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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Medicine

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

Published: June 9th, 2016

DOI:

10.3791/54026

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

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.

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.

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 US1-3. 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....

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NOTE: All xenograft therapy evaluations were done using an orthotopic tumor model for glioblastoma on a protocol approved by the Institutional Animal Care and Use Committee.

1. Isolation of Patient-derived GBM Xenograft Cells

  1. Preparation of Reagents
    1. Re-constitute collagenase-I in sterile water to a concentration of 5 mg/ml and sterile filter. Store in 1 ml aliquots at -20 °C (final concentration is 50 µg/ml in 100 ml enzyme solution).
    2. <.......

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We have shown that 3D biogel culture system supports long-term growth and function of multiple cell types. In this collaborative project, patient-derived GBM xenolines (PDX) are used for producing hundreds of microtumors. Dissociated cells (3 x 105) or neurospheres (40 - 50) were embedded in biogel beads (2 mm) and after quick gelation they are cultured in a NB-media filled custom bioreactor. Cellular viability (Calcein-AM), growth profile (MTT), and kinomic activity array-base.......

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Critical steps within the protocol predominantly relate to microtumor generation, as well as drug dosing and maintenance. Because the microtumor beads are fragile and easily torn, extreme care is needed in both developmental stages of an assay and maintenance. If an error occurs during either of these processes, experimental interpretation can be compromised, causing extension or unnecessary repetition of the experiments or even exclusion of data.

Modifications and troubleshooting, especially .......

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Supported by NIH R21 grant (PI: C. Willey, CA185712-01), Brain Tumor SPORE award (PD: G.Y. Gillespie, P20CA 151129-03) and SBIR contract (PI: R. Singh, N43CO-2013-00026).

....

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Name Company Catalog Number Comments
Collagenase-I  Sigma-Aldrich CO130
Trypsin EDTA (10X) Invitrogen 15400-054 
Neurobasal-A Life Technologies 10888-022
N-2 Supplement Life Technologies 17502-048 1x final concentration
B-27 Supplement w/o Vitamin A Life Technologies 12587-010 1x final concentration
Recombinant Human FGF-basic Life Technologies PHG0266 10 ng/mL final concentration
Recombinant Human EGF Life Technologies PGH0315 10 ng/mL final concentration
L-Glutamine Corning Cellgro Mediatech 25-005-CI 2 mM final concentration
Fungizone Omega Scientific FG-70 2.5 ug/mL final concentration
Penicillin Streptomycin Omega Scientific PS-20 100 U/mL Penicillin G, 100 ug/mL Streptomycin final concentration
Gentamicin Life Technologies 15750-060 50 ng/mL final concentration
MTT Life Technologies M6494 prepared to 5 mg/mL in PBS and sterile filtered, 1 mg/mL in well
SDS Fisher BP166 for MTT lysis buffer, prepared to 10% in 0.01M HCL, 5% in well
HCl Fisher A144SI-212 for MTT lysis buffer, prepared to 0.01M with SDS, 5 mM in well
Calcein AM Life Technologies C1430 1 mM in DMSO stock, 2 uM in PBS staining solution, 1 uM in well
Halt’s Protein Phosphatase Inhibitor cocktail  Pierce ThermoScientific 78420 1:100 ratio in MPER 
Halt's Protein Protease Inhibitor  Pierce ThermoScientific 87786 1:100 ratio in MPER
Mammalian Protein Extraction Reagent (MPER) Pierce ThermoScientific PI78501
Trypan Blue Pierce ThermoScientific 15250-061
DMSO Fisher BP231 for dissolution of calcein AM & compounds
Phosphate-Buffered Saline without Ca/Mg Lonza 17-517Q diluted to 1X with MiliQ ultrapure water and sterile filtered (for cell culture)
Dulbecco's Phosphate-Buffered Saline with Ca/Mg Corning Cellgro Mediatech 20-030-CV diluted to 1X with MiliQ ultrapure water (for pre-fixation wash)
10% Neutral Buffered Formalin Protocol 032-060
Trypan Blue Pierce ThermoScientific 15250-061
High Density Hubiogel Vivo Biosciences HDHG-5
Halt's Protein Phosphatase Inhibitor Pierce 78420
Halt's Protein Protease Inhibitor Pierce 87786
Mammalian Protein Extraction Reagent (MPER) Thermo Scientific 78501
Protein Tyrosine Kinase (PTK) Array Profiling chip PamGene 86312
PTK kinase buffer PamGene 36000 300 µl 10X PK buffer stock in 2.7 ml dH20, catalog number for PTK reagent kit
ATP PamGene 36000 catalog number for PTK reagent kit
PY20- FITC-conjugated antibody PamGene 36000 catalog number for PTK reagent kit
PTK Additive PamGene 32114
PTK-MM1 tube (10X BSA) PamGene 36000 catalog number for PTK reagent kit
Serine/Threonine Kinase (STK) Array Profiling chip PamGene 87102
STK kinase buffer PamGene 32205 catalog number for STK reagent kit
STK Primary Antibody Mix (DMAB tube) PamGene 32205 catalog number for STK reagent kit
FITC-conjugated Secondary Antibody PamGene 32203
STK-MM1 tube (100X BSA) PamGene 32205 catalog number for STK reagent kit
STK Antibody Buffer PamGene 32205 catalog number for STK reagent kit
Equipment
#11 Blades, sterile Fisher 3120030
#3 scalpel handles, sterile Fisher 08-913-5
100mm glass Petri dishes Fisher 08-748D
Semicurved forceps Fisher 12-460-318
Trypsinizing flask Fisher 10-042-12B
Magnetic stirrer Fisher 14-490-200
3/4" stir bar Fisher 14-512-125
B-D cell strainer  Fisher #352340
B-D 50ml Centrifuge tube Fisher #352098
PamStation 12 PamGene
BioNavigator 6.0 kinomic analysis software  PamGene
Evolve Kinase Assay Software PamGene
UpKin App software (upstream kinase prediction) PamGene
gentleMACS Dissociator Miltenyi Biotec 130-093-235
Rotary Cell Culture System (RCCS) Synthecon RCCS-D with 10 mL disposable HARV

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