Orthotopic mouse model of breast cancer

MDA-MB-231 cells with doxycycline inducible expression of MscL G22S and constitutive click beetle green luciferase expression (231-MscL-CBG) and MDA-MB-231 cells with constitutive click beetle green luciferase-only (231-CBG) were injected bilaterally into the 4th inguinal mammary fat pads of 6–10-week-old female NSG mice (Jackson Laboratory) on day 0. Three cohorts of mice were then studied: (1) 231-MscL-CBG mice treated with 1% sucrose in drinking water (n = 4); (2) 231-CBG mice with water containing 2 mg/ml doxycycline and 1% sucrose (n = 5); and (3) experimental group mice with 231-MscL-CBG cells and with water containing both doxycycline and sucrose as for group 2 (n = 5). The primary tumor size was tracked at the site of injection for each mouse via bioluminescence imaging (IVIS Spectrum, Perkin-Elmer). At day 43, the mice were euthanized and bioluminescence images of the liver, lung, abdomen, and spleen were taken to quantify metastases. The statistical significance testing of primary tumor growth data consisted of two-tailed student t-tests amongst all groups for each day. The statistical significance testing of the photon flux, metastasis data consisted in taking the log transform of all data points and performing a two-tailed t-test.