Abstract
IntroductionPhysiologically 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).MethodsCNS 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.ResultsMacroscopic 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.ConclusionsOur 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.
Highlights
Relevant pre-clinical ex vivo models recapitulating central nervous system (CNS) tumor micro-environmental complexity will aid development of biologically-targeted agents
The present study provides a comprehensive characterization of the Rotary Cell Culture System (RCCS) as a method of cancer cell culture to generate a tumor niche more physiologically relevant than 2D culture and drug response more representative of clinical high grade brain tumor drug response, than conventional 2D cell panels
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
Summary
Relevant pre-clinical ex vivo models recapitulating CNS tumor micro-environmental complexity will aid development of biologically-targeted agents. The lack of pediatric brain tumor models in particular, has meant that chemotherapy drugs that are currently prescribed had been initially developed for the treatment of adult central nervous system (CNS) and other solid tumors. This ignores accumulating evidence that adult and pediatric brain tumors, histologically similar are molecularly distinct [4] and harbor distinct oncogenic mutations that may provide novel therapeutic targets. As next-generation therapy moves towards specific biological and pathway-related targets, evaluation of drug efficacy and improved treatment will be achieved via novel drug discovery, and through improved pre-clinical ex vivo models that better recapitulate microenvironmental complexity [5]. Current pre-clinical drug testing models do not replicate these key features
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