ESTRO 2023 - Abstract Book

S59

Saturday 13 May

ESTRO 2023

radiation target. Similar damages can also be found in non-irradiated neighboring or distant cells, termed non-targeted effects of radiation. Recent research has shown that EVs may play a role in the non-targeted effects of radiation [Al-Mayah, et al. Mutat Res, 2015]. Therefore, we aimed to investigate the proteome of an oral squamous cell carcinoma (OSCC) cell line and their released EVs after exposure to X-ray radiation. Furthermore, we aimed to investigate the effect of these EVs after uptake in non-irradiated cancer and normal cells. Materials and Methods Human OSCC cells were irradiated with photon irradiation using doses of 0, 2 or 5 Gy. 24 hours after irradiation, EVs were isolated from the irradiated OSCC cells as described previously [Guerreiro, et al, PLoS One, 2018] by centrifugation, ultrafiltration and -exclusion chromatography and characterized by nanoparticle tracking analysis, flow cytometry and transmission electron microscopy. Irradiated OSCC cells and their associated EVs underwent liquid chromatography-mass spectrometry analysis for protein identification. Uptake studies of irradiated OSCC EVs by non-irradiated OSCC cells and oral fibroblasts were performed by staining EVs with the green fluorescent dye PKH67 and obtaining confocal images after incubation with the recipient cells (figure 1). The influence of EV uptake on proliferation and migration of the recipient cells were studied. Results Proteins involved in mediating apoptosis (CTL2, TR10A, ADA17) were overexpressed in EVs released by irradiated OSCC cells while proteins involved in DNA repair (PARP1, APTX, HAT1, among others) were upregulated in irradiated OSCC cells. Uptake of irradiated OSCC EVs did not alter the proliferative or migratory behaviour of non-irradiated OSCC cells or oral fibroblasts.

Figure 1: Uptake of EVs from irradiated OSCC cells in non-irradiated normal oral fibroblasts (A) and OSCC cells (B). Green dye is PKH67, while blue is DAPI. Conclusion Two different protein profiles, both potentially of importance for cytoprotection of X-ray irradiation, were identified in irradiated OSCC cells and their associated EVs. Our results do not exclude that non-targeted radiation effects might occur through other signalling channels than EVs released from irradiated cells. OC-0096 Influence of hypoxia on RT response in HPV+ and HPV- head and neck cancer M. Wegge 1 , R. Dok 2 , S. Nuyts 2 , L. Dubois 3 1 KU Leuve, Lab of Experimental Radiotherapy, Leuven, Belgium; 2 KU Leuven, Lab of Experimental Radiotherapy, Leuven, Belgium; 3 The M-lab, Maastricht University, Dept. of Precision Medicine, Maastricht, The Netherlands Purpose or Objective 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. Materials and Methods 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. Results 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. Conclusion 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.

OC-0097 Ferroptosis induction synergizes with radiotherapy in hypoxic human colorectal cancer.