Figure 5. Overexpression of OLIG2 induces radioresistance in OLIG2^−low MYC-amplified MB.

(A) No tumor was detected in mice transplanted with control RCMB28 tumor cells post-irradiation. Sagittal images of whole brain (bright field). Tumor cells indicated in red (mCherry), OLIG2^−GFP-lentivirus infected cells indicated in green (GFP).

(B) Large tumors were observed in mice receiving OLIG2^−GFP-lentivirus infected tumor cells post-irradiation. Sagittal images of whole brain (bright field). The arrow points to an area of apoptosis. Tumor cells indicated in red (mCherry), OLIG2^−GFP-lentivirus infected cells indicated in green (GFP).

(C) Analysis of the efficiency (GFP) of viral transduction of pWPI-OLIG2-^GFP in mCherry+ tumor cells by flow cytometry before transplantation.

(D, E) Apoptosis in the GFP⁺ (arrow) and GFP⁻ (arrowhead) tumor regions in B. In D, tissue sections were immunostained for CC-3 (violet). OLIG2^−GFP-lentivirus infected cells indicated in green (GFP). E, quantification of %CC-3⁺ cells in apoptotic and OLIG2^−GFP-lentivirus infected regions of pWPI-OLIG2^−GFP transduced irradiated RCMB28 tumors (n=3). P values determined by Student T test.

(F) Ki67 expression (violet) was detected in both GFP⁺ (square 1) and GFP⁻ (square 2) tumor regions in mice receiving OLIG2^−GFP-lentivirus infected tumor cells post-irradiation. OLIG2 expression (violet) was co-expressed with GFP.

(G) IVIS images of representative mice transplanted with control tumor cells or OLIG2^−GFP-lentivirus infected tumor cells with CSI.

(H) Survival of animals transplanted with control tumor cells or purified OLIG2^−GFP-lentivirus-infected tumor cells with or without CSI. Log-rank test p*=0.0007, OLIG2 group vs. control group; p**=0.0014, OLIG2 CSI group vs. control CSI group.

(I-M). Flow cytometry assay of OLIG2-GFP⁺ cells: isotype control (I), purification before transplantation (J), tumors without irradiation (K), and relapsed tumors post-irradiation (L). Quantification of GFP expression (M). P values determined by Student T test.

(N) Comparison of tumors from mice transplanted with OLIG2^−GFP-virus infected tumor cells without CSI (top) and relapsed tumors post-irradiation (bottom). Sagittal images of whole brain (bright field). Tumor cells indicated in red (mCherry), OLIG2^−GFP-lentivirus infected cells indicated in green (GFP).

(O) IHC of GFP (green), mCherry (red), OLIG2 (violet), Ki67 (violet), and CC3 (violet) in the tumors from mice transplanted with OLIG2-expressing RCMB28 tumor cells with (bottom panels) or without irradiation (top panels). Scale bars= 50 μm.

(P, Q). Quantification of the percentage of CC-3⁺ (P) or Ki67⁺ (Q) cells out of total mCherry+ tumor cells in nonirradiated (n=3) and irradiated (n=5) tumors.

(See also Figures S5).