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Inhibition of Hypoxic Tumor Cells using a Three-Dimensional Spheroid Model下載
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February 13, 2015
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 (VanderHeiden et al., 2009) 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 (Bennewith et al., 2011), (Brown et al., 2001). 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 masstransfer 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 (Hirschhaeuser et al., 2010).
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 1a (HIF-1a)-regulated protein that functions to maintain intracellular pH (Bennewith et al., 2011). This family of inhibitors has been shown to reduce cancer cell growth and tumor metastasis (Lou et al., 2011). 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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