As hints exist that immune stimulation by hyperthermia (HT) augments the efficacy of melanoma therapies and that tumors can be sensitized for RT with zVAD-fmk, we asked whether combinations of RT with dacarbazine (DTIC) and/or HT induce immunogenic melanoma cell death and how this is especially influenced by zVAD-fmk. that tumors can be sensitized for RT with zVAD-fmk, we asked whether combinations of RT with dacarbazine (DTIC) and/or HT induce immunogenic melanoma cell death and how this is especially influenced by zVAD-fmk. Necroptosis was inducible in poorly immunogenic B16-F10 melanoma cells and zVAD-fmk generally increased melanoma cell necrosis concomitantly with the release of HMGB1. Supernatants (SNs) of melanoma cells whose cell death was modulated with zVAD-fmk induced an upregulation of the activation markers CD86 and MHCII on macrophages. The same was seen on dendritic cells (DCs), but only when zVAD-fmk was added to multimodal tumor treatments including DTIC. DCs of MyD88 KO mice and DCs incubated with SNs containing apyrase did not increase the expression of these activation markers on their surface. The experiments revealed that zVAD-fmk decreases the tumor growth significantly and results in a significantly reduced tumor infiltration of Tregs when added to multimodal treatment of the tumor with RT, DTIC and HT. Further, a significantly increased DC and CD8+ T-cell infiltration into the tumor and in the draining lymph nodes was induced, as well as an increased expression of IFNby CD8+ T cells. However, zVAD-fmk did not further reduce tumor growth in MyD88 KO mice, mice treated with apyrase or RAG KO mice. We conclude that HMGB1, nucleotides and CD8+ T cells mediate zVAD-fmk induced anti-melanoma immune reactions in multimodal therapy settings. The cancer immune editing concept raised by Schreiber and colleagues1 and the findings that distinct chemotherapeutic agents induce immunogenic cancer cell death forms2 opened our minds that standard tumor therapies Fissinolide alone and especially in combination with further immune therapies are capable of inducing anti-tumor immune responses.3 The phenotype of the tumor cells and the tumor microenvironment are altered during therapy and, thereby, the tumor might become visible for the immune system.4 A main prerequisite for induction of anti-tumor immunity is triggering of immunogenic tumor cell death forms.5 Apoptosis is non- or even anti-inflammatory.6 In contrast, necrotic cells bear per se a high inflammatory and immunogenic potential. Damage-associated molecular patterns (DAMPs) are released because the plasma membrane of necrotic cells is disturbed.7, 8 Danger signals as the high mobility group protein B1 (HMGB1) and the nucleotide adenosine triphosphate (ATP) activate DCs, foster cross-presentation of antigens and consecutively the activation of T cells.9 DAMPs therefore link radio- and/or Fissinolide chemotherapy-induced local alterations of the tumor cells and subsequent systemic anti-tumor immune reactions.10, 11 HMGB1 is mostly passively released by therapy-induced necrotic tumor cells.12 The activation of DCs by HMGB1 is induced by its binding to TLR2 or TLR4.13, 14 HMGB1 is further required for the migration of maturing DCs. 15 The nucleotide ATP is often actively emitted and acts on purinergic receptors, especially on P2RX7.16, 17 Activation of DCs is crucial for the success of multimodal tumor treatments.18 Several preclinical and clinical studies have demonstrated that tumor cell death induced by radiochemotherapy in combination with intratumoral DC injection induces strong anti-tumor immune responses in several tumor entities.19, 20, 21 These responses can be enhanced by hyperthermia (HT). Mild HT is an additive therapy to radiotherapy (RT) and/or chemotherapy (CT) in which tumor tissue is locally heated to temperatures of 40C44?C for a time period of 1 h. HT fosters protein aggregation and aggravates radiation- and chemotherapy-induced repair of DNA damage.22 In Fissinolide addition, locally applied HT is capable of inducing systemic anti-tumor responses. 23 Melanoma is the most dangerous form of skin cancer and its response to CT and RT is poor.24 To overcome melanoma’s resistance to apoptosis, the search for multimodal treatments that aim of inducing immunogenic cell death forms is a big challenge Mouse monoclonal to HSP70 of innovative oncoimmunology,25 as much as to understand the mechanisms of therapy-induced immunogenic melanoma cell death. Nowadays, evidence has come up that necrosis as immunogenic cell death form can also occur in a programmed manner.26, 27 Necroptosis is independent of caspases and mainly occurs when caspases are not activated or inhibited.28 The.