Linking hypoxia, DNA damage and proliferation in multicellular tumor spheroids

Cross section of an immunostained tumor spheroid. Cell nuclei are labelled with DAPI (Blue). Proliferating cells are labelled in green and cells with DNA damage, as marked by y-H2AX, are labelled in red. The dotted line indicates the hypoxic zone.

Stephen Riffle, Ram Naresh Pandey, Morgan Albert, and Rashmi S. Hegde

Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, 3333 Burnet Avenue, Cincinnati, OH 45229 USA

Background

Multicellular 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.

Methods

Activation 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.

Results

DNA 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.

Conclusion

Tumor 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.