The treatment of head and neck tumors remains a challenge due to their reduced radiosensitivity. Small molecule kinase inhibitors (smKI) that inhibit the DNA damage response, may increase the radiosensitivity of tumor cells. However, little is known about how the immunophenotype of the tumor cells is modulated thereby. Therefore, we investigated whether the combination of ATM or ATR inhibitors with hypo-fractionated radiotherapy (RT) has a different impact on the expression of immune checkpoint markers (extrinsic), the release of cytokines or the transcriptome (intrinsic) of head and neck squamous cell carcinoma (HNSCC) cells.
The toxic and immunogenic effects of the smKI AZD0156 (ATMi) and VE-822 (ATRi) in combination with a hypo-fractionated scheme of 2x5Gy RT on HPV-negative (HSC4, Cal-33) and HPV-positive (UM-SCC-47, UD-SCC-2) HNSCC cell lines were analyzed as follows: cell death (necrosis, apoptosis; detected by AnxV/PI), expression of immunostimulatory (ICOS-L, OX40-L, TNFSFR9, CD70) and immunosuppressive (PD-L1, PD-L2, HVEM) checkpoint marker using flow cytometry; the release of cytokines using multiplex ELISA and the gene expression of Cal-33 on mRNA level 48 h post-RT.
Cell death was mainly induced by the combination of RT with both inhibitors, but stronger with ATRi. Further, the immune phenotype of cancer cells, not dying from combination therapy itself, is altered predominantly by RT+ATRi in an immune-stimulatory manner by the up-regulation of ICOS-L. However, the analysis of secreted cytokines after treatment of HNSCC cell lines revealed an ambivalent influence of both inhibitors, as we observed the intensified secretion of IL-6 and IL-8 after RT+ATRi. These findings were confirmed by RNAseq analysis and further the stronger immune-suppressive character of RT+ATMi was enlightened. We detected the down-regulation of a central protein of cytoplasmatic sensing pathways of nucleic acids, RIG-1, and found one immune-suppressive target, EDIL3, strongly up-regulated by RT+ATMi.
Independent of a restrictive toxicity, the combination of RT + either ATMi or ATRi leads to comprehensive and immune-modulating alterations in HNSCC. This includes pro-inflammatory signaling induced by RT + ATRi but also anti-inflammatory signals. These findings were confirmed by RNAseq analysis, which further highlighted the immune-suppressive nature of RT + ATMi.
Copyright © 2024 Meidenbauer, Wachter, Schulz, Mostafa, Zülch, Frey, Fietkau, Gaipl and Jost.

Human papilloma virus (HPV) positive head and neck squamous cell carcinoma (HNSCC) tumors respond significantly better to anticancer treatments. It is assumed to be due to a better response to radiotherapy (RT), and presumably to an increased immunogenicity. However, little is known how the immune phenotype of HNSCC tumor cells is modulated by standard treatment, namely by radiochemotherapy (RCT).
Therefore, we aimed to examine the impact of the HPV status on the immune phenotype of HNSCC cell lines following RCT with 5 × 3Gy or 1 × 19.3Gy and/or docetaxel, by analyzing cell death, release of damage-associated molecular patterns (DAMPs), surface expression of immune checkpoint molecules (ICMs) and the impact on activation of human monocyte-derived dendritic cells (hmDCs).
Cell death induction and Hsp70 release following RCT was independent of the HPV status, and RCT significantly increased the expression of the immune suppressive ICMs PD-L1, PD-L2 and HVEM. An immune stimulatory ICM, CD137, was significantly increased following RCT only on HPV-positive cell lines, as well as the release of HMGB1. Although the treatment increased cell death and modulated ICM expression in HNSCC, the hmDCs were not activated after co-incubation with treated tumor cells.
Our data with the HPV-dependent release of HMGB1 and increased expression of CD137 following RCT provide a hint for increased immunogenicity underlining the better prognosis for HPV positive tumors following RCT.
Copyright © 2023. Published by Elsevier Inc.

Therapeutic cancer vaccines targeting melanoma-associated antigens are commonly immunogenic but are rarely effective in promoting objective clinical responses. To identify critical molecules for activation of effective antitumor immunity, we have profiled autologous dendritic cell (DC) vaccines used to treat 35 patients with melanoma. We showed that checkpoint molecules induced by ex vivo maturation correlated with in vivo DC vaccine activity. Melanoma patient DCs had reduced expression of cell surface inducible T-cell costimulator ligand (ICOSL) and had defective intrinsic NF-κB signaling. Chromatin immunoprecipitation assays revealed NF-κB-dependent transcriptional regulation of ICOSL expression by DCs. Blockade of ICOSL on DCs reduced priming of antigen-specific CD8+ and CD4+ T cells from naïve donors in vitro Concentration of extracellular/soluble ICOSL released from vaccine DCs positively correlated with patient clinical outcomes, which we showed to be partially regulated by ADAM10/17 sheddase activity. These data point to the critical role of canonical NF-κB signaling, the regulation of matrix metalloproteinases, and DC-derived ICOSL in the specific priming of cognate T-cell responses in the cancer setting. This study supports the implementation of targeted strategies to augment these pathways for improved immunotherapeutic outcomes in patients with cancer.
©2020 American Association for Cancer Research.

The treatment of breast cancer by radiotherapy can be complemented by hyperthermia. Little is known about how the immune phenotype of tumor cells is changed thereby, also in terms of a dependence on the heating method. We developed a sterile closed-loop system, using either a warm-water bath or a microwave at 2.45 GHz to examine the impact of ex vivo hyperthermia on cell death, the release of HSP70, and the expression of immune checkpoint molecules (ICMs) on MCF-7 and MDA-MB-231 breast cancer cells by multicolor flow cytometry and ELISA. Heating was performed between 39 and 44 °C. Numerical process simulations identified temperature distributions. Additionally, irradiation with 2 × 5 Gy or 5 × 2 Gy was applied. We observed a release of HSP70 after hyperthermia at all examined temperatures and independently of the heating method, but microwave heating was more effective in cell killing, and microwave heating with and without radiotherapy increased subsequent HSP70 concentrations. Adding hyperthermia to radiotherapy, dynamically or individually, affected the expression of the ICM PD-L1, PD-L2, HVEM, ICOS-L, CD137-L, OX40-L, CD27-L, and EGFR on breast cancer cells. Well-characterized pre-clinical heating systems are mandatory to screen the immune phenotype of tumor cells in clinically relevant settings to define immune matrices for therapy adaption.