Dataset Information

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

ABSTRACT:

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.

SUBMITTER: Smith SJ

PROVIDER: S-EPMC3525561 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Publications

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

Smith Stuart J SJ Wilson Martin M Ward Jennifer H JH Rahman Cheryl V CV Peet Andrew C AC Macarthur Donald C DC Rose Felicity R A J FR Grundy Richard G RG Rahman Ruman R

PloS one 20121218 12

<h4>Introduction</h4>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).<h4>Methods</h4>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 an ...[more]

PMID: 23272238

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Project description:Introduction: Obesity and overweight affect more than one third of the world population, and are significant risk factors for type II diabetes, cardiovascular disease and cancer. Common obesity is usually accompanied by hyperleptinemia and leptin resistance. Purpose: Here, we investigate the molecular pathways underlying the effects of HDAC6 inhibition in reversing diet-induced obesity and interrogate potential pathways that link HDAC6 with leptin receptor signaling by conducting whole transcriptome analysis in white adipose tissues of the diet-induced obese mice that were treated with tubastatin A (an HDAC6-specific inhibitor) and in fat-specific HDAC6 knockout mice. Methods: Male 4-5 week-old C57B/6J wild type mice were fed with high fat diet for 16-20 weeks to generate diet-induced obese (DIO) mice. Around 28 weeks of age, mice were treated with daily ip injections of either tubastatin A-HCl (TubA, 25 mg/kg) or vehicle for four days. We probed the whole transcriptome in epididymal white adipose tissue (eWAT), and inguinal white adipose tissue (iWAT) following vehicle or TubA administration (eWAT.Veh, eWAT.TubA, iWAT.Veh, iWAT.TubA groups). In a separate cohort, DIO HDAC6 global knockout mice were treated with vehicle (Veh group) or TubA (TubA group). eWAT was excised for RNA extraction. In the third cohort, adipose tissue specific HDAC6 knockout mice were generated crossing adiponectin-Cre mice with HDAC6-floxed mice. These fat specific HDAC6 KO mice (AdKO) and their adiponectin-Cre controls (AdCre) were treated with vehicle for five weeks. eWAT was excised for RNA extraction.Total RNA samples from each tissue were extracted using Trizol. RNA Sequencing for the wild-type iWAT and eWAT samples from vehicle or TubA-treated mice was performed by the University of Michigan Sequencing Core, using the Illumina Hi-Seq 4000 platform. RNA sequencing for the other samples were conducted by Novogene, NovaSeq 6000 PE150, HiSeq SE50 platform. The following bioinformatics analysis have been conducted by Novogene: 1. Data Quality Control: filtering reads containing adapter or with low quality. 2. Statistics Analysis of Data Production and Quality. 3. Mapping Reads to Reference Genome. 4. Gene Expression Quantification. 5. Correlation analysis (For biological replicates only). 6. Differential Expression Analysis (two or more groups of samples). 7. GO Enrichment Analysis of Differentially Expressed Genes (DEGs) (two or more groups of samples). 8. KEGG Pathway Enrichment Analysis of Differentially Expressed Genes (DEGs) (two or more groups of samples). 9. GSEA Enrichment Analysis of Expressed Genes (two or more groups of samples). 10. Protein Protein Interaction Analysis. 11. Reactome Pathway Enrichment Analysis of Differentially Expressed Genes (DEGs) (two or more groups of samples and only for mouse samples). 12. Oncogene Functional Annotation analysis of Differentially Expressed Genes (DEGs) (two or more groups of samples and only for mouse sample). Results: Inhibitors of the cytosolic enzyme histone deacetylase 6 (HDAC6) act as potent anti-obesity agents, and leptin sensitizers. HDAC6 inhibitor TubA suppresses food intake and reduce obesity, in an HDAC6-dependent manner, resulting in up to fifty percent decrease in fat mass in DIO mice accompanied by significantly reduced hepatic steatosis and improved systemic glucose homeostasis. Conclusions: Mechanistically, peripheral -but not central- inhibition of HDAC6 confers central leptin sensitivity, and the anti-obesity effect of TubA is significantly attenuated in animals defective in the central leptin-melanocortin circuitry, including the db/db and MCR4 KO mice.

Dataset Information

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

Introduction

Methods

Results

Conclusions

Publications

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

Similar Datasets

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