Recapitulation of Tumor Heterogeneity and Molecular Signatures in a 3D Brain Cancer Model with Decreased Sensitivity to Histone Deacetylase Inhibition

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.

Glioma cell invasion is significantly enhanced in composite hydrogel matrices composed of chondroitin 4- and 4,6-sulfated glycosaminoglycans

“…Previous studies have demonstrated the role of 3D tumor microenvironment in altering the gene expression levels of IGF binding proteins, SOX family transcription factors, among others that promote cell growth and survival in glioma cells 44, 45 . In this study, we aimed to focus instead on investigating the 3D microenvironment-related factors and mechanisms that may have contributed to the enhanced migration of glioma cells when encapsulated in 3D CS-GAG matrices as observed in the in vitro microfluidics based migration assays.…”

Concise Review: 3D cell culture systems for anticancer drug screening

“…Through the use of sophisticated 3D multicellular tumor spheroid (MCTS) systems, the microenvironment, phenotype and cellular heterogeneity of tumors are effectively represented (Thoma et al, 2014). MCTS systems create a gradient of oxygen and nutrients from the outside of tumor spheroids to the core.…”

“…1), which realistically mimic in vivo tumors (Ma et al, 2012). Many research studies have shown that the genotypic profile of cells in MCTS, versus cells grown in monolayer, are more similar to in vivo tumors (Smith et al, 2012). Cells in 3D culture conditions were found to exhibit gene expression profiles different to those grown in monolayer (Luca et al, 2013;Myungjin Lee et al, 2013).…”

In Vitro Modeling of Tissue-Specific 3D Microenvironments and Possibile Application to Pediatric Cancer Research

“…An exhaustive study on brain tumor aggregates generated using the 3D RCCS TM has been presented recently by Smith and collaborators [261]. Panchalingam and coworkers [262] developed bioreactor protocols for expanding cancer stem cells from human glioblastoma specimens. Human glioblastoma-derived cells were introduced in a bioreactor with paddle impellers (NDS Technologies, Palo Alto, CA).…”