Multi-omics analysis reveals the heterogeneity and interactions among stromal and tumor cells in gastric cancer.

This study aims to investigate the heterogeneity of cancer-associated fibroblasts (CAFs) and smooth muscle cells (SMCs) and their interactions with malignant epithelium in gastric cancer (GC), with the goal of identifying potential therapeutic targets. We performed a multi-omics analysis integrating single-cell RNA sequencing and spatial transcriptomics, complemented by trajectory inference, regulon activity mapping, ligand-receptor interaction modeling, and survival association in external cohorts. Two malignant epithelial programs emerged-Tumor Cell 1 (TC 1) (wound-healing/proliferative) and Tumor Cell 1 (TC 2) (highly cycling/metabolic). In TCGA-STAD (The Cancer Genome Atlas - Stomach Adenocarcinoma), high TC 1 scores associated with worse overall survival (P < 0.01), whereas TC 2 showed a nonsignificant trend. The stroma comprised nine populations; tumors were enriched for RGS5⁺ SMCs and FAP⁺ fibroblasts, with depletion of homeostatic PI16⁺ fibroblasts. Pseudotime traced a PI16⁺→CCL11⁺→PDGFRA⁺→FAP⁺ continuum featuring late NF-κB/STAT activation and extracellular matrix (ECM)-remodeling; SMCs rewired from contractile MYH11⁺ to angiocrine RGS5⁺ states. Ligand-receptor analysis indicated asymmetric tumor-stroma crosstalk: TC 1 preferentially engaged vaso-regulatory/EGFR-immune signaling to activate CAFs and SMCs, while TC 2 amplified PDGF/MDK growth-factor circuits; stromal feedback via WNT/NRG/HGF/TGF-β reinforced malignant programs. Spatial maps confirmed EPCAM-high tumor nests encased by COL1A2/FAP⁺ fibroblastic shells interwoven with ACTA2/RGS5⁺ strands. This study highlights the critical role of CAFs and SMCs heterogeneity and their interactions within the GC TME (Tumor microenvironment). Targeting stromal pathways such as PDGF, TGF-β, and NF-κB signaling, immunomodulating CAFs, and disrupting SMC-derived vascular niches may improve therapeutic responses. Further experimental validation of ligand-receptor pairs and spatial determinants is warranted.