Single-Cell Transcriptomics Reveals Tumorigenic Lineages and Interactions Persisting and Evolving From Premalignancy to Cancer in the Stomach

Background & Aims Gastric cancer is often diagnosed at advanced stages, making the premalignant window critical for early detection. However, the epithelial changes that characterize this stage remain poorly defined. We used CTLA4-knockdown (CTLA4KD) mice, which model human CTLA4 insufficiency-associated gastric cancer, that develops autoimmune gastritis, metaplasia, and adenocarcinoma, to define molecular changes across disease progression. Methods Single-cell RNA sequencing and 40-color spectral flow cytometry were performed on gastric tissue across premalignant and cancer stages. We evaluated epithelial lineage relationships, cell-cell communication pathways, and candidate markers at the transcript and protein levels in murine and human gastric adenocarcinoma. Results We identified a metaplastic/tumorigenic population in CTLA4KD mice that emerged in premalignancy with a chief cell transcriptomic origin. Cell-cell communication analysis revealed a progressive increase in fibronectin (FN1) signaling from premalignancy through cancer, transitioning from stromal to autocrine signaling in the metaplastic/tumorigenic population. Expression of Embigin (EMB), a fibronectin receptor, increased at both transcript and protein levels in premalignancy and remained elevated in cancer. EMB⁺ epithelial cells co-expressed CD133, a cancer stem cell marker, consistent with a stem-like phenotype. EMB expression was also higher in human gastric adenocarcinoma than in matched normal tissue. Conclusions Using a model that captures the full histopathological cascade of gastric tumorigenesis, we delineate single-cell transcriptional programs that persist and evolve from premalignancy to adenocarcinoma. In-depth computational analyses identified novel markers involved in cell-cell communication during tumorigenesis, including EMB, an epithelial marker that persists from premalignancy through cancer in the murine model and validated in human gastric adenocarcinoma, suggesting EMB as a marker of premalignant progression.