Session Item

Radiotherapy (RT) is one of the main treatments for head and neck cancers (HNSCC). Low oxygen levels, also called hypoxia, is an important limiting factor of the RT response. Another important factor influencing the RT response in HNSCC is the HPV-status, with HPV+ tumors being more radiosensitive compared to HPV- ones. Although both factors shape the RT response of HNSCC individually, possible interactions between these two factors remain unclear. Hence, we aim to understand the complex interaction between hypoxia- and HPV-induced molecular alterations and their respective effects on the RT response in HNSCC.

Hypoxia fate mapped models of HNSCC were generated by introduction of a hypoxia fate mapping system in two HPV- (FADU and CAL27) and two HPV+ (SCC154 and SCC47) cell lines. This system allows tracking of hypoxic cells during their trajectory by an oxygen-dependent fluorescent switch, namely switching from DsRed (in normoxic) to GFP (in hypoxic) conditions. The system was tested in hypoxic conditions in vitro and in 3D spheroid models and was further validated by the exogenous hypoxia marker pimonidazole (hypoxyprobe). Using the hypoxia fate mapped 3D models, the influence of hypoxia on the RT response of HPV+ and HPV- HNSCC was assessed by spheroid growth delay assays.

Hypoxia fate mapped HNSCC cells 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. Next, we assessed the RT response of HPV+ and HPV- 3D hypoxia fate mapped models. HPV+ spheroids showed a slower growth in volume, delayed increase in hypoxic fraction and increased radiosensitivity compared to HPV- spheroids. To investigate the influence of hypoxia on the RT response of these models, we irradiated fate mapped spheroids containing different levels of hypoxic fraction. As expected, 2.3-fold increase in hypoxia levels resulted in 1.5-fold reduced RT response in HPV- spheroids (p<0.0001). In contrast, 2.3-fold increase in hypoxia levels did not influence the RT response of HPV+ spheroids. These results suggest that hypoxia is less detrimental for the RT response of HPV+ HNSCC cells and they underline the importance of better understanding of hypoxia- and HPV-induced molecular alterations.

These preliminary results suggest a differential effect of hypoxia on the RT response of HPV+ and HPV- HNSCC. We are further assessing this differential response and the underlying molecular mechanisms, which will be essential for the development for novel radiosensitizing strategies for HNSCC patients.