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Inhibition of Hypoxic Tumor Cells using a Three-Dimensional Spheroid Model

Comparison of PMT-based whole well detection with image-based cellular analysis

Brad Larson1, Nicky Slawny2, Wini Luty3, and Peter Banks1
1BioTek Instruments, Inc., Winooski, Vermont, USA
23D Biomatrix, Inc., Ann Arbor, Michigan, USA
3Enzo Life Sciences, Farmingdale, New York, USA

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Introduction

Discovering innovative cancer therapies requires a deep understanding of the heterogeneous microenvironment of solid tumors. The Warburg effect describes the metabolic shift from oxidative phosphorylation to aerobic glycolysis that takes place in tumors, perhaps, even before the inevitable hypoxia that occurs as the tumor outgrows its insufficient and often irregular vasculature. The culmination of metabolic changes in cancer cells contributes significantly to increased metastasis and drug-resistance, aspects known to increase patient mortality. Based on these observations, proteins that mediate metabolism and directly target hypoxic cells in primary, metastatic tumors are attractive targets for therapeutic intervention.

Spheroids, self-assembled microscale aggregates of cells, generated in hanging drop plates (HDPs) are a superior model of avascular microtumors. Because of their 3D structure, spheroids contain mass-transfer gradients of oxygen, nutrients, wastes, and therapeutic drugs that are highly comparable to what is observed in tumors within the human body. The metabolic gradients drive proliferation gradients and spheroids contain quiescent cells hypothesized to mimic drug resistant populations within tumors. Given sufficient cell numbers and time in culture, spheroids can develop hypoxic cores that can progress to necrosis, closely mimicking what is observed in vivo.

Here we show the ability to interrogate compounds that alter the hypoxic microenvironment of tumors, using spheroids grown in HDPs and a simple fluorescent dye that specifically measures the activity of hypoxic cells. For the initial test we chose an inhibitor of carbonic anhydrase IX (CA IX), a hypoxia inducible factor 1α(HIF-1α)-regulated protein that functions to maintain intracellular pH. This family of inhibitors has been shown to reduce cancer cell growth and tumor metastasis. Imaging of spheroids in the hanging drop, following treatment, was performed to observe the inhibitory effects of the compound.
The results demonstrate the validity of the 3D cell model, and the use of this method to predict potential downstream anti-metastatic effects of lead molecules.

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