Reprint of: Detection and Impact of Hypoxic Regions in Multicellular Tumor Spheroid Cultures formed by Head and Neck Squamous Cell Carcinoma Cells Lines

In solid tumors like head and neck Cancer (HNC), chronic and acute hypoxia have serious adverse clinical consequences including poorer overall patient prognosis, enhanced metastasis, increased genomic instability, and resistance to radiation-, chemo-, or immuno-therapies. However, cells in the two-dimensional monolayer cultures typically used for Cancer drug discovery experience 20%-21% O₂ levels (normoxic) which are 4-fold higher than O₂ levels in normal tissues and ≥10-fold higher than in the hypoxic regions of solid tumors. The oxygen electrodes, exogenous bio-reductive markers, and increased expression of endogenous hypoxia-regulated proteins like HIF-1α generally used to MARK hypoxic regions in solid tumors are impractical in large sample numbers and longitudinal studies. We used a novel homogeneous live-cell permeant HypoxiTRAK™ (HPTK) molecular probe compatible with high content imaging detection, analysis, and throughput to identify and quantify hypoxia levels in live HNC multicellular tumor spheroid (MCTS) cultures over time. Accumulation of fluorescence HPTK metabolite in live normoxic HNC MCTS cultures correlated with hypoxia detection by both pimonidazole and HIF-1α staining. In HNC MCTSs, hypoxic cytotoxicity ratios for the hypoxia activated prodrugs (HAP) evofosfamide and tirapazamine were much smaller than have been reported for uniformly hypoxic 2D monolayers in gas chambers, and many viable cells remained after HAP exposure. Cells in solid tumors and MCTSs experience three distinct O₂ microenvironments dictated by their distances from blood vessels or MCTS surfaces, respectively; oxic, hypoxic, or intermediate levels of hypoxia. These studies support the application of more physiologically relevant in vitro 3D models that recapitulate the heterogeneous microenvironments of solid tumors for preclinical Cancer drug discovery.

50% growth inhibitory concentration, GI(50); Abbreviations: Head and Neck Cancer, HNC; Calcein AM, CAM; Cell Titer Blue®, CTB; Cell Titer Glo®, CTG; Dimethyl sulfoxide, DMSO; Dulbecco's Mg2+ and Ca2+-free phosphate-buffered saline, PBS; Dulbecco's Modified Eagle's Medium, DMEM; Epidermal Growth Factor, EGF; Ethidium Homodimer, EHD; Evofosfamide, EFF; Extra Cellular Matrix, ECM; Federal Drug Administration, FDA; Fetal Bovine Serum, FBS; Head and neck squamous cell carcinoma, HNSCC; High Content Screening, HCS; High Throughput Screening, HTS; HypoxiTRAK™, HPTK; Hypoxia activated prodrugs, HAPs; Hypoxia-inducible factor protein 1α, HIF-1α; Image-iT™ Green Hypoxia Reagent, ITGHR; ImageXpress Micro automated imaging platform, IXM; Mean Average Fluorescence Intensity, MAFI; Mean Integrated Fluorescence Intensity, MIFI; Multicellular Tumor Spheroids, MCTSs; Multiwavelength Cell Scorning image analysis module, MWCS; National Cancer Institute, NCI; Penicillin / Streptomycin, P/S; Pimonidazole, PIMO; Relative Fluorescent units, RFUs; Relative Light Units, RLUs; Tirapazamine, TPZ; Transmitted Light, TL; Ultra-Low Attachment microtiter plates, ULA-plates; three-dimensional, 3D; two-dimensional, 2D.