Fig. 5. Safety, biodistribution, and clearance of ID Salmonella.

a Firefly luciferase-expressing ID Salmonella (2 × 10⁷ CFU/mouse) were intravenously injected into BALB/c mice with 4T1 tumors in the mammary fat pad (n = 4). Prior to imaging, mice were injected IP with 100 µl of 30 mg/ml D-luciferin. b The bacterial density in the tumors increased for 72 h and then decreased. c Biodistribution of bacteria in tumor-free BALB/c mice, 14 days after intravenous injection with 1 × 10⁷ ID Salmonella (n = 5). Densities were below detection in the lungs, kidneys, hearts, and brains. Measurements of zero bacteria in spleen (1 of 5) and liver (2 of 5) were not displayed. In spleens and livers, bacterial densities were more than 3000 times lower than in tumors (from the separate experiment in panel b using the same organ mincing technique (P = 0.0001, n = 3 mice, one mouse died prior to density measurement). d Comprehensive hematology of blood drawn from tumor-free BALB/c mice, 14 days after intravenous injection with 1 × 10⁷ ID Salmonella or saline (n = 4). No changes were observed in the number of any immune cells in the blood. e Chemistry profiling of the blood from the mice in d. There was no indication of liver damage, despite some liver colonization (c). Markers of liver damage are ALP alkaline phosphatase, ALT alanine transaminase, and AST aspartate transaminase. Data are shown as means ± SEM. Statistical comparisons in c were to a single condition performed with ANOVA followed by Dunnett’s method and asterisks indicate significance (***P < 0.001).