CD44 variant 9 expression in M2 macrophages enhances gastric cancer progression by modulating the tumor microenvironment

M2 macrophages constitute a major stromal component of gastric cancer (GC) and actively contribute to tumor progression through angiogenesis, tissue remodeling, and epithelial–mesenchymal transition (EMT). Our previous study identified CD44 variant 9 (CD44v9) expression in cytokeratin-negative stromal compartments as a marker with diagnostic and prognostic significance in gastric adenocarcinoma (GA); however, its cellular localization within the tumor microenvironment and functional relevance remain unclear. In this study, public single-cell RNA sequencing datasets from the Human Protein Atlas were analyzed to characterize CD44 expression across gastric tissue cell populations. Multiplex immunofluorescence was performed to validate the spatial distribution of CD44v9-positive cells in clinical GA specimens. CD44v9 expression was predominantly enriched in macrophages and showed significant co-localization with stromal M1 macrophages (CD68 + CD86 + CD163 − ), M2 macrophages (CD68 + CD86 − CD163 + ), and regulatory T (Treg) cells (CD3 + CD4 + FOXP3 + CD8 − ). Functionally, CD44v9 was overexpressed in THP-1-derived M2 macrophages through lentiviral transduction, and these modified cells retained their inherent M2 macrophage phenotype. CD44v9 overexpression significantly enhanced TNF-α secretion ( P < 0.05), without affecting IL-6 production ( P > 0.05). In a co-culture system, CD44v9 overexpression M2 macrophages markedly promoted GC cell proliferation, migration, and invasion. These pro-tumorigenic effects were accompanied by EMT induction, characterized by decreased E-cadherin and increased N-cadherin and Vimentin (all P < 0.05) expression, along with elevated MMP2 and MMP9 ( P < 0.05) levels in AGS cells. Collectively, these findings demonstrate that CD44v9 expression in stromal M2 macrophages promotes a pro-tumorigenic phenotype, possibly through TNF-α-mediated tumor-stroma crosstalk, thereby promoting EMT and enhancing cancer cell aggressiveness. CD44v9 expressed by M2 macrophages may represent a promising biomarker and therapeutic target in GA development.