Multiplex immunohistochemistry and flow cytometry analyses revealed distinct spatial and phenotypic heterogeneity of fibroblasts across tumor, junctional, and normal zones, with cancer-associated fibroblasts exhibiting elevated expression of activation markers (α-SMA, Calponin, Vimentin, Fibronectin) predominantly in the tumor zone, while MYH11 expression was highest in normal fibroblasts and decreased toward the tumor zone. The gene discussed is ACTA1; the disease is cancer.