Mesenchymal R-spondin 3 promotes an immunogenic tumor microenvironment with adaptive immune resistance in human gastric adenocarcinoma.

The main risk factor for gastric cancer (GC) is inflammation driven by Helicobacter pylori (H. pylori) infection. Despite growing use of immunotherapies targeting the tumor inflammatory microenvironment, responses remain suboptimal, highlighting the need to define inflammatory drivers and improve patient stratification. We assessed the role of R-spondin 3 (RSPO3), a stroma-secreted Wnt potentiator, in a Caucasian tissue microarray of 277 chemotherapy-naïve GC patients by associating RSPO3 expression with tumor inflammation, tumor stage, and survival. To mechanistically explore the role of Rspo3 in immune cell infiltration, we used conditional mouse models with stromal Rspo3 overexpression or knockout and infected them with H. pylori. We conditionally depleted the epithelial Rspo3 receptors Lgr4 and Lgr5 in mice to assess the mode of Rspo3 signaling. Findings were validated by generating a single-cell atlas from The Cancer Genome Atlas and performing gastric cancer transcriptome analysis. Tumor RSPO3 expression positively associated with mucosal T-cell infiltration and combined positive score, a phenotype specific to intestinal-type GC. In mice, stromal Rspo3 promoted T-cell infiltration with increased interferon-γ signaling and PD-L1 expression in H. pylori-driven precancerous lesions. Epithelial depletion of Lgr4/5 reduced gastric T-cell infiltration and PD-L1 expression. In mice, mesenchymal-epithelial RSPO3-LGR4/5 signaling drives inflammation and enhances immune checkpoint signaling in precancerous lesions. Positive associations between RSPO3 expression and inflammation suggest that RSPO3 signaling contributes to shaping the immune microenvironment of human intestinal-type GC.