Fig 2: α5 in fibroblasts affects the levels of ECM proteins deposition in CRC stroma. (A) Top biological processes of GO analysis with the upregulated gene clustered in ITGA5+CAFs compared with that of non-CAFs in CRC scRNA-seq database. (B) Top significant ECM genes in ITGA5+CAFs population. (C) Representative IHC staining of α5 and CAFs markers in colorectal adenocarcinoma tissues. Scale bar: 200 µm. (D) Pearson’s correlation between the expressions of α5 and CAFs or ECM markers in 29 colorectal adenocarcinoma tissues evaluated by IHC. (E) Heatmap showing association of mRNA expression in 517 colorectal adenocarcinomas extracted from TCGA database between ITGA5 and 18 ECM genes that are statistically significant (p<0.05) by Pearson’s correlation. (F) Differential mRNA expression of ECM genes in CCD-18Co cells after ITGA5 depletion evaluated by qPCR in triplicate. Each gene expression was relative to vector control (set at 1.0), statistically significant (p<0.05) by Student’s t-test. (G) Representative Masson’s trichrome staining of collagen in tumor tissues approaching tumor border following therapeutic anti-α5 and anti-PD-L1 treatment in CT26.WT tumors. Scale bar: 200 µm. (H) Quantification of per cent collagen area per field of tumor tissues using Masson’s trichrome staining. n=7 mice per group. Error bars, mean±SEM; *p<0.05, **p<0.01; ***p<0.001; ns, not significant; one-way analysis of variance. CAFs, cancer-associated fibroblasts; CRC, colorectal cancer; ECM, extracellular matrix; GO analysis, gene ontology analysis; IHC, immunohistochemistry; mRNA, messenger RNA; PD-L1, programmed death ligand 1; scRNA-seq, single-cell RNA sequencing. TCGA,

Fig 3: Targeting integrin α5 in fibroblasts potentiates colorectal cancer response to PD-L1 blockade. (A) Violin plots showing ITGA5 expression levels in CAF subclusters of fibroblasts in colorectal cancer scRNA-seq database. (B) Proportions of different fibroblast populations in normal tissues, tumor border and tumor core. (C–E) α5 deletion in fibroblasts enhances responsiveness to anti-PD-L1 treatment. Mouse MC38 cells were co-transplanted with mouse fibroblasts (vector control or α5 KO) subcutaneously into C57BL/6 mice on day 0, anti-PD-L1 antibody or isotype IgG were administrated four times every 4 days starting from day 8, followed by examining tumor growth. (C) Average tumor growth curves (n=5 mice per group; *p<0.05, two-way ANOVA with Bonferroni’s post-tests). The x axis represents days after implantation. (D) Photographs of dissected tumor samples, and (E) tumor weight on day 23 after implantation. n=5 mice per group. Error bars, mean±SEM; *p<0.05, **p<0.01, ***p<0.001; ns, not significant; one-way ANOVA. (F, G, H) Tumor regression following therapeutic anti-α5 and anti-PD-L1 treatment in CT26.WT tumors. CT26.WT cells were injected subcutaneously, and tumor volume was monitored two times a week. Mice were grouped when the tumor volume reached approximately ~100 mm3, anti-PD-L1 and anti-α5 were administered intraperitoneally two times a week from day 8 to day 21. (F) Tumor growth curves (n=7 per each group). (G) Average tumor growth curves (n=7 mice each group; mean±SEM; ns, not significant; **p<0.01; two-way ANOVA with Bonferroni’s post-tests). (H) Analysis using SynergyFinder V.3.0 showed synergistic combinations of anti-PD-L1 and anti-α5 on day 21. ANOVA, analysis of variance; CAFs, cancer-associated fibroblasts; CR, complete response; PD-L1, programmed death ligand 1; scRNA-seq, single-cell RNA sequencing.