Radiation Responses of 2D and 3D Glioblastoma Cells: A Novel, 3D-specific Radioprotective Role of VEGF/Akt Signaling through Functional Activation of NHEJ

Abstract

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.

Lineage‐specific mechanisms and drivers of breast cancer chemoresistance revealed by 3D biomimetic culture

“…Finally, profiling of treated cancer cells within the scaffold led to the identification of candidate predictive biomarkers. Several evidences indicates that engineered 3D models can be useful approaches to study and identify drug resistance mechanisms to anticancer agents (76)(77)(78)(79)(80). Here we demonstrate that the molecular changes identified through our biomimetic model are i) predictive of in vivo molecular alterations on tumor xenografts and ii) demonstrate clinical predictive potential.…”

Comparison of VEGF-A secretion from tumor cells under cellular stresses in conventional monolayer culture and microfluidic three-dimensional spheroid models

“…Furthermore, systematic comparison of the VEGF-A secretion from conventional monolayer cell culture and 3D spheroid model has not been performed due to technical limitations. Cellular stresses in 3D spheroids have been observed to potentially trigger several pro survival pathways [2,20,25,37,44], where VEGF plays a major role [45,46]. Common challenges faced in the in vitro 3D culture models include tedious sample handling, maintaining uniformity and stability to avoid structural disintegration of spheroids.…”

“…Several studies using anti-cancer agents on spheroid systems have concluded that physical properties of spheroids are related to drug efficacy [50,51]. Furthermore, recent studies have also shown that cytokine secretion profiles are different between conventional monolayer cultures and 3D culture systems [46,52]. A tactful approach capable of direct quantitative characterization of VEGF protein is highly desired to investigate responses of multicellular spheroids under specific cellular stress conditions without delving into the intercellular variations which can be further compared to the clinical observations.…”

PARP Inhibitor Resistance Mechanisms and Implications for Post-Progression Combination Therapies

“…In the setting of a BRCA2-mutated clone that developed a resistance mutation and expressed a functionally wild-type protein, VEGFR3 inhibition was sufficient to restore chemosensitivity [110]. The inhibition of VEGF and VEGFR2 in organoid models prevented Akt-mediated DNA repair, thereby preventing HR and leading to aberrant NHEJ [111]. Anti-angiogenics are also hypothesized to induce or exacerbate intratumoral hypoxia, which itself is associated with impaired HR [112][113][114][115].…”