Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

Ashley N. Gilbert; Rachael S. Shevin; Joshua C. Anderson; Catherine P. Langford; Nicholas Eustace; G. Yancey Gillespie; Raj Singh; Christopher D. Willey

doi:10.3791/54026

Method Article

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

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DOI:

10.3791/54026

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June 9th, 2016

Ashley N. Gilbert¹ , Rachael S. Shevin⁴ , Joshua C. Anderson² , Catherine P. Langford³ , Nicholas Eustace² , G. Yancey Gillespie³ , Raj Singh⁴ , Christopher D. Willey²

¹Biomedical Engineering, University of Alabama at Birmingham, ²Radiation Oncology, University of Alabama at Birmingham, ³Neurosurgery, University of Alabama at Birmingham, ⁴Vivo Biosciences, Inc.

Summary

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

Abstract

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Introduction

The most common primary intracranial malignant brain tumors are grade III astrocytomas and grade IV glioblastoma multiforme (glioblastoma or GBM). These tumors offer poor prognoses with median one-year survival between 12 - 15 months with current therapies for GBM in the US^1-3. Multimodality therapies include surgery, radiation, and chemotherapy including temozolomide (TMZ) and kinase-targeted agents. Kinase signaling is frequently dysregulated in GBM, including subsets of tumors with amplification or activating mutations in the Epidermal Growth Factor Receptor (EGFR), increases in Platelet Derived Growth Factor Receptor (PDGFR) signaling, increase....