Combined RNAi-Mediated Suppression of Rictor and EGFR Resulted in Complete Tumor Regression in an Orthotopic Glioblastoma Tumor Model
Figure 8
The combined silencing of Rictor and EGFR in vivo results in a complete inhibition of tumor growth.
U251Ng2x, U251Rictor, U251EGFR and U251EGFR/Rictor cells were implanted into the right caudate nucleus-putamen of Rag2M mice (n = 6−8). Induction of shRNA expression in mice was initiated on day 21 by dissolving 2 mg/mL doxycyline and 5% sucrose in drinking water. a) On day 49, animals were imaged by Maestro™ fluorescence imaging unit for the expression of tRFP co-expressed with the shRNA sequences upon doxycycline-induced expression. Mice were then terminated and brains were harvested, sectioned and stained for nuclei, Rictor, EGFR and p(473)-AKT and imaged for all markers in addition to tRFP by robotic fluorescence microscopy. No tumor was detected in the U251EGFR/Rictor group. b) A representative brain section from U251Ng2x, U251Rictor, U251EGFR and U251EGFR/Rictor tumor groups is shown: tRFP (red) and Hoechst (blue). c) A representative tumor section from U251Ng2x, U251Rictor and U251EGFR tumor groups is shown: nuclei (blue), rRFP (red), Rictor (yellow), EGFR (green) and p(473)-AKT (orange). d) The expression of EGFR (left axis), Rictor (right axis) and p(473)-AKT (right axis) in U251Ng2x, U251Rictor, U251EGFR tumor sections were quantified (positive staining normalized to Hoechst nuclei staining). e) Tumor sizes were estimated by quantification of tumor areas in brain sections from all groups (left axis). The expression of the proliferation marker Ki67 in the tumor (proliferating fraction) was also quantified (right axis). *p-value ≤0.05; **p-value ≤0.01; ***p-value ≤0.001 compared to control untreated cells. ‡: No tumor was detected in the U251EGFR/Rictor group.