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High linear energy transfer carbon-ion irradiation upregulates PD-L1 expression more significantly than X-rays in human osteosarcoma U2OS cells.

ABSTRACT: Programmed death ligand 1 (PD-L1) expression on the surface of cancer cells affects the efficacy of anti-PD-1/PD-L1 immune checkpoint therapy. However, the mechanism underlying PD-L1 expression in cancer cells is not fully understood, particularly after ionizing radiation (IR). Here, we examined the impact of high linear energy transfer (LET) carbon-ion irradiation on the expression of PD-L1 in human osteosarcoma U2OS cells. We found that the upregulation of PD-L1 expression after high LET carbon-ion irradiation was greater than that induced by X-rays at the same physical and relative biological effectiveness (RBE) dose, and that the upregulation of PD-L1 induced by high LET carbon-ion irradiation was predominantly dependent on ataxia telangiectasia and Rad3-related (ATR) kinase activity. Moreover, we showed that the downstream signaling, e.g. STAT1 phosphorylation and IRF1 expression, was upregulated to a greater extent after high LET carbon-ion irradiation than X-rays, and that IRF1 upregulation was also ATR dependent. Finally, to visualize PD-L1 molecules on the cell surface in 3D, we applied immunofluorescence-based super-resolution imaging. The three-dimensional structured illumination microscopy (3D-SIM) analyses revealed substantial increases in the number of presented PD-L1 molecules on the cell surface after high LET carbon-ion irradiation compared with X-ray irradiation.

SUBMITTER: Permata TBM

PROVIDER: S-EPMC8438258 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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High linear energy transfer carbon-ion irradiation upregulates PD-L1 expression more significantly than X-rays in human osteosarcoma U2OS cells.

Permata Tiara Bunga Mayang TBM Sato Hiro H Gu Wenchao W Kakoti Sangeeta S Uchihara Yuki Y Yoshimatsu Yukihiko Y Sato Itaru I Kato Reona R Yamauchi Motohiro M Suzuki Keiji K Oike Takahiro T Tsushima Yoshito Y Gondhowiardjo Soehartati S Ohno Tatsuya T Yasuhara Takaaki T Shibata Atsushi A

Journal of radiation research 20210901 5

Programmed death ligand 1 (PD-L1) expression on the surface of cancer cells affects the efficacy of anti-PD-1/PD-L1 immune checkpoint therapy. However, the mechanism underlying PD-L1 expression in cancer cells is not fully understood, particularly after ionizing radiation (IR). Here, we examined the impact of high linear energy transfer (LET) carbon-ion irradiation on the expression of PD-L1 in human osteosarcoma U2OS cells. We found that the upregulation of PD-L1 expression after high LET carbo ...[more]

PMID: 34196706

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Project description:BackgroundInterferon (IFN) consensus sequence binding protein (ICSBP) is a transcription factor induced by IFN-γ. We previously reported that ICSBP expression promotes osteosarcoma progression by enhancing transforming growth factor-β signaling. In cancer cells, programmed death-ligand 1 (PD-L1) contributes to immune escape and may also be involved in tumor progression. Because IFN-γ induces the expression of both ICSBP and PD-L1, we explored the association between ICSBP and PD-L1 expression in terms of osteosarcoma progression.MethodsThree osteosarcoma cell lines (Saos2, U2OS, and 143B) were employed. Gene expression was measured by qRT-PCR, and protein levels were assessed by immunoblotting. PD-L1 expression was evaluated in cells overexpressing ICSBP and in ICSBP knockdown cells. The effects of PD-L1 expression on cell growth were examined by MTS assays, Incucyte analysis, soft agar assays, and three-dimensional (3D) culture. Cell cycle and apoptosis were evaluated by FACS analysis of cells stained with propidium iodide (PI) and annexin V/PI, respectively. The antitumor effects of PD-L1 knockdown without or with doxorubicin treatment were evaluated in vivo in nude mice bearing ICSBP-overexpressing 143B cell xenograft. The clinical relevance of PD-L1 and ICSBP expression was evaluated immunohistochemically using a human osteosarcoma microarray and through analysis of publicly available data using Gene Expression Profiling Interactive Analysis2.ResultsICSBP overexpression upregulated PD-L1 expression in all three cell lines, whereas ICSBP knockdown decreased the PD-L1 expression. PD-L1 knockdown attenuated the cell growth and reduced colony-forming capacity in both soft agar assays and 3D culture. PD-L1 knockdown increased apoptosis and induced G2/M arrest, which was associated with decreased expression of survivin, cyclin-dependent kinase 4 (CDK4), cyclin E, and cyclin D1 expression and increased the expression of p27, phosphorylated Cdc2, and phosphorylated Wee1. PD-L1 knockdown decreased the growth of tumor xenografts and increased the doxorubicin sensitivity of ICSBP-overexpressing 143B cells both in vitro and in vivo. PD-L1 was expressed in human osteosarcoma tissues, and its expression was moderately correlated with that of ICSBP in osteosarcoma patients.ConclusionICSBP regulates PD-L1 expression in osteosarcoma cells, and PD-L1 knockdown combined with doxorubicin treatment could represent a strategy for controlling osteosarcoma expressing ICSBP.

Dataset Information

High linear energy transfer carbon-ion irradiation upregulates PD-L1 expression more significantly than X-rays in human osteosarcoma U2OS cells.

Publications

High linear energy transfer carbon-ion irradiation upregulates PD-L1 expression more significantly than X-rays in human osteosarcoma U2OS cells.

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