Therapeutic Salmonella expressing apoptin and CEA elicits combined pro-apoptotic and immune-mediated anticancer...

In the current study, we genetically engineered Salmonella Typhimurium to serve a dual purpose in cancer immunotherapy, simultaneously inducing apoptosis and activating anti-cancer immune responses. Tumor-residing tryptophan auxotrophic Salmonella, featuring trpA and trpE deletions, selectively inhabits the tumor microenvironment, promoting tumor-targeted delivery of therapeutic antigens such as apoptin (an apoptosis inducer) and carcinoembryonic antigen (CEA; related to anti-cancer immunity), while minimizing off-target effects. The therapeutic strain had reduced endotoxic responses owing to a lack of pagL in its genome. Additionally, to enhance adaptation to the tumor microenvironment, the strain underwent consecutive passaging for over five generations, increasing its potency for tumor recognition and localization. A prokaryotic and eukaryotic dual-expression plasmid was developed by incorporating the complete apoptin open reading frame into the eukaryotic region and the CEA epitope collection associated with an MHC-II agonist peptide into the prokaryotic expression cassette. Each antigen was confirmed for successful expression at the intended sizes, with functional validation performed through in vitro cytotoxicity assessment. Mouse inoculation studies demonstrated that the Salmonella therapy significantly suppresses the progression of highly aggressive 4T1 breast cancer tumors as well as metastatic dispersion in lung, liver, and spleen tissues, fostering an anti-tumoral immune environment characterized by IFN-γ and CCL2 markers. This transformative approach to Salmonella-mediated cancer immunotherapy could pave the way for targeted treatments for immune-resistant cancers and hard-to-treat malignancies.