Precision Tumor Immunotherapy via a Dual-Gated Macrophage-Bacterial Activation Platform

Immunotherapy based on live microorganisms has shown promise in preclinical studies, but its clinical translation has been hampered by limited efficacy and innegligible toxicity. Here, we developed M-BLAST (Macrophage-Bacteria encapsulation Lytic Autoactivated Synergistic Therapeutics), a dual-gated macrophage-mediated bacterial tumor-targeted delivery and in situ activation system. M-BLAST incorporates density-regulated virulence-enhanced attenuated Salmonella strains as the therapeutic core, thermally-controlled GSDMD-N-expressing macrophages as the delivery vector, and copper selenide, a photothermal material, as a heat-shock “primer.” Following systemic administration, localized near-infrared irradiation at the tumor site triggers macrophage pyroptosis, ensuring rapid and complete bacterial release. This disrupts the immunosuppressive tumor microenvironment and elicits a widespread cascading antitumor response just like a “immune bomb”, while dual-gating design of bacterial density and heat-shock ensures safety by preventing off-target activation in non-tumor regions. M-BLAST promises to enhance the therapeutic utility of living engineered bacteria for cancer while ensuring safety. Overall design: A total of 1×10^6 4T1 cells were orthotopically inoculated into the mammary fat pads of 6-week-old female BALB/c mice to establish primary breast tumor. When tumor volumes reached approximately 70 mm^3, mice were randomly allocated into experimental groups. The cohorts received intraperitoneal injections of either saline or M-BLAST cells (3.0×10^5 cells per mouse). Twelve hours post-administration, tumors in the M-BLAST+NIR group were locally exposed to 980 nm NIR irradiation (1.5 W/cm^2) for 10 minutes, while other groups remained untreated. Tumors were harvested 36 hours post-treatment. To mitigate spatial heterogeneity, entire tumors were homogenized and thoroughly mixed prior to RNA extraction. The extracted RNA was then subjected to RNA sequencing.