Radiotherapy (RT) plays a key role in the treatment of HPV+ and HPV- head and neck cancer (HNSCC). Low tissue oxygen levels, also called hypoxia, has a negative influence on the RT response resulting in a poor prognosis. Moreover, cancer cells adapt to these hypoxic circumstances by various molecular alterations leading to tumor progression, genetic instability, treatment resistance, and tumor recurrence. Unfortunately, hypoxia is a common feature in HNSCC and, despite the development of various targeting strategies, can still not be overcome. Better understanding of the hypoxia-induced molecular alterations in cancer cells is needed to develop successful hypoxia targeting agents, thereby improving the RT response and prognosis of patients with HNSCC. The role of hypoxia in tumor regrowth was assessed using hypoxia fate mapped 3D HNSCC spheroid models by spheroid growth curves. The hypoxia fate mapped models were generated by introduction of a hypoxia fate mapping system [1] in three HPV+ and three HPV- HNSCC cell lines. This system allowed tracking of hypoxic cells by an oxygen-dependent fluorescent permanent switch from red (DsRed) in normoxic to green (GFP) fluorescence in hypoxic conditions. Fate mapped spheroids were dissociated into single cells and sorted into non- (DsRed+) and (post-)hypoxic (GFP+) populations. Of both populations, the radiosensitivity and DNA damage repair capacity were assessed. Hypoxia fate mapped HNSCC spheroids showed an increase in GFP levels in hypoxic conditions. Moreover, in 3D spheroids the GFP positive fraction increased with increasing area of the spheroids. This correlated with pimonidazole (hypoxyprobe) stainings, thereby validating the hypoxia fate mapping system. In response to RT, fate mapped spheroids showed an average 1.9-fold increase of the (post-)hypoxic population in the regrowth phase, indicating that these cells play a role in tumor regrowth. Assessment of the radiosensitivity of the (post-)hypoxic cells showed an average 1.5-fold increase in clonogenic survival compared to the non-hypoxic (p<0.0001). This increased survival was not mediated by DNA double strand break induction and/or repair, since yH2AX foci did not differ between both populations. Further molecular assessments of the (post-)hypoxic cells revealed an average 1.9-fold lower percentage of micronucleated cells 48h after RT compared to non-hypoxic cells (p<0.0001) and a 2-fold higher radiosensitization by ATR/Chk inhibition, suggesting that the more aggressive behavior of these cells might be linked to a mitotic survival advantage. These results were found in both HPV+ and HPV- HNSCC, however, the HPV+ HNSCC kept their intrinsic higher radiosensitivity. Our results show an important role for radioresistant (post-)hypoxic cells in regrowth after RT, independent of HPV status. We found that the aggressive behavior of these cells might be linked to hypoxia induced molecular alterations resulting in the ability to overcome mitotic cell death. These results will help in understanding the failure of hypoxic radiosensitizers. Moreover, it will pave the way for alternative treatment strategies with a focus on targeting hypoxia driven biological alterations, resulting in a better outcome for patients with HNSCC. [ABSTRACT FROM AUTHOR]