Hypoxia Induces a Mitotic Survival Advantage After Radiotherapy in Head and Neck Cancer Cells

Hypoxia is an important cause of radiotherapy resistance in head and neck cancers. Cancer cells adapt to hypoxic conditions through various molecular alterations, leading to treatment resistance and tumor progression. A deeper understanding of these hypoxia-induced molecular alterations is essential for future development of effective hypoxia targeting and radiosensitizing therapies. In this study, we tracked (post-)hypoxic cells at single-cell level in HPV-negative and HPV-positive HNSCC models using a hypoxia fate mapping system. We found that (post-)hypoxic cells drive regrowth after radiotherapy in 3D conditions and showed an increased resistance to radiation. Transcriptomic analysis showed that post-hypoxic cells are characterized by a gene expression signature mainly defined by checkpoint regulation. Consistent with these findings, radiotherapy resistant post-hypoxic cells showed a reduced number of radiotherapy-induced micronuclei and mitotic spindle aberrations, indicating a mitotic survival advantage. Inhibition of mitotic checkpoint proteins ATR and CHK1/2 increased the radiosensitivity of post-hypoxic cells. In conclusion, our findings indicate that radiotherapy resistance in HNSCC cells is associated with mitotic survival advantage of post-hypoxic cells, independent of HPV-status.