Consensus guidelines for the detection of immunogenic cell death

none82siApoptotic cells have long been considered as intrinsically tolerogenic or unable to elicit immune responses specific for dead cell-associated antigens. However, multiple stimuli can trigger a functionally peculiar type of apoptotic demise that does not go unnoticed by the adaptive arm of the immune system, which we named "immunogenic cell death" (ICD). ICD is preceded or accompanied by the emission of a series of immunostimulatory damage-associated molecular patterns (DAMPs) in a precise spatiotemporal configuration. Several anticancer agents that have been successfully employed in the clinic for decades, including various chemotherapeutics and radiotherapy, can elicit ICD. Moreover, defects in the components that underlie the capacity of the immune system to perceive cell death as immunogenic negatively influence disease outcome among cancer patients treated with ICD inducers. Thus, ICD has profound clinical and therapeutic implications. Unfortunately, the gold-standard approach to detect ICD relies on vaccination experiments involving immunocompetent murine models and syngeneic cancer cells, an approach that is incompatible with large screening campaigns. Here, we outline strategies conceived to detect surrogate markers of ICD in vitro and to screen large chemical libraries for putative ICD inducers, based on a high-content, high-throughput platform that we recently developed. Such a platform allows for the detection of multiple DAMPs, like cell surface-exposed calreticulin, extracellular ATP and high mobility group box 1 (HMGB1), and/or the processes that underlie their emission, such as endoplasmic reticulum stress, autophagy and necrotic plasma membrane permeabilization. We surmise that this technology will facilitate the development of next-generation anticancer regimens, which kill malignant cells and simultaneously convert them into a cancer-specific therapeutic vaccine.Kepp, Oliver; Senovilla, Laura; Vitale, Ilio; Vacchelli, Erika; Adjemian, Sandy; Agostinis, Patrizia; Apetoh, Lionel; Aranda, Fernando; Barnaba, Vincenzo; Bloy, Norma; Bracci, Laura; Breckpot, Karine; Brough, David; Buqué, Aitziber; Castro, Maria G; Cirone, Mara; Colombo, Maria I; Cremer, Isabelle; Demaria, Sandra; Dini, Luciana; Eliopoulos, Aristides G; Faggioni, Alberto; Formenti, Silvia C; Fučíková, Jitka; Gabriele, Lucia; Gaipl, Udo S; Galon, Jérôme; Garg, Abhishek; Ghiringhelli, François; Giese, Nathalia A; Guo, Zong Sheng; Hemminki, Akseli; Herrmann, Martin; Hodge, James W; Holdenrieder, Stefan; Honeychurch, Jamie; Hu, Hong-Min; Huang, Xing; Illidge, Tim M; Kono, Koji; Korbelik, Mladen; Krysko, Dmitri V; Loi, Sherene; Lowenstein, Pedro R; Lugli, Enrico; Ma, Yuting; Madeo, Frank; Manfredi, Angelo A; Martins, Isabelle; Mavilio, Domenico; Menger, Laurie; Merendino, Nicolò; Michaud, Michael; Mignot, Gregoire; Mossman, Karen L; Multhoff, Gabriele; Oehler, Rudolf; Palombo, Fabio; Panaretakis, Theocharis; Pol, Jonathan; Proietti, Enrico; Ricci, Jean-Ehrland; Riganti, Chiara; Rovere-Querini, Patrizia; Rubartelli, Anna; Sistigu, Antonella; Smyth, Mark J; Sonnemann, Juergen; Spisek, Radek; Stagg, John; Sukkurwala, Abdul Qader; Tartour, Eric; Thorburn, Andrew; Thorne, Stephen H; Vandenabeele, Peter; Velotti, Francesca; Workenhe, Samuel T; Yang, Haining; Zong, Wei-Xing; Zitvogel, Laurence; Kroemer, Guido; Galluzzi, LorenzoKepp, Oliver; Senovilla, Laura; Vitale, Ilio; Vacchelli, Erika; Adjemian, Sandy; Agostinis, Patrizia; Apetoh, Lionel; Aranda, Fernando; Barnaba, Vincenzo; Bloy, Norma; Bracci, Laura; Breckpot, Karine; Brough, David; Buqué, Aitziber; Castro, Maria G; Cirone, Mara; Colombo, Maria I; Cremer, Isabelle; Demaria, Sandra; Dini, Luciana; Eliopoulos, Aristides G; Faggioni, Alberto; Formenti, Silvia C; Fučíková, Jitka; Gabriele, Lucia; Gaipl, Udo S; Galon, Jérôme; Garg, Abhishek; Ghiringhelli, François; Giese, Nathalia A; Guo, Zong Sheng; Hemminki, Akseli; Herrmann, Martin; Hodge, James W; Holdenrieder, Stefan; Honeychurch, Jamie; Hu, Hong Min; Huang, Xing; Illidge, Tim M; Kono, Koji; Korbelik, Mladen; Krysko, Dmitri V; Loi, Sherene; Lowenstein, Pedro R; Lugli, Enrico; Ma, Yuting; Madeo, Frank; Manfredi, Angelo A; Martins, Isabelle; Mavilio, Domenico; Menger, Laurie; Merendino, Nicolò; Michaud, Michael; Mignot, Gregoire; Mossman, Karen L; Multhoff, Gabriele; Oehler, Rudolf; Palombo, Fabio; Panaretakis, Theocharis; Pol, Jonathan; Proietti, Enrico; Ricci, Jean Ehrland; Riganti, Chiara; Rovere Querini, Patrizia; Rubartelli, Anna; Sistigu, Antonella; Smyth, Mark J; Sonnemann, Juergen; Spisek, Radek; Stagg, John; Sukkurwala, Abdul Qader; Tartour, Eric; Thorburn, Andrew; Thorne, Stephen H; Vandenabeele, Peter; Velotti, Francesca; Workenhe, Samuel T; Yang, Haining; Zong, Wei Xing; Zitvogel, Laurence; Kroemer, Guido; Galluzzi, Lorenz

A novel community driven software for functional enrichment analysis of extracellular vesicles data.

Bioinformatics tools are imperative for the in depth analysis of heterogeneous high-throughput data. Most of the software tools are developed by specific laboratories or groups or companies wherein they are designed to perform the required analysis for the group. However, such software tools may fail to capture "what the community needs in a tool". Here, we describe a novel community-driven approach to build a comprehensive functional enrichment analysis tool. Using the existing FunRich tool as a template, we invited researchers to request additional features and/or changes. Remarkably, with the enthusiastic participation of the community, we were able to implement 90% of the requested features. FunRich enables plugin for extracellular vesicles wherein users can download and analyse data from Vesiclepedia database. By involving researchers early through community needs software development, we believe that comprehensive analysis tools can be developed in various scientific disciplines

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field

On the pro-apoptotic mechanisms of the antitumor drugs Doxorubicin and Interferon-alpha

Anti-cancer drugs act primarily by inducing apoptosis. However, knowledge of how various substances induce apoptosis is still incomplete, and so is the basis for the great variation in cellular sensitivity to cytotoxic drugs. A detailed understanding of how anti-cancer agents induce cell death and how defects in cell death pathways promote resistance will change the way chemotherapeutic drugs are used and designed. The aim of this thesis was to investigate pro-apoptotic signaling induced by two commonly used anti-cancer drugs, Doxorubicin and Interferon-á. Doxorubicin (DXR), an anthracycline, is a major antitumor agent known to cause cellular damage via a number of mechanisms including free radical formation and inhibition of topoisomerase II. Interferon-á (IFN-á) is a pleiotropic cytokine and its ability to induce apoptosis has been proposed to be of major importance for its clinical anti-tumor activity. The results demonstrate that the mechanisms of induction of apoptosis by both drugs are strikingly similar with respect to the signaling involved. Clinically relevant concentrations of both agents induce the activation of the pro-apoptotic Bcl-2 family members Bak and Bax prior to apoptosis and anti-apoptotic Bcl-2 family members regulate this response. We could also demonstrate that Bak is activated prior to Bax by both agents. Upstream of Bak, Bax and the mitochondria, two kinases that are known to be activated by cellular stress, JNK and PKCä, are involved, both with respect to DXR and IFN-á. We demonstrated the requirement of Bak and Bax for the induction of apoptosis by DXR by using bax- as well as bak-deficient mouse embryo fibroblasts (MEFs). The BH3-only protein, Bik, which is induced in response to DXR, could be an activator of Bak and Bax. Upstream of the Bcl-2 family members, caspase-2 is activated and was found to be required for DXR-induced apoptosis in Jurkat cells. PKCä was found to be one of the critical downstream targets of caspase-2 following DXR treatment. By using chemical inhibitors against caspase-2, PKCä and JNK, our data suggest a signaling model involving caspase-2, PKCä and JNK. Survival signaling could mask the true potential of chemotherapeutic agents as demonstrated by co-incubation of a PI3K-inhibitor with DXR. Inhibition of PI3K potentiated the DXR-induced Bak, Bax activation and apoptosis in a Bcl-2 dependent but in a caspase-2, JNK and PKCä-independent manner. The upstream signaling in IFN-á-induced apoptosis was also addressed. Upstream of the mitochondria, IFN-a induces JNK phosphorylation/activation. Inhibition of JNK significantly blocked IFN-á-induced Bak and Bax activation and apoptosis, but did not affect the IFN-á-stimulated Jak/STAT signaling. This suggests that the canonical IFN-á induced pathway is not sufficient for this response. Inhibition of JNK was also found to influence the phosphorylation of the pro-apoptotic PKC family member, PKCä. Furthermore, PKCä inhibition blocked apoptosis and Bak activation induced by IFN-á. We conclude that IFN-á-induced apoptosis involves the mitochondrial pathway and the kinases JNK and PKCä. Furthermore this stress-related IFN-induced pathway is unrelated to the Jak/STAT signaling which is generally thought to mediate IFN-á's cellular responses

Cisplatin-induced nitrosylation of p53 prevents its mitochondrial translocation

The cellular response to DNA damage has been reported to involve rapid transcription-independent translocation of p53 to mitochondria. We show here that the DNA-damaging cisplatin-derived anticancer agent oxaliplatin induced both mitochondrial translocation and subsequent Bcl-xL interaction, whereas cisplatin did neither. The differential response was due to nitrosative modification of p53. Thus, cisplatin, but not oxaliplatin, induced increased expression of inducible nitric oxide synthase (iNOS). Cisplatin treatment in the presence of an iNOS inhibitor (1400W) allowed p53 mitochondrial translocation. Conversely, oxaliplatin-induced translocation of p53 was prevented by cotreatment with all exogenous NO donor. In cisplatin-treated cells, nuclear but not mitochondrial p53 showed nitrotyrosinylation that was inhibitable by 1400W. We conclude that nitrosative protein modification is more prominent in the response to cisplatin than oxaliplatin and that nitrosative modification of p53 is a major determinant of p53 subcellular location

Energy-requiring uptake of prostasomes and PC3 cell-derived exosomes into non-malignant and malignant cells

Epithelial cells lining the prostate acini release, in a regulated manner (exocytosis), nanosized vesicles called prostasomes that belong to the exosome family. Prostate cancer cells have preserved this ability to generate and export exosomes to the extracellular space. We previously demonstrated that human prostasomes have an ATP-forming capacity. In this study, we compared the capacity of extracellular vesicles (EVs) to generate ATP between normal seminal prostasomes and exosomes secreted by PC3 cells (PC3 exosomes), a prostate cancer cell line. Proteomic analyses identified enzymes of the glycolytic chain in both prostasomes and PC3 exosomes, and we found that both of them were capable of generating ATP when supplied with substrates. Notably, the net production of extracellular ATP was low for prostasomes due to a high ATPase activity contrary to an elevated net ATP level for PC3 exosomes because of their low ATPase activity. The uptake of the 2 types of EVs by normal prostate epithelial cells (CRL2221) and prostate cancer cells (PC3) was visualized and measured, demonstrating differential kinetics. Interestingly, this uptake was dependent upon an ongoing glycolytic flux involving extracellular ATP formation by EVs and/or intracellular ATP produced from the recipient cells. We conclude that the internalization of EVs into recipient cells is an energy-requiring process also demanding an active V-ATPase and the capacity of EVs to generate extracellular ATP may play a role in this process