Hypofractionated Radiotherapy Upregulates Several Immune Checkpoint Molecules in Head and Neck Squamous Cell Carcinoma Cells Independently of the HPV Status While ICOS-L Is Upregulated Only on HPV-Positive Cells

Abstract

While the treatment of squamous cell carcinoma of the head and neck (HNSCC) with radiotherapy (RT) is complemented more and more by immunotherapy in clinical trials, little is known about the impact of the human papillomavirus (HPV) status or the applied RT scheme on the immune phenotype of the tumor cells. Therefore, we aimed to examine the impact of the HPV status of four human HNSCC cell lines on cell death and the expression of immune checkpoint molecules (ICMs) after RT with either hypofractionation irradiation (5x3.0Gy) or a high single dose (1x19.3Gy) via multicolor flow cytometry and quantitative PCR at an early time point after therapy. In our study, 5x3.0Gy RT induced high numbers of early and late apoptotic cells independent of the HPV status, but necrosis was only increased in the HPV-positive UM-Scc-47 cells. Generally, the immune stimulatory ICMs (CD70, CD137-L, ICOS-L) were less affected by RT compared to the immune suppressive ones (PD-L1, PD-L2, and the herpesvirus entry mediator (HVEM)). A significant higher surface expression of the analyzed ICMs was found after hypofractionated RT compared to a single high dose; however, regardless of the HPV status, with the exception of ICOS-L. Here, HPV-positive HNSCC tumor cells showed a stronger response to 5x3.0Gy than HPV-negative ones. On the RNA level, only minor alterations of ICMs were observed following RT, with the exception of the HPV negative cell line CAL33 treated with 5x3.0Gy, where PD-L2, HVEM and CD70 were significantly increased. We conclude that the HPV status may not distinctly predict immunological responses following RT, and thus cannot be used as a single predictive marker for therapy responses in HNSCC. In contrast, the patient-specific individual expression of ICMs following RT is preferable for the targeted patient selection for immune therapy directed against distinct ICM.

Radiotherapy combined with docetaxel alters the immune phenotype of HNSCC cells and results in increased surface expression of CD137 and release of HMGB1 of specifically HPV-positive tumor cells

“…In contrast, irradiating HPV-positive cell lines resulted in a higher necrotic cell death rate, which was also recently shown by Wimmer et al. [13] . Thus indicating, that the HPV-negative cell lines killed by RT alone or in combination with docetaxel could rather suppress immune response, whereas the treated HPV-positive cell lines would stimulate an immune response, supporting better outcome of HPV-positive associated head and neck tumors.…”

“…It has been generally suggested that HPV positive HNSCC are more immunogenic compared to HPV negative ones [12] . We have just recently identified that following RT, the expression of the inducible co-stimulatory molecule ligand (ICOS-L) is upregulated only on HPV positive HNSCC cell lines [13] . As ICOS/ICOSL signaling leads to the activation, proliferation and survival of cytotoxic T cells [14] , it might foster RT-induced anti-HNSCC tumor responses.…”

Clonogenicity-based radioresistance determines the expression of immune suppressive immune checkpoint molecules after hypofractionated irradiation of MDA-MB-231 triple-negative breast cancer cells

“…In line with previous in vitro and in vivo examinations using various cancer cell lines and models, we revealed that it is not just PD-L1 which is upregulated by RT, but rather both immune inhibitory and immune stimulatory ( 50 – 53 ) ICM expression is significantly increased on the surface of TNBC cells following (hypofractionated) irradiation. This has already been demonstrated in other settings and tumor entities ( 54 , 55 ).…”

The Effect of Hyperthermia and Radiotherapy Sequence on Cancer Cell Death and the Immune Phenotype of Breast Cancer Cells

“…However, besides OX40-L, the other immune-activating ICMs (ICOS-L, CD27-L, CD137-L) examined were not strongly affected by RT and HT (not shown). This is in contrast to other tumor entities, such as, e.g., head and neck cancer, where ICOS-L is upregulated on HPV-positive cancer cells after RT [68].…”