Abstract
BackgroundMulticellular Tumor Spheroids are frequently used to mimic the regionalization of proliferation and the hypoxic environment within avascular tumors. Here we exploit these features to study the activation of DNA damage repair pathways and their correlation to developing hypoxia.MethodsActivation of DNA damage repair markers, proliferation, cell death, glycogen accumulation and developing hypoxia were investigated using immunofluorescence, immuno-histochemistry, EdU incorporation, Western blots, COMET assays, and pharmacological agents in A673 Ewing sarcoma spheroids and monolayer cultures.ResultsDNA damage marker γ-H2AX is observed in the hypoxic, peri-necrotic region of growing spheroids. While most proliferating cells are seen on the spheroid surface, there are also a few Ki-67 positive cells in the hypoxic zone. The hypoxia-induced phosphorylation of H2AX to form γ-H2AX in spheroids is attenuated by the ATM inhibitor KU55933, but not the ATR inhibitor VE-821.ConclusionTumor spheroids mimic tumor microenvironments such as the anoxic, hypoxic and oxic niches within solid tumors, as well as populations of cells that are viable, proliferating, and undergoing DNA damage repair processes under these different micro-environmental conditions. ATM, but not ATR, is the primary kinase responsible for γ-H2AX formation in the hypoxic core of A673 spheroids. Spheroids could offer unique advantages in testing therapeutics designed to target malignant cells that evade conventional treatment strategies by adapting to the hypoxic tumor microenvironment.
Highlights
Multicellular Tumor Spheroids are frequently used to mimic the regionalization of proliferation and the hypoxic environment within avascular tumors
We show that developing hypoxia in the core of growing multi-cellular tumor spheroids (MCTS) is accompanied by a distinctive distribution of proliferating cells, glycogen accumulation, Hypoxia Inducible Factor 1α (HIF1-α), activated Ataxia Telangiectasia Mutated (ATM) kinase, and induction of Phosphorylated H2AX-Serine-139 (γ-H2AX)
MCTS culture Spheroids were formed through use of the liquid overlay method wherein single cell suspensions (100 μl of A673 or Lewis lung carcinoma (LLC) cell suspensions in Dulbecco’s Modified Eagles Medium (DMEM) supplemented with 2% FBS or 10% FBS were added at a density of 5 × 104 or 5 × 103 cells ml−1 respectively) were added to 96-well plates previously coated with agar
Summary
Multicellular Tumor Spheroids are frequently used to mimic the regionalization of proliferation and the hypoxic environment within avascular tumors. We exploit these features to study the activation of DNA damage repair pathways and their correlation to developing hypoxia. The growth of solid tumors is accompanied by the development of central hypoxic or anoxic regions, an acidic extracellular pH and modified cellular glucose and energy metabolism. Tumor cells either adapt to the unfavorable microenvironment or die, leading to areas of necrosis. Continued growth of the tumor becomes dependent on the delivery of oxygen and nutrients through vascularization. A primary response of tumor cells to hypoxia is to upregulate HIF1-α and its downstream targets, principally the pro-angiogenic VEGF. Relevant consequences of tumor hypoxia include increased chemo- and radiation-resistance, increased metastatic potential, genomic instability and poorer prognosis
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