Figure 4.

Rapamycin inhibits translation of complex mRNA transcripts by highly metastatic osteosarcoma cells within the lung microenvironment.

A) Expression of ds-SL-mCherry reporter in highly metastatic murine K7M2 cells exposed to 0.1μM rapamycin or vehicle for 5 days in culture. Values represent averages from 15 images per plate × 3 plates per condition +/− SD. P-value does not reach significance using unpaired t-test with Welch’s correction.

B) Expression of ds-SL-mCherry reporter in highly metastatic human MG63.3 cells exposed to 0.1μM rapamycin or vehicle for 5 days in culture. Values represent averages of 15 images per plate × 3 plates per condition +/− SD. P-value does not reach significance using unpaired t-test with Welch’s correction.

C) In Vivo images of GFP+ ds-SL-mCherry MG63.3 lung metastases on day 27 of an experimental metastasis assay following 5 daily IP doses of vehicle or 5mg/kg rapamycin (days 22-26). Images were captured using a 5× lens on a Leica-DM IRB inverted fluorescent microscope.

D) Quantification of fraction of GFP+ tumor area expressing ds-SL-mCherry reporter in lung metastases following 5 daily IP doses of vehicle or 5mg/kg rapamycin. Values represent averages of 5 images per mouse × 3 mice per condition +/− SD. * P-value calculated using unpaired t-test with Welch’s correction < 0.05.

E) Images of PTCHD2 in GFP+ MG63.3 lung metastases on day 27 of an experimental metastasis assay following 5 daily IP doses of vehicle or 5mg/kg rapamycin (days 22-26). Images were captured using a 63× lens on a Zeiss LSM-710 laser scanning confocal microscope.

F) Quantification of fraction of GFP+ tumor area expressing PTCHD2 in lung metastases following 5 daily IP doses of vehicle or 5mg/kg rapamycin. Values represent averages of 2 images per mouse × 3 mice per condition +/− SD. * P-value calculated using unpaired t-test with Welch’s correction < 0.05.