Falong Zou

The primer sequences for RT-qPCR.

Differential expression analysis of the transcriptome of NF and CAF in OCRC, CRC and normal SMA positive tissues

Figure S10: PA does not directly promote NF-to-mCAF activation. (A) CCK-8 assay determining the optimal concentration of CAFs stimulated by PA. (B) CCK-8 assay analysis of the relationship between PA stimulation and CAFs proliferation. (C) Evaluation of changes in MMPs and TIMPs secretion in CAFs and NFs induced by PA. (D) qPCR assay was conducted to examine whether PA stimulation can promote gene expression of FN1, POSTN, and LUM (mCAFs maker) in CAFs. (E-F) Western blotting (E) and CCK-8 (F) assays confirmed whether PA could directly promote the activation of NFs to CAFs and facilitates NFs proliferation. (G) qPCR assay examined whether PA stimulation can promote mCAFs marker (FN1, POSTN, and LUM) gene expression. Statistical significance was determined by one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, no significance.

Figure S5: OCRC and CRC differed in stromal cells, with significantly more matrix CAFs in OCRC. (A) Isolated and identified the primary CAFs from fresh OCRC and CRC tissues, red: Vimentin, green: α-SMA, (Scale bars = 100μm). (B) KEGG and GO enrichment analysis of differentially expressed genes in CAFs from OCRC and CRC tissues. (C-D) Expression levels of ECM-related matrix proteins and matrix metalloproteinases in OCRC and CRC were detected by qPCR (C) and ELSIA (D). Statistical significance was determined by unpaired Student t test. *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001.

Figure S11: PA promoted CAFs function and facilitated partial NF-to-mCAF activation by stimulated CRC cells. (A) CCK-8 assay to explore the optimal concentration of CRC cells stimulated by PA. (B) Oil Red O staining visualizing lipid droplet accumulation in CRC cells following 48 hours of PA stimulation (Scale bars = 50μm). (C-E) Analysis of NF-to-mCAF activation via CRC cells under PA stimulation, including CCK-8 (C) assay, qPCR (D) assay, and cellular immunofluorescence staining (E) (Scale bars = 20μm). (F) Comparison of ECM-related matrix proteins secretion between NFs activated to CAFs and CAFs directly stimulated by PA. Statistical significance was determined by one-way and two-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, no significance.

Figure S2: OCRC and CRC differed substantially in ECM constitution, biomechanics, and collagen characteristics. (A) Masson staining of OCRC, CRC and normal tissues (Scale bars = 1mm, 50μm). (B) Quantification of collagen fiber content in OCRC, CRC and normal tissues by Masson staining (n=15). (C) Mean E-moduli measurements in OCRC, CRC and normal tissues (n=10). (D) Raman spectroscopy analysis of collagen fibers in OCRC and CRC tissues (n=10). (E) Immunohistochemistry staining and semi-quantitative analysis of CD44 in the ECM of OCRC and CRC tissues (n = 9, Scale bars = 1mm, 50μm). (F) Safranin O-fast green staining to assess chondroitin sulfate levels in OCRC and CRC tissues (Scale bars = 1mm, 50μm). (G) Analysis of collagen fiber alignment in OCRC and CRC ECM (n=15). (H) Raman spectroscopy analysis of collagen fiber orientation in OCRC and CRC ECM (n=10). (I) Immunohistochemical staining analysis of T cells (CD4, CD8) and macrophages (CD68) in the tumor microenvironments of OCRC and CRC (Scale bars = 1mm, 50μm). Statistical significance was determined by unpaired Student t test. *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001, ns, no significance.

Nontargeted metabolomics analysis of OCRC and CRC tissues

Figure S1: OCRC and CRC differed substantially in clinicopathology, with poorer prognoses in OCRC. (A) Comparative analysis of OS and DFS in CRC (n=1272) and OCRC (n=198) patients. (B) Clinicopathological characteristics of CRC (n=1272) and OCRC (n=198) patients. (C) Comparative analysis of OS and DFS in 150 matched pairs of CRC and OCRC patients. (D) Clinicopathological characteristics of 150 matched pairs of OCRC and CRC. Statistical significance was determined by Chi-Squared Test. *P <0.05, **P <0.01, ***P <0.001.

Figure S4: Functional analyses on α-SMA-positive CAFs and NFs from OCRC, CRC, and normal tissues. (A) KEGG and GO enrichment analysis of α-SMA positive cells from OCRC and CRC tissues. (B) KEGG and GO enrichment analysis of α-SMA positive cells from OCRC and Normal tissues. (C) KEGG and GO enrichment analysis of α-SMA positive cells from CRC and Normal tissues.

Figure S17: Animal models validated vanillylacetone inhibited PA-induced cytokine secretion and NF-to-CAF/mCAF activation. (A-B) Measurement of CSF-1, CXCL8, and TGF-β1 levels in CRC subcutaneous tumors and serum of C57BL/6 (A) and BALB/c (B) mice post-vanillylacetone treatment. (C) Immunofluorescence staining analysis of mCAFs in BALB/c mice subcutaneous tumors post-vanillylacetone treatment, green: FN1, red: α-SMA (Scale bars = 1mm and 20μm). (D) FN1-positive CAFs content in subcutaneous tumors from C57BL/6 and BALB/c mice post-vanillylacetone treatment. Statistical significance was determined by unpaired Student t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, no significance.