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.

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.

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.

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.

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.

Impact of radon and combinatory radon/carbon dioxide spa on pain and hypertension: Results from the explorative RAD-ON01 study.

OBJECTIVES: Therapies with low doses of radon have beneficial effects on patients suffering from chronic painful degenerative and inflammatory diseases. We already showed that this is accompanied by systemic immune modulations. We here focus on pain-reducing effects of very low doses of radon by adding carbon dioxide water and its impact on heart rate variability (HRV), blood pressure and free radicals. METHODS: 97 of 103 patients receiving radon spa (1.200 Bq/l at 34 °C or 600 Bq/l, 1 g/l CO2 at 34 °C) were monitored before and at three different time points after therapy. Individual pain perception was analyzed and the capability to process radicals. At each time point, the hypertensive patients (n = 46) were examined over 24 h for blood pressure and HRV. RESULTS: Long-term pain reduction was observed in the majority of patients. A modulation of superoxide dismutase was identified, presumably representing a priming effect for lowering radiation stress. Further, lowering of blood pressure, especially in those patients who additionally received carbon dioxide, was seen. Radon did in particular impact on HRV implying lasting relaxation effects. CONCLUSION: Radon/carbon dioxide spa efficiently reduces pain. In particular, patients simultaneously suffering from painful and cardiovascular diseases should be treated by combination of radon and CO2.

Low-Dose Radiotherapy Ameliorates Advanced Arthritis in hTNF-α tg Mice by Particularly Positively Impacting on Bone Metabolism.

Inflammation and bone erosion are central in rheumatoid arthritis (RA). Even though effective medications for control and treatment of RA are available, remission is only seen in a subset of patients. Treatment with low-dose radiotherapy (LD-RT) which has been already successfully used for amelioration of symptoms in benign diseases should be a promising approach to reduce pain, inflammation, and particularly bone erosion in patients with RA. Even though anti-inflammatory effects of LD-RT are already described with non-linear dose response relationships, and pain-reducing effects have been clinically observed, the underlying mechanisms are widely unknown. Besides immune cells many other cell types, such as fibroblast-like synoviocytes (FLS), osteoclasts, and osteoblast are present in the affected joint and might be modulated by LD-RT. For this study, these cell types were obtained from human tumor necrosis factor-α transgenic (hTNF-α tg) mice and were consecutively exposed to different doses of ionizing radiation (0.1, 0.5, 1.0, and 2.0 Gy, respectively) in vitro. In order to study the in vivo effects of LD-RT within the arthritic joint, hind paws of arthritic hTNF-α tg mice were locally irradiated with 0.5 Gy, a single dose per fraction that is known for good clinical responses. Starting at a dose of 0.5 Gy, proliferation of FLS was reduced and apoptosis significantly enhanced with no changes in necrosis. Further, expression of RANK-L was slightly reduced following irradiation with particularly 0.5 Gy. Starting from 0.5 Gy, the numbers of differentiated osteoclasts were significantly reduced, and a lower bone resorbing activity of treated osteoclasts was also observed, as monitored via pit formation and Cross Laps presence. LD-RT had further a positive effect on osteoblast-induced mineralization in a discontinuous dose response relationship with 0.5 Gy being most efficient. An increase of the gene expression ratio of OPG/RANK-L at 0.1 and 0.5 Gy and of production of OPG at 0.5 and 1.0 Gy was observed. In vivo, LD-RT resulted in less severe arthritis in arthritic hTNF-α tg mice and in significant reduction of inflammatory and erosive area with reduced osteoclasts and neutrophils. Locally applied LD-RT can, therefore, induce a beneficial micro-environment within arthritic joints by predominantly positively impacting on bone metabolism.

Combination of ionising radiation with hyperthermia increases the immunogenic potential of B16-F10 melanoma cells in vitro and in vivo.

Mild hyperthermia (HT) (41.5 °C for 30-60 min) has been shown in various cell culture systems, preclinical and clinical models to be a very potent radiosensitiser. Recent research suggests that local HT application in combination with standard tumour therapies such as radiotherapy (RT) and/or chemotherapy may not only improve local tumour control but also lead to systemic and immune mediated anti-tumour responses. Melanoma has been proven to be rather radioresistant and mostly only the addition of immunotherapy is capable of inducing beneficial anti-melanoma responses. This work therefore focuses on whether HT increases the immunogenic potential of B16-F10 mouse melanoma cells in combination with RT. The in vitro experiments revealed that combination of RT with HT resulted in an increased percentage of apoptotic and necrotic melanoma cells and an increased release of the danger signal heat shock protein 70 (Hsp70) and high mobility group box protein 1 (HMGB1). HT alone was also capable of inducing this release. We set up local irradiation and heating procedures of B16-F10 tumour-bearing C57/BL6 mice and revealed that the tumour growth of tumours treated with RT plus HT was significantly retarded compared to tumours treated only with RT. This combined treatment generated a beneficial tumour microenvironment by enhancing the infiltration of CD11c + /MHCII + /CD86+ dendritic cells, CD8+ T cells, and NK cells, and decreasing that of regulatory T cells and myeloid-derived suppressor cells. We conclude that HT in combination with RT has an immune-stimulating potential that might result in anti-tumour immunity.

The in vitro immunogenic potential of caspase-3 proficient breast cancer cells with basal low immunogenicity is increased by hypofractionated irradiation.

BACKGROUND: Radiotherapy is an integral part of breast cancer treatment. Immune activating properties of especially hypofractionated irradiation are in the spotlight of clinicians, besides the well-known effects of radiotherapy on cell cycle and the reduction of the clonogenic potential of tumor cells. Especially combination of radiotherapy with further immune stimulation induces immune-mediated anti-tumor responses. We therefore examined whether hypofractionated irradiation alone or in combination with hyperthermia as immune stimulants is capable of inducing breast cancer cells with immunogenic potential. METHODS: Clonogenic assay, AnnexinA5-FITC/Propidium iodide assay and ELISA analyses of heat shock protein 70 and high mobility group box 1 protein were applied to characterize colony forming capability, cell death induction, cell death forms and release of danger signals by breast cancer cells in response to hypofractionated radiation (4x4Gy, 6x3Gy) alone and in combination with hyperthermia (41.5 °C for 1 h). Caspase-3 deficient, hormone receptor positive, p53 wild type MCF-7 and caspase-3 intact, hormone receptor negative, p53 mutated MDA-MB231 breast cancer cells, the latter in absence or presence of the pan-caspase inhibitor zVAD-fmk, were used. Supernatants of the treated tumor cells were analyzed for their potential to alter the surface expression of activation markers on human-monocyte-derived dendritic cells. RESULTS: Irradiation reduced the clonogenicity of caspase deficient MCF-7 cells more than of MDA-B231 cells. In contrast, higher amounts of apoptotic and necrotic cells were induced in MDA-B231 cells after single irradiation with 4Gy, 10Gy, or 20Gy or after hypofractionated irradiation with 4x4Gy or 6x3Gy. MDA-B231 cells consecutively released higher amounts of Hsp70 and HMGB1 after hypofractionated irradiation. However, only the release of Hsp70 was further increased by hyperthermia. Both, apoptosis induction and release of the danger signals, was dependent on caspase-3. Only supernatants of MDA-B231 cells after hypofractionated irradiation resulted in slight changes of activation markers on dendritic cells; especially that of CD86 was upregulated and HT did not further impact on it. CONCLUSIONS: Hypofractionated irradiation is the main stimulus for cell death induction and consecutive dendritic cell activation in caspase proficient breast cancer cells. For the assessment of radiosensitivity and immunological effects of radio- and immunotherapies the readout system is crucial.

Old and new facts about hyperthermia-induced modulations of the immune system.

Hyperthermia (HT) is a potent sensitiser for radiotherapy (RT) and chemotherapy (CT) and has been proven to modulate directly or indirectly cells of the innate and adaptive immune system. We will focus in this article on how anti-tumour immunity can be induced by HT. In contrast to some in vitro assays, in vivo examinations showed that natural killer cells and phagocytes like granulocytes are directly activated against the tumour by HT. Since heat also activates dendritic cells (DCs), HT should be combined with further death stimuli (RT, CT or immune therapy) to allocate tumour antigen, derived from, for example, necrotic tumour cells, for uptake by DCs. We will outline that induction of immunogenic tumour cells and direct tumour cell killing by HT in combination with other therapies contributes to immune activation against the tumour. Studies will be presented showing that non-beneficial effects of HT on immune cells are mostly timely restricted. A special focus is set on immune activation mediated by extracellular present heat shock proteins (HSPs) carrying tumour antigens and further danger signals released by dying tumour cells. Local HT treatment in addition to further stress stimuli exerts abscopal effects and might be considered as in situ tumour vaccination. An increased natural killer (NK) cell activity, lymphocyte infiltration and HSP-mediated induction of immunogenic tumour cells have been observed in patients. Treatments with the addition of HT therefore can be considered as a personalised cancer treatment approach by specifically activating the immune system against the individual unique tumour.

Combination of ionising irradiation and hyperthermia activates programmed apoptotic and necrotic cell death pathways in human colorectal carcinoma cells.

PURPOSE: The malignancy of tumor cells can be attenuated by interfering with cell death pathways. Since hyperthermia (HT) is a very potent radiosensitizer, the influence of HT (41.5 °C for 1 hour) alone and in combination with ionising irradiation (X-ray; 5 Gy or 10 Gy) on the form of cell death as well as on the expression of proteins known to be major components in tumor cells' apoptotic and necrotic pathways were examined in colorectal tumor cells. MATERIAL AND METHODS: The expression of proteins was analysed by western blot and the relative activity of caspases-3/7 by fluorescence- based assay. Colony formation was analysed using the clonogenic assay and cell death was determined with annexin V-FITC/propidium iodide staining. RESULTS: Combining X-ray with HT led to similar activation of caspase-3/7 and p53 expression in comparison to irradiation only while the amount of the pro-apoptotic proteins PUMA and Bax was increased in HCT15 and SW480 cells. HT alone or combinations with X-ray further resulted in a temporarily increased level of the anti-apoptotic protein Bcl-2. Irradiation plus HT further led to an up-regulation of IRF-5. The levels of RIP-1, a marker for programmed necrosis, increased in tumor cells which were treated with HT and/or X-ray. Combining 5 Gy irradiation with HT compared to irradiation resulted in a significantly increased number of necrotic tumor cells and in decreased colony formation. CONCLUSION: The combined treatment of colorectal tumor cells with X-ray and HT activates distinct tumor cell pathways and fosters the early appearance of a necrotic tumor cell phenotype.

Calcium channel blocker verapamil enhances endoplasmic reticulum stress and cell death induced by proteasome inhibition in myeloma cells.

The proteasome inhibitor bortezomib is clinically approved for the treatment of multiple myeloma. However, long-term remissions are difficult to achieve, and myeloma cells often develop secondary resistance to proteasome inhibitors. We recently demonstrated that the extraordinary sensitivity of myeloma cells toward bortezomib is dependent on their extensive immunoglobulin synthesis, thereby triggering the terminal unfolded protein response (UPR). Here, we investigated whether verapamil, an inhibitor of the multidrug resistance (MDR) gene product, can enhance the cytotoxicity of bortezomib. The combination of bortezomib and verapamil synergistically decreased the viability of myeloma cells by inducing cell death. Importantly, bortezomib-mediated activation of major UPR components was enhanced by verapamil. The combination of bortezomib and verapamil resulted in caspase activation followed by poly(ADP-ribose) polymerase cleavage, whereas nuclear factor kappaB (NF-kappaB) activity declined in myeloma cells. Also, we found reduced immunoglobulin G secretion along with increased amounts of ubiquitinylated proteins within insoluble fractions of myeloma cells when using the combination treatment. Verapamil markedly induced reactive oxygen species production and autophagic-like processes. Furthermore, verapamil decreased MDR1 expression. We conclude that verapamil increased the antimyeloma effect of bortezomib by enhancing ER stress signals along with NF-kappaB inhibition, leading to cell death. Thus, the combination of bortezomib with verapamil may improve the efficacy of proteasome inhibitory therapy.

Application of hyperthermia in addition to ionizing irradiation fosters necrotic cell death and HMGB1 release of colorectal tumor cells.

Colorectal cancer is the second leading cause of death in developed countries. Tumor therapies should on the one hand aim to stop the proliferation of tumor cells and to kill them, and on the other hand stimulate a specific immune response against residual cancer cells. Dying cells are modulators of the immune system contributing to anti-inflammatory or pro-inflammatory responses, depending on the respective cell death form. The positive therapeutic effects of temperature-controlled hyperthermia (HT), when combined with ionizing irradiation (X-ray), were the origin to examine whether combinations of X-ray with HT can induce immune activating tumor cell death forms, also characterized by the release of the danger signal HMGB1. Human colorectal tumor cells with differing radiosensitivities were treated with combinations of HT (41.5 degrees C for 1h) and X-ray (5 or 10Gy). Necrotic cell death was prominent after X-ray and could be further increased by HT. Apoptosis remained quite low in HCT 15 and SW480 cells. X-ray and combinations with HT arrested the tumor cells in the radiosensitive G2 cell cycle phase. The amount of released HMGB1 protein was significantly enhanced after combinatorial treatments in comparison to single ones. We conclude that combining X-ray with HT may induce anti-tumor immunity as a result of the predominant induction of inflammatory necrotic tumor cells and the release of HMGB1.

Hyperthermia in combination with X-irradiation induces inflammatory forms of cell death.

Autoimmune diseases and cancer can be treated by influencing the immune system. Apo and nec cells are strong modulators of the immune system contributing to anti-inflammatory and pro-inflammatory responses, respectively. We examined which form of cell death was induced by HT and X-irradiation. Nec was the prominent form of cell death after combined treatment and the amount of dead cells was higher when exposing the cells to radiation before HT. Combined applications further led to an increased percentage of cells in a more radioresponsive G2 cell cycle phase. The danger signal HMGB1 is released when combining HT with radiation, a further hint that those treatments may induce inflammation and immune activation. We conclude that immune responses are appropriately adapted to the damage that has occurred and may contribute to anti-cancer immunity or chronic autoimmunity, respectively.

The role of annexin A5 in the modulation of the immune response against dying and dead cells.

Annexins are characterized by the ability to bind phospholipids of membranes in the presence of Ca2+. Annexin A5 represents a typical member of this protein family and is a natural occurring highly specific ligand for phosphatidylserine (PS). The exposure of PS is one major "eat me" signal for phagocytes of apoptotic and necrotic cells. Apoptotic cells are normally cleared via an anti-inflammatory pathway. In contrast, the uptake and removal of necrotic cells normally involves inflammation and an immune response. Interestingly, the lack of endogenous annexin A5 also leads to a reduced inflammatory potential of necrotic cells. Annexin A5 may interfere in vivo with the immunosuppressive effects of apoptotic cells since it preferentially binds PS with high affinity and inhibits apoptotic cell uptake by macrophages. In this review we focus on how defects in the clearance process can lead to chronic autoimmunity. Furthermore, the role of annexin A5 as important adjuvant for apoptotic cell-based tumour vaccines is discussed. The mechanism of how the immunogenicity of apoptotic cells can be restored by blocking their PS-dependent clearance is outlined in detail. Taken together, annexin A5 is an important modulator of the immune response against PS-exposing particles like apoptotic cells, necrotic cells, and certain viruses.