Fig. 6.

Effect of mTORC1 inhibition on established metastasis induced by T47D-RON cells. a Experimental scheme for established metastasis induced by T47D-RON cells and treatment with mTORC1 inhibitor, everolimus. T47D-RON cells were tail vein injected to NSG mice and mice were put on Doxy diet (50 ppm) to induce expression of RON. Treatment with everolimus or vehicle started at day 30 following cell injection, when metastatic lesions were detectable with in vivo IVIS imaging. Once mice developed recurrence of metastasis due to everolimus resistance, RON expression was blocked by removing doxy diet, and mice continued to be followed. b Representative IVIS images of mice over the course of treatment with everolimus or vehicle and with or without active RON expression, controlled by doxy diet. Mice were treated with everolimus (5 mg/kg) five times a week (5 days on, 2 days off), and were imaged weekly. c Graph showing quantification of metastasis in the everolimus (n = 11) vs vehicle-treated group (n = 12). Dashed line indicates the time when doxy diet was stopped to block RON expression. Data are shown as mean ± SEM, **P < 0.005 (unpaired t-test). d Representative images showing H&E, CAM5.2, RON, and pS6 (Ser 235/236) staining for metastatic lesions in the ovaries of mice treated with everolimus vs vehicle. e Status of pAKT and pERK as readouts for activation of PI3K and MAPK pathways, respectively, in metastatic lesions of ovaries through different stages of treatment with everolimus. Scale bars represent 100 μm. See also Supplementary Figure S15 and S16