Computer-aided analysis reveals metallothionein-positive cancer-associated fibroblasts promote angiogenesis in gastric adenocarcinoma.

Gastric adenocarcinoma (GC), along with its tumor microenvironment (TME), poses great challenges for clinical treatment strategies. Single-cell sequencing has become an important tool for analyzing TME heterogeneity, cell subpopulation, and gene expression patterns. 56 GC single-cell sequencing samples were analyzed, focusing on TME by delineating cancer cells, cancer-associated fibroblasts (CAFs), and macrophages. The spatial transcriptome was used to clarify the distribution characteristics of each cellular component in the tissue slice. Despite the widespread genetic mutations observed in cancer cells, certain recurrent alterations were identified in specific chromosomal regions. The heterogeneity among GC cells is profound, four cancer cell subpopulations were identified through drug sensitivity profiling. Subtype 4, although only present in some samples, demonstrates the strongest stemness and metabolic activity, possibly indicative of an early-stage cancer subpopulation. Their drug sensitivity profiles may hold promise for guiding clinical intervention. In addition, robust spatial co-localization patterns were observed between CAFs, M2 macrophages, and endothelial cells. CAFs were further categorized into six subgroups, among which a novel subgroup termed metallothionein(mt)-positive CAF (mtCAF), characterized by elevated expression of metallothionein 1X (MT1X) and subsequent vascular endothelial growth factor A (VEGFA) secretion, was identified. Immunohistochemistry preliminary confirmed the presence of this unique CAF subgroup. Additionally, M2d macrophages, besides exhibiting high VEGFA expression, also demonstrated various growth factors such as Aamphiregulin (AREG). The M2d-mtCAF axis may play an important role in GC angiogenesis. This study not only enhances our understanding of the TME heterogeneity in GC but also sheds light on the interaction between CAFs and tumor-associated macrophages (TAMs) in tumor angiogenesis.