The therapy of cancer emerged as multimodal treatment strategy. The major mode of action of locally applied radiotherapy (RT) is the induction of DNA damage that triggers a network of events that finally leads to tumor cell cycle arrest and cell death. Along with this, RT modifies the phenotype of the tumor cells and their microenvironment. Either may contribute to the induction of specific and systemic antitumor immune responses. The latter are boosted when additional immune therapy (IT) is applied at distinct time points during RT. We will focus on therapy-induced necrotic tumor cell death that is immunogenic due to the release of damage-associated molecular patterns. Immune-mediated distant bystander (abscopal) effects of RT when combined with dendritic cell-based IT and the role of fractionation of radiation in the induction of immunogenic tumor cell death will be discussed. Autologous whole-tumor-cell-based vaccines generated by high hydrostatic pressure technology will be introduced and the influence of cytokines and the immune modulator AnnexinA5 on the ex vivo generated or in situ therapy-induced vaccine efficacy will be outlined. RT should be regarded as immune adjuvant for metastatic disease and as a tool for the generation of an in situ vaccine when applied at distinct fractionation doses or especially in combination with IT to generate immune memory against the tumor. To identify the most beneficial combination and chronology of RT with IT is presumably one of the biggest challenges of innovative tumor research and therapies.

One prerequisite that radiotherapy (RT) and chemotherapy (CT) result in anti-tumor immune responses is triggering of immunogenic cell death forms such as necroptosis. The latter is inducible by inhibition of apoptosis with the pan-caspase inhibitor zVAD-fmk. The design of multimodal therapies that overcome melanoma's resistance to apoptosis is a big challenge of oncoimmunology. 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. 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 in vivo 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 IFNγ by 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.

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.