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.
Highlights
Apoptosis is non- or even anti-inflammatory.[6]
The cancer immune editing concept raised by Schreiber and colleagues[1] and the findings that distinct chemotherapeutic agents induce immunogenic cancer cell death forms[2] opened our minds that standard tumor therapies alone and especially in combination with further immune therapies are capable of inducing anti-tumor immune responses.[3]
The activation of dendritic cells (DCs) by high mobility group protein B1 (HMGB1) is induced by its binding to TLR2 or TLR4.13,14 HMGB1 is further required for the migration of maturing DCs.[15]
Summary
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 crosspresentation of antigens and consecutively the activation of T cells.[9] DAMPs link radio- and/or chemotherapyinduced 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]. HT fosters protein aggregation and aggravates radiation- and chemotherapy-induced repair of DNA damage.[22] In addition, locally applied HT is capable of inducing systemic anti-tumor responses.[23]. We examined here for the first time whether combinations of the clinically relevant single dose of RT of 2 Gy with the only for metastatic melanoma FDA-approved CT agent dacarbazine (DTIC) or combinations with HT (41.5 °C for 1 h) induce immunogenic melanoma cell death and how zVAD-fmk is capable of improving the melanoma's immunogenicity by modulating the therapy-induced melanoma cell death
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