ESTRO 2021 Abstract Book
immunogenic cell death (ICD) in human cancer cell lines. Materials and Methods
We treated the human NSCLC cell lines H1975, SW900, A549 and H460 and the human osteosarcoma cell line U2OS with ionising radiation (X-rays, 5 Gy [IR]) and ATR inhibitors (50-250 nM VE-822; 250-1250 nM AZD6738 [ATRi]), and measured checkpoint abrogation and immunogenic signalling at 0-6 days after treatment. G 2 /M checkpoint abrogation was assessed by flow cytometry. The ICD hallmark factors surface-presented calreticulin, secreted ATP and released HMGB1 were measured by an optimised flow cytometry protocol, the CellTiter-Glo assay and western blotting of medium supernatants, respectively. Endogenous type I IFN signalling following cGAS-mediated detection of cytosolic DNA from ruptured micronuclei was measured by western blotting of downstream phosphorylation of STAT1, and by IFN-β ELISA. Immunofluorescence microscopy was used to detect localisation of cGAS to micronuclei. CGAS knock-down was achieved by siRNA transfection. Results Radiation-induced type I IFN signalling – as measured by pSTAT1 levels – was observed in H1975, SW900, A549 and U2OS 3-6 days after treatment. These responses were further increased in U2OS and SW900 at 3 days and in A549 at 6 days after co-treatment with IR and ATRi. We found no response in H460. These results were confirmed by IFN-β ELISA on U2OS and H460 supernatants. si CGAS knock-down in SW900, A549 and U2OS abolished the observed response, substantiating the hypothesis of cGAS-dependent detection of cytosolic DNA from ruptured micronuclei. In agreement with the aforementioned observations, cGAS made micronuclear puncta in all cell lines except H460. Furthermore, we found increased surface-presentation of calreticulin after IR alone in H1975, but no further increase after co-treatment with IR and ATRi. IR alone also gave increased secretion of ATP from H1975, SW900, A549 and H460, with further increase for H1975, A549, H460 and U2OS after co-treatment with IR and ATRi. All cell lines showed release of HMGB1 after the combined OC-0187 Effects of proton vs photon irradiation on inflammation and senescence in salivary gland organoids D. Cinat 1,2 , L. Barazzuol 1,2 , R. Coppes 1,2 1 University Medical Center Groningen, Department of Biomedical Sciences of Cells & Systems, Groningen, The Netherlands; 2 University Medical Center Groningen, Department of Radiation Oncology, Groningen, The Netherlands Purpose or Objective Proton therapy is an innovative technique currently used as treatment against many types of tumors including head and neck cancers. Compared to conventional photon radiotherapy, proton therapy can deliver highly effective doses of radiation to the tumor while reducing the dose to the surrounding normal tissue potentially leading to fewer side effects. Besides its physical advantages, little is known about the biological impact of proton versus photon irradiation on healthy tissue and further studies are required in order to improve the treatment. Materials and Methods To investigate and compare the effects of these types of radiation, we used an established murine salivary gland organoid model that closely resembles the in vivo response. A similar strong and dose-dependent induction of senescence and senescence-related genes upon both proton and photon irradiation was observed using SA-b-Gal staining, qPCR and Western blot. RNA sequencing was performed to identify transcriptome changes in the differentially irradiated organoids at different time points and radiation doses. Results A much higher inflammatory response upon proton irradiation compared to photon irradiation was observed. In particular, we observed significant differences in genes related to cytoplasmatic dsDNA and dsRNA recognition, immunity activation, Interferon 1 (IFN1) response and tissue development. Moreover, enhanced activation of cGAS-related signalling was observed after proton irradiation. cGAS is an important protein that recognizes dsDNA released in the cytosol upon stress or DNA damage. After this recognition, cGAS promotes the expression of cytokines and IFN1 causing inflammation, immunity activation and senescence. Next to this, we observed a higher number of micronuclei containing both cGAS and dsDNA in proton compared to photon irradiated organoids, which coincided with a higher expression of Interferon stimulated genes. Interestingly, along with dsDNA also cytoplasmatic dsRNA was detected in irradiated organoids using immunofluorescence as well as a strong activation of genes related to cytosolic dsRNA sensing, both higher after proton irradiation. Along with cytoplasmatic dsDNA also the recognition of cytoplasmatic dsRNA may lead to IFN1 production and activation of an immune response. Conclusion Therefore, our study suggests that the DNA damage induced by proton irradiation might lead to more dsDNA leakage into the cytoplasm and consequently more cGAS activation. This might lead to a more pronounced activation of Interferon stimulated genes and dsRNA release that act as positive feedback for the activation of immune responses and inflammation. This enhanced cellular immune response could lead to dissimilar normal tissue response to proton versus photon irradiation. treatment. Conclusion We show that the combined treatment with radiation and ATR inhibitors can increase cGAS-dependent IFN signalling and enhance presentation of two out of three measured hallmark factors of ICD, with cell line- dependent variations in effect and time of occurrence.
Proffered papers: Proffered papers 10: Lung
OC-0188 Adjuvant treatment following irradical resection of stage I-III NSCLC: a population-based study M. Rasing 1 , M. Peters 1 , M. Aarts 2 , G. Herder 3 , A. van Lindert 4 , F. Schramel 5 , F. van der Meer 6 , J. Verhoeff 1 , P.
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