A fermented Mistletoe (Viscum album L.) extract elicits markers characteristic for immunogenic cell death driven by endoplasmic reticulum stress in vitro

Even with the recent advancements in cancer immunotherapy, treatments are still associated with debilitating side effects and unacceptable fail rates. Induction of immunogenic cell death (ICD) in tumors is a promising approach to cancer treatment that may overcome these deficiencies. Cells undergoing ICD pathways enhance the interactions between cancerous cells and immune cells of the patient, resulting in the generation of anti-cancer immunity. The goal of this therapy relies on the engagement and reestablishment of the patient's natural immune processes to target and eliminate cancerous cells systemically. The main objective of this research was to determine if non-thermal plasma could be used to elicit immunogenic cancer cell death for cancer immunotherapy. My hypothesis was that plasma induces immunogenic cancer cell death through oxidative stress pathways, followed by development of a specific anti-tumor immune response. This was tested by investigating the interactions between plasma and multiple cancerous cells in vitro and validating anti-tumor immune responses in vivo. Following plasma treatment, two surrogate ICD markers, secreted adenosine triphosphate (ATP) and surface exposed calreticulin (ecto-CRT), were emitted from all three cancerous cell lines tested: A549 lung carcinoma cell line, CNE-1 radiation-resistant nasopharyngeal cell line and CT26 colorectal cancer cell line. When these cells were co-cultured with macrophages, cells of the innate immune system, the tumoricidal activity of macrophages was enhanced, thus demonstrating the immunostimulatory activity of cells undergoing ICD. The underlying mechanisms of plasma-induced ICD were also evaluated. When plasma is generated, four major components are produced: electromagnetic fields, ultraviolet radiation, and charged and neutral reactive species. Of these, we determined that plasma-generated charged and short-lived reactive oxygen species (ROS) were the major effectors of ICD. Following plasma treatment, ROS immediately increased. When chemical attenuators of ROS were used, intracellular ROS was abrogated and emission of ICD markers were attenuated. This strongly suggests that plasma-induced ICD is associated with increased intracellular ROS. The gold-standard approach to evaluating whether a stimulus can elicit genuine ICD relies on a vaccination assay. CT26 colorectal cancer cells were treated at ICD-inducing regimes of plasma and injected into syngeneic Balb/c mice. One week later, mice were challenged with live CT26 cancer cells. Tumor progression was moderated in animals immunized with plasma-treated CT26 cells. Altogether, these provide strong evidence that plasma regimes can be adapted for a new application: ICD induction. Next, a study was conducted to test the potential of plasma to induce ICD in tumors in animals. Plasma treatment of subcutaneous tumors in mice elicited the emission of ecto-CRT and high mobility group box 1 (HMGB1), another marker of ICD, in the tumor and also recruited CD11c+ and CD45+ immune cells locally. This was followed by development of cancer-specific splenic T cells, indicating that a systemic anti-tumor response was elicited from localized plasma treatment of the tumor. Overall, this work demonstrates the development of non-thermal plasma as a novel method of inducing immunogenic cell death for cancer immunotherapy. The obtained results further our understanding of plasma-cellular interaction mechanisms and highlight the potential for clinical translation.

EMBO J 31 5, 1062–1079 (2012); published online January172012 In this issue of The EMBO Journal, Garg et al (2012) delineate a signalling pathway that leads to calreticulin (CRT) exposure and ATP release by cancer cells that succumb to photodynamic therapy (PTD), thereby providing fresh insights into the molecular regulation of immunogenic cell death (ICD).

Immunogenic cell death (ICD) in cancer represents a functionally unique therapeutic response that can induce tumor-targeting immune responses. ICD is characterized by the exposure and release of numerous damage-associated molecular patterns (DAMPs), which confer adjuvanticity to dying cancer cells. The spatiotemporally defined emission of DAMPs during ICD has been well described, whereas the epigenetic mechanisms that regulate ICD hallmarks have not yet been deeply elucidated. Here, we aimed to examine the involvement of miRNAs and their putative targets using well-established in vitro models of ICD. To this end, B cell lymphoma (Mino) and breast cancer (MDA-MB-231) cell lines were exposed to two different ICD inducers, the combination of retinoic acid (RA) and interferon-alpha (IFN-α) and doxorubicin, and to non ICD inducers such as gamma irradiation. Then, miRNA and mRNA profiles were studied by next generation sequencing. Co-expression analysis identified 16 miRNAs differentially modulated in cells undergoing ICD. Integrated miRNA-mRNA functional analysis revealed candidate miRNAs, mRNAs, and modulated pathways associated with Immune System Process (GO Term). Specifically, ICD induced a distinctive transcriptional signature hallmarked by regulation of antigen presentation, a crucial step for proper activation of immune system antitumor response. Interestingly, the major histocompatibility complex class I (MHC-I) pathway was upregulated whereas class II (MHC-II) was downregulated. Analysis of MHC-II associated transcripts and HLA-DR surface expression confirmed inhibition of this pathway by ICD on lymphoma cells. miR-4284 and miR-212-3p were the strongest miRNAs upregulated by ICD associated with this event and miR-212-3p overexpression was able to downregulate surface expression of HLA-DR. It is well known that MHC-II expression on tumor cells facilitates the recruitment of CD4+ T cells. However, the interaction between tumor MHC-II and inhibitory coreceptors on tumor-associated lymphocytes could provide an immunosuppressive signal that directly represses effector cytotoxic activity. In this context, MHC-II downregulation by ICD could enhance antitumor immunity. Overall, we found that the miRNA profile was significantly altered during ICD. Several miRNAs are predicted to be involved in the regulation of MHC-I and II pathways, whose implication in ICD is demonstrated herein for the first time, which could eventually modulate tumor recognition and attack by the immune system.

BackgroundPT-112 is a novel immunogenic small molecule1 under Phase II clinical development for cancer therapy.2–8 Besides mediating cytostatic and cytotoxic effects in numerous human and mouse cancer cells, PT-112 elicits...

Sonosensitized Aggregation-Induced Emission Dots with Capacities of Immunogenic Cell Death Induction and Multivalent Blocking of Programmed Cell Death-Ligand 1 for Amplified Antitumor Immunotherapy

Apoptotic cell death generally characterized by a morphologically homogenous entity has been considered to be essentially non-immunogenic. However, apoptotic cancer cell death, also known as type 1 programmed cell death (PCD), was recently found to be immunogenic after treatment with several chemotherapeutic agents and oncolytic viruses through the emission of various danger-associated molecular patterns (DAMPs). Extensive studies have revealed that two different types of immunogenic cell death (ICD) inducers, recently classified by their distinct actions in endoplasmic reticulum (ER) stress, can reinitiate immune responses suppressed by the tumor microenvironment. Indeed, recent clinical studies have shown that several immunotherapeutic modalities including therapeutic cancer vaccines and oncolytic viruses, but not conventional chemotherapies, culminate in beneficial outcomes, probably because of their different mechanisms of ICD induction. Furthermore, interests in PCD of cancer cells have shifted from its classical form to novel forms involving autophagic cell death (ACD), programmed necrotic cell death (necroptosis), and pyroptosis, some of which entail immunogenicity after anticancer treatments. In this review, we provide a brief outline of the well-characterized DAMPs such as calreticulin (CRT) exposure, high-mobility group protein B1 (HMGB1), and adenosine triphosphate (ATP) release, which are induced by the morphologically distinct types of cell death. In the latter part, our review focuses on how emerging oncolytic viruses induce different forms of cell death and the combinations of oncolytic virotherapies with further immunomodulation by cyclophosphamide and other immunotherapeutic modalities foster dendritic cell (DC)-mediated induction of antitumor immunity. Accordingly, it is increasingly important to fully understand how and which ICD inducers cause multimodal ICD, which should aid the design of reasonably multifaceted anticancer modalities to maximize ICD-triggered antitumor immunity and eliminate residual or metastasized tumors while sparing autoimmune diseases.

Induction of immunogenic cell death (ICD) in tumors can enhance antitumor immunity and modulate immunosuppression in the tumor microenvironment (TME). In the current study, we investigated the effect of silibinin, a natural compound with anticancer activity, and its polymer-based nanoformulations on the induction of apoptosis and ICD in cancer cells. Free and nanoparticulate silibinin were evaluated for their growth-inhibitory effects using an MTT assay. Annexin V/PI staining was used to analyze apoptosis. Calreticulin (CRT) expression was measured by flow cytometry. Western blotting was conducted to examine the levels of elf2α, which plays a role in the ICD pathway. The HSP90 and ATP levels were determined using specific detection kits. Compared to the free drug, silibinin-loaded nanocarriers significantly increased the induction of apoptosis and ICD in B16F10 cells. ICD induction was characterized by significantly increased levels of ICD biomarkers, including CRT, HSP90, and ATP. We also observed an increased expression of p-elf-2α/ elf-2α in B16F10 cells treated with silibinin-loaded micelles compared to cells that received free silibinin. Our findings showed that the encapsulation of silibinin in polymeric nanocarriers can potentiate the effects of this drug on the induction of apoptosis and ICD in B16F10 melanoma cells.

Nanoscale coordination polymers (NCPs) constructed by metal ions and organic ligands via metalligand bonds have attracted great attention for their biomedical application. Herein, a new type of NCP...

Systemic anticancer immunity can be reinstated via the induction of immunogenic cell death (ICD) in malignant cells. Thus, certain classes of cytotoxic compounds, for example, anthracyclines, oxaliplatin and taxanes are endowed with the capacity to act on cancer cells to ignite premortem stress pathways that lead to the surface exposure of calreticulin (CALR) and the cellular release of adenosine triphosphate, annexin A1, high mobility group B1 and type-1 interferons. Altogether, these alterations constitute the hallmarks of ICD. Here we report the design of a discovery pipeline for the identification of novel ICD inducers by means of a phenotypic screening platform. The use of fluorescent biosensors as proxies for the manifestation of ICD hallmarks has enabled the exploration of large collections of chemical compounds by automatized screening routines. Imaging-based assessment and phenotypic selection led to the identification of potential ICD inducers that could be validated further in vitro and in vivo, confirming that bona fide ICD inducers possess the capacity to induce immunological long-term memory and to confer resistance against rechallenge with syngeneic tumors. Machine learning algorithms analyzing the physicochemical properties of ICD inducers can assist in the preselection of compounds with potential ICD-stimulatory properties, further accelerating the screening efforts designed to develop new immunotherapeutic agents.

Optimizing Mechanisms for Induction of Immunogenic Cell Death to Improve Patient Outcome in Multiple Myeloma

Advances in Immunogenic Cell Death for Cancer Immunotherapy

Immunogenic cell death (ICD) is a crucial mechanism for triggering the adaptive immune response in cancer patients. Damage-associated molecular patterns (DAMPs) are critical factors in the detection of ICD. Chemotherapeutic drugs can cause ICD and the release of DAMPs. The aim of this study was to assess the potential for paclitaxel and platinum-based chemotherapy regimens to induce ICD in squamous cell carcinoma (SCC) cell lines. In addition, we examined the immunostimulatory effects of clinically relevant chemotherapeutic regimens utilized in the treatment of SCC. We screened for differentially expressed ICD markers in the supernatants of three SCC cell lines following treatment with various chemotherapeutic agents. The ICD markers included Adenosine Triphosphate (ATP), Calreticulin (CRT), Annexin A1 (ANXA 1), High Mobility Group Protein B1 (HMGB1), and Heat Shock Protein 70 (HSP70). A vaccination assay was also employed in C57BL/6J mice to validate our in vitro findings. Lastly, the levels of CRT and HMGB1 were evaluated in Serum samples from SCC patients. Addition of the chemotherapy drugs cisplatin (DDP), carboplatin (CBP), nedaplatin (NDP), oxaliplatin (OXA) and docetaxel (DOC) increased the release of ICD markers in two of the SCC cell lines. Furthermore, mice that received vaccinations with cervical cancer cells treated with DDP, CBP, NDP, OXA, or DOC remained tumor-free. Although CBP induced the release of ICD-associated molecules in vitro, it did not prevent tumor growth at the vaccination site in 40% of mice. In addition, both in vitro and in vivo results showed that paclitaxel (TAX) and LBP did not induce ICD in SCC cells. The present findings suggest that chemotherapeutic agents can induce an adjuvant effect leading to the extracellular release of DAMPs. Of the agents tested here, DDP, CBP, NDP, OXA and DOC had the ability to act as inducers of ICD.

Purpose: To investigate PT-112 in a human prostate cancer (PC) cell panel and assess differential sensitivity, cell death mechanism, induction of mitochondrial stress, and release of damage-associated molecular patterns (DAMPs). Background: PT-112 is a novel pyrophosphate-platinum conjugate with clinical activity in advanced solid tumors including lung, thymoma and castration-resistant PC, and in multiple myeloma. PT-112’s cancer cell death was shown previously to be independent of DNA damage. In vitro and in vivo mouse experiments have shown that PT-112 causes mitochondrial reactive oxygen species (mtROS) accumulation, DAMP release, immunogenic cell death (ICD), and T cell infiltration. Methods: Sensitivity to PT-112 was assessed in human PC cell lines (LNCap, LNCap-C4, LNCap-C4-2, DU-145, 22Rv1, VCap and PC-3) and the non-tumorigenic prostate cell line RWPE-1. We analyzed parameters involved in the cell death process such as apoptotic and necroptotic markers, mitochondrial membrane potential, and mtROS and autophagy by flow cytometry. We also evaluated PT-112’s induction of ICD markers calreticulin (CRT) cell surface exposure and ATP secretion. Finally, a possible link between HIF-1alpha expression and PT-112 sensitivity was investigated. Results: PT-112 caused growth inhibition and cancer cell death without affecting healthy RWPE-1 cells. The pan-caspase inhibitor Z-VAD-fmk significantly reduced cell death, with more mild effects seen with the RIPK1/2 inhibitor necrostatin-1 in certain cell lines. PT-112-induced cell death was accompanied by a prominent increase of mtROS and decrease in mitochondrial membrane potential, as well as by DAMP emission (ATP release and CRT exposure). PT-112 activated markers of autophagy, and there was a positive relationship between HIF-1alpha expression and the sensitivity to PT-112 in this panel. Conclusions: PT-112 was broadly active in the PC cell lines tested, while sparing benign prostate cells, indicative of PT-112 cancer cell selectivity and of activity that crosses the varied malignant prostate phenotypes, including androgen receptor positive and negative cell lines. Cell death was primarily apoptotic, as shown by the inhibitory effects of Z-VAD-fmk. Consistent with prior work reported in glycolytic murine cells, in this PC cell panel PT-112 induced mtROS accumulation and mitochondrial membrane depolarization, as well as DAMP release, demonstrating that these may be fundamental and linked responses of cancer cells to PT-112. The apparent induction of autophagy by PT-112 may be a cellular defense mechanism to drug-induced stress. The association between PT-112 sensitivity and HIF-alpha expression should be studied further, as validation of this finding could have clinical applications. Future studies will explore relationships across mitochondrial stress, ICD and HIF-1alpha in PT-112-treated cancer cells Citation Format: Ruth Soler-Agesta, Tyler D. Ames, Matthew Price, José Jimeno, Christina Y. Yim, Raquel Moreno-Loshuertos, Alberto Anel. PT-112 induces potent mitochondrial stress and immunogenic cell death in human prostate cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1115.

BackgroundIt is well accepted that the immune system efficiently contributes to positive outcomes of chemotherapeutic cancer treatment by activating immunogenic cell death (ICD). However, only a limited number of ICD-inducing compounds are well characterized at present; therefore, identification of novel ICD inducers is urgently needed for cancer drug discovery, and the need is becoming increasingly urgent.MethodsHerein, we assessed the antitumour activity of bullatacin by MTS assay and apoptosis assay. ICD biomarkers, such as calreticulin (CRT), high-mobility group protein B1 (HMGB-1), heat shock protein (HSP)70, HSP90 and ATP, were assessed by Western blotting, ELISA and flow cytometry. Western blot and qPCR assays were performed to explore the underlying mechanisms of bullatacin-induced ICD. Flow cytometry was used to detect macrophage phagocytosis.ResultsFirst, bullatacin induced apoptosis in both SW480 cells and HT-29 cells in a time-dependent manner at 10 nM, as assessed by flow cytometry. Moreover, Western blot and flow cytometry assays showed that CRT and HSP90 (biomarkers of early ICD) significantly accumulated on the cell membrane surface after approximately 6 h of treatment with bullatacin. In addition, ELISAs and Western blot assays showed that the second set of hallmarks required for ICD (HMGB1, HSP70 and HSP90) were released in the conditioned media of both SW480 and HT-29 cells after 36 h of treatment. Furthermore, qPCR and Western blot assays indicated that bullatacin triggered ICD via activation of the endoplasmic reticulum stress (ERS) signalling pathway. Finally, bullatacin promoted macrophage phagocytosis.ConclusionThis study documents that bullatacin, a novel ICD inducer, triggers immunogenic tumour cell death by activating ERS even at a relatively low concentration in vitro.

The Editorial on the Research Topic Immunogenic Cell Death in Cancer: From Benchside Research to Bedside Reality Immunogenic cell death (ICD) has emerged as a cornerstone of therapy-induced antitumor immunity (1–3). ICD is distinguished by spatiotemporally defined emission of danger signals or damage-associated molecular patterns (DAMPs) that elevate the immunogenic potential of dying cells [Garg et al.; (4)]. The important role played by DAMPs in immunity, tissue remodeling, and inflammation is discussed in details by Venereau et al. (Marco E. Bianchi lab). Most potent ICD inducers, characterized so far, elicit danger signaling through oxidative-endoplasmic reticulum stress (5). Several ICD inducers have been characterized, e.g., some chemotherapies, some physicochemical therapies (e.g., radiotherapy or photodynamic therapy/PDT), and oncolytic viruses (2, 6). Here, radiotherapy is among the first recognized immunogenic therapies [on account of “abscopal-effect” (7)]. The immunogenic potential of radiotherapy and possibilities for its combination with immune checkpoint blockers is discussed by Derer et al. (Udo S. Gaipl lab). It is noteworthy that ICD can also be achieved by various “smart” combinatorial strategies – an important point for clinically applied non-ICD inducers, discussed in details by Bezu et al. (Guido Kroemer lab). Several lines of experimental evidence have established the validity of ICD. However, the overreliance on usage of prophylactic vaccination in transplantable (heterotopic) tumor models has attracted some criticism (8). While these criticisms are valid, the field is already moving toward tumors produced orthotopically (curative/therapeutic) or in genetically engineered mouse models (GEMM) (at least for few ICD inducers, e.g., hypericin-PDT, Newcastle disease virotherapy and anthracyclines) (9–12). Moreover, the clinical existence of ICD has been proven through retrospective analysis involving cancer patient’s survival/therapy-responsiveness data (13–17). These observations have encouraged the increased usage of ICD-associated DAMPs as predictive/prognostic biomarkers – a point discussed in detail by Fucikova et al. (Radek Spisek lab). The promising results generated by systemically administered ICD inducers have also paved way for application of ICD-based dendritic cell (DC) vaccines (12). This important development has been discussed from the preclinical/clinical vantage points of various solid tumors by Vandenberk et al. (Stefaan W. van Gool lab) and lymphoma by Zappasodi et al. (Massimo Di Nicola lab). In the latter case, it is clear that the field is moving toward chimeric antigen receptor (CAR)-T cell’s application, and it will be interesting to see its combination with ICD in near future. Nevertheless, the insurmountable complexity of cancer makes it inevitable that in certain contexts, ICD may fail. This failure may stem from various factors, e.g., tumor heterogeneity (8), MHC-level heterogeneity (12), pre-established niches enriched in immunosuppressive factors or immune-checkpoints (1), stem cell-based immune-evasion (12), low mutational load, inactivating mutations/polymorphisms in certain immune-receptors (1), general ablation of danger signaling (14), and other genetic or even epigenetic causes. Several of these pro-cancerous immune-evasive mechanisms and immunotherapeutic strategies required for overcoming them are discussed in detail by Kersten et al. (Karin E. de Visser lab). The strategies for targeting epigenetic processes to improve immunotherapy are further discussed by Wachowska et al. (Jakub Golab lab). We believe that the valuable contributions of key researchers/clinicians toward this research topic/special edition have largely fulfilled its primary aim, i.e., to foster a critical discussion on experimental and clinical relevance of ICD. In fact, to further summarize and organize the fields of ICD and DAMPs, we have produced a multi-author consensus paper within this research topic that attempts to classify DAMPs and ICD inducers with an eye on translational potential of ICD (Garg et al.). This classification paper brings together >50 authors from the fields of ICD and DAMPs, and tries to reach a comprehensive accord on various terminologies related to DAMPs/ICD, the historical background of these concepts, ICD classification system (Type I vs. Type II inducers), and the relevant preclinical/clinical criteria crucial for the field(s) (Garg et al.). We hope that this consensus paper will be a useful literature resource for various researchers/clinicians. These contributions, while summarizing the status quo, have also exposed a set of major questions and challenges that still need to be addressed.