Two-color spheroid model for determining the O

Hypoxia strongly affects the growth, invasion, and therapeutic response of solid tumors, including head and neck squamous cell carcinoma (HNSCC). Despite intensive research, only a few substances have progressed to clinical trials as radiosensitizers. Therefore, new clinically relevant tumor models are needed to identify agents that overcome radiation resistance in hypoxic tumors. To study radiosensitivity of hypoxic and normoxic cells, we developed a two-color spheroid model using two HNSCC cell lines, SAS and FaDu, with GFP-labeled inner cell layers and mCherry-labeled outer layers. Optimizing the ratios of fluorescent cells enabled formation of hypoxic and normoxic zones, confirmed by pimonidazole, HIF1α, and CA IX staining. A newly established fluorescence clonogenic survival assay demonstrated transferability of results from 2D normoxia and hypoxia assays to these 3D model. The inner GFP-labeled cells showed significantly lower plating efficiency and increased radiation resistance compared to outer mCherry-labeled cells, similar to 2D hypoxic cells. To improve the model by reducing normoxia-induced HIF1α expression in the outer layer, we added physiological concentrations of ascorbic acid. Ascorbic acid also increased spheroid growth, clonogenic survival, and radioresistance under normoxia, while hypoxic responses remained unchanged. These two-layer spheroid model with distinct fluorescent labels provide a simple, robust assay to distinguish hypoxic from normoxic tumor areas in radiotherapy research. Addition of ascorbic acid further refines the physiological relevance of 3D tumor models and modulates radiosensitivity of the outer mCherry-labeled cell layer in both HNSCC models.