Integrated bulk and single-cell RNA profiling implicates MFAP5+ CAFs in potential role in peritoneal metastasis of gastric cancer.

Peritoneal metastasis (PM) is a lethal complication of advanced gastric cancer (GC), yet the stromal microenvironment driving PM remains incompletely understood. Using single-cell RNA sequencing, we profiled the stromal landscape of paired primary GC and PM tissues, identifying ten distinct carcinoma-associated fibroblast (CAF) subpopulations with diverse tissue-specific enrichment and functions. Among these, microfibril-associated protein 5-expressing CAFs (MFAP5+ CAFs) were specifically enriched in PM tissues, exhibiting a mesenchymal, pro-metastatic phenotype. Trajectory analysis revealed a phenotypic progression of CAFs during peritoneal metastasis, characterized by a shift from an immune-regulatory state toward increasingly pronounced mesenchymal features, with MFAP5+ CAFs emerging as a terminal, pro-metastatic subpopulation. Multiplex immunohistochemistry (mIHC) confirmed the selective accumulation of MFAP5+αSMA+ CAFs in PM compared to matched primary tumors, a finding further corroborated by conventional IHC. Functionally, co-culture experiments demonstrated that silencing MFAP5 in CAFs significantly impaired gastric cancer cell migration and invasion, underscoring the pro-invasive role of MFAP5+ CAFs. Bulk transcriptomic analyses of primary and metastatic GC cohorts linked elevated MFAP5 expression to poor prognosis, an immunosuppressive microenvironment characterized by reduced natural killer cell activity and increased M2 macrophage infiltration, and enrichment of extracellular matrix remodeling and invasive transcriptional programs. Furthermore, analysis of bulk RNA-seq data from peritoneal metastases revealed that MFAP5-high tumors are enriched for neutrophil extracellular trap (NET)-associated gene signatures, implicating MFAP5 in shaping a pro-metastatic immune niche. Together, these data delineate the distinct and context-dependent roles of MFAP5 in primary and metastatic gastric tumors, highlighting MFAP5+ CAFs as key drivers of peritoneal metastasis and promising therapeutic targets to hinder GC progression.