Fig. 1. Bacterial depletion reduces the tumor response to radiation.

Orthotopic E0771 mammary tumors (A-D) were treated with antibiotics (Abx) for one week prior to being treated with localized kV radiation (16 Gy) (RT). Both individual tumors (A, B), mean tumor burden ± SEM (C) and survival (D) for the indicated treatments were assessed every 3 days until endpoint. Antibiotics (Abx) were ampicillin, imipenem, cilastatin and vancomycin. Mean tumor burden/animal ± SEM and survival were also assessed in subcutaneous B16 melanomas (E, F) treated with antibiotics (Abx) for one week prior to being treated with localized kV irradiation (16 Gy). Antibiotics (Abx) were ampicillin, imipenem, cilastatin and vancomycin. E0771 mammary tumors were harvested at one week following RT and stained for bromodeoxyuridine (BrdU) and cleaved caspase 3 (CC3) to assess for proliferation and cell death respectively (G, H, left panels). Slides were scanned using the Aperio slide scanner and analyzed using the ScanScope nuclear algorithm included in the Aperio software package (G, H, right panels) to quantitate the number of BrdU (I) and CC3 (J) positive cells. Significance was determined by two-way ANOVA with post-hoc testing for tumor growth, Log-Rank test for the Kaplan-Meier survival curves and Student’s t-test with Welch’s correction for the immunohistochemistry (IHC). DNA from fecal pellets obtained from Abx treated mice were examined by quantitative PCR for bacterial 16S or fungal 18S ribosomal DNA and the fold change from untreated mice with standard error was calculated (K). Numbers (n) for each experiment are listed on the figure and are pooled data from at least two independent experiments. For all figures, significance is shown as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.