Unraveling tumor cell heterogeneity and epithelial-mesenchymal plasticity in gastric adenocarcinoma: an integrative multi-omics framework evaluating PVR/CD155 as a tumor cell-intrinsic EMT-associated target.

BACKGROUND: The tumor microenvironment (TME) of gastric adenocarcinoma is exceptionally heterogeneous, and epithelial-mesenchymal transition (EMT) is a central mechanism promoting local invasion and metastatic spread. Even so, how EMT-programmed cells are spatially arranged within tumor tissue, how they interact with neighboring immune populations, and which molecular nodes might be exploited therapeutically remain incompletely defined. METHODS: We built a large-scale, multi-layered analytical pipeline that combined single-cell transcriptomics (approximately 250,000 cells drawn from two independent patient cohorts), Visium-based spatial transcriptomics processed through Bayesian cell2location deconvolution, niche-level SpaTopic modeling, deep-learning histology analysis (ResNet50 feature extraction paired with CellProfiler-derived morphometrics), and ensemble survival modeling spanning over one hundred algorithmic combinations. Gaussian mixture modeling was used to define EMT-high cell states, the Scissor framework was applied to link fibroblast subsets to patient mortality, and a multi-tier filtering scheme was used to nominate druggable candidate genes. The top candidate, PVR/CD155, was interrogated experimentally by RT-qPCR, Western blotting, immunohistochemistry, and siRNA knockdown in AGS cells. We further evaluated PVR druggability by molecular docking, a 100-nanosecond molecular dynamics trajectory, and MM/GBSA binding-energy estimation using PP-121 as a candidate ligand. RESULTS: Sub-clustering identified seven fibroblast subclusters: Fib_APOD, Fib_COL4A1, Fib_SLPI, Fib_COL1A1, Fib_CCL4, Fib_STMN1, and Fib_S100B. The SLPI-high subset preferentially localized to peritoneal metastases. Phenotype-guided Scissor mapping nominated a survival-associated fibroblast program enriched within COL4A1-expressing fibroblast states. PVR/CD155 was prioritized as an EMT-linked candidate gene; single-cell expression profiling showed that PVR was most frequently detected in endothelial, epithelial and fibroblast compartments, with low detection in lymphoid and plasma cells. PVR/CD155 was significantly upregulated in gastric cancer cell lines and tumor tissues. PVR/CD155 knockdown in AGS cells decreased proliferation, migration and invasion, supporting a tumor cell-intrinsic functional role rather than establishing PVR as a stromal immune biomarker. Molecular docking and dynamics simulations identified PP-121 as a candidate PVR-binding ligand for future biochemical validation. CONCLUSION: The proposed framework connects single-cell resolution with spatial and histological data, produces validated prognostic tools and nominates PVR/CD155 as a tumor cell-intrinsic EMT-associated target candidate for gastric cancer. Stromal or immune regulatory roles of PVR remain plausible but require direct compartment-specific and functional validation.