Recapitulation of tumor heterogeneity and molecular signatures in a 3D brain cancer model with decreased sensitivity to histone deacetylase inhibition

INTRODUCTION Physiologically relevant pre-clinical ex vivo models recapitulating CNS tumor micro-environmental complexity will aid development of biologically-targeted agents. We present comprehensive characterization of tumor aggregates generated using the 3D Rotary Cell Culture System (RCCS). METHODS CNS cancer cell lines were grown in conventional 2D cultures and the RCCS and comparison with a cohort of 53 pediatric high grade gliomas conducted by genome wide gene expression and microRNA arrays, coupled with immunohistochemistry, ex vivo magnetic resonance spectroscopy and drug sensitivity evaluation using the histone deacetylase inhibitor, Vorinostat. RESULTS Macroscopic RCCS aggregates recapitulated the heterogeneous morphology of brain tumors with a distinct proliferating rim, necrotic core and oxygen tension gradient. Gene expression and microRNA analyses revealed significant differences with 3D expression intermediate to 2D cultures and primary brain tumors. Metabolic profiling revealed differential profiles, with an increase in tumor specific metabolites in 3D. To evaluate the potential of the RCCS as a drug testing tool, we determined the efficacy of Vorinostat against aggregates of U87 and KNS42 glioblastoma cells. Both lines demonstrated markedly reduced sensitivity when assaying in 3D culture conditions compared to classical 2D drug screen approaches. CONCLUSIONS Our comprehensive characterization demonstrates that 3D RCCS culture of high grade brain tumor cells has profound effects on the genetic, epigenetic and metabolic profiles of cultured cells, with these cells residing as an intermediate phenotype between that of 2D cultures and primary tumors. There is a discrepancy between 2D culture and tumor molecular profiles, and RCCS partially re-capitulates tissue specific features, allowing drug testing in a more relevant ex vivo system

Extracellular vesicle-transported Semaphorin3A promotes vascular permeability in glioblastoma

International audienceGlioblastoma are malignant highly vascularized brain tumours, which feature large oedema resulting from tumour-promoted vascular leakage. The pro-permeability factor Semaphorin3A (Sema3A) produced within glioblastoma has been linked to the loss of endothelial barrier integrity. Here, we report that extracellular vesicles (EVs) released by patient-derived glioblastoma cells disrupt the endothelial barrier. EVs expressed Sema3A at their surface, which accounted for in vitro elevation of brain endothelial permeability and in vivo vascular permeability, in both skin and brain vasculature. Blocking Sema3A or its receptor Neuropilin1 (NRP1) hampered EV-mediated permeability. In vivo models using ectopically and orthotopically xenografted mice revealed that Sema3A-containing EVs were efficiently detected in the blood stream. In keeping with this idea, sera from glioblastoma multiforme (GBM) patients also contain high levels of Sema3A carried in the EV fraction that enhanced vascular permeability, in a Sema3A/ NRP1-dependent manner. Our results suggest that EV-delivered Sema3A orchestrates loss of barrier integrity in glioblastoma and may be of interest for prognostic purposes