Fig. 1: FSTL3 as a prognostic factor predominantly expressed in CCAFs and associated with VM formation in colon cancer.

A, B Venn diagram showed the intersection of fibroblast-related genes (A) and prognosis-related genes (B) by CAF_EPIC and CAF_MCPcount methods in TCGA and GSE39582 cohorts. C Volcano plot showed genes significantly upregulated (Red labeled) and downregulated (Green labeled) in VM group using data from GSE240789 cohort. D Venn diagram showed the intersection of fibroblast-related genes, prognosis related genes and VM-related genes. E Violin plot showed the expression levels of FSTL3 and SERPINE1 different cell subtypes in GSE166555 cohort. F The significance distribution of gene for the difference between non_VM and VM tissues, as well as for the prognostic analysis. G The heatmap displayed the expression of FSTL3 among different cell types in four different scRNA-seq cohorts on the TISCH online database. H IF staining of FAP and FSTL3 in colon cancer tissues and the quantification of FSTL3 intensity in stroma area and tumor area (Scale bar =100 μm, n = 3, biologically independent samples). I IF staining of FAP, a-SMA, and FSTL3 in primary CCAFs (Scale bar =100 μm). J Histogram comparing the proportion of CD34 (n = 87), D2-40 (n = 128) positivity between the high- and low- expression groups of FSTL3. K CD31/PAS IHC staining for the tissue from ZHSU. The black arrow represents VM (−) which is CD31⁺PAS^-, and the red arrow represents VM (+) which is CD31^-PAS⁺. Both tubes contain red blood cells (Scale bar =100 μm, n = 157). L, M Histogram comparing the proportion of VM positivity (L) and the blood vessel intensity (M) between the high- and low-expression groups of FSTL3. N IF staining of FAP and FSTL3, along with CD31/PAS IHC staining, was performed on colon cancer tissue and its adjacent tumor tissue, two adjacent slices from a continuous section were used for the IF and IHC staining to ensure the results correspond to the same area, the black arrow indicates CD31⁺ blood vessels, while the red arrow and white dashed line represent VM; Scale bar =10 μm. O Bar graph comparing plasma FSTL3 protein levels in patients with colon cancer (n = 30) and colon adenomas (n = 10). P Kaplan–Meier survival curves for PFS in ZHSU cohort stratified by FSTL3 expression levels; Q Kaplan–Meier survival curves on PFS of the ZHSU cohort stratified by FSTL3 expression and VM status. All statistics are expressed as mean ± SD. Statistical significance was calculated by two-tailed t test in (E), (H), (M), and (O). The Chi-Squared test was applied for categorial data in (J) and (L). Statistical analysis of survival time was performed using log-rank tests in (P) and Gehan–Breslow–Wilcoxon test in (Q).