Modulation of PD‑L1 expression by standard therapy in head and neck cancer cell lines and exosomes

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 ligand‑1 (PD‑L1) is a key player in immunotherapy. Tumor cells, and exosomes derived therefrom, are carriers of PD‑L1 and efficiently suppress immune responses. The aim of the present study was to analyze the influence of established therapies on PD‑L1 expression of HNSCC cell lines and their exosomes. The HNSCC cell lines, UM‑SCC‑11B, UM‑SCC‑14C and UM‑SCC‑22C 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 PD‑L1 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 PD‑L1 were detected using western blotting and semi‑quantitative 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 UM‑SCC‑11B and UM‑SCC‑22B showed strong expression of pERK1/2 and PD‑L1, respectively. RT upregulated the PD‑L1 expression in UM‑SCC‑11B and UM‑SCC‑14C and in exosomes from all three cell lines. CT alone induced PD‑L1 expression in all cell lines. CRT induced the expression of PD‑L1 in all HNSCC cell lines and exosomes from UM‑SCC‑14C and UM‑SCC‑22B. The data indicated a potential co‑regulation of PD‑L1 and activated ERK1/2, most evident in UM‑SCC‑14C. Exosomes from irradiated UM‑SCC‑14C cells protected the unirradiated cells from apoptosis by Caspase 3/7 downregulation. The present study suggested a tumor cell‑mediated regulation of PD‑L1 upon platinum‑based CRT in HNSCC and in exosomes. A co‑regulation of PD‑L1 and MAPK signaling response was hypothesized.

Keywords: MAPKs; cetuximab; exosomes; head and neck squamous cell carcinomas; programmed death ligand‑1; small extracellular vesicles.

Figure 1

Immunohistochemical staining for basal expression of target proteins. All cell lines were examined by immunohistochemistry for basal expression of the target proteins and, as expected, showed heterogeneous expression with different expression levels of pERK1/2, panERK1/2 as well as PD-L1, as shown. UM-SCC-11 showed the strongest expression levels of pERK1/2 while PD-L1 and panERK1/2 were expressed most markedly in UM-SCC-22B. Magnification: ×200, scale bar, 50 µm. p, phosphorylated; PD-L1, programmed cell death ligand-1.

Figure 2

Impact of fractionated irradiation and Cetux on expression levels of pERK1/2, tERK1/2 and PD-L1 in UM-SCC-11B, UM-SCC-14C and UM-SCC-22B cells. Cells were irradiated in the linear accelerator by applying a fractionated scheme with 5×2 Gy on days 3-7 after seeding and/or treated with Cetux (5 µg/ml) on days 3, 5 and 7. Mock-treated cells served as control. Cells were harvested on day 10. Western blots of pERK1/2 and tERK1/2 (upper panel) and PD-L1 (lower panel) are shown. GAPDH was used as a loading control. UM-SCC-14C cells showed an association of pERK1/2 and PD-L1 expression levels with distinct basal and postradiogenic inhibition by Cetux. EGFR inhibited pERK1/2 expression in-22B cells to a lesser extent, however, MEK/ERK signaling by blockade of the upstream EGF receptor was not consequently impeded in all three cell lines. Cetux, cetuximab; p, phosphorylated; t, total; PD-L1, programmed cell death ligand-1. ^*P<0.05.

Figure 3

Impact of fractionated irradiation and cisplatin on expression levels of pERK1/2, tERK1/2 and PD-L1. Cells were irradiated in the linear accelerator by applying a fractionated scheme with 5×2 Gy on days 3-7 after seeding and/or treated with cisplatin with either 1 µM or 5 µM on days 3, 5 and 7. Mock-treated cells served as control. Cells were harvested on day 10. Western blots of pERK1/2 and tERK1/2 (upper panel) and PD-L1 (lower panel) are shown. GAPDH was used as a loading control. All three cell lines showed a strong activation of ERK1/2 by the higher concentration of cisplatin teamed with fractionated RT as well as a strong induction of PD-L1 after cisplatin treatment which was supra-additive when combined with irradiation. p, phosphorylated; t, total; PD-L1, programmed cell death ligand-1. ^*P<0.05; ^**P<0.01.

Figure 4

Immunohistochemical staining of ex vivo HNSCC tissue cultures. (A) Workflow of the experimental setting. After surgical resection, vital tumor tissues were cut into 2-3-mm-thick slides and kept in culture for up to 10 days. After experimental treatment samples were formalin-fixed and paraffin-embedded. Tissue sections were analyzed by hematoxylin and eosin staining and immunohistochemical staining. The correlation of experimental data with clinical outcome most likely offers the perspective of personalized therapy approaches. (B) Representative immunohistochemistry staining of PD-L1 in ex vivo tumor tissues with or without cisplatin treatment (3×80 µM). Left panel: Moderate immunostaining of PD-L1 in an untreated oropharyngeal SCC sample, scored with a TPS of 5%. Right panel: Distinct induction of PD-L1 after cisplatin treatment in corresponding samples from the same tumor, scored with a TPS of 25%. (C) Left panel: Low basal expression levels of the proliferation marker Ki-67 in untreated controls of the same OPSCC. Right panel: Further reduction of Ki-67 positive cells after cisplatin treatment. Scale bar, 50 µm. Parts of the figure were drawn by using pictures from Servier Medical Art (http://smart.servier.com/), licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/). HNSCC, head and neck squamous cell carcinoma; PD-L1, programmed cell death ligand-1; SCC, squamous cell carcinoma; TPS, tumor proportion score; OPSCC, oropharyngeal squamous cell carcinoma.

Figure 5

Morphology, size distribution, concentration and protein profiles of exosomes from HNSCC cell lines. Supernatants from HNSCC cell lines were collected and exosomes were isolated on mini-size exclusion chromatography columns. (A) Exosomes from UM-SCC-11B (11B), UM-SCC-14C (14C) and UM-SCC-22B (22B) show the typical vesicular shape and size in TEM images. Scale bar, 100 nm. (B) Western blotting was performed after loading 10 µg exosome preparation per lane and show the exosome markers TSG101, CD63 and CD9 and ApoA1 and Grp94 were used as purity control. (C) Nanoparticle tracking analysis was performed to detect median sizes and particle concentrations of the isolated particles. Representative pictures are shown of representative exosome preparations. HNSCC, head and neck squamous cell carcinoma.

Figure 6

Protein profiles of exosomes from (un)treated HNSCC cell lines UM-SCC-11B, UM-SCC-14C and UM-SCC-22B. The HNSCC cell lines were treated with 5×2 Gy radiotherapy (RT), 1 µM cisplatin (Cis1) and as combination therapy (Cis1+RT). As a control (Ctrl), untreated cell lines were used. (A) Western blotting of exosomes from HNSCC cell lines were performed using 10 mg exosome preparation per lane. Expression levels of PD-L1, TRAIL and TSG101 were detected for the different treatment conditions and compared. (B) Semi-quantitative densitometry of western blot bands of PD-L1 and TRAIL were related as mean integrated pixel values (image intensity band area) were related to TSG101 and compared for the different therapeutic conditions. HNSCC, head and neck squamous cell carcinoma; PD-L1, programmed cell death ligand-1.

Figure 7

Proliferation and apoptosis of untreated HNSCC cell lines by exosomes from HNSCC following different treatment conditions. Exosomes were isolated from (un)treated HNSCC cell lines and co-incubated with matching untreated HNSCC cell lines for 24 h. The treatment conditions were the following: 5×2 Gy RT alone and CRT (Cis1+RT). (A) The proliferation of the HNSCC cell lines was detected using an MTS assay displaying a significant reduction of proliferation by exosomes from UM-SCC-11B treated with CRT. (B) Apoptosis was indirectly measured using the Caspase 3/7 assay. Exosomes from the irradiated HNSCC cell lines UM-SCC-11B and UM-SCC-22B induced apoptosis in the untreated HNSCC cell lines, while the exosomes from the irradiated UM-SCC-14C reduced the apoptosis. The graphs show means (bars) with standard error means (whiskers) of three independent experiments. ^*P<0.05; ^**P<0.01. HNSCC, head and neck squamous cell carcinoma; RT, radiotherapy; Cis1, cisplatin; CRT, chemoradiotherapy.