ABSTRACT
Although checkpoint inhibitors (CPI) have recently extended the treatment options and improved clinical response of advanced stage head and neck squamous cell carcinoma (HNSCC), treatment success remains unpredictable. Programmed cell death ligand1 (PDL1) is a key player in immunotherapy. Tumor cells, and exosomes derived therefrom, are carriers of PDL1 and efficiently suppress immune responses. The aim of the present study was to analyze the influence of established therapies on PDL1 expression of HNSCC cell lines and their exosomes. The HNSCC cell lines, UMSCC11B, UMSCC14C and UMSCC22C were treated with fractionated radiotherapy (RT; 5x2 Gy), cisplatin (CT) and cetuximab (Cetux) as monotherapy, or combined therapy, chemoradiotherapy (CRT; RT and CT) or radioimmunotherapy (RT and Cetux). The expression of PDL1 and phosphorylated (p)ERK1/2 as a mediator of radioresistance were assessed using western blotting, immunohistochemistry and an ex vivo vital tissue culture model. Additionally, exosomes were isolated from concentrated supernatants of the (un)treated HNSCC cell lines by size exclusion chromatography. Exosomal protein expression levels of PDL1 were detected using western blotting and semiquantitative levels were calculated. The functional impact of exosomes from the (un)treated HNSCC cell lines on the proliferation (MTS assay) and apoptosis (Caspase 3/7 assay) of the untreated HNSCC cell lines were measured and compared. The HNSCC cell lines UMSCC11B and UMSCC22B showed strong expression of pERK1/2 and PDL1, respectively. RT upregulated the PDL1 expression in UMSCC11B and UMSCC14C and in exosomes from all three cell lines. CT alone induced PDL1 expression in all cell lines. CRT induced the expression of PDL1 in all HNSCC cell lines and exosomes from UMSCC14C and UMSCC22B. The data indicated a potential coregulation of PDL1 and activated ERK1/2, most evident in UMSCC14C. Exosomes from irradiated UMSCC14C cells protected the unirradiated cells from apoptosis by Caspase 3/7 downregulation. The present study suggested a tumor cellmediated regulation of PDL1 upon platinumbased CRT in HNSCC and in exosomes. A coregulation of PDL1 and MAPK signaling response was hypothesized.