Jitka Fucikova

Consensus guidelines for the detection of immunogenic cell death

Apoptotic 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.

Trial watch: Dendritic cell-based interventions for cancer therapy.

Dendritic cells (DCs) occupy a privileged position at the interface between innate and adaptive immunity, orchestrating a large panel of responses to both physiological and pathological cues. In particular, whereas the presentation of antigens by immature DCs generally results in the development of immunological tolerance, mature DCs are capable of priming robust, and hence therapeutically relevant, adaptive immune responses. In line with this notion, functional defects in the DC compartment have been shown to etiologically contribute to pathological conditions including (but perhaps not limited to) infectious diseases, allergic and autoimmune disorders, graft rejection and cancer. Thus, the possibility of harnessing the elevated immunological potential of DCs for anticancer therapy has attracted considerable interest from both researchers and clinicians over the last decade. Alongside, several methods have been developed not only to isolate DCs from cancer patients, expand them, load them with tumor-associated antigens and hence generate highly immunogenic clinical grade infusion products, but also to directly target DCs in vivo. This intense experimental effort has culminated in 2010 with the approval by the US FDA of a DC-based preparation (sipuleucel-T, Provenge®) for the treatment of asymptomatic or minimally symptomatic metastatic castration-refractory prostate cancer. As an update to the latest Trial Watch dealing with this exciting field of research (October 2012), here we summarize recent advances in DC-based anticancer regimens, covering both high-impact studies that have been published during the last 13 mo and clinical trials that have been launched in the same period to assess the antineoplastic potential of this variant of cellular immunotherapy.

Human Tumor Cells Killed by Anthracyclines Induce a Tumor-Specific Immune Response

Immunogenic cell death is characterized by the early surface exposure of chaperones including calreticulin and HSPs, which affect dendritic cell (DC) maturation and the uptake and presentation of tumor antigens. It has also been shown that it is characterized by the late release of high mobility group box 1 (HMGB1), which acts through Toll-like receptor 4 (TLR4) and augments the presentation of antigens from dying tumor cells to DCs. Most of the data on immunogenic tumor cell death were obtained using mouse models. In this study, we investigated the capacity of clinically used chemotherapeutics to induce immunogenic cell death in human tumor cell lines and primary tumor cells. We found that only anthracyclines induced a rapid translocation of calreticulin, HSP70, and HSP90 to the cell surface and the release of HMGB1 12 hours after the treatment. The interaction of immature DCs with immunogenic tumor cells led to an increased tumor cell uptake and induces moderate phenotypic maturation of DCs. Killed tumor cell-loaded DCs efficiently stimulated tumor-specific IFN-γ-producing T cells. DCs pulsed with killed immunogenic tumor cells also induced significantly lower numbers of regulatory T cells than those pulsed with nonimmunogenic tumor cells. These data indicate that human prostate cancer, ovarian cancer, and acute lymphoblastic leukemia cells share the key features of immunogenic cell death with mice tumor cells. These data also identify anthracyclines as anticancer drugs capable of inducing immunogenic cell death in sensitive human tumor cells.

Combinatorial strategies for the induction of immunogenic cell death.

1 Equipe 11 labellisée par la Ligue Nationale contre le Cancer, Centre de Recherche des Cordeliers, Paris, France, 2 U1138, INSERM, Paris, France, Metabolomics and Cell Biology Platforms, Gustave Roussy Campus Cancer, Villejuif, France, 4 Faculté de Medecine, Université Paris-Sud, Le Kremlin-Bicêtre, France, 5 Department of Chemistry, University of Coimbra, Coimbra, Portugal, 6 Laboratory for General Biochemistry and Physical Pharmacy, Faculty of Pharmacy, Ghent University, Ghent, Belgium, 7 Laboratory of Molecular and Cellular Therapy, Vrije Universiteit Brussel, Jette, Belgium, 8 Sotio a.c., Prague, Czech Republic, 9 Department of Immunology, 2nd Faculty of Medicine, University Hospital Motol, Charles University, Prague, Czech Republic, Gustave Roussy Campus Cancer, Villejuif, France, 11 Université Paris Descartes, Paris, France, 12 Université Pierre et Marie Curie, Paris, France, 13 Pôle de Biologie, Hopitâl Européen George Pompidou, AP-HP, Paris, FranceThe term “immunogenic cell death” (ICD) is commonly employed to indicate a peculiar instance of regulated cell death (RCD) that engages the adaptive arm of the immune system. The inoculation of cancer cells undergoing ICD into immunocompetent animals elicits a specific immune response associated with the establishment of immunological memory. Only a few agents are intrinsically endowed with the ability to trigger ICD. These include a few chemotherapeutics that are routinely employed in the clinic, like doxorubicin, mitoxantrone, oxaliplatin, and cyclophosphamide, as well as some agents that have not yet been approved for use in humans. Accumulating clinical data indicate that the activation of adaptive immune responses against dying cancer cells is associated with improved disease outcome in patients affected by various neoplasms. Thus, novel therapeutic regimens that trigger ICD are urgently awaited. Here, we discuss current combinatorial approaches to convert otherwise non-immunogenic instances of RCD into bona fide ICD.

Molecular and Translational Classifications of DAMPs in Immunogenic Cell Death

The immunogenicity of malignant cells has recently been acknowledged as a critical determinant of efficacy in cancer therapy. Thus, besides developing direct immunostimulatory regimens, including dendritic cell-based vaccines, checkpoint-blocking therapies, and adoptive T-cell transfer, researchers have started to focus on the overall immunobiology of neoplastic cells. It is now clear that cancer cells can succumb to some anticancer therapies by undergoing a peculiar form of cell death that is characterized by an increased immunogenic potential, owing to the emission of the so-called “damage-associated molecular patterns” (DAMPs). The emission of DAMPs and other immunostimulatory factors by cells succumbing to immunogenic cell death (ICD) favors the establishment of a productive interface with the immune system. This results in the elicitation of tumor-targeting immune responses associated with the elimination of residual, treatment-resistant cancer cells, as well as with the establishment of immunological memory. Although ICD has been characterized with increased precision since its discovery, several questions remain to be addressed. Here, we summarize and tabulate the main molecular, immunological, preclinical, and clinical aspects of ICD, in an attempt to capture the essence of this phenomenon, and identify future challenges for this rapidly expanding field of investigation.

Trial Watch: Immunogenic cell death inducers for anticancer chemotherapy

The term “immunogenic cell death” (ICD) is now employed to indicate a functionally peculiar form of apoptosis that is sufficient for immunocompetent hosts to mount an adaptive immune response against dead cell-associated antigens. Several drugs have been ascribed with the ability to provoke ICD when employed as standalone therapeutic interventions. These include various chemotherapeutics routinely employed in the clinic (e.g., doxorubicin, epirubicin, idarubicin, mitoxantrone, bleomycin, bortezomib, cyclophosphamide and oxaliplatin) as well as some anticancer agents that are still under preclinical or clinical development (e.g., some microtubular inhibitors of the epothilone family). In addition, a few drugs are able to convert otherwise non-immunogenic instances of cell death into bona fide ICD, and may therefore be employed as chemotherapeutic adjuvants within combinatorial regimens. This is the case of cardiac glycosides, like digoxin and digitoxin, and zoledronic acid. Here, we discuss recent developments on anticancer chemotherapy based on ICD inducers.

Distinct patterns of intratumoral immune cell infiltrates in patients with HPV-associated compared to non-virally induced head and neck squamous cell carcinoma

Human papillomavirus (HPV) infection is one of the most important etiologic causes of oropharyngeal head and neck squamous cell carcinoma (HNSCC). Patients with HPV-positive HNSCC were reported to have a better clinical outcome than patients with HPV-negative cancers. However, little is known about the possible causes of different clinical outcomes. In this study, we analyzed a detailed immune profile of tumor samples from HNSCC patients with respect to their HPV status. We analyzed the characteristics of immune cell infiltrates, including the frequency and distribution of antigen-presenting cells and naïve, regulatory and effector T cells and the cytokine and chemokine levels in tumor tissue. There was a profound difference in the extent and characteristics of intratumoral immune cell infiltrates in HNSCC patients based on their HPV status. In contrast to HPV-negative tumor tissues, HPV-positive tumor samples showed significantly higher numbers of infiltrating IFNγ+ CD8+ T lymphocytes, IL-17+ CD8+ T lymphocytes, myeloid dendritic cells and proinflammatory chemokines. Furthermore, HPV-positive tumors had significantly lower expression of Cox-2 mRNA and higher expression of PD1 mRNA compared to HPV-negative tumors. The presence of a high level of intratumoral immune cell infiltrates might play a crucial role in the significantly better response of HPV-positive patients to standard therapy and their favorable clinical outcome. Furthermore, characterization of the HNSCC immune profile might be a valuable prognostic tool in addition to HPV status and might help identify novel targets for therapeutic strategies, including cancer immunotherapy.

High hydrostatic pressure induces immunogenic cell death in human tumor cells

Recent studies have identified molecular events characteristic of immunogenic cell death (ICD), including surface exposure of calreticulin (CRT), the heat shock proteins HSP70 and HSP90, the release of high‐mobility group box protein 1 (HMGB1) and the release of ATP from dying cells. We investigated the potential of high hydrostatic pressure (HHP) to induce ICD in human tumor cells. HHP induced the rapid expression of HSP70, HSP90 and CRT on the cell surface. HHP also induced the release of HMGB1 and ATP. The interaction of dendritic cells (DCs) with HHP‐treated tumor cells led to a more rapid rate of DC phagocytosis, upregulation of CD83, CD86 and HLA‐DR and the release of interleukin IL‐6, IL‐12p70 and TNF‐α. DCs pulsed with tumor cells killed by HHP induced high numbers of tumor‐specific T cells. DCs pulsed with HHP‐treated tumor cells also induced the lowest number of regulatory T cells. In addition, we found that the key features of the endoplasmic reticulum stress‐mediated apoptotic pathway, such as reactive oxygen species production, phosphorylation of the translation initiation factor eIF2α and activation of caspase‐8, were activated by HHP treatment. Therefore, HHP acts as a reliable and potent inducer of ICD in human tumor cells.

Physical modalities inducing immunogenic tumor cell death for cancer immunotherapy

The concept of immunogenic cancer cell death (ICD), as originally observed during the treatment with several chemotherapeutics or ionizing irradiation, has revolutionized the view on the development of new anticancer therapies. ICD is defined by endoplasmic reticulum (ER) stress response, reactive oxygen species (ROS) generation, emission of danger-associated molecular patterns and induction of antitumor immunity. Here we describe known and emerging cancer cell death-inducing physical modalities, such as ionizing irradiation, ultraviolet C light, Photodynamic Therapy (PDT) with Hypericin, high hydrostatic pressure (HHP) and hyperthermia (HT), which have been shown to elicit effective antitumor immunity. We discuss the evidence of ICD induced by these modalities in cancer patients together with their applicability in immunotherapeutic protocols and anticancer vaccine development.

Dynamics of T-cell infiltration during the course of ovarian cancer: the gradual shift from a Th17 effector cell response to a predominant infiltration by regulatory T-cells.

The type of immune cells that are present within the tumor microenvironment can play a crucial role in the survival of patients. However, little is known about the dynamics of the tumor‐infiltrating immune cells during disease progression. We studied the immune cells that infiltrated the tumor tissues of ovarian cancer patients at different stages of disease. The early stages of development of ovarian carcinomas were characterized by a strong Th17 immune response, whereas in stage II patients, recruitment of high numbers of Th1 cells was observed. In disseminated tumors (Stages III–IV), we detected a dominant population of Helios+ activated regulatory T cells (Tregs) along with high numbers of monocytes/macrophages and myeloid dendritic cells (mDCs). Tumor‐infiltrating Tregs had markedly lower expression of CCR4 than circulating Tregs, and the numbers of tumor‐infiltrating Tregs significantly correlated with the levels of CCL22 in ovarian tumor cell culture supernatants, suggesting their recruitment via a CCR4/CCL22 interaction. CCL22 was mainly produced by tumor cells, monocytes/macrophages and mDCs in the primary ovarian tumors, and its expression markedly increased in response to IFNγ. Taken together, the specific recruitment of Tregs, probably triggered by inflammatory stimuli, leads to a significant immune suppression in the advanced stages of ovarian cancer.