Effects of X-ray Irradiation on Extracellular Vesicles Isolated from Head and Neck Cancer Cells
Inga Solgard Juvkam,
Norway
OC-0095
Abstract
Effects of X-ray Irradiation on Extracellular Vesicles Isolated from Head and Neck Cancer Cells
Authors: Inga Solgard Juvkam1, Olga Zlygosteva2, Mateusz Sitarz3, Eirik Malinen4,5, Nina Edin2, Hilde Galtung1, Tine Søland1,6
1University of Oslo, Institute of Oral Biology, Oslo, Norway; 2University of Oslo, Department of Physics, Oslo, Norway; 3Aarhus University Hospital , Danish Center for Particle Therapy, Aarhus, Denmark; 4University of Oslo , Department of Physics, Oslo, Norway; 5Oslo University Hospital, Department of Medical Physics, Oslo, Norway; 6Oslo University Hospital, Department of Pathology, Oslo, Norway
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Purpose or Objective
Extracellular vesicles (EVs) are membrane-bound vesicles released from cells through different biogenesis pathways. EVs contain nucleic acids, lipids and proteins from the releasing cell that can be taken up by recipient cells and alter their function. Thus, EVs are important in cellular communication in physiological and pathological processes and can be used as potential biomarkers in diseases such as cancer. Ionizing radiation used in cancer therapy causes direct damages to the 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.
Material 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.