Tumor Stroma Content Regulates Penetration and Efficacy of Tumor-targeting Bacteria

Bacteria-based cancer therapy (BBCT) strains grow selectively in primary tumors and metastases, colonize solid tumors independent of genetics, and kill cells resistant to standard molecular therapy. Clinical trials of BBCT in solid tumors have not reported any survival advantage yet, partly due to the limited bacterial colonization. Collagen, abundant in primary and metastatic solid tumors, has a well-known role in hindering intratumoral penetration of therapeutics. Nevertheless, the effect of collagen content on the intratumoral penetration and antitumor efficacy of BBCT is rarely unexplored. We hypothesized that the presence of collagen limits the penetration and, thereby, the antitumor effects of tumor-selective Salmonella. Typhimurium VNP20009 cheY⁺. We tested our hypothesis in low and high collagen content tumor spheroid models of triple-negative murine breast cancer. We found that high collagen content significantly hinders bacteria transport in tumors, reducing bacteria penetration and distribution by ~7-fold. The higher penetration of bacteria in low collagen-content tumors led to an overwhelming antitumor effect (~73% increase in cell death), whereas only a 28% increase in cell death was seen in the high collagen-content tumors. Our mathematical modeling of intratumoral bacterial colonization delineates the role of growth and diffusivity, suggesting an order of magnitude lower diffusivity in the high collagen-content tumors dominates the observed outcomes. Finally, our single-cell resolution analysis reveals a strong spatial correlation between bacterial spatial localization and collagen content, further corroborating that collagen acts as a barrier to bacterial penetration despite S. Typhimurium VNP20009 cheY⁺ motility. Understanding the effect of collagen on BBCT performance could lead to engineering more efficacious BBCT strains capable of overcoming this barrier to colonization of primary tumors and metastases.