Figure 1. Cytotoxic effects of axitinib and G47Δ-mIL12 in mouse and human cells in vitro.
A, B. Dose response curves for axitinib (A) and G47Δ-mIL12 (B) in 005 GSCs after 4 days and MBMEC after 5 days; each graph represents an average of 2–4 experiments performed in triplicate. For G47Δ-mIL12 experiments (B), cells were seeded in their respective media with ‘no heparin’, virus was added 0 hr (for MBMEC) or 5 hr (for 005) post-seeding, and viability measured by MTS assay. C, D. Axitinib reduced matrigel-based tube formation in 005 GSCs. C. Microscopic images of representative wells imaged at 20× are shown. Bar = 100 µm. D. Quantification of branching point counts from 3–5 random fields/well. The numbers of tube branches were significantly reduced after axitinib treatment (0.03µM or 0.3µM) compared to mock treatment. Experiment was performed in triplicate. Tukey’s multiple comparison test was used to compare the different treatment groups; ****P < 0.0001. E. Cytotoxic effects of axitinib in human GSCs in vitro. Dose response curves for axitinib in MGG50, MGG85, and MGG123 GSCs after 6 days, as measured by MTS assay; each graph represents an average of three experiments performed in triplicate. F. In vitro limiting dilution study. 005 cells were plated in 96-well plate. Cells were continuously exposed to a non-toxic concentration of axitinib (300 nM) and/or G47Δ-mIL12 (moi 0.1) for 12 days and number of wells (n=30 wells total) containing spheres were recorded and plotted using ELDA: Extreme Limiting Dilution Analysis form, as described (35). Estimated stem cell frequencies were; 1 in 4.3 for mock, 1 in 11.7 for axitinib, 1 in 5.9 for virus, and 1 in 20.4 for combination. Treatment groups were compared to each other using a Chi Square test: mock vs. axitinib, p=7.9 × 10−15; mock vs. virus, p=0.024; mock vs. combination, p=1,1 × 10−25; axitinib vs. virus, p=5.1 × 10−6; axitinib vs. combination, p=0.0006; virus vs. combination, p=5.6 × 10−13.