Glioblastoma is resistant to conventional treatments and has dismal prognosis. Despite promising in vitro data, molecular targeted agents have failed to improve outcomes in patients, indicating that conventional two-dimensional (2D) in vitro models of GBM do not recapitulate the clinical scenario. Responses of primary glioblastoma stem-like cells (GSC) to radiation in combination with EGFR, VEGF, and Akt inhibition were investigated in conventional 2D cultures and a three-dimensional (3D) in vitro model of GBM that recapitulates key GBM clinical features. VEGF deprivation had no effect on radiation responses of 2D GSCs, but enhanced radiosensitivity of GSC cultures in 3D. The opposite effects were observed for EGFR inhibition. Detailed analysis of VEGF and EGF signaling demonstrated a radioprotective role of Akt that correlates with VEGF in 3D and with EGFR in 2D. In all cases, positive correlations were observed between increased radiosensitivity, markers of unrepaired DNA damage and persistent phospho-DNA-PK nuclear foci. Conversely, increased numbers of Rad51 foci were observed in radioresistant populations, indicating a novel role for VEGF/Akt signaling in influencing radiosensitivity by regulating the balance between nonhomologous end-joining and homologous recombination-mediated DNA repair. Differential activation of tyrosine kinase receptors in 2D and 3D models of GBM explains the well documented discrepancy between preclinical and clinical effects of EGFR inhibitors. Data obtained from our 3D model identify novel determinants and mechanisms of DNA repair and radiosensitivity in GBM, and confirm Akt as a promising therapeutic target in this cancer of unmet need.

Purpose Low-dose whole lung radiotherapy (LDLR) has been proposed as a treatment for patients with acute respiratory distress syndrome associated with SARS-CoV-2 infection and clinical trials are underway. There is an urgent need for preclinical evidence to justify this approach and inform dose, scheduling and mechanisms of action. Materials and methods Female C57BL/6 mice were treated with intranasal bleomycin sulphate (7.5 or 11.25 units/kg, day 0), then exposed to whole lung radiation therapy (0.5, 1.0, 1.5 Gy or sham, day 3). Bodyweight was measured daily and lung tissue harvested for histology and flow cytometry on day 10. Computed tomography (CT) lung imaging was performed pre-radiation (day 3) and pre-endpoint (day 10). Results Bleomycin caused pneumonitis of variable severity which correlated with weight loss. LDLR at 1.0 Gy was associated with a significant increase in the proportion of mice recovering to 98% of initial bodyweight and a proportion of these mice exhibited less severe histopathological lung changes. Mice experiencing moderate initial weight loss were more likely to respond to LDLR than those experiencing severe initial weight loss. Additionally, LDLR (1.0 Gy) significantly reduced bleomycin-induced increases in interstitial macrophages, CD103+ dendritic cells and neutrophil-DC hybrids. Overall, bleomycin-treated mice exhibited significantly higher percentages of non-aerated lung in left than right lungs and LDLR (1.0 Gy) limited further reductions in aerated lung volume in right but not left lungs. LDLR at 0.5 and 1.5 Gy did not improve bodyweight, flow cytometric or radiological readouts of bleomycin-induced pneumonitis. Conclusions Our data support the concept that LDLR can ameliorate acute inflammatory lung injury, identify 1.0 Gy as the most effective dose and provide evidence that it is more effective in the context of moderate than severe pneumonitis. Mechanistically, LDLR at 1.0 Gy significantly suppressed bleomycin-induced accumulation of pulmonary interstitial macrophages, CD103+ dendritic cells and neutrophil-DC hybrids.

PurposeLow-dose whole lung radiotherapy (LDLR) has been proposed as a treatment for patients with acute respiratory distress syndrome associated with SARS-CoV-2 infection and clinical trials are underway. There is an urgent need for preclinical evidence to justify this approach and inform dose, scheduling and mechanisms of action.Materials and methodsFemale C57BL/6 mice were treated with intranasal bleomycin sulphate (7.5 or 11.25 units/kg, day 0), then exposed to whole lung radiation therapy (0.5, 1.0, 1.5 Gy or sham, day 3). Bodyweight was measured daily and lung tissue harvested for histology and flow cytometry on day 10. Computed tomography (CT) lung imaging was performed pre-radiation (day 3) and pre-endpoint (day 10).ResultsBleomycin caused pneumonitis of variable severity which correlated with weight loss. LDLR at 1.0 Gy was associated with a significant increase in the proportion of mice recovering to 98% of initial bodyweight and a proportion of these mice exhibited less severe histopathological lung changes. Mice experiencing moderate initial weight loss were more likely to respond to LDLR than those experiencing severe initial weight loss. Additionally, LDLR (1.0 Gy) significantly reduced bleomycin-induced increases in interstitial macrophages, CD103+ dendritic cells and neutrophil-DC hybrids. Overall,bleomycin-treated mice exhibited significantly higher percentages of non-aerated lung in left than right lungs and LDLR (1.0 Gy) prevented further reductions in aerated lung volume in right but not left lungs. LDLR at 0.5 and 1.5 Gy did not modulate bodyweight or flow cytometric readouts of bleomycin-induced pneumonitis.ConclusionsOur data support the concept that LDLR can ameliorate acute inflammatory lung injury, identify 1.0 Gy as the most effective dose and provide preliminary evidence that it is more effective in the context of moderate than severe pneumonitis. Mechanistically, LDLR at 1.0 Gy significantly suppressed bleomycin-induced accumulation of pulmonary interstitial macrophages, CD103+ dendritic cells and neutrophil-DC hybrids.

<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>Statistical analysis of MK2206 efficacy study</p>