Fig. 1. The intracellular lifestyle of Salmonella is controlled by flhDC.
a The design goals were to genetically engineer a bacterial vehicle that (1) synthesizes a protein drug (yellow/purple), (2) actively invades into cancer cells, and (3) releases drug, which escapes Salmonella vacuoles (SCVs, red). b, c Ninety-six hours after intratumoral injection of 2 × 106 CFU of intracellular-reporting Salmonella into subcutaneous 4T1 tumors in BALB/c mice, more bacteria (red) were intracellular (green; black arrows) than extracellular (white arrows; P < 0.0001; n = 1258 bacteria in 4 mice). d) In monolayer culture, Salmonella (light blue, arrows) invade cancer cells (red). Extracellular bacteria were removed with gentamicin (an invasion assay). e Knockout ΔflhD Salmonella were transformed with PBAD-flhDC. Uninduced bacteria (flhDC-) minimally invaded cancer cells. Induction with 20 mM arabinose (flhDC+) promoted invasion (black arrows). f Re-expression of flhDC significantly increased invasion (P = 0.0012; n = 3 independent biological samples). g In three-dimensional tumor-on-a-chip devices (top left), flhDC+ Salmonella, with a green invasion reporter (top right), invaded more cells than flhDC− controls (P < 0.0001; n = 6 chambers for flhDC+ and n = 4 for flhDC-). Data are shown as means ± SEM. Statistical comparisons in c, f, and g, and are two-tailed, unpaired Student’s t-tests with asterisks indicating significant differences (**P < 0.01; ****P < 0.0001). Images in b, d, and e are representative of 4, 52, and 6 independent biological samples. Scale bars in b, d, and e are 10 µm, and in g the scale bars are 100 µm.