Engineered Salmonella delivering the NMDAR antagonist conantokin G synergizes with PD-L1 blockade to enhance colorectal cancer regression.

Engineered attenuated Salmonella typhimurium has been explored as a tumor-targeting platform for localized therapeutic delivery. Here, we developed a strain capable of secreting the N-methyl-D-aspartate receptor (NMDAR) antagonist conantokin G (Con G) via the flagellar type III secretion system (T3SS). The engineered bacteria preferentially accumulated in tumor tissues and induced robust colorectal cancer cell death characterized by membrane disruption, leading to significant tumor suppression in vitro and in vivo with minimal systemic toxicity. Bulk RNA-seq analysis confirmed induction of immunogenic cell death pathways and concurrent suppression of Treg-associated gene expression. Salmonella colonization was associated with increased tumor PD-L1 expression, likely driven by infection-induced inflammatory signaling, which provided a rationale for combination with α-PD-L1 therapy. Combination treatment enhanced tumor regression, prolonged survival, and achieved complete tumor clearance in a subset of mice. Analysis of 106 clinical colorectal cancer specimens further demonstrated that high expression of NR2B, a subunit of NMDAR, is associated with advanced disease stage and poor prognosis. Together, these findings establish NR2B as a clinically relevant therapeutic target and highlight engineered bacterial delivery of Con G as a strategy to sensitize colorectal cancer to immune checkpoint blockade.