Emission Of Damage-associated Molecular Patterns Research Articles

ER-Targeting Cyanine Dye as an NIR Photoinducer to Efficiently Trigger Photoimmunogenic Cancer Cell Death.

Endoplasmic reticulum (ER) stress, caused by overproduction of reactive oxygen species (ROS), has been shown to be responsible for immunogenic cell death (ICD). Seeking ROS generator targeting ER is an optimal solution to efficiently induce ER stress. Despite clear indications of demand for ER-targeting photosensitizer, the alternative chemical tools remain limited. Herein, the first ER-localizable ICD photoinducer using thio-pentamethine cyanine dye (TCy5) to induce ER stress under mild near-infrared (NIR) irradiation has been developed. Within the ICD photoinducer design, polyfluorinated TCy5-Ph-3F possesses a selective tropism to ER accumulation and superior ROS generation capability in both normoxia and hypoxia conditions, which benefit from its low singlet-triplet gaps. Under NIR irradiation, cancer cells stained by TCy5-Ph-3F will lead to ER stress and induce massive emission of damage-associated molecular patterns, including calreticulin and heat-shock protein 70 exposure, high mobility group box 1 efflux, and adenosine triphosphate secretion. Dendritic cells maturation and CD8+ T cells activation in vivo also highlight the effectiveness. Therefore, the growth of abscopal tumors was substantially suppressed by the primary tumor treated with TCy5-Ph-3F and NIR irradiation. These results confer practical applicability that could provide a guideline for designing efficient ICD photoinducers, which will enable expanding organic molecular applications for cancer immunotherapy.

Induction of Immunogenic Cell Death by Photodynamic Therapy Mediated by Aluminum-Phthalocyanine in Nanoemulsion.

Photodynamic therapy (PDT) has been clinically employed to treat mainly superficial cancer, such as basal cell carcinoma. This approach can eliminate tumors by direct cytotoxicity, tumor ischemia, or by triggering an immune response against tumor cells. Among the immune-related mechanisms of PDT, the induction of immunogenic cell death (ICD) in target cells is to be cited. ICD is an apoptosis modality distinguished by the emission of damage-associated molecular patterns (DAMP). Therefore, this study aimed to analyze the immunogenicity of CT26 and 4T1 treated with PDT mediated by aluminum-phthalocyanine in nanoemulsion (PDT-AlPc-NE). Different PDT-AlPc-NE protocols with varying doses of energy and AlPc concentrations were tested. The death mechanism and the emission of DAMPs–CRT, HSP70, HSP90, HMGB1, and IL-1β–were analyzed in cells treated in vitro with PDT. Then, the immunogenicity of these cells was assessed in an in vivo vaccination-challenge model with BALB/c mice. CT26 and 4T1 cells treated in vitro with PDT mediated by AlPc IC50 and a light dose of 25 J/cm2 exhibited the hallmarks of ICD, i.e., these cells died by apoptosis and exposed DAMPs. Mice injected with these IC50 PDT-treated cells showed, in comparison to the control, increased resistance to the development of tumors in a subsequent challenge with viable cells. Mice injected with 4T1 and CT26 cells treated with higher or lower concentrations of photosensitizer and light doses exhibited a significantly lower resistance to tumor development than those injected with IC50 PDT-treated cells. The results presented in this study suggest that both the photosensitizer concentration and light dose affect the immunogenicity of the PDT-treated cells. This event can affect the therapy outcomes in vivo.

REIC/Dkk-3 Gene Therapy Induces Immunogenic Cell Death in a Mouse Model of Malignant Mesothelioma.

The adenovirus vector- carrying reduced expression in immortalized cell (REIC) gene (Ad-REIC) increases endoplasmic reticulum stress chaperone GRP78/BiP expression and induces the JNK-mediated apoptotic pathway. We aimed to determine whether Ad-REIC-induced apoptotic cell death can trigger immunogenic cell death (ICD). We examined the emission of damage-associated molecular patterns in vitro and the vaccination effect in vivo. We determined the immunological changes in the tumour microenvironment by putative ICD inducers and the combined effects of immune checkpoint blockade therapies. Ad-REIC induced the release of high-mobility group box 1 and adenosine triphosphate and the translocation of calreticulin in murine mesothelioma AB12 cells. The vaccination effect was elicited by Ad-REIC treatment in vivo. The effect of Ad-REIC was potentiated by anti-cytotoxic T-lymphocyte-associated protein 4 antibody treatment in a murine mesothelioma AB1-HA cell model. Ad-REIC induces ICD in malignant mesothelioma.

Novel porphyrazine-based photodynamic anti-cancer therapy induces immunogenic cell death

The immunogenicity of dying cancer cells determines the efficacy of anti-cancer therapy. Photodynamic therapy (PDT) can induce immunogenic cell death (ICD), which is characterized by the emission of damage-associated molecular patterns (DAMPs) from dying cells. This emission can trigger effective anti-tumor immunity. Only a few photosensitizers are known to induce ICD and, therefore, there is a need for development of new photosensitizers that can induce ICD. The purpose of this work was to analyze whether photosensitizers developed in-house from porphyrazines (pz I and pz III) can induce ICD in vitro and in vivo when used in PDT. We indetified the optimal concentrations of the photosensitizers and found that, at a light dose of 20 J/cm2 (λex 615–635 nm), both pz I and pz III efficiently induced cell death in cancer cells. We demonstrate that pz I localized predominantly in the Golgi apparatus and lysosomes while pz III in the endoplasmic reticulum and lysosomes. The cell death induced by pz I-PDT was inhibited by zVAD-fmk (apoptosis inhibitor) but not by ferrostatin-1 and DFO (ferroptosis inhibitors) or by necrostatin-1 s (necroptosis inhibitor). By contrast, the cell death induced by pz III-PDT was inhibited by z-VAD-fmk and by the necroptosis inhibitor, necrostatin-1 s. Cancer cells induced by pz I-PDT or pz III-PDT released HMGB1 and ATP and were engulfed by bone marrow-derived dendritic cells, which then matured and became activated in vitro. We demonstrate that cancer cells, after induction of cell death by pz I-PDT or pz III-PDT, are protective when used in the mouse model of prophylactic tumor vaccination. By vaccinating immunodeficient mice, we prove the role of the adaptive immune system in protecting against tumours. All together, we have shown that two novel porphyrazines developed in-house are potent ICD inducers that could be effectively applied in PDT of cancer.

Bip inhibition in glioma stem cells promotes radiation-induced immunogenic cell death

Tumor regression in sites distant to the irradiated field are thought to be associated with emission of damage-associated molecular patterns (DAMPs) molecules and generation of immunogenic cell death (ICD). Glioma stem cells (GSCs) are resistant to high doses of radiation, and ultimately select the outgrowth of a more aggressive tumor. This study showed high-dose IR triggered fewer DAMPs molecules exposure and release in GSCs comparing to matched non-GSCs. Downregulation of binding immunoglobulin protein (Bip) promoted IR-mediated endoplasmic reticulum stress to generate DAMPs molecules by PERK and IRE1-α phosphorylation, and increased dendritic cells mature and effector T lymphocytes activation. GSCs treated with Bip knockdown and IR efficiently prevented tumor generation, and reduced post-radiotherapy tumor recurrence. These data suggest that Bip plays a critical role in inhibition of IR-induced ICD in GSCs, and Bip inhibition may be a promising strategy on adjuvant therapy by ameliorating tumor immune microenvironment.

PKR and GCN2 stress kinases promote an ER stress-independent eIF2α phosphorylation responsible for calreticulin exposure in melanoma cells

ABSTRACTThe immunogenic cell death (ICD) process represents a novel therapeutic approach to treat tumours, in which cytotoxic compounds promote both cancer cell death and the emission of damage-associated molecular patterns (DAMPs) from dying cells, to activate the immune system against the malignancy. Therefore, we explored the possibility to stimulate the key molecular players with a pivotal role in the execution of the ICD program in melanoma cells. To this aim, we used the pro-ICD agents mitoxantrone and doxorubicin and found that both agents could induce cell death and stimulate the release/exposure of the strictly required DAMPs in melanoma cells: i) calreticulin (CRT) exposure on the cell membrane; ii) ATP secretion; iii) type I IFNs gene up-regulation and iv) HMGB1 secretion, highlighting no interference by oncogenic BRAF. Importantly, although the ER stress-related PERK activation has been linked to CRT externalization, through the phosphorylation of eIF2α, we found that this stress pathway together with PERK were not involved in melanoma cells. Notably, we identified PKR and GCN2 as key mediators of eIF2α phosphorylation, facilitating the translocation of CTR on melanoma cells surface, under pro-ICD drugs stimulation. Therefore, our data indicate that pro-ICD drugs are able to stimulate the production/release of DAMPs in melanoma cells at least in vitro, indicating in this approach a potential new valuable therapeutic strategy to treat human skin melanoma malignancy.

Cell death and immunity in cancer: From danger signals to mimicry of pathogen defense responses.

The immunogenicity of cancer cells is an emerging determinant of anti-cancer immunotherapy. Beyond developing immunostimulatory regimens like dendritic cell-based vaccines, immune-checkpoint blockers, and adoptive T-cell transfer, investigators are beginning to focus on the immunobiology of dying cancer cells and its relevance for the success of anticancer immunotherapies. It is currently accepted that cancer cells may die in response to anti-cancer therapies through regulated cell death programs, which may either repress or increase their immunogenic potential. In particular, the induction of immunogenic cancer cell death (ICD), which is hallmarked by the emission of damage-associated molecular patterns (DAMPs); molecules analogous to pathogen-associated molecular patterns (PAMPs) acting as danger signals/alarmins, is of great relevance in cancer therapy. These ICD-associated danger signals favor immunomodulatory responses that lead to tumor-associated antigens (TAAs)-directed T-cell immunity, which paves way for the removal of residual, treatment-resistant cancer cells. It is also emerging that cancer cells succumbing to ICD can orchestrate "altered-self mimicry" i.e. mimicry of pathogen defense responses, on the levels of nucleic acids and/or chemokines (resulting in type I interferon/IFN responses or pathogen response-like neutrophil activity). In this review, we exhaustively describe the main molecular, immunological, preclinical, and clinical aspects of immunosuppressive cell death or ICD (with respect to apoptosis, necrosis and necroptosis). We also provide an extensive historical background of these fields, with special attention to the self/non-self and danger models, which have shaped the field of cell death immunology.

Immunogenic Apoptotic Cell Death and Anticancer Immunity.

For many years it has been thought that apoptotic cells rapidly cleared by phagocytic cells do not trigger an immune response but rather have anti-inflammatory properties. However, accumulating experimental data indicate that certain anticancer therapies can induce an immunogenic form of apoptosis associated with the emission of damage-associated molecular patterns (DAMPs), which function as adjuvants to activate host antitumor immune responses. In this review, we will first discuss recent advances and the significance of danger signaling pathways involved in the emission of DAMPs, including calreticulin, ATP, and HMGB1. We will also emphasize that switching on a particular signaling pathway depends on the immunogenic cell death stimulus. Further, we address the role of ER stress in danger signaling and the classification of immunogenic cell death inducers in relation to how ER stress is triggered. In the final part, we discuss the role of radiotherapy-induced immunogenic apoptosis and the relationship of its immunogenicity to the fraction dose and concomitant chemotherapy.

Immunogenic cell death.

Currently, it is widely acknowledged that a proactive anticancer immunosurveillance mechanism takes part in the rejection of neoplastic lesions before they progress towards a benign or malignant tumour. However in cases of very aggressive neoplastic lesions consisting of cells with high mutational diversity, cancer cell variants might be formed that are capable of evading host defence systems against uncontrolled proliferation and anticancer immunosurveillance. This is mainly accomplished through the exhibition of low immunogenicity, which is a particularly important stumbling block in the revival of long-lasting as well as stable anticancer immunity. Recently, it has emerged emphatically that inciting a cancer cell death routine, associated with the activation of danger signalling pathways evoking emission of damage-associated molecular patterns (DAMPs), markedly increases the immunogenicity of dying cancer cells. This cell death pathway has been termed "immunogenic cell death" (ICD). In the present review we introduce this concept and discuss its characteristics in detail. We also discuss in detail the various molecular, immunological and operational determinants of ICD.

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.