Radiation combined with hyperthermia induces HSP70-dependent maturation of dendritic cells and release of pro-inflammatory cytokines by dendritic cells and macrophages.

PURPOSE: Hyperthermia (HT) treatment of cancer patients was revived over the last years and has been proven to be beneficiary for many cancer entities when applied temperature controlled in multimodal treatments. We examined whether a combination of ionizing irradiation (X-ray) and HT (41.5°C; 1 h) can induce the release of heat shock protein (HSP) 70 by tumor cells and thereby lead to the activation of dendritic cells and macrophages. MATERIAL AND METHODS: Extracellular HSP70 was detected in supernatants (SN) of treated colorectal tumor cells by ELISA. Maturation of dendritic cells (DC) after contact with the SN was measured by flow-cytometry. Phagocytosis assays were conducted to get hints about the immune stimulating potential of the tumor cells after the respective treatments. RESULTS: An increased surface expression of HSP70 was observed after X-ray or X-ray plus HT while the amount of extracellular HSP70 was only increased when HT was given additionally. A high up-regulation of the co-stimulation molecule CD80 and the chemokine receptor CCR7 on DC was measured after contact with SN of X-ray plus HT treated cells. This was dependent on extracellular HSP70. Combined treatments further led to significantly increased phagocytosis rates of macrophages and DC and increased pro-inflammatory cytokine (IL-8 and IL-12) secretion. CONCLUSION: X-ray combined with HT induces HSP70 dependent activation of immune cells and might generate a tumor microenvironment beneficial for cure.

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.

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.

AnnexinA5 renders dead tumor cells immunogenic--implications for multimodal cancer therapies.

Multimodal tumor therapies should aim not only to kill the tumor cells, but also to stimulate a specific immune response to keep residual tumor (stem) cells and metastases under control. Apoptotic cells are mostly noninflammatory or even anti-inflammatory while necrotic cells stimulate the immune system. Whether the immunogenicity of apoptotic tumor cells can be increased by interfering with their swift and phosphatidylserine (PS)-dependent clearance by macrophages was examined. AnnexinA5 (AnxA5) is a naturally occurring highly specific ligand for PS. Proteins of the annexin family are characterized by a selective affinity for phospholipids in the presence of Ca2+ ions. The phagocytosis by macrophages of irradiated, apoptotic tumor cells (ITC) was partially inhibited when the ITC were preincubated with AnxA5. Activated macrophages secreted higher amounts of TNFalpha and IL-1beta after contact with ITC plus AnxA5 in comparison with ITC alone, while the amount of TGF-beta was decreased. Macrophages of AnxA5-deficient mice showed an increased phagocytosis of dead cells. Wild-type mice, where endogenous AnxA5 is present, displayed a significantly faster decline in size of allogeneic tumors in comparison with AnxA5-deficient animals. The addition of AnxA5 to ITC vaccines increased the percentage of tumor-free mice in syngeneic tumor protection and tumor cure assays. AnxA5 alone led to a retard of syngeneic tumor growth that was, however, less pronounced in comparison to treatment of the tumor with ionizing irradiation. In conclusion, AnxA5 disturbs the PS-dependent clearance by macrophages of dying tumor cells, leading to the accumulation of the latter, to the occurrence of secondary necrotic cells, and to an increased uptake of the dead cells by dendritic cells. Tumor cure appendages with dead tumor cells should be performed with AnxA5 as an immune stimulator and could be combined with irradiation, chemotherapy, and hyperthermia to induce immunogenic cell death forms in vivo or ex vivo.

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.