Stroma content in triple-negative breast cancer spheroid models regulates penetration and efficacy of tumor-targeting Salmonella typhimurium.

Bacteria-based cancer therapy (BBCT) has not yet conferred clinical survival advantages in solid tumors, partly due to limited colonization. Collagen is well-known to hinder the penetration and efficacy of many therapeutic modalities in solid tumors. Nevertheless, the effect of collagen on BBCT efficacy remains largely unexplored. We hypothesized that collagen limits the distribution and, thereby, the efficacy of tumor-selective Salmonella Typhimurium VNP20009. By comparing high and low collagen-content triple-negative breast cancer spheroid models, we found that high collagen content reduces bacterial distribution by ~4.5-fold and antitumor effect by 61%. Mathematical modeling of bacteria intratumoral distribution shows that a 10-fold lower diffusivity in collagen-rich tumors is responsible for the observed outcomes. Single-cell resolution imaging corroborates these findings, revealing bacteria accumulation behind collagen-rich regions, wherein collagen acts as a physical barrier for motile bacteria. Discovering the interplay between collagen content and BBCT performance opens new opportunities for engineering BBCT strains with improved efficacy.