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.

Comparison of clinicians' and researchers' ratings of proposed diagnostic criteria for compulsive buying-shopping disorder within a Delphi study.

Diagnostic criteria for compulsive buying shopping disorder were recently proposed based on a Delphi consensus study including 138 experts from 35 countries. The present study represents a secondary analysis of those data. To provide further support for the validity of expert responses in the Delphi study, the sample was retrospectively divided into clinician and researcher subgroups. The two groups were compared with respect to demographic variables, their importance ratings of clinical features, possible diagnostic criteria, differential diagnoses and specifiers of compulsive buying shopping disorder. Researchers reported less years of treating/assessing individuals with compulsive buying shopping disorder and stated that they have treated/assessed individuals with compulsive buying shopping disorder less often in the last 12 months than clinicians. Responses from the two groups concerning the importance ratings of possible diagnostic criteria of compulsive buying shopping disorder converged with only few minor differences with small to moderate group effects. However, even for those criteria, the consensus threshold (≥75% agreement with the proposed criterion) was reached in both groups. The lack of differences in the responses of the two groups indicates good validity for the proposed diagnostic criteria. Future research should address the clinical applicability and diagnostic validity of the criteria.

Detailed in vitro analyses of the impact of multimodal cancer therapy with hyperthermia and radiotherapy on the immune phenotype of human glioblastoma cells.

PURPOSE: Improvements of heat-delivery systems have led to hyperthermia (HT) being increasingly recognized as an adjunct treatment modality also for brain tumors. But how HT affects the immune phenotype of glioblastoma cells is only scarcely known. MATERIALS AND METHODS: We therefore investigated the effect of in vitro HT, radiotherapy (RT), and the combination of both (RHT) on cell death modalities, immune checkpoint molecule (ICM) expression and release of the danger signal HSP70 of two human glioblastoma cell lines (U87 and U251) by using multicolor flow cytometry and ELISA. Hyperthermia was performed once or twice for 60-minute sessions reaching temperatures of 39 °C, 41 °C, and 44 °C, respectively. RT was administered with 5 x 2 Gy. RESULTS: A hyperthermia chamber for cell culture t-flasks regulating the temperature via a contact sensor was developed. While the glioblastoma cells were rather radioresistant, particularly in U251 cells, the combination of RT with HT significantly increased the percentage of apoptotic and necrotic cells for all temperatures examined and for both, single and double HT application. In line with that, an increased release of HSP 70 was seen only in U251 cells, mainly following treatment with HT at temperatures of 44 °C alone or in combination with RT. In contrast, immune suppressive (PD-L1, PD-L2, HVEM) and immune stimulatory (ICOS-L, CD137-L and Ox40-L) ICMs were significantly increased mostly on U87 cells, and particularly after RHT with 41 °C. CONCLUSIONS: Individual assessment of the glioblastoma immune cell phenotype with regard to the planned treatment is mandatory to optimize multimodal radio-immunotherapy protocols including HT.

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

Hyperthermia (HT) is an accepted treatment for recurrent breast cancer which locally heats the tumor to 39-44 °C, and it is a very potent sensitizer for radiotherapy (RT) and chemotherapy. However, currently little is known about how HT with a distinct temperature, and particularly, how the sequence of HT and RT changes the immune phenotype of breast cancer cells. Therefore, human MDA-MB-231 and MCF-7 breast cancer cells were treated with HT of different temperatures (39, 41 and 44 °C), alone and in combination with RT (2 × 5 Gy) in different sequences, with either RT or HT first, followed by the other. Tumor cell death forms and the expression of immune checkpoint molecules (ICMs) were analyzed by multicolor flow cytometry. Human monocyte-derived dendritic cells (moDCs) were differentiated and co-cultured with the treated cancer cells. In both cell lines, RT was the main stressor for cell death induction, with apoptosis being the prominent cell death form in MCF-7 cells and both apoptosis and necrosis in MDA-MB-231 cells. Here, the sequence of the combined treatments, either RT or HT, did not have a significant impact on the final outcome. The expression of all of the three examined immune suppressive ICMs, namely PD-L1, PD-L2 and HVEM, was significantly increased on MCF-7 cells 120 h after the treatment of RT with HT of any temperature. Of special interest for MDA-MB-231 cells is that only combinations of RT with HT of both 41 and 44 °C induced a significantly increased expression of PD-L2 at all examined time points (24, 48, 72, and 120 h). Generally, high dynamics of ICM expression can be observed after combined RT and HT treatments. There was no significant difference between the different sequences of treatments (either HT + RT or RT + HT) in case of the upregulation of ICMs. Furthermore, the co-culture of moDCs with tumor cells of any treatment had no impact on the expression of activation markers. We conclude that the sequence of HT and RT does not strongly affect the immune phenotype of breast cancer cells. However, when HT is combined with RT, it results in an increased expression of distinct immune suppressive ICMs that should be considered by including immune checkpoint inhibitors in multimodal tumor treatments with RT and HT. Further, combined RT and HT affects the immune system in the effector phase rather than in the priming phase.

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.

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.

Cell-in-cell phenomenon: leukocyte engulfment by non-tumorigenic cells and cancer cell lines.

BACKGROUND: Research on cell-in-cell (CIC) phenomena, including entosis, emperipolesis and cannibalism, and their biological implications has increased in recent years. Homotypic and heterotypic engulfment of various target cells by numerous types of host cells has been studied in vitro and in tissue sections. This work has identified proteins involved in the mechanism and uncovered evidence for CIC as a potential histopathologic predictive and prognostic marker in cancer. Our experimental study focused on non-professional phagocytosis of leukocytes. RESULTS: We studied the engulfment of peripheral blood mononuclear cells isolated from healthy donors by counting CIC structures. Two non-tumorigenic cell lines (BEAS-2B, SBLF-9) and two tumour cell lines (BxPC3, ICNI) served as host cells. Immune cells were live-stained and either directly co-incubated or treated with irradiation or with conventional or microwave hyperthermia. Prior to co-incubation, we determined leukocyte viability for each batch via Annexin V-FITC/propidium iodide staining. All host cells engulfed their targets, with uptake rates ranging from 1.0% ± 0.5% in BxPC3 to 8.1% ± 5.0% in BEAS-2B. Engulfment rates of the cancer cell lines BxPC3 and ICNI (1.6% ± 0.2%) were similar to those of the primary fibroblasts SBLF-9 (1.4% ± 0.2%). We found a significant negative correlation between leukocyte viability and cell-in-cell formation rates. The engulfment rate rose when we increased the dose of radiotherapy and prolonged the impact time. Further, microwave hyperthermia induced higher leukocyte uptake than conventional hyperthermia. Using fluorescent immunocytochemistry to descriptively study the proteins involved, we detected ring-like formations of diverse proteins around the leukocytes, consisting, among others, of α-tubulin, integrin, myosin, F-actin, and vinculin. These results suggest the involvement of actomyosin contraction, cell-cell adhesion, and the α-tubulin cytoskeleton in the engulfment process. CONCLUSIONS: Both non-tumorigenic and cancer cells can form heterotypic CIC structures by engulfing leukocytes. Decreased viability and changes caused by microwave and X-ray irradiation trigger non-professional phagocytosis.

In Vitro Examinations of Cell Death Induction and the Immune Phenotype of Cancer Cells Following Radiative-Based Hyperthermia with 915 MHz in Combination with Radiotherapy.

Multimodal tumor treatment settings consisting of radiotherapy and immunomodulating agents such as immune checkpoint inhibitors are more and more commonly applied in clinics. In this context, the immune phenotype of tumor cells has a major influence on the anti-tumor immune response as well as the composition of the tumor microenvironment. A promising approach to further boost anti-tumor immune responses is to add hyperthermia (HT), i.e., heating the tumor tissue between 39 °C to 45 °C for 60 min. One key technique is the use of radiative hyperthermia systems. However, knowledge is limited as to how the frequency of the used radiative systems affects the immune phenotype of the treated tumor cells. By using our self-designed in vitro hyperthermia system, we compared cell death induction and expression of immune checkpoint molecules (ICM) on the tumor cell surface of murine B16 melanoma and human MDA-MB-231 and MCF-7 breast cancer cells following HT treatment with clinically relevant microwaves at 915 MHz or 2.45 GHz alone, radiotherapy (RT; 2 × 5 Gy or 5 × 2 Gy) alone or in combination (RHT). At 44 °C, HT alone was the dominant cell death inductor with inactivation rates of around 70% for B16, 45% for MDA-MB-231 and 35% for MCF-7 at 915 MHz and 80%, 60% and 50% at 2.45 GHz, respectively. Additional RT resulted in 5-15% higher levels of dead cells. The expression of ICM on tumor cells showed time-, treatment-, cell line- and frequency-dependent effects and was highest for RHT. Computer simulations of an exemplary spherical cell revealed frequency-dependent local energy absorption. The frequency of hyperthermia systems is a newly identified parameter that could also affect the immune phenotype of tumor cells and consequently the immunogenicity of tumors.

Proposed diagnostic criteria for compulsive buying-shopping disorder: A Delphi expert consensus study.

BACKGROUND AND AIMS: Consensus in acknowledging compulsive buying-shopping disorder (CBSD) as a distinct diagnosis has been lacking. Before research in this area can be advanced, it is necessary to establish diagnostic criteria in order to facilitate field trials. METHODS: The study consisted of the following phases: (1) operationalization of a broad range of potential diagnostic criteria for CBSD, (2) two iterative rounds of data collection using the Delphi method, where consensus of potential diagnostic criteria for CBSD was reached by an international expert panel, and (3) interpretation of findings taking into account the degree of certainty amongst experts regarding their responses. RESULTS: With respect to diagnostic criteria, there was clear expert consensus about inclusion of the persistent and recurrent experience of (a) intrusive and/or irresistible urges and/or impulses and/or cravings and/or preoccupations for buying/shopping; (b) diminished control over buying/shopping; (c) excessive purchasing of items without utilizing them for their intended purposes, (d) use of buying-shopping to regulate internal states; (e) negative consequences and impairment in important areas of functioning due to buying/shopping; (f) emotional and cognitive symptoms upon cessation of excessive buying/shopping; and (g) maintenance or escalation of dysfunctional buying/shopping behaviors despite negative consequences. Furthermore, support was found for a specifier related to the presence of excessive hoarding of purchased items. CONCLUSIONS: The proposed diagnostic criteria can be used as the basis for the development of diagnostic interviews and measures of CBSD severity.

Defining Metaniches in the Oral Cavity According to Their Microbial Composition and Cytokine Profile.

The human oral microbiota consists of over 700 widespread taxa colonizing the oral cavity in several anatomically diverse oral niches. Lately, sequencing of the 16S rRNA genes has become an acknowledged, culture-independent method to characterize the oral microbiota. However, only a small amount of data are available concerning microbial differences between oral niches in periodontal health and disease. In the context of periodontitis, the cytokine expression in the gingival crevicular fluid has been studied in detail, whereas little is known about the cytokine profile in hard and soft tissue biofilms. In order to characterize oral niches in periodontal health, the oral microbiota and cytokine pattern were analyzed at seven different sites (plaque (P), gingival crevicular fluid (GCF), saliva (S), tongue (T), hard palate (HP), cheek (C) and sublingual area (U)) of 20 young adults using next-generation sequencing and multiplex immunoassays. Site-specific microbial compositions were detected, which clustered into three distinct metaniches ("P-GCF", "S-T-HP" and "C-U") and were associated with niche-/metaniche-specific cytokine profiles. Our findings allow the definition of distinct metaniches according to their microbial composition, partly reflected by their cytokine profile, and provide new insights into microenvironmental similarities between anatomical diverse oral niches.

Differences of the Immune Phenotype of Breast Cancer Cells after Ex Vivo Hyperthermia by Warm-Water or Microwave Radiation in a Closed-Loop System Alone or in Combination with Radiotherapy.

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.

Immune biological rationales for the design of combined radio- and immunotherapies.

Cancer immunotherapies are promising treatments for many forms of cancer. Nevertheless, the response rates to, e.g., immune checkpoint inhibitors (ICI), are still in low double-digit percentage. This calls for further therapy optimization that should take into account combination of immunotherapies with classical tumor therapies such as radiotherapy. By designing multimodal approaches, immune modulatory properties of certain radiation schemes, additional immune modulation by immunotherapy with ICI and hyperthermia, as well as patient stratification based on genetic and immune constitutions have to be considered. In this context, both the tumor and its microenvironment including cells of the innate and adaptive immune system have to be viewed in synopsis. Knowledge of immune activation and immune suppression by radiation is the basis for well-elaborated addition of certain immunotherapies. In this review, the focus is set on additional immune stimulation by hyperthermia and restoration of an immune response by ICI. The impact of radiation dose and fractionation on immune modulation in multimodal settings has to be considered, as the dynamics of the immune response and the timing between radiotherapy and immunotherapy. Another big challenge is the patient stratification that should be based on matrices of biomarkers, taking into account genetics, proteomics, radiomics, and "immunomics". One key aim is to turn immunological "cold" tumors into "hot" tumors, and to eliminate barriers of immune-suppressed or immune-excluded tumors. Comprehensive knowledge of immune alterations induced by radiation and immunotherapy when being applied together should be utilized for patient-adapted treatment planning and testing of innovative tumor therapies within clinical trials.