title: Modulation of radiochemoimmunotherapy-induced B16 melanoma cell death by the pan-caspase inhibitor zVAD-fmk induces anti-tumor immunity in a HMGB1-, nucleotide- and T-cell-dependent manner.
abstract: 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.
authors.affiliation: Department of Radiation Oncology, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Germany.
authors.forename: N
authors.initials: N
authors.lastname: Werthmöller
authors.affiliation: Department of Radiation Oncology, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Germany.
authors.forename: B
authors.initials: B
authors.lastname: Frey
authors.affiliation: Department of Radiation Oncology, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Wunderlich
authors.affiliation: Department of Radiation Oncology, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Fietkau
authors.affiliation: Department of Radiation Oncology, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Germany.
authors.forename: U S
authors.initials: US
authors.lastname: Gaipl
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000627:therapeutic use*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000276:immunology* / Q000528:radiation effects; D001081:Apyrase / Q000627:therapeutic use; D051940:B7-2 Antigen / Q000096:biosynthesis / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D061945:Caspase Inhibitors / Q000627:therapeutic use; D045744:Cell Line, Tumor; D059248:Chemoradiotherapy; D003131:Combined Modality Therapy; D003606:Dacarbazine / Q000627:therapeutic use; D003713:Dendritic Cells / Q000276:immunology; D024243:HMGB1 Protein / Q000378:metabolism*; D000949:Histocompatibility Antigens Class II / Q000096:biosynthesis / Q000276:immunology; D018398:Homeodomain Proteins / Q000235:genetics; D006979:Hyperthermia, Induced; D007371:Interferon-gamma / Q000096:biosynthesis / Q000276:immunology; D008213:Lymphocyte Activation / Q000276:immunology; D017737:Macrophages, Peritoneal / Q000276:immunology; D008546:Melanoma, Experimental / Q000188:drug therapy / Q000473:pathology* / Q000532:radiotherapy; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics; D011839:Radiation, Ionizing; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D051940:B7-2 Antigen; D061945:Caspase Inhibitors; C497965:Cd86 protein, mouse; D024243:HMGB1 Protein; C582808:HMGB1 protein, mouse; D000949:Histocompatibility Antigens Class II; D018398:Homeodomain Proteins; C507780:Myd88 protein, mouse; D053594:Myeloid Differentiation Factor 88; D014409:Tumor Necrosis Factor-alpha; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C064658:RAG-1 protein; D003606:Dacarbazine; D007371:Interferon-gamma; D001081:Apyrase