Evaluation of Tumor-colonizing Salmonella Strains using the Chick Chorioallantoic Membrane Model

The chick embryo chorioallantoic membrane (CAM) tumor model is a valuable preclinical model for studying the tumor colonizing process of Salmonella enterica serovar Typhimurium. It offers advantages such as cost-effectiveness, rapid turnaround, reduced engraftment issues, and ease of observation. In this study, we explored and validated the applicability of the partially immune-deficient CAM tumor model. Herein, we demonstrate that Salmonella preferentially colonizes tumors and directly causes tumor cell death. Bacterial migration, tumor colonization, and intra-tumor distribution did not require flagellar-mediated motility. The vast majority of Salmonella that colonized the CAM tumor were extracellular. Thus, tumor invasion was independent of both SPI1- and SPI2-encoded type three secretion systems. Surprisingly, the extracellular residence of Salmonella on CAM tumors did not require biofilm formation. We evaluated our wild-type parental strain compared to the attenuated clinical strain VNP20009 and discovered a reduced tumor colonization capability of VNP20009. The inability to effectively colonize CAM tumors potentially explains the reduced anti-tumor efficacy of VNP20009. Our work establishes the xenograft CAM model as an informative and predictive screening platform for studying tumor-colonizing Salmonella. IMPORTANCECancer has a major impact on society, as it poses a significant health burden to human populations worldwide. Salmonella Typhimurium has demonstrated promise in cancer treatment by exerting direct tumoricidal effects and enhancing host-mediated anti-tumor immunity in xenograft mouse studies. A general understanding of its pathogenesis and the relative ease of genetic manipulation support the development of attenuated strains for therapeutic use. Alternative in ovo models such as the CAM tumor model present a suitable screening platform to accelerate the development of therapeutic strains. It allows for rapid evaluation of Salmonella strains to assess their efficacy and potential as oncolytic agents. The present study establishes that the in ovo tumor model can be utilized as a preclinical tool for evaluating oncolytic Salmonella, bridging the gap between in vitro and in vivo screening.