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.