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.

Contribution of RIP3 and MLKL to immunogenic cell death signaling in cancer chemotherapy

“…31,51 A recent study suggested that B16F10 melanoma cells can be driven into ICD in the presence of the pan-caspase inhibitor z-VAD-FMK. 54 Hence, the current results altogether suggest that there is not a single ICD pathway but multiple distinct signaling cascades that can culminate in ICD.…”

Necroptosis: Mechanisms and Relevance to Disease

“…Further supporting this contention, B16 mouse melanoma cells growing in syngeneic mice exhibit an improved sensitivity to radiation therapy in the presence of Z-VAD-fmk, promoting necroptosis over apoptotic RCD and hence favoring the release of immunostimulatory DAMPs and consequent DC maturation (189). Moreover, the antineoplastic effects of Newcastle disease virus (NDV) in an orthotopic GL261 mouse glioblastoma model are associated with CALR exposure on the surface of dying cells and HMGB1 release, depend on a functional immune system, and can be abolished by Nec-1 (190).…”

A fate worse than death: apoptosis as an oncogenic process

“…Supporting this idea, some studies have shown a positive effect of caspase-inhibition in combination with chemo-or radiotherapy 119,120 . These potentiating effects may be due to various mechasnisms including inhibition of tumour vascularization and enhanced anti-tumour immunity 119,120 . Nevertheless, inhibition of caspase function may have untoward and potentially unwanted effects.…”