Effect of micro- and nanoplastic particles on human macrophages.

Micro- and nanoplastics (MNPs) are ubiquitous in the environment, resulting in the uptake of MNPs by a variety of organisms, including humans, leading to particle-cell interaction. Human macrophages derived from THP-1 cell lines take up Polystyrene (PS), a widespread plastic. The question therefore arises whether primary human macrophages also take up PS micro- and nanobeads (MNBs) and how they react to this stimulation. Major aim of this study is to visualize this uptake and to validate the isolation of macrophages from peripheral blood mononuclear cells (PBMCs) to assess the impact of MNPs on human macrophages. Uptake of macrophages from THP-1 cell lines and PBMCs was examined by transmission electron microscopy (TEM), scanning electron microscopy and live cell imaging. In addition, the reaction of the macrophages was analyzed in terms of metabolic activity, cytotoxicity, production of reactive oxygen species (ROS) and macrophage polarization. This study is the first to visualize PS MNBs in primary human cells using TEM and live cell imaging. Metabolic activity was size- and concentration-dependent, necrosis and ROS were increased. The methods demonstrated in this study outline an approach to assess the influence of MNP exposure on human macrophages and help investigating the consequences of worldwide plastic pollution.

Effects of serial radon spa therapy on pain and peripheral immune status in patients suffering from musculoskeletal disorders- results from a prospective, randomized, placebo-controlled trial.

In this randomized, placebo-controlled cross-over trial we aimed to investigate if radon spa therapy exerts more pain relief than exposure to warm water alone. In addition, immunological parameters were assessed in both treatment groups. In the RAD-ON02 trial, 116 patients suffering from musculoskeletal disorders (MSDs) received either serial radon spa or solely warm water baths. Pain intensity was assessed by determination of different pain parameters on a visual analogue scale and by pressure point dolorimetry at baseline and at weeks 4, 12 and 24. The longitudinal immune status of the patients was analyzed by a flow cytometry-based assay from peripheral blood at the time points of pain assessments. There were no side effects attributable to radon exposure observed. However, radon spa was superior to warm water applications at week 4 in terms of pain reduction. Pain and morning stiffness at the time of assessment were significantly reduced after radon spa (p<0.001, p<0.01) but not after warm water baths. The dolorimetry resulted in a significantly higher exerted pressure strength in patients after radon spa (p<0.001), but not after warm water applications. During the long-term follow-up, both treatment modalities reduced pain to a similar degree and pain modulation was not distorted by the participants' intake of analgesics. No significant changes in the immune status attributable specifically to radon were found, even though the increase in regulatory T cell counts occurs earlier after radon baths than after sole warm water baths and a higher level of significance is reached after radon spa at week 24. Serial radon spa has additive pain-relieving effects. The immunological parameters assessed in our study appear not to be directly linked to the pain reduction caused by radon exposure, at least in MSD patients with predominantly degenerative diseases. Clinical trial registration: https://www.clinicaltrialsregister.eu/ctr-search/search?query=rad-on02, identifier 2016-002085-31; https://drks.de/search/de/trial, identifier DRKS00016019.

Machine Learning-assisted immunophenotyping of peripheral blood identifies innate immune cells as best predictor of response to induction chemo-immunotherapy in head and neck squamous cell carcinoma - knowledge obtained from the CheckRad-CD8 trial.

PURPOSE: Individual prediction of treatment response is crucial for personalized treatment in multimodal approaches against head-and-neck squamous cell carcinoma (HNSCC). So far, no reliable predictive parameters for treatment schemes containing immunotherapy have been identified. This study aims to predict treatment response to induction chemo-immunotherapy based on the peripheral blood immune status in patients with locally advanced HNSCC. METHODS: The peripheral blood immune phenotype was assessed in whole blood samples in patients treated in the phase II CheckRad-CD8 trial as part of the pre-planned translational research program. Blood samples were analyzed by multicolor flow cytometry before (T1) and after (T2) induction chemo-immunotherapy with cisplatin/docetaxel/durvalumab/tremelimumab. Machine Learning techniques were used to predict pathological complete response (pCR) after induction therapy. RESULTS: The tested classifier methods (LDA, SVM, LR, RF, DT, and XGBoost) allowed a distinct prediction of pCR. Highest accuracy was achieved with a low number of features represented as principal components. Immune parameters obtained from the absolute difference (lT2-T1l) allowed the best prediction of pCR. In general, less than 30 parameters and at most 10 principal components were needed for highly accurate predictions. Across several datasets, cells of the innate immune system such as polymorphonuclear cells, monocytes, and plasmacytoid dendritic cells are most prominent. CONCLUSIONS: Our analyses imply that alterations of the innate immune cell distribution in the peripheral blood following induction chemo-immuno-therapy is highly predictive for pCR in HNSCC.

Immune modulation during anti-cancer radio(immuno)therapy.

Cancer can affect all human organs and tissues and ranks as a prominent cause of death as well as an obstruction to increasing life expectancy. A notable breakthrough in oncology has been the inclusion of the immune system in fighting cancer, potentially prolonging life and providing long-term benefits. The concept of "immunotherapy" has been discussed from the 19th and early 20th centuries by Wilhelm Busch, William B. Coley and Paul Ehrlich. This involves distinct approaches, including vaccines, non-specific cytokines and adoptive cell therapies. However, despite the advances made in recent years, questions on how to select the best therapeutic options or how to select the best combinations to improve clinical outcomes are still relevant for scientists and clinicians. More than half of cancer patients receive radiotherapy (RT) as part of their treatment. With the advances in RT and immunotherapy approaches, it is reasonable to consider how to enhance immunotherapy with radiation and vice versa, and to investigate whether combinations of these therapies would be beneficial. In this chapter, we will discuss how the immune system responds to cancer cells and different cancer therapies with a focus on combination of RT and immunotherapy (radioimmunotherapy, RIT).

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.

PURPOSE: 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). METHODS: 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). RESULTS: 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. CONCLUSION: 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.

Broadband microwave spiral applicator (105-125 MHz) for in vitro examinations of hyperthermia-induced tumor cell death forms - first analyses with human breast cancer cells.

PURPOSE: Local tumor heating with microwave applicators has been used in multimodal breast cancer therapies. This hyperthermia allows to target small regions while marginally affecting healthy tissue. However, most preclinical examinations only use simplified heating methods. Microwave applicators employed for deep heating to provide the greatest depth of penetration operate in the tens to hundreds frequency. Therefore, we aimed to adapt and test a clinically often used broadband spiral applicator (105-125 MHz) for hyperthermia with clinically wanted temperatures of 41 and 44 °C in in vitro settings with human breast cancer cell lines and with simulations. MATERIAL AND METHODS: A clinically used spiral-microwave applicator (105-125 MHz) was the basis for the construction, simulation, and optimization of the in vitro HT set-up under stationary conditions. Microwave effects on tumor cell death of two human breast cancer cell lines (hormone-receptor positive MCF-7 and triple-negative MDA-MB-231) were compared with conventional heating in a contact-heating chamber. Cell death forms were analyzed by AnnexinV/Propidium iodide staining. RESULTS: An in vitro spiral applicator microwave-based heating system that is effective at applying heat directly to adherent breast cancer cells in cell culture flasks with medium was developed. Simulations with COMSOL proved appropriate heat delivery and an optimal energy coupling at a frequency of 111 ± 2.5 MHz. Apoptosis and necrosis induction and significantly higher cell death rates than conventional heating at both temperatures were observed, and MCF-7 showed higher death rates than MDA-MB-231 tumor cells. CONCLUSIONS: Well-characterized in vitro heating systems are mandatory for a better understanding of the biological effects of hyperthermia in tumor therapies and to finally determine optimized clinical treatment schemes.

Deep Learning and Registration-Based Mapping for Analyzing the Distribution of Nodal Metastases in Head and Neck Cancer Cohorts: Informing Optimal Radiotherapy Target Volume Design.

We introduce a deep-learning- and a registration-based method for automatically analyzing the spatial distribution of nodal metastases (LNs) in head and neck (H/N) cancer cohorts to inform radiotherapy (RT) target volume design. The two methods are evaluated in a cohort of 193 H/N patients/planning CTs with a total of 449 LNs. In the deep learning method, a previously developed nnU-Net 3D/2D ensemble model is used to autosegment 20 H/N levels, with each LN subsequently being algorithmically assigned to the closest-level autosegmentation. In the nonrigid-registration-based mapping method, LNs are mapped into a calculated template CT representing the cohort-average patient anatomy, and kernel density estimation is employed to estimate the underlying average 3D-LN probability distribution allowing for analysis and visualization without prespecified level definitions. Multireader assessment by three radio-oncologists with majority voting was used to evaluate the deep learning method and obtain the ground-truth distribution. For the mapping technique, the proportion of LNs predicted by the 3D probability distribution for each level was calculated and compared to the deep learning and ground-truth distributions. As determined by a multireader review with majority voting, the deep learning method correctly categorized all 449 LNs to their respective levels. Level 2 showed the highest LN involvement (59.0%). The level involvement predicted by the mapping technique was consistent with the ground-truth distribution (p for difference 0.915). Application of the proposed methods to multicenter cohorts with selected H/N tumor subtypes for informing optimal RT target volume design is promising.

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

Only a subset of patients with triple-negative breast cancer (TNBC) benefits from a combination of radio- (RT) and immunotherapy. Therefore, we aimed to examine the impact of radioresistance and brain metastasizing potential on the immunological phenotype of TNBC cells following hypofractionated RT by analyzing cell death, immune checkpoint molecule (ICM) expression and activation of human monocyte-derived dendritic cells (DCs). MDA-MB-231 triple-negative breast cancer tumor cells were used as model system. Apoptosis was the dominant cell death form of brain metastasizing tumor cells, while Hsp70 release was generally significantly increased following RT and went along with necrosis induction. The ICMs PD-L1, PD-L2, HVEM, ICOS-L, CD137-L and OX40-L were found on the tumor cell surfaces and were significantly upregulated by RT with 5 x 5.2 Gy. Strikingly, the expression of immune suppressive ICMs was significantly higher on radioresistant clones compared to their respective non-radioresistant ones. Although hypofractionated RT led to significant cell death induction and release of Hsp70 in all tumor cell lines, human monocyte-derived DCs were not activated after co-incubation with RT-treated tumor cells. We conclude that radioresistance is a potent driver of immune suppressive ICM expression on the surface of TNBC MDA-MB-231 cells. This mechanism is generally known to predominantly influence the effector phase, rather than the priming phase, of anti-tumor immune responses.

Deep learning for automatic head and neck lymph node level delineation provides expert-level accuracy.

Background: Deep learning-based head and neck lymph node level (HN_LNL) autodelineation is of high relevance to radiotherapy research and clinical treatment planning but still underinvestigated in academic literature. In particular, there is no publicly available open-source solution for large-scale autosegmentation of HN_LNL in the research setting. Methods: An expert-delineated cohort of 35 planning CTs was used for training of an nnU-net 3D-fullres/2D-ensemble model for autosegmentation of 20 different HN_LNL. A second cohort acquired at the same institution later in time served as the test set (n = 20). In a completely blinded evaluation, 3 clinical experts rated the quality of deep learning autosegmentations in a head-to-head comparison with expert-created contours. For a subgroup of 10 cases, intraobserver variability was compared to the average deep learning autosegmentation accuracy on the original and recontoured set of expert segmentations. A postprocessing step to adjust craniocaudal boundaries of level autosegmentations to the CT slice plane was introduced and the effect of autocontour consistency with CT slice plane orientation on geometric accuracy and expert rating was investigated. Results: Blinded expert ratings for deep learning segmentations and expert-created contours were not significantly different. Deep learning segmentations with slice plane adjustment were rated numerically higher (mean, 81.0 vs. 79.6, p = 0.185) and deep learning segmentations without slice plane adjustment were rated numerically lower (77.2 vs. 79.6, p = 0.167) than manually drawn contours. In a head-to-head comparison, deep learning segmentations with CT slice plane adjustment were rated significantly better than deep learning contours without slice plane adjustment (81.0 vs. 77.2, p = 0.004). Geometric accuracy of deep learning segmentations was not different from intraobserver variability (mean Dice per level, 0.76 vs. 0.77, p = 0.307). Clinical significance of contour consistency with CT slice plane orientation was not represented by geometric accuracy metrics (volumetric Dice, 0.78 vs. 0.78, p = 0.703). Conclusions: We show that a nnU-net 3D-fullres/2D-ensemble model can be used for highly accurate autodelineation of HN_LNL using only a limited training dataset that is ideally suited for large-scale standardized autodelineation of HN_LNL in the research setting. Geometric accuracy metrics are only an imperfect surrogate for blinded expert rating.

The various functions and phenotypes of macrophages are also reflected in their responses to irradiation: A current overview.

Macrophages are a vital part of the innate immune system that are involved in healthy biological processes but also in disease modulation and response to therapy. Ionizing radiation is commonly used in the treatment of cancer and, in a lower dose range, as additive therapy for inflammatory diseases. In general, lower doses of ionizing radiation are known to induce rather anti-inflammatory responses, while higher doses are utilized in cancer treatment where they result, next to tumor control, in rather inflammatory responses. Most experiments that have been carried out in ex vivo on macrophages find this to be true, however in vivo, tumor-associated macrophages, for example, show a contradictory response to the respective dose-range. While some knowledge in radiation-induced modulations of macrophages has been collected, many of the underlying mechanisms remain unclear. Due to their pivotal role in the human body, however, they are a great target in therapy and could potentially aid in better treatment outcome. We therefore summarized the current knowledge of macrophage mediated radiation responses.

Prospective Evaluation of CD45RA+/CCR7- Effector Memory T (TEMRA) Cell Subsets in Patients with Primary and Secondary Brain Tumors during Radiotherapy of the Brain within the Scope of the Prospective Glio-CMV-01 Clinical Trial.

Radiotherapy (RT) of the brain is a common treatment for patients with high-grade gliomas and brain metastases. It has previously been shown that reactivation of cytomegalovirus (CMV) frequently occurs during RT of the brain. This causes neurological decline, demands antiviral treatment, and is associated with a worse prognosis. CMV-specific T cells are characterized by a differentiated effector memory phenotype and CD45RA+ CCR7- effector memory T (TEMRA) cells were shown to be enriched in CMV seropositive individuals. In this study, we investigated the distribution of TEMRA cells and their subsets in the peripheral blood of healthy donors and, for the first time, prospectively within the scope of the prospective Glio-CMV-01 clinical trial of patients with high-grade glioma and brain metastases during radiation therapy as a potential predictive marker. First, we developed a multicolor flow cytometry-based assay to monitor the frequency and distribution of TEMRA cells in a longitudinal manner. The CMV serostatus and age were considered as influencing factors. We revealed that patients who had a reactivation of CMV have significantly higher amounts of CD8+ TEMRA cells. Further, the distribution of the subsets of TEMRA cells based on the expression of CD27, CD28, and CD57 is highly dependent on the CMV serostatus. We conclude that the percentage of CD8+ TEMRA cells out of all CD8+ T cells has the potential to serve as a biomarker for predicting the risk of CMV reactivation during RT of the brain. Furthermore, this study highlights the importance of taking the CMV serostatus into account when analyzing TEMRA cells and their subsets.

Low-dose radiotherapy of osteoarthritis: from biological findings to clinical effects-challenges for future studies.

Osteoarthritis (OA) is one of the most common and socioeconomically relevant diseases, with rising incidence and prevalence especially with regard to an ageing population in the Western world. Over the decades, the scientific perception of OA has shifted from a simple degeneration of cartilage and bone to a multifactorial disease involving various cell types and immunomodulatory factors. Despite a wide range of conventional treatment modalities available, a significant proportion of patients remain treatment refractory. Low-dose radiotherapy (LDRT) has been used for decades in the treatment of patients with inflammatory and/or degenerative diseases and has proven a viable option even in cohorts of patients with a rather poor prognosis. While its justification mainly derives from a vast body of empirical evidence, prospective randomized trials have until now failed to prove the effectiveness of LDRT. Nevertheless, over the decades, adaptions of LDRT treatment modalities have evolved using lower dosages with establishment of different treatment schedules for which definitive clinical proof is still pending. Preclinical research has revealed that the immune system is modulated by LDRT and very recently osteoimmunological mechanisms have been described. Future studies and investigations further elucidating the underlying mechanisms are an essential key to clarify the optimal patient stratification and treatment procedure, considering the patients' inflammatory status, age, and sex. The present review aims not only to present clinical and preclinical knowledge about the mechanistic and beneficial effects of LDRT, but also to emphasize topics that will need to be addressed in future studies. Further, a concise overview of the current status of the underlying radiobiological knowledge of LDRT for clinicians is given, while seeking to stimulate further translational research.

Identification and characterization of circular RNAs as novel putative biomarkers to predict anti-PD-1 monotherapy response in metastatic melanoma patients - Knowledge from two independent international studies.

Melanoma is the most aggressive skin malignancy with high morbidity. Anti-programmed cell death protein 1 (PD-1) monotherapy has been applied in metastatic melanoma. However, still most of the patients do not respond to anti-PD-1 and the availability of the present approved biomarkers therefore is limited. Here we combined the transcriptomic and clinical data of 163 advanced melanoma patients receiving anti-PD-1 from NIH Melanoma Genome Sequencing Project (phs000452, 122 patients) as the training and internal validation cohort, and Melanoma Institute Australia cohort (PRJEB23709, 41 patients) as the external validation cohort, respectively. Circular RNAs (circRNAs) are an evolutionarily conserved novel class of noncoding endogenous RNAs (ncRNAs) found in the eukaryotic transcriptome and were used based on RNAseq data for our analyses. 74,243 circular RNAs (circRNAs) were identified with NCLscan and CIRCexplorer2. Thereof, 70 circRNAs significantly associated with progression-free survival and overall survival. Further, a prognostic circRNAs signature consisting of HSA_CIRCpedia_1497, HSA_CIRCpedia_12559, HSA_CIRCpedia_43640, HSA_CIRCpedia_43070, and HSA_CIRCpedia_21660 could be determined with LASSO regression. This signature was a prognostic factor of overall survival and progression-free survival among the analyzed advanced melanoma patients. The concordance indexes (C-index of OStraining: 0.61, C-index of PFStraining: 0.68) also confirmed its credibility and accuracy. First enrichment analysis indicated that immune response and pathways related to tumor immune microenvironment were enriched. In conclusion, we succeeded to construct and validate novel prognostic circRNAs signature for advanced melanoma patients treated with anti-PD-1 immunotherapy.

The Emerging Evidence Supporting Integration of Deep Regional Hyperthermia With Chemoradiation in Bladder Cancer.

For decades, the antineoplastic potential of hyperthermia alone or in combination with radiotherapy and/or chemotherapy has been subject of intensive preclinical and clinical research in various tumor entities. The clinical evidence on the beneficial effects of additional hyperthermia in combination with intravesical Mitomycin C for superficial non-muscle-invasive bladder cancer as well as for deep regional microwave hyperthermia techniques applied during an external beam radiotherapy or chemoradiation treatment for more advanced tumors are summarized. In some series, deep regional hyperthermia in combination with an initial transurethral resection and Cisplatin-based chemoradiation increased the 5-year overall survival rates up to 20%. The presented data justifies a fresh irrespective chance for mild regional hyperthermia in the context of new progressive prospective trials on multimodality treatment for bladder preservation.

Tumor-specific radiosensitizing effect of the ATM inhibitor AZD0156 in melanoma cells with low toxicity to healthy fibroblasts.

PURPOSE: Despite new treatment options, melanoma continues to have an unfavorable prognosis. DNA damage response (DDR) inhibitors are a promising drug class, especially in combination with chemotherapy (CT) or radiotherapy (RT). Manipulating DNA damage repair during RT is an opportunity to exploit the genomic instability of cancer cells and may lead to radiosensitizing effects in tumors that could improve cancer therapy. METHODS: A panel of melanoma-derived cell lines of different origin were used to investigate toxicity-related clonogenic survival, cell death, and cell cycle distribution after treatment with a kinase inhibitor (KI) against ATM (AZD0156) or ATR (VE-822, berzosertib), irradiation with 2 Gy, or a combination of KI plus ionizing radiation (IR). Two fibroblast cell lines generated from healthy skin tissue were used as controls. RESULTS: Clonogenic survival indicated a clear radiosensitizing effect of the ATM inhibitor (ATMi) AZD0156 in all melanoma cells in a synergistic manner, but not in healthy tissue fibroblasts. In contrast, the ATR inhibitor (ATRi) VE-822 led to additive enhancement of IR-related toxicity in most of the melanoma cells. Both inhibitors mainly increased cell death induction in combination with IR. In healthy fibroblasts, VE-822 plus IR led to higher cell death rates compared to AZD0156. A significant G2/M block was particularly induced in cancer cells when combining AZD0156 with IR. CONCLUSION: ATMi, in contrast to ATRi, resulted in synergistic radiosensitization regarding colony formation in melanoma cancer cells, while healthy tissue fibroblasts were merely affected with respect to cell death induction. In connection with an increased number of melanoma cells in the G2/M phase after ATMi plus IR treatment, ATMi seems to be superior to ATRi in melanoma cancer cell treatments when combined with RT.

Normofractionated irradiation and not temozolomide modulates the immunogenic and oncogenic phenotype of human glioblastoma cell lines.

PURPOSE: Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor, with an overall poor prognosis after diagnosis. Conventional treatment includes resection, chemotherapy with temozolomide (TMZ), and concomitant radiotherapy (RT). The recent success of immunotherapy approaches in other tumor entities, particularly with immune checkpoint inhibitors, could not be clinically transferred to GBM treatment so far. Therefore, preclinical analyses of the expression of both immune-suppressive and immune-stimulatory checkpoint molecules following treatment of human glioblastoma cells with RT and/or temozolomide is needed to design feasible radio(chemo)immunotherapy trials for GBM in the future. METHODS: Five human glioblastoma cell lines (H4, HROG-06, U118, U138, U251) were analyzed regarding their clonogenic survival and cell death forms after chemotherapy (CT) with TMZ and/or normofractionated RT (5â€¯× 2 Gy) via multicolor flow cytometry. Further, the tumor cell surface expression of immune-activating (OX40L, CD137L, CD70, and ICOSL) and immune-suppressive (PD-L1, PD-L2, HVEM) checkpoint molecules and of an oncogenic molecule (EGFR) were measured via multicolor flow cytometry after CT and RT alone or after RCT. RESULTS: Normofractionated RT and not TMZ was the trigger of induction of predominantly necrosis in the glioblastoma cells. Notably, clonogenicity did not correlate with cell death induction by RT. The basal expression level of immune-suppressive PD-L1, PD-L2, and HVEM varied in the analyzed glioblastoma cells. RT, but not TMZ, resulted in a significant upregulation of PD-L1 and PD-L2 in all tumor cells investigated. Also, the expression of HVEM was increased after RT in most of the GBM cell lines. In contrast, normofractionated RT individually modulated expression of the stimulating immune checkpoint molecules CD70, CD137L, OX40L, and ICOSL1. The oncogenic factor EGFR was significantly increased by irradiation in all examined cell lines, albeit to a different extent. None of the investigated molecules were downregulated after the treatments. CONCLUSION: Normofractionated radiotherapy modulates the immunogenic as well as the oncogenic phenotype of glioblastoma cells, partly individually. Therefore, not only PD-L1 and PD-L2, but also other immunogenic molecules expressed on the surface of glioblastoma cells could serve as targets for immune checkpoint blockade in combination with RT in the future.