In Vivo CRISPR Screening Identifies the Glutamate Receptor GRIA2 as Promoting Peritoneal Metastasis of Gastric Cancer via Calcium‐Dependent β‐Catenin Activation

ABSTRACT Peritoneal metastasis is the most lethal manifestation of gastric cancer, with a median survival of less than one year, highlighting the need for new therapeutic targets. Through an in vivo genome‐wide CRISPR/Cas9 screen, we identified GRIA2, an AMPA‐type glutamate receptor subunit, as a key driver of peritoneal metastasis. GRIA2 promotes gastric cancer cell migration, invasion, stemness, and adhesion to mesothelial cells in a glutamate‐dependent manner. Mechanistically, glutamate activates GRIA2, enhancing its interaction with GSK‐3β and inducing calcium influx, inhibiting GSK‐3β kinase activity and stabilizing β‐catenin, thereby activating the Wnt/β‐catenin signaling pathway. Single‐cell RNA sequencing revealed that cancer‐associated fibroblasts are the primary source of glutamate in the peritoneal microenvironment, which establishes a paracrine axis that enhances GRIA2‐driven metastasis. Pharmacological inhibition of AMPA receptors with NBQX and Selurampanel suppressed peritoneal metastasis in both cell line‐derived and patient‐derived organoid xenograft (PDOX) mouse models. In clinical analysis, GRIA2 expression in peritoneal metastases correlated with the levels of β‐catenin and phosphorylated GSK‐3β (serine 9), with high GRIA2 expression predicting poor prognosis. These findings suggest that GRIA2 is a novel therapeutic target, and AMPA receptor antagonists are promising agents for treating gastric cancer peritoneal metastasis.