An emerging role for nanomaterials in increasing immunogenicity of cancer cell death

In the last decade, it has become clear that anti-cancer therapy is more successful when it can also induce an immunogenic form of cancer cell death (ICD). ICD is an umbrella term covering several cell death modalities, including apoptosis and necroptosis. In general, ICD is characterized by the emission of damage-associated molecular patterns (DAMPs) and/or cytokines/chemokines, leading to the induction of strong anti-tumor immune responses. In experimental cancer therapy, new observations indicate that the immunogenicity of dying cancer cells can be improved by the use of biomaterials. In this review, after a brief overview of the basic principles of the concept of ICD and discussion of the potential use of DAMPs as biomarkers of therapy efficacy, we discuss an emerging role of nanomaterials as a promising strategy to modulate the immunogenicity of cancer cell death. We address how nanocarriers can be used to increase the immunogenicity of ICD and then turn our attention to their dual action. Nanocarriers can be used to increase the immunogenicity of dying cancer cells and to reduce the side effects of chemotherapy. Future studies will show whether biomaterials are truly an optimal strategy to modulate the immunogenicity of dying cancer cells and will provide the insights needed for the development of novel treatment strategies for cancer.     

Immunogenic cell death in cancer therapy: Present and emerging inducers

In the tumour microenvironment (TME), immunogenic cell death (ICD) plays a major role in stimulating the dysfunctional antitumour immune system. Chronic exposure of damage‐associated molecular patterns (DAMPs) attracts receptors and ligands on dendritic cells (DCs) and activates immature DCs to transition to a mature phenotype, which promotes the processing of phagocytic cargo in DCs and accelerates the engulfment of antigenic components by DCs. Consequently, via antigen presentation, DCs stimulate specific T cell responses that kill more cancer cells. The induction of ICD eventually results in long‐lasting protective antitumour immunity. Through the exploration of ICD inducers, recent studies have shown that there are many novel modalities with the ability to induce immunogenic cancer cell death. In this review, we mainly discussed and summarized the emerging methods for inducing immunogenic cancer cell death. Concepts and molecular mechanisms relevant to antitumour effects of ICD are also briefly discussed.     

Immunogenic cell death-inducing metal complexes: From the benchtop to the clinic

The immune system presents a complex array of processes designed to maintain homeostasis in malignant cellular growth. Malignancy is the result of a breakdown in immune surveillance by cancer cells evading immune recognition. Significant efforts have been made in modulating immune checkpoint signaling cascades to bypass the resulting immune evasion and establish an anticancer effect. More recently, it was discovered that a form of regulated cell death can involve the stimulation of immune response as its downstream effect and subsequently re-establish immune surveillance. This mechanism, known as immunogenic cell death (ICD), is being exploited as a target to prevent tumor relapse and prevent cancer metastasis. It is now appreciated that metal-based compounds play a key role in ICD activation due to their unique biochemical properties and interactions within cancer cells. With fewer than 1% of known anticancer agents documented as ICD inducers, recent efforts have been made to identify novel entities capable of stimulating a more potent anticancer immune response. While the recent reviews by us or others focus primarily on either discussing the chemical library of ICD inducers or intricate detailing of biological pathways associated with ICD, this review aims to bridge these two topics as a concise summary. Furthermore, early clinical evidence and future directions of ICD are briefly summarized.     

Multimodal immunogenic cancer cell death as a consequence of anticancer cytotoxic treatments

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.     

Danger signalling during cancer cell death: origins,plasticity and regulation

Accumulating data indicates that following anti-cancer treatments, cancer cell death can be perceived as immunogenic or tolerogenic by the immune system. The former is made possible due to the ability of certain anti-cancer modalities to induce immunogenic cell death (ICD) that is associated with the emission of damage-associated molecular patterns (DAMPs), which assist in unlocking a sequence of events leading to the development of anti-tumour immunity. In response to ICD inducers, activation of endoplasmic reticulum (ER) stress has been identified to be indispensable to confer the immunogenic character of cancer cell death, due to its ability to coordinate the danger signalling pathways responsible for the trafficking of vital DAMPs and subsequent anti-cancer immune responses. However, in recent times, certain processes apart from ER stress have emerged (e.g., autophagy and possibly viral response-like signature), which have the ability to influence danger signalling. In this review, we discuss the molecular nature, emerging plasticity in the danger signalling mechanisms and immunological impact of known DAMPs in the context of immunogenic cancer cell death. We also discuss key effector mechanisms modulating the interface between dying cancer cells and the immune cells, which we believe are crucial for the therapeutic relevance of ICD in the context of human cancers, and also discuss the influence of experimental conditions and animal models on these.     

The intrinsic immunogenic properties of cancer cell lines,immunogenic cell death,and how these influence host antitumor immune responses

Modern cancer therapies often involve the combination of tumor-directed cytotoxic strategies and generation of a host antitumor immune response. The latter is unleashed by immunotherapies that activate the immune system generating a more immunostimulatory tumor microenvironment and a stronger tumor antigen-specific immune response. Studying the interaction between antitumor cytotoxic therapies, dying cancer cells, and the innate and adaptive immune system requires appropriate experimental tumor models in mice. In this review, we discuss the immunostimulatory and immunosuppressive properties of cancer cell lines commonly used in immunogenic cell death (ICD) studies being apoptosis or necroptosis. We will especially focus on the antigenic component of immunogenicity. While in several cancer cell lines the epitopes of endogenously expressed tumor antigens are known, these intrinsic epitopes are rarely determined in experimental apoptotic or necroptotic ICD settings. Instead by far the most ICD research studies investigate the antigenic response against exogenously expressed model antigens such as ovalbumin or retroviral epitopes (e.g., AH1). In this review, we will argue that the immune response against endogenous tumor antigens and the immunopeptidome profile of cancer cell lines affect the eventual biological readouts in the typical prophylactic tumor vaccination type of experiments used in ICD research, and we will propose additional methods involving immunopeptidome profiling, major histocompatibility complex molecule expression, and identification of tumor-infiltrating immune cells to document intrinsic immunogenicity following different cell death modalities.Subject terms: Cancer models, Antigen-presenting cells, Immune cell death     

Which cell death modality wins the contest for photodynamic therapy of cancer?

Photodynamic therapy (PDT) was discovered more than 100 years ago. Since then, many protocols and agents for PDT have been proposed for the treatment of several types of cancer. Traditionally, cell death induced by PDT was categorized into three types: apoptosis, cell death associated with autophagy, and necrosis. However, with the discovery of several other regulated cell death modalities in recent years, it has become clear that this is a rather simple understanding of the mechanisms of action of PDT. New observations revealed that cancer cells exposed to PDT can pass through various non-conventional cell death pathways, such as paraptosis, parthanatos, mitotic catastrophe, pyroptosis, necroptosis, and ferroptosis. Nowadays, immunogenic cell death (ICD) has become one of the most promising ways to eradicate tumor cells by activation of the T-cell adaptive immune response and induction of long-term immunological memory. ICD can be triggered by many anti-cancer treatment methods, including PDT. In this review, we critically discuss recent findings on the non-conventional cell death mechanisms triggered by PDT. Next, we emphasize the role and contribution of ICD in these PDT-induced non-conventional cell death modalities. Finally, we discuss the obstacles and propose several areas of research that will help to overcome these challenges and lead to the development of highly effective anti-cancer therapy based on PDT.Subject terms: Cancer immunotherapy, Cell death and immune response     

Regulated lytic cell death in breast cancer

Breast cancer (BC) is a very common cancer among women and one of the primary causes of death in women worldwide. Because BC has different molecular subtypes, the challenges associated with targeted therapy have increased significantly, and the identification of new therapeutic targets has become increasingly urgent. Blocking apoptosis and inhibiting cell death are important characteristics of malignant tumours, including BC. Under adverse conditions, including exposure to antitumour therapy, inhibition of cell death programmes can promote cancerous transformation and the survival of cancer cells. Therefore, inducing cell death in cancer cells is fundamentally important and provides new opportunities for potential therapeutic interventions. Lytic forms of cell death, primarily pyroptosis, necroptosis and ferroptosis, are different from apoptosis owing to their characteristic lysis, that is, the production of cellular components, to guide beneficial immune responses, and the application of lytic cell death (LCD) in the field of tumour therapy has attracted considerable interest from researchers. The latest clinical research results confirm that lytic death signalling cascades involve the BC cell immune response and resistance to therapies used in clinical practice. In this review, we discuss the current knowledge regarding the various forms of LCD, placing a special emphasis on signalling pathways and their implications in BC, which may facilitate the development of novel and optimal strategies for the clinical treatment of BC.     

Many faces of DAMPs in cancer therapy

A new concept of immunogenic cell death (ICD) has recently been proposed. The immunogenic characteristics of this cell death mode are mediated mainly by molecules called ‘damage-associated molecular patterns'' (DAMPs), most of which are recognized by pattern recognition receptors. Some DAMPs are actively emitted by cells undergoing ICD (e.g. calreticulin (CRT) and adenosine triphosphate (ATP)), whereas others are emitted passively (e.g. high-mobility group box 1 protein (HMGB1)). Recent studies have demonstrated that these DAMPs play a beneficial role in anti-cancer therapy by interacting with the immune system. The molecular pathways involved in translocation of CRT to the cell surface and secretion of ATP from tumor cells undergoing ICD are being elucidated. However, it has also been shown that the same DAMPs could contribute to progression of cancer and promote resistance to anticancer treatments. In this review, we will critically evaluate the beneficial and detrimental roles of DAMPs in cancer therapy, focusing mainly on CRT, ATP and HMGB1.     

The Achilles’ heel of cancer: targeting tumors via lysosome-induced immunogenic cell death

Interest in the lysosome’s potential role in anticancer therapies has recently been appreciated in the field of immuno-oncology. Targeting lysosomes triggers apoptotic pathways, inhibits cytoprotective autophagy, and activates a unique form of apoptosis known as immunogenic cell death (ICD). This mechanism stimulates a local and systemic immune response against dead-cell antigens. Stressors that can lead to ICD include an abundance of ROS which induce lysosome membrane permeability (LMP). Dying cells express markers that activate immune cells. Dendritic cells engulf the dying cell and then present the cell’s neoantigens to T cells. The discovery of ICD-inducing agents is important due to their potential to trigger autoimmunity. In this review, we discuss the various mechanisms of activating lysosome-induced cell death in cancer cells specifically and the strategies that current laboratories are using to selectively promote LMP in tumors.Subject terms: Cancer immunotherapy, Immunotherapy, Tumour immunology, Apoptosis, Adaptive immunity     

Oleandrin,a cardiac glycoside,induces immunogenic cell death via the PERK/elF2α/ATF4/CHOP pathway in breast cancer

Chemotherapeutic agents have been linked to immunogenic cell death (ICD) induction that is capable of augmenting anti-tumor immune surveillance. The cardiac glycoside oleandrin, which inhibits Na⁺/K⁺-ATPase pump (NKP), has been shown to suppress breast cancer growth via inducing apoptosis. In the present study, we showed that oleandrin treatment triggered breast cancer cell ICD by inducing calreticulin (CRT) exposure on cell surface and the release of high-mobility group protein B1 (HMGB1), heat shock protein 70/90 (HSP70/90), and adenosine triphosphate (ATP). The maturation and activation of dendritic cells (DCs) were increased by co-culturing with the oleandrin-treated cancer cells, which subsequently enhanced CD8⁺ T cell cytotoxicity. Murine breast cancer cell line EMT6 was engrafted into BALB/c mice, and tumor-bearing mice were administered with oleandrin intraperitoneally every day. Oleandrin inhibited tumor growth and increased tumor infiltrating lymphocytes including DCs and T cells. Furthermore, the differential mRNA expression incurred by oleandrin was investigated by mRNA sequencing and subsequently confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Mechanistically, oleandrin induced endoplasmic reticulum (ER) stress-associated, caspase-independent ICD mainly through PERK/elF2α/ATF4/CHOP pathway. Pharmacological and genetic inhibition of protein kinase R-like ER kinase (PERK) suppressed oleandrin-triggered ICD. Taken together, our findings showed that oleandrin triggered ER stress and induced ICD-mediated immune destruction of breast cancer cells. Oleandrin combined with immune checkpoint inhibitors might improve the efficacy of immunotherapy.Subject terms: Tumour immunology, Preclinical research     

IAPs: guardians of RIPK1

Deregulation of innate immune signalling and cell death form the basis of most human disease pathogenesis. Inhibitor of APoptosis (IAP) protein-family members are frequently overexpressed in cancer and contribute to tumour cell survival, chemo-resistance, disease progression and poor prognosis. Although best known for their ability to regulate caspases, IAPs also influence ubiquitin-dependent pathways that modulate innate immune signalling by activation of NF-κB. Recent advances in our understanding of the molecular mechanisms through which IAPs influence cell death and innate immune responses have provided new insights into novel strategies for treatment of cancer. In this review we discuss our current understanding of IAP-mediated NF-κB signalling, as well as elaborate on unexpected insights into the involvement of IAPs in regulating the 'Ripoptosome', a novel intrinsic cell death-inducing platform. We propose an evolutionarily conserved concept whereby IAPs function as guardians of killer platforms such as the apoptosome in Drosophila and the Ripoptosome in mammals.     

PD-1/PD-L1 pathway: Basic biology and role in cancer immunotherapy

Over the course of past few years, cancer immunotherapy has been accompanied with promising results. However, preliminary investigations with respect to immunotherapy concentrated mostly on targeting the immune checkpoints, nowadays, emerge as the most efficient strategy to raise beneficial antitumor immune responses. Programmed cell death protein 1 (PD-1) plays an important role in subsiding immune responses and promoting self-tolerance through suppressing the activity of T cells and promoting differentiation of regulatory T cells. PD-1 is considered as an immune checkpoint and protects against autoimmune responses through both induction of apoptosis in antigen-specific T cells and inhibiting apoptosis in regulatory T cells. Several clinical trials exerting PD-1 monoclonal antibodies as well as other immune-checkpoint blockades have had prosperous outcomes and opened new horizons in tumor immunotherapy. Nonetheless, a bulk of patients have failed to respond to these newly emerging immune-based approach and the survival rate was not satisfying. Additional strategies, especially combination therapies, has been initiated and been further promising. Attempts to identify novel and well-suited predictive biomarkers are also sensed. In this review, the promotion of cancer immunotherapy targeting PD-1 immunoinhibitory pathway is discussed.     

Inducers of immunogenic cancer cell death

Recently, cytokine-based pro-tumourigenic signalling has been found to play a major role in the immune system's pro-tumourigenic activity. On the other hand, other recent findings have shown that immunogenic cancer cell death triggered by certain anticancer modalities might reset the dysfunctional immune system towards the activation of a long-lasting protective anti-tumour response. Therefore, using inducers of immunogenic cell death (ICD) that can prevent or impede tumour-promoting cytokine signalling is one of the best ways of instigating or restoring efficient anti-tumour immunity. In this review it is discussed, how the different ICD inducers interact with the immune system and influence cytokine-based pro-tumourigenic signalling. We believe that it is crucial to discover or develop new anti-cancer therapeutic modalities that can induce ICD and impede tumour-promoting cytokine signalling.     

Capsaicin as an inducer of damage-associated molecular patterns (DAMPs) of immunogenic cell death (ICD) in human bladder cancer cells

Few conventional cytotoxic anticancer therapeutics induce immunogenic cell death (ICD). This means that they induce tumor cells to undergo apoptosis while eliciting the emission of a spatiotemporal-defined combination of damage-associated molecular patterns (DAMPs) decoded by the immune system to activate antitumor immunity effective for long-term therapeutic success. The neurotoxin capsaicin (CPS) can induce both cancer cell apoptosis and immune-mediated tumor regression. In the present study, we investigated whether CPS is capable of eliciting the emission of ICD hallmarks in human bladder cancer cell lines undergoing apoptosis. We demonstrated that CPS induces pre- and early apoptotic cell surface exposure of calreticulin (CRT), HSP90, and HSP70 as well as ATP release. Moreover, CRT exposure was prevented by inhibition of endoplasmic reticulum–Golgi traffic by brefeldin A. Furthermore, high-mobility group box 1, HSP90, and HSP70 were passively released at late apoptotic stages. We provide the first evidence that CPS is an inducer of ICD hallmarks, suggesting CPS as a novel potential immunogenic cytotoxic agent.     

Pharmacological inhibitors of anaplastic lymphoma kinase (ALK) induce immunogenic cell death through on-target effects

Immunogenic cell death (ICD) is clinically relevant because cytotoxicants that kill malignant cells via ICD elicit anticancer immune responses that prolong the effects of chemotherapies beyond treatment discontinuation. ICD is characterized by a series of stereotyped changes that increase the immunogenicity of dying cells: exposure of calreticulin on the cell surface, release of ATP and high mobility group box 1 protein, as well as a type I interferon response. Here, we examined the possibility that inhibition of an oncogenic kinase, anaplastic lymphoma kinase (ALK), might trigger ICD in anaplastic large cell lymphoma (ALCL) in which ALK is activated due to a chromosomal translocation. Multiple lines of evidence plead in favor of specific ICD-inducing effects of crizotinib and ceritinib in ALK-dependent ALCL: (i) they induce ICD stigmata at pharmacologically relevant, low concentrations; (ii) can be mimicked in their ICD-inducing effects by ALK knockdown; (iii) lose their effects in the context of resistance-conferring ALK mutants; (iv) ICD-inducing effects are mimicked by inhibition of the signal transduction pathways operating downstream of ALK. When ceritinib-treated murine ALK-expressing ALCL cells were inoculated into the left flank of immunocompetent syngeneic mice, they induced an immune response that slowed down the growth of live ALCL cells implanted in the right flank. Although ceritinib induced a transient shrinkage of tumors in lymphoma-bearing mice, irrespective of their immunocompetence, relapses occurred more frequently in the context of immunodeficiency, reducing the effects of ceritinib on survival by approximately 50%. Complete cure only occurred in immunocompetent mice and conferred protection to rechallenge with the same ALK-expressing lymphoma but not with another unrelated lymphoma. Moreover, immunotherapy with PD-1 blockade tended to increase cure rates. Altogether, these results support the contention that specific ALK inhibition stimulates the immune system by inducing ICD in ALK-positive ALCL.Subject terms: Cancer, Cancer models     

Autophagy-mediated anti-tumoral activity of imiquimod in Caco-2 cells

Imiquimod (IMQ) is recognized as a topical immune response modifier compound that enhances immune responses with anti-viral and anti-tumoral activities. Its anti-tumoral effects have been previously demonstrated in a variety of cancer cells, and were identified as indirect responses mediated by the immune modulation of cutaneous dendritic cells. Recently, the pro-apoptotic activities of IMQ occurring via the modulation of bcl-2 family have been reported in several tumor cells. In this study, we first observed IMQ-initiated autophagy determined by vesicular organelle formation and the generation of LC3-II in Caco-2 human colonic adenocarcinoma cells, which expressing functional TLR7. Additionally, IMQ-induced autophagy resulted in cell death occurring independently of molecular changes of apoptotic markers. Loxoribine also induced autophagy and autophagy-induced cell death at less potent than IMQ. Moreover, the activation of autophagy by rapamycin induced enhanced cell death in TNF-alpha-treated Caco-2 cells, which were autophagy and cell death-resistant. Our results led us to conclude that IMQ exerts a direct effect on the anti-tumoral activity of Caco-2 cells via autophagy-induced cell death. In conclusion, the modulation of autophagy might be applied in a potential cancer therapy for the treatment of colon cancer cells.     

Molecular mechanisms of ATP secretion during immunogenic cell death

The immunogenic demise of cancer cells can be induced by various chemotherapeutics, such as anthracyclines and oxaliplatin, and provokes an immune response against tumor-associated antigens. Thus, immunogenic cell death (ICD)-inducing antineoplastic agents stimulate a tumor-specific immune response that determines the long-term success of therapy. The release of ATP from dying cells constitutes one of the three major hallmarks of ICD and occurs independently of the two others, namely, the pre-apoptotic exposure of calreticulin on the cell surface and the postmortem release of high-mobility group box 1 (HMBG1) into the extracellular space. Pre-mortem autophagy is known to be required for the ICD-associated secretion of ATP, implying that autophagy-deficient cancer cells fail to elicit therapy-relevant immune responses in vivo. However, the precise molecular mechanisms whereby ATP is actively secreted in the course of ICD remain elusive. Using a combination of pharmacological screens, silencing experiments and techniques to monitor the subcellular localization of ATP, we show here that, in response to ICD inducers, ATP redistributes from lysosomes to autolysosomes and is secreted by a mechanism that requires the lysosomal protein LAMP1, which translocates to the plasma membrane in a strictly caspase-dependent manner. The secretion of ATP additionally involves the caspase-dependent activation of Rho-associated, coiled-coil containing protein kinase 1 (ROCK1)-mediated, myosin II-dependent cellular blebbing, as well as the opening of pannexin 1 (PANX1) channels, which is also triggered by caspases. Of note, although autophagy and LAMP1 fail to influence PANX1 channel opening, PANX1 is required for the ICD-associated translocation of LAMP1 to the plasma membrane. Altogether, these findings suggest that caspase- and PANX1-dependent lysosomal exocytosis has an essential role in ATP release as triggered by immunogenic chemotherapy.     

Crosstalk between ER stress and immunogenic cell death

Preclinical and clinical findings suggest that tumor-specific immune responses may be responsible – at least in part – for the clinical success of therapeutic regimens that rely on immunogenic cell death (ICD) inducers, including anthracyclines and oxaliplatin. The molecular pathways whereby some, but not all, cytotoxic agents promote bona fide ICD remain to be fully elucidated. Nevertheless, a central role for the endoplasmic reticulum (ER) stress response has been revealed in all scenarios of ICD described thus far. Hence, components of the ER stress machinery may constitute clinically relevant druggable targets for the induction of ICD. In this review, we will summarize recent findings in the field of ICD research with a special focus on ER stress mechanisms and their implication for cancer therapy.