Comparative single-cell transcriptomics of orthotopic and subcutaneous gastric tumors reveal immune and stromal heterogeneity.

Preclinical cancer models often use subcutaneous (SC) implantation, which fails to recapitulate the native tumor microenvironment (TME) of orthotopic (ORT) sites. To resolve these differences, we used single-cell RNA sequencing (scRNA-seq) on paired SC and ORT implants of the CKP syngeneic gastric cancer model. Histopathological differences were minimal, but scRNA-seq revealed profound TME divergence. ORT tumors displayed robust stromal activation, coordinated fibroblast and endothelial signaling, and an immune compartment marked by higher T/NK cell activation and IgA-biased B cell plasma programs, reflecting a physiological mucosal environment. In contrast, SC tumors had higher overall T cell infiltration but showed markedly increased CD8+ T cell exhaustion and an enriched oxidative tumor program. Our findings provide critical guidance: SC models are optimal for high-throughput and exhaustion-focused assays, whereas ORT models are indispensable for studying organ-specific immune and stromal biology with translational fidelity.