ARTICLE ABSTRACTPeritoneal mesothelial cells (PMC) cover organ surfaces in the abdominal cavity. In this study, lineage tracing revealed that the PMCs guide cancer cell invasion in the gastric wall and in peritoneal metastatic lesions. Serosal PMCs covering the stomach surface entered the gastric wall to create a novel niche that favored gastric cancer cell invasion. PMC infiltration was induced by incorporation of cancer cell–derived, Wnt3a-containing extracellular vesicles. Infiltrated PMCs in turn promoted subserosal invasion of cancer cells. Mutual attraction between cancer cells and PMCs accelerated tumor invasion in the gastric wall, and PMC-led cancer cell invasion in disseminated tumors within the abdominal wall and diaphragm. Addition of the carboxyl terminus of Dickkopf-1 attenuated directional invasion of PMCs toward cancer cells both in vitro and in the gastric wall in vivo. PMCs were sensitive to the aldehyde dehydrogenase (ALDH) inhibitor disulfiram (DSF), as ALDH activity is elevated in PMCs. Wnt3a upregulated ALDH, and addition of DSF inhibited the invasive properties of PMCs, whereas DSF pretreatment suppressed gastric infiltration of PMCs and subserosal invasion by cancer cells. Our results suggest that stabilization of PMCs may become an effective therapy for the prevention of local invasion and metastasis of gastric cancer. Cancer Res; 77(3); 684–95. ©2016 AACR.