Engineered *Salmonella* delivering **conantokin G** synergizes with **PD-L1 blockade** to achieve complete colorectal cancer regression.

Colorectal cancer (CRC) remains a significant global health challenge, with advanced stages often resistant to conventional therapies. While immune checkpoint blockade (ICB) targeting PD-1/PD-L1 has revolutionized cancer treatment, many CRC patients show limited response. A critical gap exists in sensitizing these tumors to immunotherapy. N-methyl-D-aspartate receptors (NMDARs), particularly the NR2B subunit, are increasingly implicated in cancer progression, offering a novel, underexplored therapeutic target. Localized delivery of therapeutics via engineered bacteria like Salmonella presents a promising strategy to overcome systemic toxicity and enhance tumor-specific efficacy.

Researchers engineered an attenuated Salmonella typhimurium strain to secrete the NMDAR antagonist conantokin G (Con G) via its flagellar type III secretion system (T3SS). This engineered bacterium was tested for its ability to target and suppress colorectal cancer in vitro and in vivo. The study evaluated tumor accumulation, cell death mechanisms, and systemic toxicity. They also investigated the combination of the engineered Salmonella-Con G delivery with α-PD-L1 therapy in a murine model of colorectal cancer, assessing tumor regression, survival, and immune modulation. Additionally, 106 clinical colorectal cancer specimens were analyzed for NR2B expression and its correlation with disease stage and prognosis.

The engineered Salmonella preferentially accumulated in tumor tissues, inducing 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. > Combination treatment with engineered Salmonella-Con G and α-PD-L1 therapy significantly 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, establishing NR2B as a clinically relevant therapeutic target.