Quorum-sensing Salmonella selectively trigger protein expression within tumors

Significance Nonpathogenic Salmonella localize to tumors and can be engineered to secrete anticancer proteins, but tumor-specific expression is essential to prevent systemic toxicity. To reduce unwanted side effects in healthy tissue, we integrated Salmonella with a quorum-sensing (QS) switch that only initiates drug expression in the tightly packed colonies present within tumors. Using an in vitro 3D-tumor-on-a-chip device and in vivo mouse models, we show that QS Salmonella specifically initiates protein expression within cancerous tissue while remaining uninduced in livers. Protein expression was triggered when inducer molecules from enough close neighbors reached a critical concentration. Because of these selective qualities, QS Salmonella are a promising tool for tumor-specific delivery of therapeutic proteins. Salmonella that secrete anticancer proteins have the potential to eliminate tumors, but nonspecific expression causes damage to healthy tissue. We hypothesize that Salmonella, integrated with a density-dependent switch, would only express proteins in tightly packed colonies within tumors. To test this hypothesis, we cloned the lux quorum-sensing (QS) system and a GFP reporter into nonpathogenic Salmonella. Fluorescence and bacterial density were measured in culture and in a tumor-on-a-chip device to determine the critical density necessary to initiate expression. QS Salmonella were injected into 4T1 tumor-bearing mice to quantify GFP expression in vivo using immunofluorescence. At densities below 0.6 × 1010 cfu/g in tumors, less than 3% of QS Salmonella expressed GFP. Above densities of 4.2 × 1010 cfu/g, QS Salmonella had similar expression levels to constitutive controls. GFP expression by QS colonies was dependent upon the distance to neighboring bacteria. No colonies expressed GFP when the average distance to neighbors was greater than 155 µm. Calculations of autoinducer concentrations showed that expression was sigmoidally dependent on density and inversely dependent on average radial distance. Based on bacterial counts from excised tissue, the liver density (0.0079 × 1010 cfu/g) was less than the critical density (0.11 × 1010 cfu/g) necessary to initiate expression. QS Salmonella are a promising tool for cancer treatment that will target drugs to tumors while preventing damage to healthy tissue.