us to compare the immune TME at primary and metastatic sites. We found no difference in the infiltration of THBS1-expressing cells between primary tumors and liver metastases in WT mice (Fig. 3l). However, increased infiltration of CD8+ T cells was more evident in metastasis in Thbs1-/- mice (Fig. 3m and Supplementary Fig. 5a), suggesting a stronger induction of antitumor immunity in metastasis by THBS1-deficiency. FACS analyses of WT tumors demonstrated that most T lymphocytes, including CD8+ T cells, CD4+ T cells, and Tregs, and myeloid populations, including DCs, TAMs, monocytes, and neutrophils, were similar in both lesions (Fig. 3n, o). Notably, we observed a significantly lower number of PMN-MDSCs in metastases than in primary sites, whereas the number of MO-MDSCs was comparable between lesions (Fig. 3p). Because the low incidence and small size of metastasis in MTO-bearing Thbs1-/- mice hampered sufficient sample collection from metastasis for FACS analyses, we performed immunofluorescence to determine the immune TME of metastasis in WT and Thbs1-/- mice, which revealed no significant change in monocytic lineages and neutrophils (Supplementary Fig. 5b, c); however, the number of PMN-MDSCs in metastasis was significantly lower in both genotypes (Fig. 3q and Supplementary Fig. 5d). Based on these findings, we hypothesized that the change in the proportion of PMN-MDSCs might account for the difference in the degree of antitumor immunity between lesions. To directly examine the suppressive function of PMN-MDSCs and MO-MDSCs in WT and Thbs1-/- tumors, we isolated MDSCs using a MACS-based MDSC isolation kit and co-cultured them with T cells stimulated with anti-CD3/CD28 antibodies (Supplementary Fig. 5e). Both PMN- and MO-MDSCs from WT mice showed reduced numbers of CD69+ active CD8+ T cells and increased numbers of CTLA4+ exhausted cells (Fig. 3r), confirming their immunosuppressive functions. Notably, while PMN-MDSCs from Thbs1-/- mice exhibited no change in suppressive activity, this activity was abolished in MO-MDSCs from Thbs1-/- mice (Fig. 3r), suggesting that THBS1 mainly contributes to the function of monocytic lineages. Together with the lower accumulation of PMN-MDSCs in metastasis, these results suggest that the THBS1-mediated immunosuppressive function of the monocytic lineage is more critical for metastasis formation and explain why the impact of THBS1 loss is more evident in metastasis. In support of this notion, an increase in the infiltration of active TCF7+ CD8+ cells was more pronounced in metastases than in primary tumors of Thbs1-/- mice (Fig. 3s and Supplementary Fig. 5f).

THBS1-CD47 and THBS1-CD36 axes contribute to metastasis development
Although THBS1 activates latent TGFβ in physiological state22,24–28, analyses of RNA-seq data revealed an increased enrichment of TGFβ signaling in tumors in Thbs1-/- mice (Supplementary Fig. 6a, b). Furthermore, these tumors did not show a reduction in the levels of phospho-SMAD3, a representative downstream mediator of TGFβ signaling, and cancer-associated fibroblast (CAF) marker αSMA (Supplementary Fig. 6c, d). To test whether THBS1 targets CD47 or CD36 to generate an immunosuppressive TME, we used Cd47-/- and Cd36-/- mice for orthotopic implantation of MTO (Fig. 4a). Tumors in Cd47-/- and Cd36-/- mice demonstrated increased CD8+ cell infiltration and apoptosis compared to those in WT mice, although the extent of the increase was less than that in tumors in Thbs1-/- mice (Fig. 4b, c). Reflecting the difference in CD8+ T cell infiltration, MTO-bearing Cd47-/- and Cd36-/- mice showed reduced liver metastases, although to a slightly lesser extent than MTO-bearing Thbs1-/- mice (Fig. 4d, e). Furthermore, reduced levels of lymph node metastases were observed in Cd47-/- mice, like those observed in Thbs1-/- mice, but not in Cd36-/- mice (Fig. 4e). Enhanced vascularization was observed only in tumors in Cd36-/- and Thbs1-/- mice (Fig. 4b, c), suggesting that the inhibition of angiogenesis was predominantly attributed to the action of THBS1-CD36. A previous report demonstrated that THBS1-CD47 signaling directly inhibits TCR-mediated T cell activation22, while the THBS1-CD36 axis has been shown to reduce the inflammatory response by promoting IL10 secretion in myeloid cells18. However, IL10 levels were comparable between tumors in WT and Cd36-/- mice (Fig. 4f). A previous report has shown that CD36 signaling in intratumoral CD8+ T cells attenuates the function of these cells29. Considering that THBS1 directly restricts CD3/CD28-mediated T cell activation (Fig. 2n, o), we hypothesized that both THBS1-CD47 and THBS1-CD36 inhibit antitumor immunity via a direct mechanism in CD8+ T cells. Consistent with this notion, a substantial infiltration of CD47+ CD8+ and CD36+ CD8+ T cells was observed in tumors in WT mice (Fig. 4g). To examine the impact of CD47 or CD36 deficiency on CD8+ T cells, we stimulated T cells isolated from Cd47-/- and Cd36-/- mice with anti-CD3/CD28 antibodies in the presence of THBS1 (Supplementary Fig. 6e, f). Notably, THBS1-mediated inhibitory effect was abolished in both CD47-/- CD8+ T cells and CD36-/- CD8+ T cells (Fig. 4h, i), suggesting that THBS1 suppresses T cell activity through binding with CD47 and CD36 on CD8+ T cells. To validate the role of THBS1-CD36 in restricting vascularization, we performed a tube formation assay using mouse C166 endothelial cells. THBS1 dampened the tube-forming activity of C166, which reverted to normal levels when C166 was co-treated with sulfosuccinimidyl oleate, an irreversible inhibitor of CD36, but not with the anti-CD47 antibody (Fig. 4j). These data indicated the critical role of the THBS1-CD47 and THBS1-CD36 axes in establishing an immunosuppressive microenvironment essential for tumor metastasis. Independent of its immunomodulatory function, THBS1 confers hypovascular characteristics to tumors by interacting with CD36.

Tumor-infiltrating monocyte-like cells are primary producers of THBS1
Next, we aimed to determine the cellular source of THBS1 in the TME of CRC. Co-immunofluorescence revealed a strong colocalization of THBS1 and CD11b in orthotopic MTO tumors (Fig. 5a). To elucidate the role of THBS1 produced by myeloid cells, we used LysM-Cre;Thbs1f/f (LysM;Thbs1Δ/Δ) mice, in which Thbs1 was selectively deleted from lysozyme M (LysM)-expressing myeloid cells for orthotopic implantation (Fig. 5b). Analysis of LysM-Cre;Rosa26-LSL-EYFP (LysM;EYFP) mice, in which LysM-expressing cells are labeled with yellow fluorescent protein (YFP), confirmed that THBS1 was expressed primarily by YFP+ monocyte/macrophage lineages (Fig. 5c). Tumors in LysM;Thbs1Δ/Δ mice exhibited a substantial reduction in THBS1 expression (Fig. 5d and Supplementary Fig. 2h). However, we observed that Thbs1-knockdown MTO showed no significant difference in growth and metastatic formation in the splenic injection model (Supplementary Fig. 4b–g), suggesting no obvious contribution of tumor epithelium-derived THBS1 to tumor progression. Taken together, these findings indicate that TME-derived THBS1 plays a critical role in promoting CRC metastasis by restraining antitumor immunity.

THBS1-expressing monocyte-like cells are derived from BM
To demonstrate that THBS1-expressing monocyte-like cells were recruited from the BM, we compared the BM of MTO-bearing mice with that of untreated healthy mice and found increased expression of Thbs1 and Cd11b in the BM cells of MTO-bearing mice (Fig. 6a). FACS analysis revealed an increase in the number of CD11b+ cells in the BM and peripheral blood of MTO-bearing mice (Fig. 6b). Furthermore, CD45+CD11b+ cells in the BM and peripheral blood demonstrated the highest Thbs1 expression (Fig. 6c). Notably, patients with CRC exhibited higher levels of THBS1 in blood serum and primary CRC than those with non-cancerous lesions (Supplementary Fig. 8a, b). These results suggest that tumor formation at the primary site induces the BM to produce THBS1-expressing myeloid lineages that further migrate to the primary lesion. To demonstrate this, we transplanted BM from green fluorescent protein (GFP)-transgenic mice into WT mice and generated GFP-BM chimeric mice for MTO implantation (Supplementary Fig. 8c). EPCAM-GFP+ BM-derived cells, that accounted for 21.2±5.1% of the total cell population in tumors (Supplementary Fig. 8d), had the highest expression of Thbs1 and Vcan compared to that in EPCAM+ tumor epithelium or EPCAM-GFP- residential stromal cells and also compared to that in MTO or MC38 cells (Fig. 6d, e and Supplementary Fig. 8e). Furthermore, orthotopic tumors in GFP-BM chimeric mice showed strong co-localization of THBS1 and GFP (Fig. 6f). To examine the impact of THBS1 restoration on the tumor-suppressive phenotypes of Thbs1-/- mice, WT-BM was transplanted into Thbs1-/- mice to generate WT-BM Thbs1-/- chimeric mice, followed by MTO implantation (Supplementary Fig. 8f). Notably, orthotopic tumors in WT-BM Thbs1-/- chimeric mice exhibited THBS1 levels equivalent to those in the WT control (Fig. 6g and Supplementary Fig. 8g). In addition, the reduced metastases and increased positivity for CD31, CD8, and C-Cas3 observed in the primary tumors of Thbs1-/- mice reverted to WT levels after transplantation of WT-BM (Fig. 6h–j and Supplementary Fig. 8g). Together with the scRNA-seq results, these data confirmed that BM-derived monocyte-like cells are the primary producers of THBS1, which generates an immunosuppressive TME in mesenchymal tumors; this is also consistent with the contribution of THBS1 to the function of MO-MDSC (Fig. 3r).

CXCL12-CXCR4 axis is critical for recruitment of THBS1-expressing cells
Aggressive CRC with a mesenchymal phenotype is characterized by a high density of CAFs, which release an array of chemokines depending on the epithelial subtype6,16,32,33. Given that BM cells in mice with mesenchymal MTO tumors demonstrated higher Thbs1 expression than those in mice with immunogenic MC38 tumors (Fig. 6k), we sought to identify the CAF-derived factors that recruit THBS1-expressing cells from the BM. Similar to a previous report, TCGA analysis identified several chemokines specifically upregulated in CMS4 CRC (Fig. 6l and Supplementary Fig. 8h, i)6. Among them, CXCL12 and CCL2 are potent myeloid attractants expressed by CAFs6,32,33. Cxcl12 expression was enriched in MTO-derived tumors, whereas that of Ccl2 was enriched in MC38-derived tumors (Fig. 6m). In addition, THBS1-high human CRC showed prominent expression of CXCL12 and its principal receptor, CXCR4 (Fig. 6n). These data suggest that CXCL12 is responsible for the recruitment of THBS1-expressing monocyte-like cells. Consistent with this, we observed the highest Thbs1 expression in CD45+CD11b+CXCR4+ cells in the BM of MTO-bearing mice (Fig. 6o). Furthermore, consistent with the role of monocyte-like cells as primary producers of THBS1, FACS analyses identified CD45+CD11b+CXCR4+Ly6C+CCR2+ cells as the dominant population for THBS1 expression in both primary and metastatic sites, which was supported by the colocalization of Ly6C, CXCR4, and THBS1 in tumors (Fig. 6p, q and Supplementary Fig. 8j). To further confirm this in vivo, MTO-bearing WT and GFP-BM chimeric mice were administered LIT-927, a selective neutraligand of CXCL12 (Fig. 6r). This treatment impaired the recruitment of GFP+ cells and reduced THBS1 levels in the primary tumors of MTO-bearing GFP-BM chimeric mice (Fig. 6s and Supplementary Fig. 8k, l). Consistent with the results in Thbs1-/- mice, CXCL12 inhibition suppressed the metastatic burden in MTO-bearing mice with enhanced vascular formation, CD8+ cell infiltration, and apoptosis (Fig. 6t, u and Supplementary Fig. 8k, l). Analysis of the SMC dataset revealed fibroblasts as the primary cluster expressing CXCL12 in CRC (Supplementary Fig. 8m). Taken together, these results indicate that the primary lesion stimulates the BM to enhance the recruitment of THBS1-expressing cells via the action of CXCL12 produced by CAF, highlighting the interplay between CAF and myeloid signatures in mesenchymal CRC.

THBS1 loss partially improves aggressive CRC response to current therapies
Currently, the beneficial effects of ICI are limited to microsatellite instability-high (MSI-H) CRC5,34. Consistent with the aggressive characteristics of THBS1-high CRC, analyses of TCGA and TMA revealed increased THBS1 expression in MSS CRC (Fig. 7a, b and Supplementary Fig. 1m). In contrast, anti-angiogenic therapy targeting vascular endothelial growth factor (VEGF) or its receptor (VEGFR) is an essential part of the current armamentarium. Given that the absence of THBS1 altered the TME from immunosuppressive to antitumor and induced hypervascularity (Fig. 2), we questioned whether these microenvironmental alterations induce therapeutic vulnerability to these treatments. To this end, Thbs1-/- mice were treated with an anti-PD1 antibody or anti-VEGFR2 antibody following MTO implantation (Fig. 7c). Both treatments partially reduced primary tumor size with sustained reductions of liver and lymph node metastases in Thbs1-/- mice, but not in WT mice (Fig. 7c–h). ICI treatment enhanced CD8+ T cell infiltration and cell death, whereas VEGF inhibition enhanced apoptosis and reduced angiogenesis in primary tumors of Thbs1-/- mice (Supplementary Fig. 9a–c).

Increased tomato lectin perfusion in tumors in Thbs1-/- mice suggested that the enhanced blood vessels induced by THBS1-deficiency were functional (Fig. 7i, j), implying the possibility of improved drug uptake. Therefore, we examined the combined effect of THBS1 inhibition and CRC chemotherapy, including folinic acid, fluorouracil, and oxaliplatin (FOLFOX) (Fig. 7k). FOLFOX treatment reduced primary tumor size in both WT and Thbs1-/- mice, but metastases were significantly suppressed in Thbs1-/- mice (Fig. 7k–p). Immunostaining revealed the most pronounced apoptosis in FOLFOX-treated Thbs1-/- mice (Supplementary Fig. 9d). Collectively, these results demonstrate that the loss of THBS1 results in a partially increased susceptibility of aggressive CRC to currently available treatments.

Methods
Humansamplesandethicsapproval
Paraffin-embeddedtissuesectionsofCRCandadenomawereobtainedfromsurgicallyresectedCRCsandendoscopicallyresectedadenomas.Serum samples were obtained from 37 patients (sex, male: n=19, female: n=18; age, 36–91 (median=72); Stage, 0: n=5, 1: n=12, 2: n=8, 3: n=12) with CRCs and 19 patients with benign tumors(sex, male: n=15, female: n=4; age, 57–89 (median=72); histology, adenoma: n=15, sessile serrated lesion: n=4). None of the patients had undergone preoperative radiation or chemotherapy. Written informed consent was obtained from above patients with the protocol approved by the Ethics Committee of Kyoto University Graduate School and Faculty of Medicine. TMAs of 331 surgically resected human CRC samples (sex, male: n=169, female: n=162; age: 21–96 (median=68)) at Osaka Metropolitan University Hospital were applied for survival analyses, univariable and multivariable analyses. The Ethics Committee of Kyoto University and Osaka Metropolitan University approved the use of patient TMA samples for this experiment without requiring written informed consent. Informed consent was obtained in the form of opt-out on the website. Baseline characteristics are shown in Supplementary Fig. 1l. Participant compensation was not provided for both cohorts. Among these TMA samples, 118 CRCs were previously annotated to CMS4 (n=30) or non-CMS4 (CMS1–3) by immunostaining-based CMS classifier4. CRC samples in TMA were classified into THBS1-low (n=102), -intermediate (n=122) and -high (n=107) according to staining intensity of THBS1. To evaluate fibrosis, Masson’s trichrome staining (Sigma-Aldrich) was performed. CD14, CD11b, CD68 and CD8 were stained in 164 CRC samples in the TMA. Opal multiplex IHC (NEL810001KT; Akoya) was performed to assess the loss of the mismatch repair (MMR) proteins, MLH1, PMS2, MSH2, and MSH6 in TMA (n=301) using goat anti-mouse IgG1 (1:500, PA1-74421; Thermo Fisher) as a secondary antibody. Non-detection of nuclear staining of any of the MMR proteins in the tumor cells was interpreted as MMR-deficient (MSI-H) (MSI-H, n=97; MSS, n=204). The adjacent stromal cells were used as internal controls. The following primary antibodies were used for immunohistochemical analyses: THBS1 (1:500, MA5-13398; Thermo Fisher), CD14 (1:500, 75181; Cell Signaling), CD11b (1:1000, ab133357; Abcam), CD68 (1:500, 76437; Cell Signaling), CD8 (1:200, 85336; Cell Signaling), EPCAM (1:100, ab71916; Abcam) and NLRP3 (1:100, NBP2-12446; Novus Bio), MLH1 (1:25, 47954; Cell Signaling), PMS2 (1:100, 556415; BD Biosciences), MSH2 (1:100, NA27; Calbiochem), MSH6 (1:100, 610919; BD Biosciences). RNAscope for THBS1 (probe no. 426581; ACD) were performed in primary tumor site and metastatic liver tumor site of human CRC sample. All images were collected from each stained slide on a BZ-X710 microscope (KEYENCE). The number of whole cells and positively stained cells in IHC images were measured using the QuPath v.0.3.247, and the whole area and positively stained area in IF images were measured by ImageJ/Fiji software v.2.3.0/1.53f48.

Mice
Thbs1-deficient(Thbs1-/-(#006141)), LysM-Cre (#004781) and Rosa26-EYFP (#006148) mice were purchased from Jackson Laboratories. C57BL/6 mice, GFP transgenic mice (C57BL/6-Tg (CAG-EGFP)) mice were purchased from Japan SLC, Inc. Systemic CD47-deficient (CD47-/-) (CD47f/f; E2A-Cre) mice were generated by crossing CD47f/f mice with E2A-Cre mice49. Thbs1f/f mice were generated as previously described39. CD36-deficient (CD36-/-) mice were kindly provided by Dr. Freeman (Harvard Medical School). All mouse strains were generated in a C57BL/6 background and were born and maintained under pathogen-free conditions. These mice were maintained in 14 hr light/10 h dark cycle, and the housing temperature and humidity were maintained were 24 °C and 50%, respectively. Animal handling and experimental procedures conformed to institutional guidelines and were approved by the animal research committee of Kyoto University (Kyoto, Japan) and performed in accordance with Japanese government regulations. 8–10 weeks old male mice were used in all experiments.

Generation of bone marrow (BM) chimeric mice
For the generation of GFP-BM chimeric mice, GFP transgenic mice were euthanized, and their BM cells were collected from their femur and humerus. Collected BM cells were admixed with the Red Blood Cell Lysis Solution (130-094-183; Miltenyi Biotec) for depletion of red blood cells, and diluted in HBSS. Recipient mice (C57BL/6 or Thbs1-/-) were exposed to 10 Gy total body irradiation (TBI) and intravenously injected the collected donor BM cells via tail vein. For the generation of WT-BM Thbs1-/- chimeric mice, BM cells were collected from C57BL/6 mice and transplanted to Thbs1-/- mice. The recipient mice were bred with 2mg/ml neomycin-supplemented drinking water for 6 weeks after BM transplantation, followed by orthotopic implantation.

Cell and organoid culture
Mouse tumor organoids (MTO: kindly provided by Dr. Batlle’s laboratory) were cultured in advanced DMEM supplemented with 1% penicillin-streptomycin, Glutamax (gibco), N-2 Supplement (gibco) and B-27 Supplement (gibco)17. MC38 cells (kindly provided by Dr. Honjo’s laboratory) were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. C166 cells were purchased from ATCC (CRL-2581) and cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. OVA53 cells (kindly provided by Dr. Takehito Sato) were cultured in RPMI supplemented with 10% FBS and 1% penicillin-streptomycin23. Isolated CD3+ T cells and PMN-/MO-MDSC were cultured in RPMI supplemented with 10% FBS and 1% penicillin-streptomycin.

Orthotopic implantation of organoids to murine rectum
Eight to ten weeks old mice were anaesthetized by isoflurane inhalation and placed in a supine position. Rectal mucosae were exposed by insertion of two blunt-ended hemostats into the anus. A 20μl cell suspension of 105 cells of MTO or MC38 admixed with matrigel (Corning) was directly injected into the colonic mucosae. Mice implanted with MTOs were euthanized 8–9 weeks after injection. Mice implanted with MC38 were euthanized 5 weeks after injection because the MC38-bearing mice died at around 5 weeks after implantation. Orthotopic primary lesions, liver and lymph node metastases were scored macroscopically as well as histologically. The maximal tumor burden permitted by the ethics committee was 10% of body weight. We ensured that each time mice were sacrificed, the maximal tumor burden did not exceed this limit.

Splenic injection of organoids
Eight to ten weeks old mice were anaesthetized by isoflurane inhalation and placed in a right lateral decubitus position. A 100ml cell suspension of 105 cells of MTO was injected into spleen, followed by partial splenectomy of injected lesion. Mice were euthanized 4 weeks after injection and liver metastasis were scored macroscopically.

Bioluminescence imaging
Bioluminescence imaging was used to track luciferase expression of orthotopically implanted MTO. For ex vivo imaging, primary tumor, liver and lymph node were resected from tumor-bearing mice 2 min after intravenous injection of luciferin (5mg/mouse; 88294, Thermo Scientific) and imaged using IVIS Lumina II (PerkinElmer). For in vivo imaging, mice were imaged using IVIS Lumina II thirty minutes after intraperitoneal injection of luciferin (5mg/mouse; 88294, Thermo Scientific).

BM and peripheral blood cells collection
Non-treated healthy mice or mice orthotopically implanted with MTO or MC38 were anaesthetized by isoflurane inhalation and their blood was collected from abdominal aorta. Collected blood was immediately mixed with 100U heparin (FUJIFILM Wako). After the mice were euthanized, BM cells were collected from femur and humerus. Collected blood or BM were admixed with the Red Blood Cell Lysis Solution (130-094-183; Miltenyi Biotec). Total RNA was extracted from whole blood cells, BM cells or FACS-sorted cells (CD45+CD11b+CXCR4+, CD45+CD11b+CXCR4-, CD45+CD11b-, and CD45- cells) using the RNeasy Mini Kit (QIAGEN), followed by qRT-PCR analyses (Supplementary Fig. 10e). The number of CD45+CD11b+ cells was counted by flow cytometry by a FACSAria II (BD Biosciences). The following primary antibodies were used for flow cytometry: CD45 (1:70, 552848; BD Biosciences), CD11b (1:70, 553310; BD Biosciences) and CXCR4 (1:70, 146511; Biolegend).

In vivo treatments
MTOs were orthotopically implanted in the rectum or injected to the spleen of 8–10 weeks old C57BL/6 and Thbs1-/- mice. For CD8+ T cell depletion, mice were intraperitoneally injected with αCD8 antibody (200μg; BE0061, BioXcell) or control IgG (200μg; BE0090, BioXcell) twice per week from 5 weeks to 8 weeks after orthotopic implantation of MTOs in the rectum or from 0 weeks to 4 weeks after splenic injection of MTOs. For CXCL12 inhibition, mice were intraperitoneally injected with LIT-927 (25mg/kg; S8813, Selleck) three times per week for 8 weeks after orthotopic implantation of MTOs. For neutralizing antibody treatment, mice were treated with intraperitoneal injection of αPD-1 antibody (200μg; BE0146, BioXcell), DC101 (αVEGFR2 antibody (800μg; BE0060, BioXcell)) or control IgG (200μg; BE0089, BioXcell) twice per week for 3 weeks starting at 5 weeks after implantation of MTOs. For FOLFOX treatment, mice were treated sequentially with intraperitoneal injection of oxaliplatin (6mg/kg; HY-17371, MedChem Express), followed by 5-FU (50mg/kg; F6627, Sigma-Aldrich) and levoleucovorin (90mg/kg; HY-13667, MedChem Express) on a weekly basis for 3 weeks as previously described50. In the treatment experiments (αPD-1 antibody, αVEGFR2 antibody, and FOLFOX), the mice with less than 7mm or more than 12mm in tumor diameter at the beginning of drug treatment were defined as outliers and removed from the further analyses. The change in diameter of primary tumors from the beginning to the end of treatment was measured.

In vitro stimulation of T cells
To check the mechanism of THBS1 to regulate T cell activity, splenocytes were collected from C57BL/6 or Cd47-/- or Cd36-/- mouse with the Tumor Dissociation Kit (130-096-730; Miltenyi Biotec) and were admixed with the Red Blood Cell Lysis Solution (130-094-183; Miltenyi Biotec). CD3+ T cells were sorted from the collected splenocytes by MACS using CD3ε MicroBead Kit (130-094-973; Miltenyi Biotec). Sorted CD3+ T cells were seeded at 105 cells/well with the presence or absence of recombinant THBS1 (12.5μg/ml, 25μg/ml or 62.5μg/ml for WT T cell, 25μg/ml for Cd47-/- or Cd36-/- T cell; 7859-TH-050, R&D systems) simultaneously stimulated using anti-CD3/CD28 antibodies (11452D; Thermo Fisher) in 96 well plate. For analysis of IFNg activity, protein transport inhibitor (554724; BD Biosciences) was added when T cells were seeded in the plate. After 24 h of seeding, CD3+ T cells were collected and stained with following antibodies, fixed with Cytofix (554714; BD Biosciences), followed by flow cytometry by a FACSAria II (BD Biosciences). Active CD8+ T cells were selected as CD3+CD8+CD69+, CD3+CD8+IFNg+ or CD3+CD8+TCF7+ and inactive CD8+ T cell were selected as CD3+CD8+PDCD1+ or CD3+CD8+CTLA4+ (Supplementary Fig. 10b). The following primary antibodies were used for flow cytometry: CD3 (1:70, 555275; BD Biosciences), CD8 (1:70, 100706; Biolegend), CD69 (1:70, 104512; Biolegend), IFNg (1:70, 563376; BD Biosciences), TCF7 (1:70, 566692; BD Biosciences), PDCD1 (1:70, 135224; Biolegend) and CTLA4 (1:70, 106314; Biolegend).

Cytotoxicity assay
For cytotoxicity assay, OVA-expressing MTOs were generated by lentivirus infection (pLV[Exp]-Puro-EF1A>{Ovalbumin(ns)}: T2A:EGFP(VB900133-6569mtk); VectorBuilder). Effector T cells for OVA-expressing cells were obtained by stimulation of OVA53 cells by anti-CD3/CD28 antibodies (11452D; Thermo Fischer) overnight. OVA-expressing MTOs were dissociated by Tripsin-EDTA (0.25%) (Thermo Fischer) and labeled with CFSE using 7-AAD/CFSE Cell-Mediated Cytotoxicity Assay Kit (600120; Cayman Chemical). 104 cells of CFSE labeled OVA-expressing MTO and stimulated OVA53 cells (102, 5×102, 103, 5×103, 104 or 2×104 cells) were co-cultured in the presence or absence of recombinant THBS1 (25μg/ml; 7859-TH-050, R&D systems) in 24 well plate for 20h. After co-incubation, dead cells were stained with 7-AAD, followed by flow cytometry using a FACSAria II (BD Biosciences) (Supplementary Fig. 10c). Cytotoxicity was analyzed by the proportion of 7-AAD+ cells in CFSE+ cells. The proportion was statically analyzed in each cell number condition of OVA cells.

MDSC assay
For the examination of immunosuppressive function of MDSC, PMN-MDSC and MO-MDSC were collected from primary tumors of WT or Thbs1-/- mouse implanted MTO to the rectum using Myeloid-Derived Suppressor Cell isolation Kit (130-094-538; Miltenyi Biotec). Each MDSC (104 cells/well) and CD3+ T cells (104 cells/well) collected from healthy WT mouse as described in (In vitro stimulation of T cells) were co-cultured with the stimulation by anti-CD3/CD28 antibodies (11452D; Thermo Fischer). After co-incubation, cells were collected and analyzed by flow cytometry using a FACSAria II (BD Biosciences) (Supplementary Fig. 10f). Immunosuppressive potency of each MDSC was analyzed by the proportion of CD69+ cells or CTLA4+ cells in CD8+ cells. The following primary antibodies were used for flow cytometry: CD3 (1:70, 555275; BD Biosciences), CD8 (1:70, 100706; Biolegend), CD69 (1:70, 104512; Biolegend) and CTLA4 (1:70, 106314; Biolegend).

Tube formation assay
To examine the potential function of CD36 in vascularization, tube formation assay was performed using Endothelial Tube Formation Assay (CBA-200; Cell Biolabs). 50μl of ECM gel was added to each well of 96 well plate and incubated for 30min. 5×104 cells of Mouse endothelial cells, C166 were harvested in each well in the presence or absence of recombinant THBS1 (25μg/ml; 7859-TH-050, R&D systems), simultaneously treated with anti CD47 antibody (10μg/ml; BE0270, BioXcell) or sulfosuccinimidyl oleate (SSO) (20μM, 25μM, 50μM, 100μM, 200μM; 11211, Cayman Chemical). Images were obtained microscopically after incubation for 2h. Cell images were obtained using microscope IX73 (Olympus). Quantification of tube area was performed by ImageJ/Fiji software v.2.3.0/1.53f48.

Generation of Thbs1-knockdown MTO
To examine the potential role of THBS1 expressed in tumor epithelium, Thbs1-knockdown MTOs were generated by induction of shRNA for Thbs1 using lentivirus (shThbs1#1 (LVM(VB900138-8684hkq)-K1), shThbs1#2 (LVM(VB900120-5533jmq)-K1), scramble shRNA control (LVM(VB010000-0009mxc)-K1); VectorBuilder). The knockdown efficiency was confirmed by qRT-PCR for Thbs1. Proliferation potency of each organoid was analyzed by luciferase assay. For luciferase assay, 104 cells of each organoid were seeded in matrigel in each well and cultured for 5 days and luminescence intensity was measured by SpectraMax iD5 (Molecular Devices) 10 min after 250μg of luciferin (88294; Thermo Scientific) was added in each well every day. To examine potential role of THBS1 expressed in tumor epithelium for metastasis, splenic injection of Thbs1-knockdown MTO (shThbs1#2) or control MTO (Scramble shRNA) to WT mice was performed (n=6).

THBS1 ELISA
Human serum samples were collected from whole blood of patients with adenomas and CRCs by using EDTA tubes and centrifugation, and the concentration of THBS1 in the serum was analyzed by Human Thrombospondin-1 Quantikine ELISA Kit (DTSP10; R&D systems). Colorimetry was performed on a microplate reader Sunrise (TECAN).

Histology, immunohistochemistry, and immunofluorescence
Tissues were fixed 2–5 days in masked formalin 2A (Japan tanner) and embedded in paraffin. Paraffin-embedded tissues were cut into 5μm sections. H&E staining was performed with standard protocols. Masson’s trichrome staining was carried out following manufacturer’s instructions (HT15-1KT; Sigma). For immunostaining, sections were deparaffinized, rehydrated, and then treated for antigen retrieval. After blocking in Protein Block Serum-Free solutions (DAKO), tissues were incubated with primary antibody overnight at 4°C followed by incubation with biotinylated (1:200, BA-5000, BA-1000, BA-2000, BA-4000; Vector Laboratories) or fluorophore-conjugated secondary antibody. For immunohistochemistry, endogenous peroxidase was quenched in 3% H2O2 in methanol for 10 min at room temperature. For immunohistochemistry, antibodies were visualized with avidin-biotin complex (Vectastain Elite; Vector Laboratories) using diaminobenzidine (DAB) (Dako) as the chromogen. For immunofluorescence analysis (double staining), sections were incubated with Alexa Flour 488 or 594 conjugated secondary antibodies (1:200, A21207, A21203, A21202, A21206; Thermo fisher) for 1h at room temperature, followed by counterstaining with Hoechst (Thermo Fisher). For immunofluorescence staining, the signal was also visualized using the Opal 3-Plex Manual Kit (Akoya Biosciences), which allows simultaneous detection of multiple targets in the same image. Secondary antibodies (undiluted, ImmPRESS Reagent (MP-7401, MP-7404; Vector Laboratories), EnVision+ System-HRP Labelled Polymer (K4001; Dako)). Fluorophores Opal 520, Opal 570, and Opal 690 were used, and the sections were counterstained with Spectral DAPI. To investigate lectin perfusion, tumor sections were isolated from the mice orthotopically implanted MTO to the rectum 1h after intravenous injection of biotinylated tomato lectin (100μg/mouse, B-1175; Vector Laboratories) and were incubated with HRP-conjugated secondary antibodies (1:200, Vectastain Elite ABC HRP kits) for 1h at room temperature and visualized with DAB (Dako). RNAscope was performed for detection of expression of Thbs1 in MTO tumor sample using Thbs1-probes (No. 457891 (ACD)). The following primary antibodies were used for immunostaining: THBS1 (1:500, MA5-13398; Thermo Fisher), CD31 (1:100, 7769; Cell Signaling), CD8 (1:2000, ab209775; Abcam), CD4 (1:1000 (using Opal system), ab183685; Abcam), CD11c (1:1000 (using Opal system), 97585; Cell Signaling), C-Cas3 (1:100, 9664; Cell Signaling), CK19 (1:500, ab52625; Abcam), CD11b (1:1000, ab133357; Abcam), GFP (1:500, ab6673; Abcam), NLRP3 (1:100, NBP2-12446; Novus Bio), αSMA (1:100, ab5694; Abcam), pSMAD3 (1:200, ab52903; Abcam), EPCAM (1:100, ab71916; Abcam), Ly6C (1:100 (using Opal system) 128002; Biolegend), Ly6G (1:1000 (using Opal system, 87048; Cell Signaling), F4/80 (1:2500 (using Opal system), 70076; Cell Signaling), FOXP3 (1:1000 (using Opal system), 12653; Cell Signaling), TCF7 (1:1500 (using Opal system), MA514965; Thermo Fisher), CD36 (1:1500 (using Opal system), ab202909; Abcam), CD47 (1:1000 (using Opal system), SC-53050; Santa Cruz), CXCR4 (1:4000 (using Opal system), ab181020; Abcam) and CTLA4 (1:1000 (using Opal system), ab237712; Abcam). For staining of EYFP, GFP antibody (1:500, ab6673; Abcam) was used. All images were collected from each stained slide on a BZ-X710 microscope (KEYENCE). The number of whole cells and positively stained cells in IHC images were measured using the QuPath47, and the whole area and positively stained area in IF images were measured by ImageJ/Fiji software v.2.3.0/1.53f48.

Flow cytometry analysis
Flow cytometry experiments were performed on BM cells, blood cells, tumor-consisting cells. BM cells and blood cells were collected from healthy mice or WT or Thbs1-/- mice orthotopically implanted MTO or MC38 in the rectum. Tumor-consisting cells were collected from primary tumors from WT or Thbs1-/- mice orthotopically inoculated MTO in the rectum or metastatic liver tumors from WT mice injected MTO to the spleen. For collecting tumor-consisting cells, fresh tumors were dissociated with Tumor Dissociation Kit (130-096-730; Miltenyi Biotec) and red blood cells were removed with using Red Blood Cell Lysis Solution (130-094-183; Miltenyi Biotec). The collected cells were blocked with mouse Fc Block (1:50, anti CD16/CD32 antibody; 553142, BD Biosciences) for 10min on ice and then incubated with the following antibodies for 30min on ice: CD45 (1:70, 552848; BD Biosciences), CD11b (1:70, 553310; BD Biosciences), CXCR4 (1:70, 146511; BioLegend), EPCAM (1:70, 48-5791-82; Invitrogen), CD45 (1:70, 557659; BD Biosciences) (1:70, 103106; Biolegend), CD3 (1:70, 555275; BD Biosciences) (1:70, 100236; Biolegend), CD4 (1:70, 100406; Biolegend), CD8 (1:70, 553035; BD Biosciences) (1:70, 100706; Biolegend), CD69 (1:70, 104512; Biolegend), CD11c (1:70, 117318; Biolegend), CD11b (1:70, 553310; BD Biosciences), F4/80 (1:70, 565410; BD Biosciences), Ly6C (1:70, 560595; BD Biosciences), Ly6G (1:70, 562737; BD Biosciences), PDCD1 (1:70, 135224; Biolegend), CTLA4 (1:70, 106314; Biolegend) and CCR2 (1:70, 150610; Biolegend). For intracellular staining, fixation and permeabilization was performed using BD Cytofix/CytopermTM Fixation/Permeabilization Kit (554714; BD Biosciences) for staining of IFNg and Transcription Factor Buffer Set (562574; BD Biosciences) for staining of FOXP3 and TCF7, followed by incubation for 30 minuntes with the following antibodies: FOXP3 (1:70, 562996; BD Biosciences), IFNg (1:70, 563376; BD Biosciences), TCF7 (1:70, 566692; BD Biosciences) and Cy7/Alexa Flour 700-conjugated THBS1 (1:70). For generation of Cy7/Alexa Flour 700-conjugated anti THBS1 antibody, Cy7 Conjugation Kit (ab102882) or Alexa FlourR 700 Conjugation Kit (Fast)-Lightning-LinkR (ab269824; Abcam) was used with anti THBS1 antibody (MA5-13398; Thermo Fischer). Flow cytometry was performed on a FACSAria II (BD Biosciences) and the data was analyzed using FlowJo v.10. THBS1 positive cell sorting strategy is shown in Supplementary Fig. 10f. Each cellular population was determined as the following markers: CD8+ T cell (CD3+CD8+), CD4+ T cell (CD3+CD4+), Treg (CD3+FOXP3+), Dendritic cell (CD45+CD11c+), TAM (CD11b+F4/80+), monocyte (CD45+CD11c-CD11b+Ly6C+Ly6G-F4/80-), neutrophil (CD45+CD11c-CD11b+Ly6C+Ly6G+F4/80-), PMN-MDSC (CD45+CD11c-CD11b+Ly6C+Ly6G+), MO-MDSC (CD45+CD11c-CD11b+Ly6C+Ly6G-) (Supplementary Fig. 10a, d).

Immunoblotting analysis
Tumors induced by orthotopic implantation were lysed in RIPA buffer (ab156034; Abcam) with phosphatase and protease inhibitors (5872; Cell Signaling) using gentleMACS Dissociator (Miltenyi Biotec). Protein concentration in lysates were determined by using Protein Assay Dye Reagent Concentration (5000006; Bio-Rad). Cell extracts were denatured, subjected to SDS-PAGE and transferred to membranes (Trans Blot Turbo, 1704156; Bio-Rad). After blocking with Blocking One (03953-95; Nacalai), the membranes were incubated with the following antibodies overnight at 4°C: pSMAD3 (1:1000, 9520; Cell Signaling), SMAD3 (1:1000, 9513; Cell Signaling) and β-actin (1:4000, A1978; Sigma Aldrich). After 2h incubation with the appropriate horseradish peroxidase-conjugated antibodies (1:10000, 7074/7076; Cell Signaling), the immune complexes were detected by chemiluminescence (34580; Thermo Fisher) on an Amersham Imager 600 (GE Healthcare).

RNA extraction and qRT-PCR analysis
Total RNA from samples was extracted using the RNeasy Mini Kit (QIAGEN). Complementary DNA (cDNA) was obtained using the ReverTra Ace qRT-PCR RT Kit (TOYOBO). Real-time PCR was performed in duplicate using the SYBR Green PCR Master Mix (Roche Diagnostics) on a LightCycler 96 (Roche Diagnostics). Gene expression values for each sample were normalized to the 18S RNA. qRT-PCR primers are listed in Supplementary Table 5.

RNA-Seq and analysis
For RNA-seq, total RNA was extracted from the tumors generated by orthotopic implantation of MTO in the rectum of WT or THBS1-/- mice using the RNeasy Mini Kit (QIAGEN). RNA-seq was performed by Macrogen, Inc on the Novaseq 6000 platform with 2×100 base pair paired-end sequencing using SMART-Seq v4 Ultra Low Input RNA kit and TruSeq RNA Sample Prep Kit v2. Adaptors and low-quality bases were trimmed from the reads using Trimmomatic (version 0.39) with default parameters. Reads were mapped to the Mus musculus reference genome build mm10 using STAR (version 2.7.3a) and counted by RSEM (version v1.3.1). Read count data were normalized using the iDEGES/edgeR method. Normalized count data were used for gene set enrichment analysis (GSEA) and differentially expressed genes were determined using TCC (TagCount Comparison) with a false discovery rate cutoff value <0.05. GSEA was performed using GSEA software (http://www.gsea-msigdb.org/gsea/index.jsp).

Single-cell RNA sequencing
Tumors generated by orthotopic implantation of MTO in the rectum of WT and Thbs1-/- mice (n=2 mice per group) were processed into single-cell suspension using Tumor Dissociation Kit (Miltenyi Biotec). Dead cells were removed by Dead Cell Removal Kit (Miltenyi Biotec). scRNA-seq libraries were generated using the Chromium Single Cell 3′ v3 Reagent (10× Genomics). Cells were loaded onto the 10× Chromium Controller (10× Genomics) using Chromium Next GEM Chip G single cell kit (10× Genomics) at a concentration of 1,000 cells per μl as described in the manufacturer’s protocol (10× Chromium Single Cell 3′ Reagent Kits User Guide (v3.1 Chemistry Dual Index)). 5,000 cells from each WT mouse (W1, W2: n=2) and 10,000 cells from each Thbs1-/- mouse (T1, T2: n=2) were loaded. Generation of gel beads in emulsion (GEMs), barcoding, GEM-RT clean-up, complementary DNA amplification and library construction were all performed as per the manufacturer’s protocol. Individual sample quality was checked using a TapeStation (Agilent Technology). The final library pool was sequenced by a DNBSEQ-G400RS (MGI). The Cell Ranger version 6.1.1 software suite from 10× Genomics was used to process, align and summarize unique molecular identifier (UMI) counts. Estimated number of cells of T1, T2, W1 and W2 were 9875, 6791, 5071, and 5268 respectively. Mean depth of T1, T2, W1 and W2 were 31720, 52408, 67364, and 58361 reads per cell and 800, 1088, 1308, and 1446 median genes per cell, respectively. Additional data processing and analysis was performed using Seurat version 4.2.351 in R version 4.2.3. Cells expressing fewer than 200 genes and cells expressing greater than 20% mitochondrial related genes were removed. The genes expressed in fewer than five cells were excluded. The expression set of selected 8209 cells from T1, 4907 cells from T2, 3852 cells from W1, 3963 cells from W2 was converted into Seurat object. Each sample was normalized using SCTransform52. For integration, the Seurat’s SelectIntegrationFeatures function was used in the merged object to select genes taken as input in anchors identification procedure by the Seurat’s PrepSCTIntegration function and the Seurat’s FindIntegrationAnchors function. The Seurat’s IntegrateData function was used to integrate all objects. Dimensionality reduction was performed by RunPCA function and RunUMAP function. All cells were classified into three compartments, epithelium (Epcam), Immune (Ptprc), or stroma (Pecam1, Dcn or Rgs5). Immune compartment was extracted and re-normalized. Then it was devided into B cells (Cd79a), T/NK cells (Cd3d or Nkg7) or Myeloid cells (Lyz2, Itgam or S100a8). Cd8a positive cluster was extracted from T/NK and proceeded to further analysis. Representative genes for clusters of myeloid cells and CD8+ T cells are shown in Supplementary Fig. 3d, g, respectively. Representative genes for stem-like signature, dysfunction signature and M2-macrophage-related signature are shown in Supplementary Table 6. Information of recipient mouse genotype in published single cell transcriptome data of orthotopic MTO tumor (GSE154863) was provided by the original author (A.D.). Cells of WT mice were used for analysis. Cells expressing greater than 7% mitochondrial related genes were removed. The myeloid cell cluster was detected as described above and in Fig. 5j.

Bioinformatics analysis
CRC samples in TCGA were stratified according to CMS subtypes (n: CMS1=85, CMS2=132, CMS3=78, CMS4=184) based on previous report using CMScaller package5,53. MSI-H and MSS groups in TCGA were stratified by MSIsensor (n: MSI-H=88, MSS=494)54. Bulk iCMS classification was performed according to previous report16 using R 4.2.3 and CMScaller package53. Briefly, gene expression quantities standardized with FPKM was log transformed and iCMS was determined using nearest template prediction. Adjusted p-value<0.05 was considered as significant. IMF classification was conducted with applying iCMS, MSI, and fibrotic transcriptome (CMS4), and CRC samples in TCGA were stratified into five IMF classes: i2_MSS_NF (n=117), i2_MSS_F (n=101), i3_MSI (n=81), i3_MSS_NF (n=73) and i3_MSS_F (n=51). The “CMS4_UP” and “CMS4_DOWN” signatures were generated, as previously described4,17. Representative genes for “CMS4_UP” and “CMS4_DOWN” signatures are shown in Supplementary Table 6. For analysis of CXCL12-expressing cells in single cell transcriptomic dataset of human CRCs (SMC), we extracted B cell (CD79A), epithelial cell (EPCAM), mast cell (KIT), myeloid (CD68), T cell (CD3D) and stromal cells (DCN) compartment in accordance with original author’s annotation30 and re-clustered stromal cells into five subpopulations: fibroblast (COL1A2, PDPN, DCN), smooth muscle cell (MYH11), pericyte (MCAM, RGS5), endothelial cell (PECAM1, LYVE1) and enteric glial cell (S100B). For analysis of myeloid cells, myeloid cell compartment was extracted in accordance with original author’s annotation30. To correct batch effect, myeloid cell object was split by sample ID with Seurat’s SplitObject function and normalized with SCTransform method. Then data was integrated as described above. Dimensionality reduction was performed by RunPCA function and RunUMAP function. Re-integrated myeloid cells were stratified into six clusters according to expression of known marker genes. The marker genes used here are shown in Fig. 5m.

Statistics and reproducibility
Pairwise differences were measured using two-tailed independent student’s t-tests. If the data did not meet this test, a Mann-Whitney U-test was used. Statistical significance between groups of 3 or more was determined by a one-way or two-way ANOVA, followed by the Tukey’s multiple comparison test. Data are presented as the mean±SEM. Survival was measured by the Kaplan-Meier method and analyzed by Log-rank (Mantel-Cox) test. Outlier was identified and removed using the ROUT method (Q=1%). Statical correlation was measured using the Pearson Correlation Coefficient (two-tailed, confidence interval (CI)=95%). Logistic regression analysis was employed to estimate univariate and multivariate odds ratio and 95% CI. Statistical analysis was performed using GraphPad Prism 9 or JMP. Values of P<0.05 were considered as significantly different.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "with expertise in colorectal cancer\nIn this manuscript “Tumor-infiltrating monocyte-like cells create immunosuppressive and\ntherapy-resistant microenvironment via THBS1 production in colorectal cancer”, the authors\npresent a model in which tumor CXCL12/CCR4 stimulates bone marrow monocytes to\nexpress THBS1 that is then able to bind CD47/CD36 cells in the tumor bed and create an\nimmunosuppressive TME. At least, that is how I interpret their data. They use mouse tumor\norganoid models (MTO), generated from Apc, Trp53, Kras, Tgfbr2 GEM, and MC38 mice\ngenerated from carcinogen-induced colon cancer in C57BL/6. Tumors are injected into the\nrectum of syngeneic mice and straight THBS1 knock-out mice in which THBS1 is eliminated\nin all cells.\nThis reviewer would like clarification of some of the experimental details.\nWhat cells in the TME express THBS1? RNAscope would answer this question. If THBS1 is\nexpressed in the tumors themselves, this would complicate using the THBS1 knock-out\nmice. Might need to show by IHC that THBS1 is not present in tumors. A query of the protein\natlas can give validity to the claim that monocytes are the main actors for THBS1 effects.\nAlong with CD14, it would be helpful to have other myeloid lineage markers – for example,\nCD68, CD11B. The authors should mention that CMS4 is associated with an inflammatory\nsignature and stromal activation but show that THBS1-/- mice have GSEA data with\nincreased enrichment in inflammatory or immune response - might need to explain this and\nhow these aren’t in opposition for the reader.\nTHBS1 has pleiotropic effects. The authors seem focused on the immunosuppressive effects.\nThe authors might consider performing co-culture experiments with a T cell killing assay\nwith tumor cells incubated with or without THBS1.\nCan the authors confirm that CD47 and CD36 are only receptors for THBS1? And what cells\nexpress these receptors? This is necessary for their claims that CD47 and CD36 knock-out\nmice phenocopy the THBS1 knock-out mice. Showing a difference in activation of\ndownstream signaling based on addition or absence of the ligand might be helpful, as well\nas colocalization studies/in vitro results looking at the markers as well as vascularization\nassays might enhance this mechanistic insight.\nThe authors mention THBS1 was increased in CMS4 and CCS3 CRC subtypes. Colorectal\nCancer Subtypes (CCS) classification is not widely used. Can the authors state the value of\nusing this classification system in this report? They should update their CMS classification (I\nJonito et al., Nature Genetics 54:963-975, 2022).\nThe authors are encouraged to examine whether THBS1 is increased in MSS vs MSI-H CRC.", "reviewer_2": "with expertise in colorectal cancer, metastasis\nThis manuscript by Omatsu et al. investigated the promoting role of THBS1 in the colorectal\ncancer by forming the immunosuppressive and vascular-rich tumor environment. The\nauthors first identified that high THBS-1 expression in tumor stroma was correlated with\npoor survival in human CRC patients. Then they verified those observations in mouse CRC\nmodels. By using Thbs1 knock out mice, they found that loss of TME-derived THBS1\nsuppressed CRC liver and lymph node metastasis. Next, they performed ScRNA-seq analysis\nfrom the MTO tumors from WT and Thbs1 knock out mice, revealed that Thbs1 deficiency\ncan block T cell exhaustion and increase the stem cell-like CD8+ T cells. By using Cd36 knock\nout and Thbs1 knock out mice, they concluded that THBS1-CD47 and THBS1-CD36 axes were\nboth responsible for the formation of immunosuppressive microenvironment, while THBS1-\nCD36 can further influence the vascular structure. Finally, they identified the primary source\nof THBS1, and demonstrated that THBS1-expressing myeloid cells were recruited from bone\nmarrow to TME via CXCL12-CXCR4.\nThe authors are to be recommended on a very extensive array of experimentation, the use\nof different transgene mice and the numerous innovative approaches. However, the data\nfor supporting the contribution of myeloid cell-derived THBS1 in CRC are limited, and there\nare some confusing points that either explanation or further experimentation.\n1. The authors found that THBS1 expression is mainly in the stroma of CRC samples by IHC\nstaining in Fig.1a, while the mRNA expression of THBS1 were analyzed in Fig.1d by using\nTCGA datasets, in which were mainly tumor tissues. Therefore, the bioinformatic analysis in\nTCGA is not representative, also for the survival analysis. It is better to use your own TMA\narray to do the survival analysis.\n2. Fig.1i, the authors revealed the relationship between THBS1 expression and\nmasson/CD14 by showing IHC staining. For the same CRC samples, it is better to do the\nstaining on the consecutive sections, which will be more representative.\n3. The study focus on the TME-derived THBS1 in CRC. The CRC cells can also produce THBS1,\nthe role of tumor cell-derived THBS1 should be investigated and excluded. How is the THBS1\nexpression in tumor cells? The expression of THBS1 in organoid and MC38 cells should be\ndetected. Whether the tumor cell-derived THBS1 can contribute CRC progression?\n4. For the bioluminescence imaging，such as. Fig.2e, 5e,6i, 6r, 7d, 7i, 7q， the authors\nshowed the primary lesion(white), lymph node (orange), and liver (yellow) metastasis. How\ndid the authors define the signaling are in the lymph node or liver? It is better to combine\nCT with IVIS imaging to better define the lymph node and live. Or to isolate the lymph node\nand liver, to do the ex -vivo imaging, to check the luciferase signaling from lymph node and\nliver.\n5. Fig.3j, the authors performed the stimulation experiment on CD3+T cells with and\nwithout recombinant THBS1 treatment (25ug/ml). How did the authors define the\nconcentration of recombinant THBS1? Is this concentration-dependent effect? Why using\nthe CD3+T cells? It is better to isolate the CD8+T cells to perform this experiment. For the\nactivation of T cells, normally both anti-CD3 and anti-CD28 were used. Why did the authors\nuse anti-CD3 only, which is not enough to activate T cells? In addition, the protein\nexpression of IFN-g and CTLA4 should be checked by flow cytometry, which makes more\nsense.\n6. Fig.6f, the magnified region should be marked in the figure, to make it more visible and\nobvious.\n7. Sup Fig.4c, the full WB blot should be provided, and the molecular weight should be\nmarked.\n8. Sup Fig5a, Sup Fig.6h, the scale bars for THBS1 and CD31 staining were different.", "reviewer_3": "with expertise in bioinformatics, immunology\nOmatsu, M, et al, present a study of the immunosuppressive mechanisms in an aggressive\nsubtype of colo-rectal cancer (CRC), integrating mouse models, scRNAseq of a limited set of\nhuman patient samples, and bulk RNAseq and IHC of large human patient cohorts. In\nparticular, they demonstrate a central role for thrombospondin-1 (THBS-1) produced by\nmonocytes recently recruited from the bone marrow to the tumor site. THBS-1 expression is\nshown to be associated with poor prognosis in human tumors. Mouse experiments show a\nclear and strong effect on vascularization, T cell infiltration, necrosis, and metastasis and a\npotential, although not striking, effect on efficacy of immune checkpoint blockade,\nchemotherapy, and anti-angiogensis therapy.\nIn summary, this manuscript reveals a clear mechanism in a frequent, aggressive cancer for\nrecruitment of bone marrow derived myeloid populations, their contribution to generating\nan immunosuppressive microenvironment, and their relationship with metastasis. This\ncould be appropriate for Nature Communications however there are a few major issues (see\nbelow) that need to be addressed before this is clear.\nMajor comments\n1. The correlative analyses in Figure 1 and Supplementary Figure 1 connecting THBS1\nexpression with eg prognosis, CRC subtype, gene expression profiles, etc need to more\nclearly demonstrate how much THBS1 independently drives these patterns versus just being\na marker of the CMS4 subtype.\nAdditionally, much more justification/information is needed for the splitting of samples by\ntiers of THBS1 expression. In Fig 1 d-f, why is it not just by median ? In Fig 1 o, why are\nintermediate and high grouped together ?\n2. The timing in Figure 2 is not clear. If tumor is implanted at week 8, what does week 0\nrefer to ? This is particularly important for interpreting the survival analysis in panel h.\nGiven the strong effects of Thbs1-/- early in Figure 2, especially e-g, the KM curves in panel h\nare very surprising. Can the authors explain why there is just a modest delay and then all the\nThbs1-/- die as rapidly as the WT ? Are they dying of the disease in a different way ?\n3. In all the scRNAseq analyses, more extensive explanation/justification must be provided\nfor all cluster annotations (eg with heatmaps, differential gene expression analysis, marker\ngenes, or other approaches).\n4. The results in Fig 7 on THBS1 inhibition in combination with other treatments do not\ndemonstrate a compelling synergistic effect and therefore should not be highlighted in the\ntitle and abstract. Relatedly, survival analysis and tumor growth kinetics are standard\nanalyses in these types of in vivo tumor treatment models – why are they not included here\n? The evidence connecting THBS1 to metastatic activity (eg Fig 2g, 4e, 5f, 6h and to a lesser\nextent 6s), conversely, is much more compelling and perhaps does warrant highlighting\nearly and clearly.\nMinor comments\n1. (line 108) “These results suggest that the stroma-infiltrating cells are the primary source\nof THBS1 and demonstrate a strong association between high THBS1 level, aggressive\nphenotype, and poor prognosis in human CRCs” and Fig. 1c\nThe expression of THBS1 appears higher in the normal colon than in the tumor epithelium.\nIs it significantly different? Can you provide a possible explanation? Additionally, the violin\nplots should be replaced by individual data points since n is relatively low. Finally, at this\npoint in the manuscript it is demonstrated that THBS1 expression comes from the stroma,\nbut not that it is from infiltrating cells.\n2. Fig. S 1d, e\nWhy are there different n for the THBS1 high and low categories between these two plots\nfrom the same dataset/cohort?\n3. Fig. 2h,i,j\nCan these be re-sized to avoid having the awkward “x10” above the vertical axes in the plot\nat far right? It is currently confusing to compare with results of similar experiment in panel\nc.\n4. Fig. 3c\nAlthough the statistical test (Mann-Whitney, aka Wilcoxon) indicates the differential\nexpression is significant, the violin plots are not convincing. Perhaps another type of\nvisualization would be more useful? Ideally, validation by FACS would be even better.\n5. (line 197) and Fig. 3k\nPlease provide a citation for these genes as IFNg target/marker genes. Similar for all\nsignatures/gene sets analyzed in the paper.\n6. (line 231-232) and Figure 4\nThe phenocopies between Thbs1-/-, CD36-/-, and CD47-/- are clear. However, attributing\nthis directly to interactions between THBS1 and these proteins and referring to “THBS1-\nCD47 and THBS1-CD36 axes” seems premature without double mutants since, at least a\npriori, other receptors for THBS1 or other ligands for CD47 and CD36 could explain these\neffects through parallel rather than direct mechanisms.\n7. Fig. 5i-k\nThese same genes should also be shown in the mouse scRNAseq data.\n8. Fig. S 5b\nAs with major comment 1, provide these plots divided by CRC categories.\n9. Fig. 6b\nPlease provide the total number of cells in each condition in addition to the percentage to\nrule out eg a decrease in lymphocytes.\n10. (lines 303-304) Fig. 6l and Supplementary Figure 6g\nPlease provide statistical tests that these genes are specifically upregulated in mesenchymal\nCMS4 CRCs.\n11. (lines 303, 307) Fig. 6m and Supplementary Figure 6g\nIt is confusing that CCL2 is high in CMS4 from TCGA but then lower in the MTO compared to\nMC38. Can the authors provide any hypotheses?\n12. Fig. 7\nWhy does the data for the WT and Thbs1-/- control conditions appear identical in panels f\nand s, but different from same conditions in panel n? All violin plots in this figure should be\nreplaced by individual data points. The evidence that combination therapy is synergistic or\neven additive here, outside of potentially panels e and r, is not compelling.", "reviewer_4": "with expertise in cancer immunology, myeloid cells\nOmatsu et al report that a high expression of thrombospondin-1 (THBS1) positively\ncorrelates with the mesenchymal characteristics, immunosuppression, and poor prognosis\nin CRCs. They found that bone marrow derived monocyte-like cells recruited by the action of\nCXCL12 are the primary source of THBS1\ncontributing to the creation of a therapy-refractory environment by inducing cytotoxic T cell\nexhaustion and impairing vascularization. According to the authors, THBS1 loss in the TME\nrenders tumors sensitive to immune checkpoint inhibitors and anti-cancer drugs. However,\nthere are several critical points that need to be addressed to consider this study suitable for\npublication in Nature Communications:\n1. The authors observed a substantial increase in CD8+ T cell infiltration and cleaved\ncaspase-3 (C-Cas3)+ apoptotic cells in orthotopic tumors from Thbs1-/- mice. Treatment\nwith anti-CD8 antibody effectively depleted CD8+ T cells and, concomitantly, decreased\napoptotic cells in the primary tumors (Fig. 2i,j), confirming the cytotoxic activity of the\ninfiltrating CD8+ T cells. How do the authors explain that there is no significant difference in\ntumor growth in KO mice? If THBS1 contributes to immunosuppression, why are T cells not\nable to kill tumors in KO mice?\n2. Thbs1-/- mice showed increased vascular formation, thus I was wondering if there is a\nchange in the myeloid compartment that is known to contribute to the immunosuppressive\nTME. No changes in immunosuppressive myeloid cells infiltration may explain why T cells are\nstill ineffective in killing tumors in those KO mice.\n3. The authors showed reduced metastatic lesions, therefore it would be critical to compare\nthe immune TME and THBS1 levels in primary tumor vs metastatic lesions. Such differences\nin immune composition and THBS1 levels may explain why there is reduction in metastasis\nlesions only.\n4. The authors posited that increased infiltrating CD8+ T cells in the orthotopic primary site\nmay account for the reduction of metastatic burden in MTO-bearing Thbs1-/- mice. It is also\npossible that there is an increase of CD8 T cells in metastatic sites that may help to reduce\nmetastatic burden. Thus, what is the frequency of T cells in metastatic lesions in those KO\nmice? The authors showed that loss of THBS1 blocks T cell exhaustion and increases stem-\nlike CD8+ T cells, what is the phenotype of T cells in primary vs metastasis?\n5. BM-derived monocyte-like cells are the primary producers of THBS1 in stroma-rich\nintestinal tumors. THBS1 affects the activation of T cells in vitro, therefore monocyte like\ncells may suppress T cell activation via the release of THBS1. Since myeloid cells used\ndifferent mechanisms to suppress T cells, I was wondering if myeloid cell derived THBS1\ncontributes to the immunosuppressive activity of these cells. Moreover, what is the\nexpression of other suppressive markers in WT vs KO mice?\n6. The authors found the highest Thbs1 expression in CD45+CD11b+CXCR4+ cells in BM of\nMTO-bearing mice, suggesting that THBS1-expressing CD11b+ cells are recruited via the\naction of CXCL12-CXCR4 signaling. The authors suggest that these cells\n(CD45+CD11b+CXCR4+) are monocyte like cells. However other myeloid cells expressed\nthese markers. Therefore to better understand the myeloid cell composition, the authors\nshould characterize CD45+CD11b+CXCR4+ cells in primary tumors and metastasis sites.\n7. CD8+ T cells in 176 Thbs1-/- tumors exhibited a decrease in dysfunctional markers such as\nPdcd1 and Ctla4. How do the authors explain that anti-PD1 therapy may work in this model?\nDo the mice survive longer? Thbs1-/- tumors and combo with anti-PD1 showed similar\nreduction in mets. However, tumors are smaller in KO mice following treatment with anti-\nPD1. How do the authors explain that a reduction in primary tumor does not reflect in a\nsignificant decrease in number of mets? I have observed a similar trend in fig7m and s."}, "author_response": "RESPONSES TO REVIEWERS' COMMENTS:\nPlease note that Reviewer’s comments are in blue.\nReviewer #1: with expertise in colorectal cancer\nWe thank the reviewers for their constructive and insightful comments, which have helped us\nimprove our manuscript. We have addressed all the concerns and comments raised and have\nincluded new data as requested. We hope that, with this extensive revision, the reviewers will find\nour paper suitable for publication in Nature Communications.\nIn response to the reviewers' request to clarify “some of the experimental details”, we have\nincluded new experimental data that further validates the conclusions of our study. Please see\nbelow.\nSpecific comments:\n1. What cells in the TME express THBS1? RNAscope would answer this question. If THBS1 is\nexpressed in the tumors themselves, this would complicate using the THBS1 knock-out mice. If\nTHBS1 is expressed in the tumors themselves, this would complicate using the THBS1 knock-out\nmice. Might need to show by IHC that THBS1 is not present in tumors. A query of the protein atlas can\nhelp validity to the claim that monocytes are the main actors for THBS1 effects.\nFollowing the reviewer’s comment, we have performed RNAscope analyses of both human CRC\nsamples and mouse MTO orthotopic tumors. RNAscope demonstrated that THBS1 was clearly\nexpressed by infiltrating cells in the tumor stroma and not by tumor cells in both human CRCs\nand mouse MTO tumors (new Fig. 1b and Supplementary Fig. 2c). This observation was\nsupported by double IF analyses showing that THBS1 did not co-localize with EPCAM+ tumor\ncells (new Supplementary Fig. 1a and 2d). We have followed the reviewer’s suggestion and\nexamined the human protein atlas (https://www.proteinatlas.org/ENSG00000137801-\nTHBS1/single+cell+type) which showed the highest expression of THBS1 in monocytes, followed\nby macrophages and Kupffer cells (see the figure below). These data, together with the results\nfrom FACS (new Fig. 6d and 6p) and scRNA-seq analyses of human and mouse CRCs (new Fig.\n5h–m and Supplementary Fig. 3c, 3d, and 7c), demonstrate that monocytes are the primary\nmediators of THBS1's effects on tumorigenesis. This was further supplemented by the orthotopic\ntumor phenotype in LysM-cre;Thbs1/ mice, which showed that monocyte/macrophage-specific\nloss of THBS1 activated the antitumor immune response and suppressed metastasis (new Fig.\n5b–g and Supplementary Fig. 7a, 7b).\n2. Along with CD14, it would be helpful to have other myeloid lineage markers – for example,\nCD68, CD11B.\nFollowing this reviewer’s comment, we have performed IHC for other myeloid lineage makers\nCD68 and CD11B in TMA of human CRC and found clear correlative relationship between\nexpression of THBS1 and that of CD68 and CD11b (new Supplementary Fig. 1j).\nThe authors should mention that CMS4 is associated with an inflammatory signature and stromal\nactivation but show that THBS1-/- mice have GSEA data with increased enrichment in\ninflammatory or immune response - might need to explain this and how these aren’t in opposition\nfor the reader.\nThank you for pointing this out. According to the original paper on consensus molecular subtyping,\nCMS1 CRC is characterized by an active immune response with high infiltration of effector\nimmune cells, whereas CMS4 CRC is characterized by increased stromal activation and immune\ninfiltration (Guinney J et al., Nat Med, 2015, Fig. 3i). This suggests that the TME in both CMS1\nand CMS4 show immune cell infiltration and enrichment of inflammatory signatures, distinct from\nthat of CMS2 and CMS3, which display few or no infiltrating immune cells. However, in contrast\nto the highly immunogenic TME of CMS1 CRC, which contains a large number of effector immune\ncells such as cytotoxic CD8+ T cells, the inflamed TME of CMS4 CRC includes a substantial\nnumber of immunosuppressive cells such as MDSCs, TAMs, and regulatory T cells (Guinney et\nal., Nat Med, 2015; Dienstmann et al., Nat Rev Cancer, 2017), indicating that the types of\ninflammation in CMS1 (immune active) and CMS4 (immunosuppressed) are completely different.\nOrthotopic tumors in Thbs1-/- mice exhibited increased infiltration of CD8+ T cells (new Fig. 2f,3m\nand Supplementary Fig. 2n,5a) and increased enrichment of gene signatures, including the IFN\ngamma response (GSEA data, Fig. 2c and Supplementary Fig. 3i), suggesting that THBS1\ninhibition switches the state of inflammation from “immunosuppression” to “immune active.” In the\nresponse to this comment, we have explained in more detail the characteristics of inflammation\nin the TME of mesenchymal CMS4 in revised manuscript and avoided confusing descriptions that\ncould have misled the reader.\n3. THBS1 has pleiotropic effects. The authors seem focused on the immunosuppressive effects.\nThe authors might consider performing co-culture experiments with a T cell killing assay with\ntumor cells incubated with or without THBS1.\nAs suggested by the reviewer, we performed a T cell-killing assay using T cells and OVA-\nexpressing MTO. THBS1 significantly attenuated the cytotoxic activity of CD8+ T cells to tumor\ncells (Fig. 2q).\n4. Can the authors confirm that CD47 and CD36 are only receptors for THBS1? And what cells\nexpress these receptors? This is necessary for their claims that CD47 and CD36 knock-out\nmice phenocopy the THBS1 knock-out mice. Showing a difference in activation of\ndownstream signaling based on addition or absence of the ligand might be helpful, as well\nas colocalization studies/in vitro results looking at the markers as well as vascularization\nassays might enhance this mechanistic insight.\nUnder physiological conditions, THBS1 interacts with multiple receptors, including CD47,\nCD36, and TGFbeta; however, in the context of mesenchymal CRC, we propose that THBS1\nexerts its immunosuppressive function by binding to CD47 and CD36 on CD8+ T cells. Regarding\nthe cell types expressing CD47 and CD36, scRNA-seq of human CRC demonstrated that CD47\nexpression was almost ubiquitous in a variety of immune, epithelial, and stromal cells, whereas\nthat of CD36 was observed in immune and stromal cells (see figure below). Among immune cells,\nCD36 is primarily expressed by myeloid cells, and also by some CD8+ T cells. Double\nimmunofluorescence staining revealed that infiltrating CD8+ T cells in MTO tumors expressed\nboth CD47 and CD36 (new Fig. 4g).\nFollowing the reviewer’s comment, we performed in vitro experiments on T cells isolated from WT,\nCd47-/- and Cd36-/- mice to examine the differences in the activation of downstream signaling in\nthe presence or absence of the ligand (THBS1) (new Fig. 4h and 4i). Notably, the loss of CD47\nsignificantly blocked the suppressive effect of THBS1 on the activation of CD8+ T cells (new Fig.\n4h), which is in agreement with a previous report demonstrating that THBS1-CD47 signaling\ndirectly inactivates T cells by suppressing TCR signaling (Li Z et al., J Immunol, 2001). With regard\nto CD36, we first checked the changes in macrophages of orthotopic MTO tumors because a\nprevious report demonstrated that THBS1 acts on macrophages to suppress lung inflammation\nby binding to macrophage CD36, which further inhibits T cell function by secreting IL-10 (Akdis\nCA et al., Immunology, 2001). This is consistent with the fact that myeloid cells are the primary\ncells that express CD36. However, there was no change in IL10 expression in orthotopic tumors\nin either Thbs1-/- or Cd36-/- mice compared to that in the control (new Fig. 4f and Supplementary\nFig. 2r). Furthermore, there was no distinct difference in TAM population or expression of TAM-\nrelated genes between WT and Thbs1-/- mouse tumors (Supplementary Fig. 2p, 3e, 3f). These\ndata suggest that macrophage/IL10 is not involved in the immunosuppressive activity induced by\nthe THBS1-CD36 axis in this model. Notably, the THBS1-induced suppressive effect on T cell\nactivation was also impaired in CD36-deficient CD8+ T cells (Fig. 4i). These data indicate that\nboth CD47 and CD36 are involved in the immunosuppressive function of THBS1, wherein THBS1\ndirectly suppresses CD8+ T cell activity.\nAs suggested by the reviewer, we performed a vascularization assay. Consistent with our in vivo\ndata (new Fig. 2d and Supplementary Fig. 2m) and previous reports (Bagavandoss P et al., BBRC,\n1990; Dawson DW et al., J Cell Biol, 1997; Jiménez B et al., Nat Med, 2000), the addition of\nTHBS1 blocked the vascular formation of endothelial cells (C166) in vitro. Notably, the\nsimultaneous use of a CD36 inhibitor (but not a CD47 inhibitor) blocked the inhibitory effect of\nTHBS1 on angiogenesis (Fig. 4j). We understand that neither Cd47-/- nor Cd36-/- mice were able\nto completely phenocopy Thbs1-/- mice because both CD47 and CD36 are multifaceted and\nfunction differently depending on their binding partners. Other THBS1 receptors or ligands for\nCD47 and CD36 may explain these effects through parallel rather than direct mechanisms. A\nrecent study demonstrated that fatty acids suppress antitumor immune activity by binding to CD36\non CD8+ T cells (Ma, X et al., Cell Metab. 2021). However, our new findings that the loss of either\nCD47 or CD36 impairs THBS1-induced immunosuppression in CD8+ T cells provide a\nmechanistic insight into how THBS1 contributes to the formation of the immunosuppressive and\nangiogenic TME. We have included these new data in the revised manuscript.\n5. The authors mention THBS1 was increased in CMS4 and CCS3 CRC subtypes. Colorectal\nCancer Subtypes (CCS) classification is not widely used. Can the authors state the value of using\nthis classification system in this report? They should update their CMS classification (I Jonito et\nal., Nature Genetics 54:963-975, 2022).\nThank you for this helpful suggestion. As the reviewer has suggested, the CCS classification has\nnot been widely used because it was integrated into the CMS classification (Guinney J et al., Nat\nMed, 2015). In addition, considering that the dissection of cellular and molecular characteristics\nof individual cell types are necessary to precisely understand the biology of each classified CRC,\nthe updated CMS classification reported by Jonito et al., who used scRNA-seq for their\nclassification, should be included in our analysis. Therefore, we replaced the CCS classification\nwith the updated CMS classification (referred to as the IMF classification). Importantly, we\ndiscovered that i3-MSS-F CRC, a subtype with the poorest prognosis, exhibited the highest\nTHBS1 expression, followed by i2-MSS-F CRC. These data further support our hypothesis that\nTHBS1 plays a pivotal role in the progression of aggressive CRC with a mesenchymal phenotype.\nThe new data are shown in Fig. 1d.\n6. The authors are encouraged to examine whether THBS1 is increased in MSS vs MSI-H CRC.\nOur bioinformatics analyses of TCGA samples showed that MSS CRC exhibited higher THBS1\nexpression than MSI-H CRC (Fig. 7a). Since MSS CRC is a mixed cohort, including both stroma-\nactive (THBS1 high) and stroma-inactive CRC (THBS1 low), the result becomes more evident\nwhen we sub-group the samples following the IMF classification (see the above comment #5).\nTHBS1 expression in i3-MSS-F CRC was higher than that in MSI-H CRC (Fig. 1d). These data\nare consistent with our hypothesis that THBS1 induces an immunosuppressive TME in CRC with\na mesenchymal phenotype. This was further supplemented by the analyses of our own TMA\ncohorts that demonstrated the association of THBS1-high CRC with a low frequency of MSI-H,\nand multivariable logistic regression analysis, which showed that high THBS1 expression was\nindependently correlated with MSS (new Fig. 7b and Supplementary Fig. 1m).\nReviewer #2: with expertise in colorectal cancer, metastasis\nWe are pleased to note that the reviewer is of the opinion that “the authors are to be recommended\non a very extensive array of experimentation, the use of different transgene mice and the\nnumerous innovative approaches.”\nHowever, data supporting the contribution of myeloid cell-derived THBS1 in CRC are limited, and\nthere are some confusing points for explanation or further experimentation”.\nThank you for your valuable comment and suggestion. We hope that our extensive revisions and\nnew data will clarify our message and satisfy your concerns.\n1. The authors found that THBS1 expression is mainly in the stroma of CRC samples by IHC\nstaining in Fig.1a, while the mRNA expression of THBS1 were analyzed in Fig.1d by using TCGA\ndatasets, in which were mainly tumor tissues. Therefore, the bioinformatic analysis in TCGA is\nnot representative, also for the survival analysis. It is better to use your own TMA array to do the\nsurvival analysis.\nThank you for this helpful suggestion. We understand the concern raised by the reviewer;\nhowever, we still find it worth using the TCGA dataset in our analyses, particularly as a\nsupplement to the results from our own TMA cohort. Although TCGA samples did not discriminate\nbetween tumor and non-tumor cells (including stroma and infiltrating immune cells), the gene\nexpression profile of bulk tumor tissues still reflected the distinctive characteristics of whole\ntumors (not just tumor cells). For example, the consensus molecular subtyping (CMS)\nclassification, which is based on the mRNA expression of all cell types in bulk tumor tissues,\nhas been widely used to study the role of molecules expressed in the TME. In particular, CMS4 CRC,\nwhich is highly desmoplastic and contains a large number of stromal components, exhibits high\nexpression of stroma-related genes. This indicates that gene expression profiles in bulk tissues\ncan reflect the characteristics of stroma-rich, aggressive CRC and that THBS1 may play a role in\nthis. We agree with the reviewer that drawing solid conclusions based solely on analyses of bulk\ntissues in TCGA is not an adequate approach and that it is essential to reinforce the data by\ncombining it with other approaches, including TMA analyses and single-cell transcriptomes, as\nwe have done in this study. To respond to this reviewer, we have moved TCGA analysis data\n(Fig. 1d in the previous version) to Supplementary Fig. 1b and 1c and have also included the\nanalyses using the updated CMS classification (new Fig. 1d; I Jonito et al., Nat Genet, 2022), in\nwhich gene expression analyses at single-cell levels were applied to TCGA samples, which may\nfurther strengthen the data presented here. Furthermore, we improved the analysis of our TMA\nsamples for multiple prognostic factors and survival (new Fig. 1m, 1n, new Supplementary Fig.\n1l, 1m and Supplementary Tables 1–4).\n2. Fig.1i, the authors revealed the relationship between THBS1 expression and masson/CD14 by\nshowing IHC staining. For the same CRC samples, it is better to do the staining on the consecutive\nsections, which will be more representative.\nFollowing this comment, we performed histological analyses with IHC using consecutive sections\nto strengthen our conclusion (new Fig. 1g and Supplementary Fig. 1j). We observed a correlation\nbetween THBS1 expression and the expression of other markers (Masson’s trichrome, CD14,\nCD68, and CD11b).\n3. The study focus on the TME-derived THBS1 in CRC. The CRC cells can also produce THBS1,\nthe role of tumor cell-derived THBS1 should be investigated and excluded. How is the THBS1\nexpression in tumor cells? The expression of THBS1 in organoid and MC38 cells should be\ndetected. Whether the tumor cell-derived THBS1 can contribute CRC progression?\nThank you for the suggestion. To compare THBS1 expression levels in the tumor epithelium and\nstroma, we performed RNAscope analysis on both human CRC samples and mouse MTO\northotopic tumors. RNAscope analysis demonstrated that THBS1 was strongly expressed by\ninfiltrating cells in the tumor stroma and was not detectable in the tumor cells (new Fig. 1b and\nSupplementary Fig. 2c). Although not detectable by IHC and RNAscope, quantitative RT-PCR\nanalyses revealed a very low level of THBS1 expression in (EPCAM+) MTO cells (new\nSupplementary Fig. 8e; approximately 5% of that in BM-derived cells). MC38 cells did not exhibit\nsignificant THBS1 expression (new Supplementary Fig. 8e). To investigate the potential\ncontribution of low levels of THBS1 produced by MTO cells, we generated THBS1 knockdown\nMTO (Thbs1 KD) and compared them with shNT MTO (scramble). Thbs1 KD MTO showed no\nchange in growth or metastatic potential in the splenic injection experiment (new Supplementary\nFig. 4b–g). These data clearly support our notion that TME-derived THBS1 contributes to the\nprogression of CRC with a mesenchymal phenotype.\n4. For the bioluminescence imaging, such as. Fig.2e, 5e,6i, 6r, 7d, 7i, 7q, the authors showed the\nprimary lesion(white), lymph node (orange), and liver (yellow) metastasis. How did the authors\ndefine the signaling are in the lymph node or liver? It is better to combine CT with IVIS imaging to\nbetter define the lymph node and live. Or to isolate the lymph node and liver, to do the ex -vivo\nimaging, to check the luciferase signaling from lymph node and liver.\nThe bioluminescence signal results were verified by macroscopic and histological findings to\nconfirm whether the positive signals represented lymph nodes or liver metastases. In addition, ex\nvivo IVIS imaging confirmed metastasis to the liver and lymph nodes and demonstrated positive\nsignals (new Supplementary Fig. 4a). However, we did not always confirm this using ex vivo\nimaging in the experiments in which we applied bioluminescence imaging. Therefore, we have\navoided discriminating between lymph nodes and liver metastases on imaging in the revised\nmanuscript. We have now denoted it as just “distant metastasis” instead of “lymph node\nmetastasis” or “liver metastasis.”\n5. Fig.3j, the authors performed the stimulation experiment on CD3+T cells with and without\nrecombinant THBS1 treatment (25ug/ml). How did the authors define the concentration of\nrecombinant THBS1? Is this concentration-dependent effect? Why using the CD3+T cells? It is\nbetter to isolate the CD8+T cells to perform this experiment. For the activation of T cells, normally\nboth anti-CD3 and anti-CD28 were used. Why did the authors use anti-CD3 only, which is not\nenough to activate T cells?\nThank you for the suggestion. For the experiment indicated (Fig. 3j in the previous manuscript),\nwe referred to a previous report that demonstrated an inhibitory effect of THBS1 on T cell\nactivation (Li Z et al., J Immunol, 2001), in which the authors used anti-CD3 to stimulate T cells\nand the concentration of recombinant THBS1 was set in the range of 0 to 62.5 ug/ml. They found\nthe inhibitory effect of THBS1 at a concentration above 12.5 ug/ml and used mainly 30 ug/ml to\nstimulate Jurkat T cells. We tested 12.5, 25, and 62.5 ug/ml and found that 25 ug/ml of THBS1\nshowed a consistent inhibitory effect on T cells isolated from mice. We agree with the reviewer\nthat it would be better to use anti-CD3 and anti-CD28 as stimuli and analyze CD8+ T cells in this\nexperiment. We analyzed CD8+ T cells sorted by flow cytometry and found that THBS1 treatment\nsuppressed CD8+ T cell activation, which was mediated by anti-CD3 and anti-CD28 antibodies in\na concentration-dependent manner (new Fig. 2m–o).\nIn addition, the protein expression of IFN-g and CTLA4 should be checked by flow cytometry,\nwhich makes more sense.\nFollowing this comment, we used flow cytometry to examine the expression of IFNg, CTLA4, and\nother molecules. The new data are included in new Fig. 2n and 2o.\n6. Fig.6f, the magnified region should be marked in the figure, to make it more visible and\nobvious.\nThe magnified region of the corresponding figure has been marked in the revised version of the\nmanuscript (new Fig. 6f).\n7. Sup Fig.4c, the full WB blot should be provided, and the molecular weight should be marked.\nFull WB has been included and the molecular weight is marked in the revised version of the\nmanuscript (new Supplementary Fig. 6c).\n8. Sup Fig5a, Sup Fig.6h, the scale bars for THBS1 and CD31 staining were different.\nWe corrected the scale bars in the corresponding figures (new Supplementary Fig. 7a and 8k).\nReviewer #3: with expertise in bioinformatics, immunology\nWe are pleased to see that this reviewer appreciates the impact of our study (“this manuscript\nreveals a clear mechanism in a frequent, aggressive cancer for recruitment of bone marrow\nderived myeloid populations, their contribution to generating an immunosuppressive\nmicroenvironment, and their relationship with metastasis”) and finds that “This could be\nappropriate for Nature Communications”.\nHowever, “there are a few major issues (see below) that need to be addressed before this is\nclear”.\nWe would like to thank the reviewers for their insightful suggestions, which helped us improve our\nstudy. We have revised the manuscript and incorporated new data in response to the reviewers’\ncomments. We hope that the reviewer will find our revisions satisfactory and suitable for\npublication.\nMajor comments\n1. The correlative analyses in Figure 1 and Supplementary Figure 1 connecting THBS1\nexpression with eg prognosis, CRC subtype, gene expression profiles, etc need to more clearly\ndemonstrate how much THBS1 independently drives these patterns versus just being a marker\nof the CMS4 subtype.\nThank you for this helpful suggestion. We performed univariate and multivariate analyses to\ninvestigate the association between THBS1 expression and prognosis (overall mortality, cancer-\nrelated death, and recurrence), CRC subtype (clinical stage (I–III), invasiveness (T1–4), primary\ntumor location, lymph node metastasis (N0–3), lymphatic invasion, venous invasion,\nundifferentiated/mucinous histology, and MSI/MSS), and gene expression profile (CMS4 or not).\nUnivariate analysis (new Supplementary Fig. 1l) demonstrated a significant association between\nTHBS1 expression and aggressive characteristics (advanced clinical stage, lymph node\nmetastasis, positive undifferentiated histology, and MSS) and a poorer prognosis (high incidence\nof recurrence, increased mortality, and cancer-related death). Importantly, multivariate logistic\nregression analyses (new Supplementary Fig. 1m and Supplementary Tables 1–4) revealed that\nhigher THBS1 expression independently correlated with recurrence (OR = 2.48 (1.19–5.18), P =\n0.046), lymph node met positive (OR = 2.32 (1.27–4.22), P = 0.019), undifferentiated histology\n(OR = 5.72 (1.44–22.7), P = 0.022), and MSS (OR = 2.94 (1.50-5.74), P = 0.004). Regarding the\nassociation with patient survival, multivariate logistic regression analyses defined high THBS1\nexpression as an independent predictor of overall mortality (OR = 2.69 (1.21–5.99), P = 0.03) and\ncancer-related death (OR = 4.29 (1.52–12.0), P = 0.01), whereas multivariate Cox regression\nanalysis revealed no significant difference (HR = 1.19 (0.85–1.67), P = 0.291). The TMA samples\ncomprised only surgically resected samples (stages I–III) and did not include stage IV CRC with\nmetastases, in which THBS1 plays an important role, which might have hindered the acquisition\nof more obvious results. However, the Kaplan-Meier curves revealed a correlation between higher\nTHBS1 expression and lower overall, disease-specific, and recurrence-free survival rates\n(new Fig. 1m and 1n). Although there were some limitations to our analyses, these results clearly\nsuggest a possible role of THBS1 in independently driving the aggressive patterns of\nmesenchymal CRC.\nAdditionally, much more justification/information is needed for the splitting of samples by\ntiers of THBS1 expression. In Fig 1 d-f, why is it not just by median ?\nWe downloaded the survival data from the Human Protein Atlas\n(https://www.proteinatlas.org/humanproteome/pathology/method) and Prognoscan\n(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2689870/), which are widely used web-based\nresources for survival analyses (we apologize for the lack of clarity in the previous manuscript).\nWe used the cut-off automatically set by these websites based on the FPKM value of each gene.\nThe data from these websites have been well-accepted and published in several papers (e.g.,\nGoto Y et al., Nat Commun. 2015 6:6153; Liu Y et al., Cancer Discov. 2013 3:870-9). However,\nin response to the concern raised by this reviewer, we have reanalyzed the survival of these\ndatasets by splitting samples just by the median to obtain the same n for both THBS1 high and\nlow groups. Using this new approach, we found that high THBS1 expression was associated with\na poor prognosis, and we have replaced the survival data in the revised manuscript (new Fig. 1f\nand Supplementary Fig.1f).\nIn Fig 1 o, why are intermediate and high grouped together ?\nIn response to the reviewer’s concern, we have separated the THBS1 high and THBS1\nintermediate groups and show the data of all three groups as high (n=107), intermediate (n=122),\nand low (n=102) in the revised figure. The log-rank test revealed significant differences in OS,\nDSS, and DFS. Please note that we have increased the number of TMA samples in the revised\nmanuscript. These new data have been included in new Fig. 1m and 1n.\n2. The timing in Figure 2 is not clear. If tumor is implanted at week 8, what does week 0 refer to\n?\nIn Figure 2 of the previous manuscript, “week 0” refers to the birth of the mice used for the\nexperiments. In the revised manuscript, we set “week 0” as the timing of tumor cell implantation\nin all figures to avoid any confusion.\nGiven the strong effects of Thbs1-/- early in Figure 2, especially e-g, the KM curves in panel h are\nvery surprising. Can the authors explain why there is just a modest delay and then all the Thbs1-\n/- die as rapidly as the WT ? Are they dying of the disease in a different way ?\nAlthough Thbs1-/- mice showed a clear reduction in metastases and an increase in apoptotic cells\nin tumors, there was sustained growth of the primary tumors. In the revised manuscript, we have\nextended our analyses to include a larger number of mice. We observed longer survival in Thbs1-\n/- mice implanted with MTO in the rectum (new Fig. 3d). However, growing primary tumors\neventually killed most of the Thbs1-/- mice, although Thbs1-/- mice did not show distant metastases\nor peritoneal dissemination, which frequently occurred and killed WT mice in the experiment.\n3. In all the scRNAseq analyses, more extensive explanation/justification must be provided for all\ncluster annotations (eg with heatmaps, differential gene expression analysis, marker genes, or\nother approaches).\nAs requested, we have provided a more extensive explanation by including heat maps and marker\ngenes in the revised manuscript for all scRNA-seq analyses (new Fig. 2, Fig. 5, Supplementary\nFig.3 and Methods).\n4. The results in Fig 7 on THBS1 inhibition in combination with other treatments do not\ndemonstrate a compelling synergistic effect and therefore should not be highlighted in the title\nand abstract.\nWe agree with this observation. As suggested by the reviewer, the most compelling result of\nThbs1-/- mice in this study is the suppression of metastasis. We toned down our claim regarding\nthe synergistic effects of THBS1 inhibition in combination with other treatments in the revised\nmanuscript, including the title and abstract.\nRelatedly, survival analysis and tumor growth kinetics are standard analyses in these types of in\nvivo tumor treatment models – why are they not included here ?\nThe evidence connecting THBS1 to metastatic activity (eg Fig 2g, 4e, 5f, 6h and to a lesser extent\n6s), conversely, is much more compelling and perhaps does warrant highlighting early and clearly.\nIn response to the comments of the reviewer, the tumor growth kinetics of all treatment\nexperiments have been included in the revised manuscript (new Fig. 7f and 7n). Survival data\nwere not available for these experiments because the treatment duration was too short to analyze\nits effect on survival. The treatment duration was set by referring to a previously published study\nin which similar treatments were performed (Martinez-Ordonez et al., Cancer Cell, 2023). We also\nobserved that many mice, including both WT and Thbs1-/- mice, became sick and some died when\ntreated with anti-PD1 or anti-VEGFR antibodies for more than 4 weeks, probably due to the\nanaphylaxis induced by those antibodies. Repeated (4–5 times) administration of anti-PD1 and\nanti-PDL1 monoclonal antibodies was reported to induce fatal xenogeneic hypersensitivity\nreactions (IgG mediated pathway of anaphylaxis) specifically in a murine cancer model (Mall et\nal., Oncoimmunology, 2016). This phenomenon is not restricted to anti-PD1/PDL1 antibodies,\nand other monoclonal antibodies also induce IgG mediated anaphylaxis when used in mice\n(Murphy et al., Blood, 2014). We anticipate that this fatal adverse effect would make it difficult for\nus to interpret the survival data even if we extend the treatment duration.\nAs suggested by the editor and the reviewer(s), the most compelling result of Thbs1-/- mice is\nsuppressed metastasis. Therefore, considering the limited space of this study, we focused more\non investigating the role of THBS1 in metastatic activity than the synergistic effect of combination\ntreatment. We have extensively analyzed the differences in the immune TME between liver\nmetastases and primary tumors and have highlighted how THBS1 inhibition affects the immune\nTME of liver metastases compared to that of primary tumors (new Fig. 3 and Supplementary Fig.\n5) instead of expanding our analyses on the synergistic effect of combination treatment. Using\nthis approach, we believe that we have significantly improved the manuscript.\nMinor comments\n1. (line 108) “These results suggest that the stroma-infiltrating cells are the primary source of\nTHBS1 and demonstrate a strong association between high THBS1 level, aggressive phenotype,\nand poor prognosis in human CRCs” and Fig. 1c\nThe expression of THBS1 appears higher in the normal colon than in the tumor epithelium.\nIs it significantly different? Can you provide a possible explanation? Additionally, the violin\nplots should be replaced by individual data points since n is relatively low. Finally, at this\npoint in the manuscript it is demonstrated that THBS1 expression comes from the stroma,\nbut not that it is from infiltrating cells.\nYes, there was a significant difference between the normal colon and the tumor epithelium.\nHowever, normal colon samples include both the epithelium and stroma, which complicates the\ninterpretation of the results. Furthermore, n of the “normal colon” group was just 8, which was\nlower than other groups. Therefore, we have removed the “normal colon” group from the analysis\nin the revised manuscript. In addition, as requested by the reviewer, the violin plots have been\nreplaced with individual data points (new Fig. 1c).\nFinally, at this point in the manuscript it is demonstrated that THBS1 expression comes from the\nstroma, but not that it is from infiltrating cells.\nAccording to this comment, we have changed our description from “stroma-infiltrating cells” to\n“stroma” in the corresponding part of the revised manuscript.\n2. Fig. S 1d, e\nWhy are there different n for the THBS1 high and low categories between these two plots\nfrom the same dataset/cohort?\nAs explained above (major comment #1), the splitting of the samples was based on a web-based\nresource in the previous version of the manuscript. We have now split the data by the median to\nhave the same n for comparing the THBS1 high and low groups (new Supplementary Fig. 1f).\n3. Fig. 2h,i,j\nCan these be re-sized to avoid having the awkward “x10” above the vertical axes in the plot\nat far right? It is currently confusing to compare with results of similar experiment in panel\nc.\nWe resized these graphs to avoid confusion (new Fig. 3f and 3h).\n4. Fig. 3c\nAlthough the statistical test (Mann-Whitney, aka Wilcoxon) indicates the differential\nexpression is significant, the violin plots are not convincing. Perhaps another type of\nvisualization would be more useful? Ideally, validation by FACS would be even better.\nAs substantial number of samples with “zero” expression were included in both groups for these\ngenes, the results seemed unconvincing, as the reviewer suggested. Therefore, we have\nremoved these data from the revised manuscript. In response to this reviewer’s comment, we\nperformed FACS analyses to validate the results and found a reduction in CTLA4 but not in\nPDCD1 (new Supplementary Fig. 3h). Consistent with this result, double-immunostaining\ndemonstrated a reduction of CTLA4+ exhausted CD8+ T cells and increase in TCF7+ active CD8+\nT cells in Thbs1-/- tumors (new Fig. 2p). This was further supported by in vitro experiments,\nwhich demonstrated that THBS1 directly increased the proportion of CTLA4+CD8+ cells in a dose-\ndependent manner (new Fig. 2o).\n5. (line 197) and Fig. 3k\nPlease provide a citation for these genes as IFNg target/marker genes. Similar for all\nsignatures/gene sets analyzed in the paper.\nWe have provided a citation for the IFN gamma signature genes in the Methods section, which\nwere obtained from the Hallmark Collection of MSigDB.\n6. (line 231-232) and Figure 4\nThe phenocopies between Thbs1-/-, CD36-/-, and CD47-/- are clear. However, attributing\nthis directly to interactions between THBS1 and these proteins and referring to “THBS1-\nCD47 and THBS1-CD36 axes” seems premature without double mutants since, at least a\npriori, other receptors for THBS1 or other ligands for CD47 and CD36 could explain these\neffects through parallel rather than direct mechanisms.\nDouble immunofluorescence confirmed that CD8+ T cells infiltrating orthotopic MTO tumors\nexpressed both CD47 and CD36. In vitro experiments analyzing CD8+ T cells from WT, CD47-/-,\nor CD36-/- mice revealed that loss of either CD47 or CD36 significantly impaired the suppressive\neffect of THBS1 on activation of CD8+ T cells (new Fig. 4h and 4i). These data suggest that both\nCD47 and CD36 are involved in the immunosuppressive function of THBS1 to directly suppress\nCD8+ T cell activity. However, we agree that we cannot exclude the possibility that other receptors\nfor THBS1 or ligands for CD47 and CD36 could explain these effects through parallel mechanisms.\nWe revised the description in the revised manuscript to avoid the overstatement.\n7. Fig. 5i-k\nThese same genes should also be shown in the mouse scRNAseq data.\nAs requested, we have provided the plots of mouse scRNA-seq data of orthotopic MTO tumors\n(new Fig. 5h–j and Supplementary Fig. 3c–d,7c). Please note that marker genes to define human\nmonocytes and mouse monocytes are different (Geissmann F, et al, Immunity, 2003:19(1):71-82;\nZiegler-Heitbrock L, et al. Blood. 2010:116(16):e74-80; Zilionis R, et al. Immunity.\n2019:50(5):1317-1334 e10). In our analysis, monocyte-like cells showed a gene expression\npattern of classical monocytes (CD14+, CD16- in human CRC; Ly6Chi, CCR2+, CX CR1int in mouse\n2010:70(14):5728-5739, Franklin R.A. et al. Science. 2014:344(6186):921-925).\n8. Fig. S 5b\nAs with major comment 1, provide these plots divided by CRC categories.\nAs requested, we have provided plots for each CRC category (Fig. 5p).\n9. Fig. 6b\nPlease provide the total number of cells in each condition in addition to the percentage to\nrule out eg a decrease in lymphocytes.\nWe have included the total number of cells in the figure (Fig. 6b).\n10. (lines 303-304) Fig. 6l and Supplementary Figure 6g\nPlease provide statistical tests that these genes are specifically upregulated in mesenchymal\nCMS4 CRCs.\nIn response to the comments from the reviewer, we have reanalyzed the TCGA dataset and\nprovided the data of statistical tests for genes that were specifically upregulated in mesenchymal\nCMS4 CRCs (new Supplementary Fig. 8i).\n11. (lines 303, 307) Fig. 6m and Supplementary Figure 6g\nIt is confusing that CCL2 is high in CMS4 from TCGA but then lower in the MTO compared to\nMC38. Can the authors provide any hypotheses?\nThank you for this comment. The importance of CCL2 in MC38-derived tumor models has been\nreported in several papers (e.g., Zhao L et al., Hepatology. 2013:57(2):829-39; Tu W et al., Cell\nDeath Dis. 2021 Sep 27;12(10):882). Together with our bioinformatics data showing that CCL2 is\nupregulated in both CMS1 and CMS4 compared to the other groups (CMS2 and CMS3; new\nSupplementary Fig. 8h), we anticipated that CCL2 expression would be potentially induced by a\ncertain inflammatory condition, regardless of whether it is an antitumor or pro-tumor immune\nresponse. We observed that CCL2 expression was downregulated in MTO-derived tumors.\nAlthough MTO is an excellent model of CMS4 CRC (Tauriello DVF, Nature, 2017), it may\nrepresent a subtype of CMS4 CRC in which CCL2 may not be involved. Recent updates on the\nCMS classification (IMF classification; I Jonito et al., Nature Genetics 54:963-975, 2022)\ncategorized CMS4 CRC into two distinct groups: i3-MSS-F CRC and i2-MSS-F CRC, suggesting\nthat CMS4 is still a heterogeneous group. This may indicate that not all CMS4 showed high CCL2\nexpression, as MTO did not. To robustly examine the role of CCL2 in CMS4 CRC, a panel of\nCMS4 CRC organoids (ideally derived from human CMS4 CRCs) would be required for the\northotopic model, which will be achieved in future studies. Due to the limited space of the paper,\nwe were unable to discuss this in depth; however, we have emphasized that CMS4 is not a\ncoherent group and may create a different array of chemokines depending on its epithelial\nsubtype in the revised manuscript.\n12. Fig. 7\nWhy does the data for the WT and Thbs1-/- control conditions appear identical in panels f\nand s, but different from same conditions in panel n?\nWe apologize for the inappropriate presentation of data. We performed a treatment experiment\nwith anti-PD1 and anti-VEGFR antibodies as one experiment and compared it with one control\ncondition (i.e., the experiment was performed with three conditions: control vs. anti-PD1 ab-\ntreated vs. anti-VEGFR ab-treated). We have corrected the description in the text and presented\nthe figures.\nAll violin plots in this figure should be replaced by individual data points.\nAs requested by the reviewer, we have replaced all violin plots with by individual data points in\nthe figure in the revised manuscript.\nThe evidence that combination therapy is synergistic or even additive here, outside of potentially\npanels e and r, is not compelling.\nWe agree with the reviewer’s comments. We toned down our claim regarding the synergistic\neffects of THBS1 inhibition in combination with other treatments in the revised manuscript.\nReviewer #4: with expertise in cancer immunology, myeloid cells\nWe thank the reviewers for their valuable comments and suggestions. Following the reviewer’s\ncomments, we have extensively revised the manuscript to reveal the role of THBS1 in the\nregulation of the immune TME, with a particular focus on the difference between primary tumor\nand metastasis, which provides great insight into why THBS1 inhibition primarily suppresses\nmetastasis. We believe that we have addressed all the concerns raised by the reviewer and hope\nour revisions are found satisfactory and suitable for publication.\n1. The authors observed a substantial increase in CD8+ T cell infiltration and cleaved\ncaspase-3 (C-Cas3)+ apoptotic cells in orthotopic tumors from Thbs1-/- mice. Treatment with\nanti-CD8 antibody effectively depleted CD8+ T cells and, concomitantly, decreased apoptotic\ncells in the primary tumors (Fig. 2i,j), confirming the cytotoxic activity of the infiltrating CD8+ T\ncells. How do the authors explain that there is no significant difference in tumor growth in KO\nmice? If THBS1 contributes to immunosuppression, why are T cells not able to kill tumors in KO\nmice?\nWe thank the reviewer for this valuable comment. Primary tumors in Thbs1-/- mice exhibited an\nincreased number of C-Cas3+ apoptotic cells and extensive necrotic areas compared to those in\nWT mice, which was consistent with the substantial increase in CD8+ T cells infiltrating the primary\ntumors. However, the size of the primary tumor was comparable between WT and Thbs1-/- mice.\nIncreased angiogenic activity in Thbs1-/- mice may explain this controversy in part. However, these\ndata rather indicated that THBS1-mediated immunosuppression was more pronounced in\nmetastatic lesions than in primary tumor sites. Of note, our additional data on comparison of the\nimmune TME revealed that although both primary and metastatic sites in Thbs1-/- mice showed\nincreased CD8+ T cell infiltration, the degree of increase was greater in metastasis than in primary\ntumor (new Fig. 3m and Supplementary Fig. 5a), suggesting the stronger induction of antitumor\nimmunity in metastasis by THBS1-deficiency. We propose that this different immune activity\nbetween lesions was attributed to the difference of MDSC accumulation in these lesions (new Fig.\n3p,q and Supplementary Fig. 5d). Thus, THBS1 inhibition mainly affected the development of\nmetastasis rather than primary lesions. We have described these issues in more detail in our\nresponses to the following comments (please see #2, #3, and #4).\n2. Thbs1-/- mice showed increased vascular formation, thus I was wondering if there is a change\nin the myeloid compartment that is known to contribute to the immunosuppressive TME. No\nchanges In immunosuppressive myeloid cells infiltration may explain why T cells are still\nineffective in killing tumors in those KO mice.\nWe performed extensive analyses to study the immune phenotypes of primary tumors and\nmetastases. scRNA-seq suggested that there was a change only in the CD8+ T-cell population\nand not in other populations, including the myeloid compartments, in the immune TME of primary\ntumors. These results were confirmed by FACS (Supplementary Fig.2p). Further, we have\nprovided data on the M2 macrophage-related gene signature, which represents the\nimmunosuppressive function of the myeloid lineage, and their marker genes showed no significant\ndifferences between WT and Thbs1-/- mice (new Supplementary Fig. 2r and 3f). Thus, as the\nreviewer suggested, while CD8+ T cells were increased and more active in the primary tumors of\nThbs1-/- mice, these changes were still insufficient to reduce the primary tumor size.\n3. The authors showed reduced metastatic lesions, therefore it would be critical to compare\nthe immune TME and THBS1 levels in primary tumor vs metastatic lesions. Such differences\nin immune composition and THBS1 levels may explain why there is reduction in metastasis\nlesions only.\nThis is a critical question, and we have made a huge effort to address it (see the new Fig. 3l–s\nand Supplementary Fig. 5a–d). Following the reviewer’s suggestion, we first compared THBS1\nlevels in primary tumors and metastases of MTO-bearing WT mice. However, THBS1 levels were\ncomparable between the two groups, indicating that THBS1 levels do not explain why metastasis\nwas more strongly suppressed at the primary site in Thbs1-/- mice (new Fig. 3l). Notably, a\ncomparison of the immune TME revealed a change in the number of infiltrating CD8+ T cells\nbetween primary and metastatic sites. Although both sites in Thbs1-/- mice showed increased\nCD8+ T cell infiltration, the degree of increase was greater in metastasis than in primary tumors,\nsuggesting a stronger induction of antitumor immunity in metastasis by THBS1-deficiency (new\nFig. 3m and Supplementary Fig. 5a). This point has been discussed further in the following section\n(#4), but it provides a clear explanation as to why the impact of THBS1 deficiency on\ntumorigenesis was more evident in metastasis. We next sought to figure out the underlying\nmechanism and found that metastatic tumors contained a significantly smaller number of\nCD45+CD11c-CD11b+Ly6C+Ly6G+PMN-MDSC compared to primary tumors in WT mice while the\nnumber of CD45+CD11c-CD11b+Ly6C+Ly6G- MO-MDSC was comparable between lesions (new\nFig. 3p,q and Supplementary Fig. 5d). This suggests that while both PMN- and MO-MDSC work\ntogether to suppress antitumor immune activity in primary tumors, MO-MDSC play a dominant\nrole in metastasis, at least in this model. Together with the fact that the monocyte lineage was a\nprimary source of THBS1 (new Fig. 5) and that THBS1 loss affected the suppressive function of\nMO-MDSC but not of PMN-MDSCs (see answer #5; new Fig. 3r), these data explain why the\nimpact of THBS1 inhibition was more pronounced on metastasis. We understand that there are\nlimitations to our analysis. The low incidence and small size of metastases in MTO-bearing Thbs1-\n/- mice hampered sufficient sample collection from metastases for most assays, including FACS.\nHowever, we believe that we have successfully addressed the fundamental question of why\nTHBS1 loss has a greater impact on metastasis than on the primary site in an adequate manner.\n4. The authors posited that increased infiltrating CD8+ T cells in the orthotopic primary site\nmay account for the reduction of metastatic burden in MTO-bearing Thbs1-/- mice. It is also\npossible that there is an increase of CD8 T cells in metastatic sites that may help to reduce\nmetastatic burden. Thus, what is the frequency of T cells in metastatic lesions in those KO\nmice? The authors showed that loss of THBS1 blocks T cell exhaustion and increases stem-like CD8+ T cells, what is the phenotype of T cells in primary vs metastasis?\nWe thank the reviewer for the helpful suggestions. As anticipated, immune phenotyping of the\nTME in primary and metastatic tumors by IHC/IF revealed that the increase in infiltrating CD8+ T\ncells was significantly and substantially greater in metastases than in primary sites in Thbs1-/- \nmice (new Fig. 3m and Supplementary Fig. 5a). We also performed splenic injection of MTO and\nfound that liver metastasis development was strongly suppressed in Thbs1-/- mice in this system\n(new Fig. 3i–k). THBS1 loss-mediated antitumor activity in the splenic injection model was\nabolished when CD8+ T cells were depleted (new Fig. 3i–k), further validating the importance of\ninfiltrating CD8+ T cells, particularly in metastatic lesions, for the antitumor immune response.\nConsistent with this notion, while CD8+ T cells in both primary tumors and metastases of Thbs1-/- \nmice showed high expression of the stem-like marker TCF7, metastases contained a higher\nnumber of TCF7-expressing CD8+ T cells than primary sites, indicating that the impact of THBS1\ninhibition on preventing CD8+ T cell exhaustion was more evident in metastasis (new Fig. 3s and\nSupplementary Fig. 5f).\n5. BM-derived monocyte-like cells are the primary producers of THBS1 in stroma-rich intestinal\ntumors. THBS1 affects the activation of T cells in vitro, therefore monocyte like cells may suppress\nT cell activation via the release of THBS1. Since myeloid cells used different mechanisms to\nsuppress T cells, I was wondering if myeloid cell derived THBS1 contributes to the\nimmunosuppressive activity of these cells.\nAs suggested by the reviewer, our data suggest that THBS1 contributes to the\nimmunosuppressive function of tumor-infiltrating myeloid-derived cells (MDSCs). Our in vitro\nexperiments using recombinant THBS1 demonstrated that THBS1 directly inhibited T cell\nactivation and that loss of receptor (either CD47 or CD36) impaired the THBS1-mediated\nsuppressive activity (new Fig. 2m–o, 4h and 4i). These data clearly suggest THBS1 produced by\nmyeloid cells affects the activation of T cells through binding to its receptors CD47 and CD36. To\nfurther clarify the association of THBS1 function and monocytic myeloid-lineage, we performed\nMDSC assays and investigated whether THBS1 inhibition affects the activity of PMN-MDSCs,\nMO-MDSC, or both. We isolated PMN- and MO-MDSC from WT and Thbs1-/- mice and measured\ntheir suppressive effects on CD8+ T cell activation induced by treatment with anti-CD3 and anti-\nCD28 antibodies. While THBS1 loss did not change the suppressive activity of PMN-MDSCs,\nit significantly impaired that of MO-MDSC (new Fig. 3r), which is consistent with our notion that the\nmonocyte lineage among myeloid cell compartments produces THBS1 and indicates that THBS1\ncontributes to the immunosuppressive activity of MO-MDSC. We also found that the accumulation\nof PMN-MDSCs was reduced in metastases compared to that in primary sites, whereas MO-\nMDSC accumulation was not different between primary and metastatic sites (new Fig. 3p,q and\nSupplementary Fig. 5d), suggesting that MO-MDSC play an important role in suppressing the\nantitumor immune response in metastasis, which is consistent with the fact that the impact of\nTHBS1 loss was more pronounced in metastatic lesions.\nMoreover, what is the expression of other suppressive markers in WT vs KO mice?\nIn response to the reviewer’s comment, we have examined other immunosuppressive markers\nby FACS, IHC/IF, and scRNA-seq analyses and found no significant difference in the infiltration\nof lymphocytes, including Tregs and suppressive myeloid cells (new Fig.2e, 3n–q and\nSupplementary Fig. 2o–q,3c,3e,5b–d). These results were further reinforced by the data in which\nthe macrophage-related suppressive gene signature and representative suppressive marker\nexpression were not significantly different between WT and Thbs1-/- tumors (new Supplementary\nFig. 2r and 3f).\n6. The authors found the highest Thbs1 expression in CD45+CD11b+CXCR4+ cells in BM of\nMTO-bearing mice, suggesting that THBS1-expressing CD11b+ cells are recruited via the action\nof CXCL12-CXCR4 signaling. The authors suggest that these cells\n(CD45+CD11b+CXCR4+) are monocyte like cells. However other myeloid cells expressed\nthese markers. Therefore to better understand the myeloid cell composition, the authors\nshould characterize CD45+CD11b+CXCR4+ cells in primary tumors and metastasis sites.\nIn response to the reviewer’s concern, we examined THBS1 expression in CD45+CD11b+CXCR4+\ncells from primary tumors and metastasis. FACS analyses revealed that\nCD45+CD11b+CXCR4+Ly6C+CCR2+ monocyte-like cells in tumors were the primary population of\nTHBS1-expressing cells at both primary and metastatic sites, compared to other cell populations,\nincluding CD45+CD11b+CXCR4+Ly6C+CCR2-, CD45+CD11b+CXCR4+Ly6C-,\nCD45+CD11b+CXCR4-, CD45+CD11b-, and CD45- cells. These data were further supported by\nOPAL-IF, which showed the co-localization of Ly6C, CXCR4, and THBS1 at both primary and\nmetastatic sites. These new data have been included in Fig. 6p,q, and Supplementary Fig. 8j.\n7. CD8+ T cells in 176 Thbs1-/- tumors exhibited a decrease in dysfunctional markers such as\nPdcd1 and Ctla4. How do the authors explain that anti-PD1 therapy may work in this model?\nFig. 3c in the previous version of the manuscript showed a statistically significant reduction in the\nmRNA expression of Pdcd1 and Ctla4. However, as suggested by the other reviewer, these\nresults did not appear convincing because the scRNA-seq data for these genes included\nsubstantial number of samples with \"zero\" expression in both groups. Therefore, we re-evaluated\nthe expression of these markers in Thbs1-/- tumors by FACS and found a significant reduction in\nCTLA4 but not in PDCD1 (new Supplementary Fig. 3h). Based on this, anti-PD1 therapy could be\nexpected to be more effective in Thbs1-/- tumors, but the combined effect of THBS1 inhibition and\nanti-PD1 ab was not compelling, despite significantly reducing the growth rate of primary tumors\n(new Fig. 7g). This could be because the infiltration of CD8+ T cells in the primary tumors of Thbs1-\n/- mice was not sufficient to kill all tumor cells, particularly in primary sites where PMN-MDSCs,\nwhich were not affected by THBS1 loss in their suppressive function (new Fig. 3r), could\ncompensate for the impaired function of MO-MDSCs (due to THBS1 loss).\nDo the mice survive longer?\nSurvival data were not available from this experiment, since the duration of treatment was too\nshort to analyze the effect on survival. The treatment duration was set by referring to a previously\npublished study in which similar treatments were performed (Martinez-Ordonez et al., Cancer\nCell, 2023). We also observed that many mice, including both WT and Thbs1-/- mice, became sick\nand some died when treated with anti-PD1 or anti-VEGFR antibodies for more than 4 weeks,\nprobably due to the anaphylaxis induced by those antibodies. Repeated (4–5 times)\nadministration of anti-PD1 and anti-PDL1 monoclonal antibodies was reported to induce fatal\nxenogeneic hypersensitivity reactions (IgG mediated pathway of anaphylaxis) specifically in a\nmurine cancer model (Mall et al., Oncoimmunology, 2016). This phenomenon is not restricted to\nanti-PD1/PDL1 antibodies, and other monoclonal antibodies also induce IgG mediated\nanaphylaxis when used in mice (Murphy et al., Blood, 2014). We anticipate that this fatal adverse\neffect would make it difficult for us to interpret the survival data even if we extend the treatment\nduration.\nThbs1-/- tumors and combo with anti-PD1 showed similar reduction in mets. However, tumors are\nsmaller in KO mice following treatment with anti-PD1. How do the authors explain that a reduction\nin primary tumor does not reflect in a significant decrease in number of mets? I have observed a\nsimilar trend in fig7m and s.\nBecause THBS1 inhibition did not reduce PDCD1 expression at the primary site, as previously\ndescribed (new Supplementary Fig. 3h), it is theoretically possible that a combination of THBS1\ninhibition and an anti-PD1 antibody exhibits a synergistic effect in both primary tumors and\nmetastases. However, in regards of metastasis, although the combination tended to reduce the\nnumber of both liver and lymph node metastases, the reduction was not statistically significant\n(new Fig. 7h). This could be because THBS1 inhibition itself strongly suppresses metastasis,\nmaking it difficult to observe a clear effect of the combination treatment on metastasis. This could\nalso explain why a clear difference in metastatic burden was not observed with other treatments,\nincluding anti-VEGFR antibodies and FOLFOX (new Fig. 7h,7p). These data strongly suggest,\nas suggested by the editor and the reviewer(s), that the most compelling result of Thbs1-/- mice is\nsuppressed metastasis. Therefore, considering the limited space of the paper, we focused more\non investigating the role of THBS1 in metastatic activity than on demonstrating the synergistic\neffect of the combination treatment. In the revised manuscript, we have toned down the claim on\nthis part and extensively improved our analyses to reveal the difference in immune phenotypes\nbetween primary and metastatic tumors.\nREVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors have addressed my previous concerns.\nThe manuscript is much improved.\nReviewer #2 (Remarks to the Author):\nThe authors have addressed all of my concerns, I do not have further concerns.\nReviewer #3 (Remarks to the Author):\nThe authors have done an excellent job of responding to my comments in the first round,\nparticularly concerning the survival analyses and the relative focus on metastasis vs drug\nsensitivity. The manuscript is significantly improved and I only have a few minor points.\nOtherwise I am supportive of publication.\nMinor comments:\n1. I would suggest explicitly adding the n in every figure panel analyzing cohorts, even if it is\nredundant (ie the same cohort is analysed in multiple panels). For example, Supplementary\nFigure 1 panels b, c, h, i, and k. This just makes it easier for readers in my opinion.\n2. Page 6, line 128. The reference to “Thbs1-/- tumors” is confusing. If I am not mistaken,\nthe MTO malignant cells in this experiment are Thbs1 proficient, but the recipient mouse is\nThsb1 -/-. Clarifying the language here would be helpful. The same issue comes up\nrepeatedly in this paragraph as well as for the CD47 and CD36 -/- mice on page 11.\n3. Specifying the concentration of THBS1 in figure 4h would help to compare with figure\n2n,o.\n4. The ex vivo T cell activation and cytotoxicity studies in Fig 2 m, n, o, and q are very\ninteresting and broadly consistent with the in vivo findings. However, the strong block of\nactivation ex vivo by THBS1 seems at odds with the strong enrichment for\nexhausted/dysfunctional CD8 T cells (which generally is associated with chronic TCR\nactivation) in THBS1 high tumors in vivo. It would be good to address this in the discussion,\nperhaps in relation to the strong, acute activation of the ex vivo protocol compared to the\nmore long-term and likely lower-level TCR triggering by endogenous antigens in vivo.\nReviewer #4 (Remarks to the Author):\nThe authors addressed all reviewer concerns. No further experiments/clarifications are\nrequired\nRESPONSES TO REVIEWERS' COMMENTS:\nWe would like to express our gratitude to all the reviewers for their meticulous review and insightful\nsuggestions, which have greatly contributed to improving the manuscript.\nReviewer #1 (Remarks to the Author): The authors have addressed my previous concerns. The\nmanuscript is much improved.\nWe are pleased to note that the reviewers have found that our revised manuscript has adequately\naddressed all the concerns raised. Thank you so much for the reviewers’ invaluable suggestions.\nReviewer #2 (Remarks to the Author): The authors have addressed all of my concerns, I do not\nhave further concerns”.\nWe genuinely appreciate the reviewers’ positive feedback regarding our additional research. the\nreviewers’ invaluable comments and suggestions have significantly contributed to enhancing the\nmanuscript.\nReviewer #3 (Remarks to the Author): The authors have done an excellent job of responding to\nmy comments in the first round, particularly concerning the survival analyses and the relative\nfocus on metastasis vs drug sensitivity. The manuscript is significantly improved and I only have\na few minor points. Otherwise I am supportive of publication.”)\nWe are pleased to note that this reviewer also acknowledges the enhancements made to our\nrevised manuscript. We have revised the manuscript in response to the reviewers’ comments.\nMinor comments\n1. I would suggest explicitly adding the n in every figure panel analyzing cohorts, even if it is\nredundant (ie the same cohort is analysed in multiple panels). For example, Supplementary\nFigure 1 panels b, c, h, i, and k. This just makes it easier for readers in my opinion.\nThank you for this helpful suggestion. We have added the n in every figure panel analyzing\ncohorts (new Fig. 1d, 1j, 2l, 5n, 5p, 6n, 7a and Supplementary Fig. 1b, 1c, 1g-1i, 1k, 3j).\n2. Page 6, line 128. The reference to “Thbs1-/- tumors” is confusing. If I am not mistaken, the\nMTO malignant cells in this experiment are Thbs1 proficient, but the recipient mouse is Thsb1 -/-\n. Clarifying the language here would be helpful. The same issue comes up repeatedly in this\nparagraph as well as for the CD47 and CD36 -/- mice on page 11.\nIn response to the reviewer’s concern, we have revised the reference from “Thbs1-/- tumors” to\n“tumors in Thbs1-/- mice” through the whole manuscript to avoid any confusion. The description\nof “Cd47-/- tumors” and “Cd36-/- tumors” has been revised in the same way.\n3. Specifying the concentration of THBS1 in figure 4h would help to compare with figure 2n,o.\nAs requested, we have provided the information about concentration of THBS1 in Fig. 4h.\n4. The ex vivo T cell activation and cytotoxicity studies in Fig 2 m, n, o, and q are very interesting\nand broadly consistent with the in vivo findings. However, the strong block of activation ex vivo\nby THBS1 seems at odds with the strong enrichment for exhausted/dysfunctional CD8 T cells\n(which generally is associated with chronic TCR activation) in THBS1 high tumors in vivo. It would\nbe good to address this in the discussion, perhaps in relation to the strong, acute activation of the\nex vivo protocol compared to the more long-term and likely lower-level TCR triggering by\nendogenous antigens in vivo.\nThank you for pointing this out. As suggested by the reviewer, because of a difference in the\nexperimental models, THBS1 exerts its suppressive effect on CD8+ T cell activity in a different\nway between ex vivo/in vitro (strong block of activation) and in vivo conditions (induce\nexhaustion/dysfunction). We have included the discussion about this issue in the reviesed\nmanuscript.\nReviewer #4 (Remarks to the Author): The authors addressed all reviewer concerns. No further\nexperiments/clarifications are required.\nThank you very much for your valuable comments and suggestions for enhancing our manuscript."}]

Decision letter