Jia Chen, Peng Xu, Qingyuan Wang, Juncheng Dai, Shuang Liang, Sheng Yang, Guiping Xie, Xiang Li, Yaohui Wang, Hongjin Hua, Xiaochun Ping, Jiajia Shen, Lizong Shen
Background The immunomodulatory roles of cancer-associated fibroblasts (CAFs) in gastric cancer have been increasingly recognized. This study aimed to identify specific CAF subpopulations and to elucidate the mechanisms underlying their immunosuppressive effects. Methods We developed a single-cell RNA sequencing (scRNA-seq) protocol to enrich CAFs and matched normal fibroblasts (NFs) from stomach tissues. Bioinformatics analyses, along with in vitro and in vivo experiments, were performed to functionally validate a distinct CAF subset. Results We profiled 8,322 high-quality fibroblasts from nine patient-matched tumor and adjacent normal tissue pairs. A unique CAF subset, CD146 + CAFs, was identified. CD146 + CAFs were significantly enriched in the tumor microenvironment and associated with poor survival and M2-like macrophage infiltration. Tumor cells promoted CD146 + CAFs expansion via JAG1-NOTCH3 and LGALS3-CD146 signaling. Mechanistically, NOTCH3 acted as a master regulator in CD146 + CAFs. Tumor cell-derived JAG1 engaged NOTCH3 on CD146 + CAFs, which triggered PI3K/AKT-mediated CD146 transcription and enhanced COL4A1 secretion. Functionally, CD146 + CAF-derived COL4A1 bound macrophage CD44/IL7R to drive macrophage M2-like polarization, thereby suppressing CD8 + T-cell function. Therapeutic blockade of CD146 or COL4A1 in vivo impaired M2 polarization, reinvigorated CD8 + T-cell activity, and restrained tumor growth. Conclusions Our findings underscore the pivotal role of CD146 + CAFs in shaping an immunosuppressive microenvironment and highlight CD146 + CAFs and COL4A1 as promising candidates for targeted therapy in gastric cancer.