<p>Erlotinib radiosensitisation of 2D GSC is VEGF-independent. A-D, Clonogenic survival of R10 GSC grown in 3D (A) and 2D (B-D) conditions and irradiated with single doses of X-rays (0-6 Gy; n=3) 2 hours after treatment of erlotinib (1microM) or vehicle (DMSO) in the presence (+VEGF) or absence (-VEGF) of VEGF (3 ng/ml). Mean {plus minus} SD of 3 independent experiments is shown; curves are fitted to a linear quadratic model. Erlotinib significantly radioprotected 3D VEGF-deprived cells (two-way ANOVA; R10 3D (-) VEGF vehicle vs 3D (-) VEGF plus erlotinib p < 0.05). No significant effect of erlotinib was observed in the presence of VEGF in 3D cells. Erlotinib radiosensitised 2D G7, E2 and R10 cells irrespective of VEGF presence (B-D). E, Quantification of gH2AX foci per nucleus in G7 3D cells treated with erlotinib in the absence of VEGF, or with VEGF after radiation treatment (24 h). Graph represents mean of medians from 3 independent experiments. p values calculated by t test (* p<0.01; ** p<0.001). F and G, Quantification of pDNA-PKcs (upper graph) and Rad51 foci (lower graph) per nucleus following radiation treatment. Graph represents mean of medians from 3 independent experiments. p values calculated by t test (* p <0.01; ** p <0.001).H, Quantification of DNA-PKcs and EGFR colocalisation per nucleus in E2 3D GSC. Approximately 40 nuclei were quantified for each condition. Box and whisker plots represent median number of signal per nucleus, p values calculated by Mann Whitney U test (*p<0.05; **p<0.005).</p>
<p>Radiosensitisation of G1 and G7 3D GSC by PARP inhibitor olaparib and ATM inhibitor KU55933. A, Determination of secreted VEGF by Elisa assay of media recovered from G7, E2 and R10 3D cells grown at hypoxic (1% O2) and normoxic (20% O2) conditions. Media was collected 48 h after seeding. Data represents means {plus minus} SD from 3 independent experiments performed in duplicate. B, Clonogenic survival efficiency of G1 and G7 GSC grown on 3D conditions after receiving 0 or 3 Gy in a 96-well clonogenic assay. C-E, Erlotinib (C), olaparib (D) and KU55933 (E) dose response (0.1 microM to 10 microM) at 0 and 3 Gy in G1 and G7 3D GSC. Cells were treated with respective inhibitor 2 h prior to ionizing radiation. Values were normalized to vehicle (DMSO) against their respective radiation dose. Curves were fitted with a median enhancement effect model. Statistical analysis with one-way ANOVA, *p<0.05; **p<0.005.</p>
<p>2D and 3D GSC exhibit similar levels of CENPF positive cells and radiation response to MK-2206. A, Representative immunofluorescent images of G7 and E2 cells grown in 2D or 3D conditions for CENPF. B, Percentage of cells showing CENPF staining per field as in (A). Graph depicts mean {plus minus}SD of three independent experiments. C, Representative immunofluorescent images for gammaH2AX foci of E2 GSC grown in 2D conditions fixed with 2% PFA at the indicated time points following ionising radiation (5 Gy). D, Percentages of cells displaying micronuclei. An average of 350 cells/condition/experiment were identified randomly and scored. Mean {plus minus} SEM of 3 independent experiments. P values calculated by t test. Aberrant DNA damage repair in VEGF-deprived GSC. E, Quantification of pDNA-PKcs (Thr2609) foci per nucleus following radiation treatment. Graph represents mean of medians from 3 independent experiments. p values calculated by t test (* p<0.01; ** p<0.001). F, Representative immunofluorescent images of G7 GSC grown in 3D conditions for BRCA2 foci at 3 hours following ionising radiation (5 Gy) in the presence or absence of VEGF. G, Percentage of nuclei exhibiting Rad51 foci. Positive Rad51 nuclei (>3 Rad51 foci per nucleus) were quantified and expressed as the percentage of total cells per image field. 3 fields (~ 30-50 cells per field) from 4 independent experiments were quantified. Graph depicts mean {plus minus}SD.</p>
<p>Tables depicting dose modifying factors (DMF) at surviving fractions of 0.37% and 0.1% survival were calculated for each treatment combination. A, G7, E2 and R10 cells grown in 2D or 3D conditions grown in the presence of VEGF [(+) VEGF], or the absence of VEGF [(-) VEGF] followed by radiation at indicated doses (0-5 Gy). DMF at 0.37 and 0.1 for (+) VEGF compared to (-) VEGF conditions. B, G7, E2 and R10 cells grown in 2D or 3D conditions were treated with DMSO or erlotinib followed by radiation treatment as in A. DMF at 0.37 and 0.1 for erlotinib compared to DMSO conditions. C, G7 and E2 cells were incubated with MK2206, Erlotinib and their combination for 2D conditions or {plus minus}VEGF and MK2206 for 3D conditions and irradiated as in A. DMF calculated for treatments v DMSO for 2D cells; (-) VEGF, (-) VEGF plus MK2206, or (+) VEGF plus MK2206 v (+) VEGF for 3D cells. D, E2 cells were transfected with either siScramble (siScr) or siRNAs against Akt1 and Akt3 for 48 hrs followed by radiation treatment as in A. DMF calculated for siAkt vs siScr. E, Cell lysates from E2 cells transfected with siRNA against DNA-PKcs (siDNAPKcs) or siScr for 48 hours followed by radiation treatment as in A. DMF calculated for treatments v DMSO_siScr.</p>
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