Tumor sample gene expression data were obtained from The Cancer Genome Atlas (TCGA), including 287 colon adenocarcinoma (COAD) samples, 95 rectum adenocarcinoma (READ) samples, 517 lung adenocarcinoma (LUAD) samples, 501 lung squamous cell carcinoma (LUSC) samples, and 97 triple-negative breast cancer (TNBC) samples. Tumor purity and estimation of CD4+ or CD8+ infiltration in COAD, READ, LUAD, LUSC, and BRCA were performed as previously described (41). Wnt pathway genes were downloaded from the Broad Institute for analyses at the following link: http://software.broadinstitute.org/gsea/msigdb/cards/KEGG_WNT_SIGNALING_PATHWAY.

In COAD, Spearman correlation analysis was conducted to determine the correlation of each Wnt pathway gene with the Treg cell markers CD4, CD25, and FOXP3 across the 287 samples. Subsequently, hierarchical clustering was conducted according to the correlation coefficient of each Wnt pathway gene and Treg cell marker. A subset of Wnt pathway genes was found to be positively correlated with the expression of all three Treg cell markers. The expression correlation between genes within this subset and Treg cell markers was then calculated in the READ, LUAD, LUSC, and TNBC tumor samples. To build a heat map of the expression of these Wnt pathway genes in COAD, the TCGA COAD tumor samples were first stratified according to Treg cell infiltration (determined by the expression level of Treg cell markers CD4, CD25, and FOXP3). Expression of the Wnt pathway genes was then plotted for tumors with high Treg cell infiltration (Treg-hi; top quartile of Treg cell marker expression), tumors with low Treg cell infiltration (Treg-lo; bottom quartile of Treg cell marker expression), and others (either top or bottom quartile of Treg cell marker expression).

APC mutation data were obtained from TCGA cbioportal (www.cbioportal.org), and CIBERSORT (https://cibersort.stanford.edu) was used to estimate tumor immune cell infiltration. COAD samples were separated into two APC mutated and nonmutated cohorts, and t test was used to evaluate CD8+ T cell and Treg cell infiltration difference between APC mutated and nonmutated samples.

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