Integrative multi-omics analysis identifies stromal-immune crosstalk as a determinant of immunotherapy efficacy and establishes a prognostic signature in gastric cancer.

Immune checkpoint inhibitors like pembrolizumab exhibit variable efficacy in metastatic gastric cancer (GC). This study aimed to identify molecular drivers of pembrolizumab response, explore mechanisms of immune checkpoint inhibitors (ICIs) efficacy, and develop a prognostic signature. Transcriptomic analysis of pembrolizumab-treated GC (TIGER database) identified 165 response-associated differentially expressed genes (DEGs). Functional annotation and single-cell RNA sequencing (scRNA-seq) data from the Gene Expression Omnibus (GEO) revealed that responder-upregulated genes (R-DEGs) were enriched in immune activation pathways and mainly localized to CD8 + T/NK cells. In contrast, non-responder-upregulated genes (D-DEGs) were linked to extracellular matrix (ECM) remodeling and mainly expressed in fibroblasts/endothelial cells. CellChat analysis demonstrated that key DEGs mediate immune-stromal crosstalk via MHC-I and collagen/laminin signaling. A prognostic signature (Lasso-StepCox[forward] Riskscore; LSR: APOD, APOH, BATF2, GJA1, MAGED1, SLC5A1, SLCO2A1, VWF, VCAN) was derived and validated in four independent GC cohorts from the GEO and Cancer Genome Atlas (TCGA) database. Multi-omics analyses showed that LSR-high tumors exhibited aggressive clinicopathological features, increased stromal components, reduced cytotoxic immune infiltration, diminished tumor mutational burden (TMB), and poorer prognosis. Immunohistochemistry (IHC) and spatial transcriptomics in GC showed that stromal VWF/VCAN expression correlates with reduced CD8⁺ T cell granzyme B expression, suggesting T cell dysfunction. High VWF expression in GC predicted poor survival, and a combined VWF/VCAN score showed enhanced prognostic stratification. This study highlights stromal-immune crosstalk as a driver of pembrolizumab resistance and provides a signature as a clinical tool for prognosis and personalized therapy in metastatic GC.