Ecto-Calreticulin Expression After Particle Irradiation Versus Conventional Photon Irradiation in Human Cancer Cell Lines

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Radiotherapy can activate anti-tumor immune responses by triggering immunogenic cell death (ICD) of tumor cells. ICD is characterized by the release of danger-associated molecular patterns (DAMPs). Calreticulin (CRT) is one of the most potent DAMPs. The cell surface translocation of CRT (ecto-CRT) serves as an “eat me” signal to antigen-presenting cells (APCs), which plays a pivotal role in activating anti-tumor immunity. However, there is limited information describing the impact of proton and carbon-ion irradiation on ecto-CRT expression. Thus, we investigated ecto-CRT expression in multiple human cancer cell lines irradiated by proton and carbon-ion in comparison to conventional photon irradiation. This study examined four human cancer cell lines: A549 (lung adenocarcinoma), U251 MG (glioma), Tca8113 (tongue squamous carcinoma) and CNE-2 (nasopharyngeal carcinoma). Cell lines were irradiated to 0,2,4,10Gy with proton, carbon ion, or photon. The expression levels of ecto-CRT were analyzed by flow cytometry at 12h, 24h and 48h post-irradiation. Mean Fluorescence Intensity (MFI) of all samples were calculated by cytometry analysis software. There was a dose-dependent increase of ecto-CRT expression after photon, proton or carbon-ion irradiation in all four cell lines. Higher dose irradiation induced more ecto-CRT expression (p<0.05). The expression levels of ecto-CRT were significantly increased at 48h post-irradiation rather than 12h or 24h post-irradiation (p<0.05). When comparing at the same relative biological effectiveness (RBE) dose, 10GyE, there was no significant difference of ecto-CRT expression changes at 48h post-irradiation with proton and carbon-ion in comparison to photon. The MFI fold changes of 10GyE dose at 48h post-irradiation compared to control group (0Gy) for A549, U251, Tca8113 and CNE-2 were 1.63-1.73, 1.29-1.79,1.54-1.69 and 2.21-2.59, respectively. Proton and carbon-ion as well as photon irradiation can enhance the ecto-CRT expression with a dose-dependent manner in human cancer cell lines.