Targeting immunogenic cell death in cancer

Immunogenic cell death (ICD) is a type of cancer cell death triggered by certain chemotherapeutic drugs, oncolytic viruses, physicochemical therapies, photodynamic therapy, and radiotherapy. It involves the activation of the immune system against cancer in immunocompetent hosts. ICD comprises the release of damage‐associated molecular patterns (DAMPs) from dying tumor cells that result in the activation of tumor‐specific immune responses, thus eliciting long‐term efficacy of anticancer drugs by combining direct cancer cell killing and antitumor immunity. Remarkably, subcutaneous injection of dying tumor cells undergoing ICD has been shown to provoke anticancer vaccine effects in vivo. DAMPs include the cell surface exposure of calreticulin (CRT) and heat‐shock proteins (HSP70 and HSP90), extracellular release of adenosine triphosphate (ATP), high‐mobility group box‐1 (HMGB1), type I IFNs and members of the IL‐1 cytokine family. In this review, we discuss the cell death modalities connected to ICD, the DAMPs exposed during ICD, and the mechanism by which they activate the immune system. Finally, we discuss the therapeutic potential and challenges of harnessing ICD in cancer immunotherapy.

Abbreviations

ATP

adenosine triphosphate

BAK

BCL‐2 homologous antagonist killer

BAX

BCL‐2‐associated X protein

BCL‐2

B‐cell lymphoma 2

BID

BH3‐interacting domain death agonist

c‐FLIP

cellular FLICE‐like inhibitory protein

cGAMP

cyclic guanosine monophosphate–adenosine monophosphate

cGAS

cyclic GMP‐AMP synthase

CRT

calreticulin

CXCL10

chemokine C‐X‐C motif ligand 10

DAMPs

damage‐associated molecular patterns

DCs

dendritic cells

DISC

death‐inducing signaling complex

ER

endoplasmic reticulum

FADD

FAS‐associated protein with death domain

FASL

FAS ligand

GSDMD

gasdermin D

GSDMD^NT

N‐terminal fragment of gasdermin D

GSDME

gasdermin E

HMGB1

high‐mobility group box‐1

HSP

heat‐shock proteins

Hyp‐PDT

hypericin‐based photodynamic therapy

ICD

immunogenic cell death

IFN

interferon

IFNAR

IFN‐α and IFN‐β receptors

IL

interleukin

IRF3

interferon regulatory factor 3

ISGs

IFN‐stimulated genes

LPS

lipopolysaccharide

MAPK

mitogen‐activated protein kinase

MHC

major histocompatibility complex

MLKL

mixed‐lineage kinase‐like

MOMP

mitochondrial outer membrane permeabilization

mtDNA

mitochondrial DNA

NF‐κB

nuclear factor kappa‐light‐chain‐enhancer of activated B cells

NK cells

natural killer cells

NLR

NOD‐like receptor

NLRP3

NOD‐like receptor family, pyrin domain‐containing 3 protein

P2RX7

purinergic receptor P2X 7

PD‐L1

programmed death ligand

PRRs

pattern recognition receptors

PS

phosphatidyl serine

RCD

regulated cell death

RIPK1

receptor‐interacting serine/threonine protein kinase 1

RIPK3

receptor‐interacting serine/threonine protein kinase 3

ROS

reactive oxygen species

STING

stimulator of interferon genes

tBID

truncated form of BID

TBK1

TANK‐binding kinase 1

TLR

Toll‐like receptor

TNF

tumor necrosis factor

TRAIL

TNF‐related apoptosis‐inducing ligand

ZBP

Z‐DNA‐binding protein

     

化疗与肿瘤免疫原性细胞死亡

化疗耐药是临床难题,研究发现某些蒽环类化合物和奥沙利铂等化疗药物不仅诱导肿瘤细胞凋亡,而且可以引起免疫原性细胞死亡(ICD),通过诱导肿瘤细胞自噬,释放3类信号:钙网蛋白暴露在细胞表面,刺激树突状细胞(DC)吞噬;三磷酸腺苷释放,招募DC进入肿瘤灶;高迁徙率族蛋白B1促进DC与垂死肿瘤细胞形成稳定结合,诱导机体产生特异性的T细胞抗肿瘤免疫.深入理解化疗诱导的ICD,对于完善化疗方案具有重要意义.     

Concepts and mechanisms underlying chemotherapy induced immunogenic cell death: impact on clinical studies and considerations for combined therapies

Chemotherapy has historically been thought to induce cancer cell death in an immunogenically silent manner. However, recent studies have demonstrated that therapeutic outcomes with specific chemotherapeutic agents (e.g. anthracyclines) correlate strongly with their ability to induce a process of immunogenic cell death (ICD) in cancer cells. This process generates a series of signals that stimulate the immune system to recognize and clear tumor cells. Extensive studies have revealed that chemotherapy-induced ICD occurs via the exposure/release of calreticulin (CALR), ATP, chemokine (C–X–C motif) ligand 10 (CXCL10) and high mobility group box 1 (HMGB1). This review provides an in-depth look into the concepts and mechanisms underlying CALR exposure, activation of the Toll-like receptor 3/IFN/CXCL10 axis, and the release of ATP and HMGB1 from dying cancer cells. Factors that influence the impact of ICD in clinical studies and the design of therapies combining chemotherapy with immunotherapy are also discussed.     

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.     

Molecular determinants of immunogenic cell death elicited by anticancer chemotherapy

The success of some chemo- and radiotherapeutic regimens relies on the induction of immunogenic tumor cell death and on the induction of an anticancer immune response. Cells succumbing to immunogenic cell death undergo specific changes in their surface characteristics and release pro-immunogenic factors according to a defined spatiotemporal pattern. This stimulates antigen presenting cells such as dendritic cells to efficiently take up tumor antigens, process them, and cross-prime cytotoxic T lymphocytes, thus eliciting a tumor-specific cognate immune response. Such a response can also target therapy-resistant tumor (stem) cells, thereby leading, at least in some instances, to tumor eradication. In this review, we shed some light on the molecular identity of the factors that are required for cell death to be perceived as immunogenic. We discuss the intriguing observations that the most abundant endoplasmic reticulum protein, calreticulin, the most abundant intracellular metabolite, ATP, and the most abundant non-histone chromatin-binding protein, HMGB1, can determine whether cell death is immunogenic as they appear on the surface or in the microenvironment of dying cells.     

An atypical caspase-independent death pathway for an immunogenic cancer cell line

REGb cell line, a highly immunogenic tumor cell variant isolated from a rat colon cancer, yields regressive tumors when injected into syngeneic hosts. We previously demonstrated that REGb tumor immunogenicity was related to the capacity of releasing dead cells in vivo. Also, in vitro, REGb cell monolayers release dead cells, especially when cultured in serum-free medium. In the current study, we show that the release of dead cells results from an atypical death process associating features of necrosis and apoptosis. In spite of features considered as hallmarks of caspase-dependent apoptosis, including chromatin fragmentation and DNA oligonucleosomal cleavage, caspases are not activated and caspase inhibitors are ineffective to prevent REGb cell death. In contrast with a number of other types of cell death, the spontaneous death of REGb cells in culture depends on de novo protein synthesis as this death is blocked by low doses of the mRNA translation inhibitor cycloheximide. This unusual mode of cell death that associates necrotic and apoptotic features could provide optimal conditions for triggering a specific immune response.     

微波消融治疗骨肉瘤诱导免疫原性细胞死亡的机制研究

目的 探讨是否经微波消融导致肿瘤细胞死亡引起免疫原性肿瘤抗原释放,从而诱导肿瘤特异性免疫反应。方法 3种分别来源于小鼠、大鼠和人类骨肉瘤细胞系作为微波消融模型。在体外消融和原位消融过程中,通过对不同灭活时间及不同实验组设计,检测具有免疫原性分子的表达水平变化。结果 将经体外消融的肿瘤细胞或上清液作为肿瘤疫苗,再植入荷瘤动物模型后,肿瘤细胞生长得到了显著抑制。该效应的产生与疫苗诱导的特异性CD₈(+) T细胞密切相关。效应细胞作用的发挥是通过释放IFN-γ和TNF-α细胞因子来实现,该过程又启动了Fas/FasL介导的细胞凋亡机制。结论 本研究表明经微波消融处理的骨肉瘤细胞可用于诱导特定的抗肿瘤免疫效应。微波原位消融技术联合免疫治疗可望为那些因对化疗不敏感患者提供更多治疗选择。     

免疫原性细胞死亡相关分子的表达机制及对免疫的调节

研究表明,用部分化学药物和放射线等手段治疗肿瘤,肿瘤发生免疫原性细胞死亡（ICD）,随后细胞表面高表达损伤相关分子模式(DAMPs),如钙网蛋白（CRT）、三磷酸腺苷（ATP）、热休克蛋白(HSP)、高迁移率族蛋白B 1（HMGB1）信号分子,增强肿瘤细胞的免疫原性,招募树突状细胞(DC)到肿瘤床并提高其功能,激活特异性的细胞毒性T淋巴细胞(CTL)对肿瘤的攻击。ICD及其DAMPs为肿瘤治疗提供了新的治疗依据和手段,监测化疗前后肿瘤细胞免疫原性的变化,将化疗和免疫治疗有机结合,可提高肿瘤的治疗效果。本文对ICD相关分子的表达机制及对机体免疫的调节等进行综述。     

Treatment of osteosarcoma with microwave thermal ablation to induce immunogenic cell death

Microwave ablation (MWA) has been used as a classical hyperthermic ablation method for decades with the intention to induce direct killing of tumor cells or modulation of tumor architecture. The purpose of this study was to explore whether MWA induced tumor cell death could generate an immunogenic source of tumor antigens and elicit tumor-specific immune responses, taking an alternative antitumor effects. Three kinds of osteosarcoma cell lines, respectively derived from mice, rats and human, were selected as ablation models. In vitro and in situ tumor ablation were both performed to detect the “damage-associated molecular patterns” (DAMPs) exposure level. Active ablated products vaccination resulted in complete protection in both mouse and rat tumor-bearing models, which was mediated primarily by vaccine-elicited CD8⁺ T cells. These effector cells functioned by releasing IFN-γ and TNF-α in the presence of target cells, which may trigger FasL-directed cell apoptosis. These data suggest that MWA-processed osteosarcoma cells could be applied to generate specific antitumor effects, especially for in situ ablation. Hence, MWA could be used in combination with immunotherapy, especially for patients who have failed chemotherapy or who have limited treatment options.     

Capsaicin triggers immunogenic PEL cell death,stimulates DCs and reverts PEL-induced immune suppression

Capsaicin, the pungent alkaloid of red pepper has been extensively studied for its many properties, especially the anti-inflammatory and anti-oxidant ones. It binds to vanilloid receptor 1, although it has been reported to be able to mediate some effects independently of its receptor. Another important property of Capsaicin is the anticancer activity against highly malignant tumors, alone or in combination with other chemotherapeutic agents. In this study, we found that Capsaicin induced an apoptotic cell death in PEL cells correlated with the inhibition of STAT3. STAT3 pathway, constitutively activated in PEL cells, is essential for their survival. By STAT3 de-phosphorylation, Capsaicin reduced the Mcl-1 expression level and this could represent one of the underlying mechanisms leading to the Capsaicin-mediated cell death and autophagy induction. Next, by pharmacological or genetic inhibition, we found that autophagy played a pro-survival role, suggesting that its inhibition could be exploited to increase the Capsaicin cytotoxic effect against PEL cells. Finally, we show that Capsaicin induced DAMP exposure, as for an immunogenic cell death, directly promoted DC activation and, more importantly, that it counteracted the immune-suppression, in terms of DC differentiation, mediated by the PEL released factors.     

CD47 agonist peptide PKHB1 induces immunogenic cell death in T‐cell acute lymphoblastic leukemia cells

T‐cell acute lymphoblastic leukemia (T‐ALL) has a poor prognosis derived from its genetic heterogeneity, which translates to a high chemoresistance. Recently, our workgroup designed thrombospondin‐1‐derived CD47 agonist peptides and demonstrated their ability to induce cell death in chronic lymphocytic leukemia. Encouraged by these promising results, we evaluated cell death induced by PKHB1 (the first‐described serum‐stable CD47‐agonist peptide) on CEM and MOLT‐4 human cell lines (T‐ALL) and on one T‐murine tumor lymphoblast cell‐line (L5178Y‐R), also assessing caspase and calcium dependency and mitochondrial membrane potential. Additionally, we evaluated selectivity for cancer cell lines by analyzing cell death and viability of human and murine non‐tumor cells after CD47 activation. In vivo, we determined that PKHB1‐treatment in mice bearing the L5178Y‐R cell line increased leukocyte cell count in peripheral blood and lymphoid organs while recruiting leukocytes to the tumor site. To analyze whether CD47 activation induced immunogenic cell death (ICD), we evaluated damage‐associated molecular patterns (DAMP) exposure (calreticulin, CRT) and release (ATP, heat shock proteins 70 and 90, high‐mobility group box 1, CRT). Furthermore, we gave prophylactic antitumor vaccination, determining immunological memory. Our data indicate that PKHB1 induces caspase‐independent and calcium‐dependent cell death in leukemic cells while sparing non‐tumor murine and human cells. Moreover, our results show that PKHB1 can induce ICD in leukemic cells as it induces CRT exposure and DAMP release in vitro, and prophylactic vaccinations inhibit tumor establishment in vivo. Together, our results improve the knowledge of CD47 agonist peptides potential as therapeutic tools to treat leukemia.     

Circulating immunogenic cell death biomarkers HMGB1 and RAGE in breast cancer patients during neoadjuvant chemotherapy

Neoadjuvant chemotherapy in breast cancer patients aims at preoperative reduction of tumor volume for better resection results and prognosis. As not all patients respond to neoadjuvant therapy, predictive biomarkers are needed for more efficient individual management. In prospectively collected sera of 51 consecutive locally confined breast cancer (LBC) patients receiving preoperative, neoadjuvant chemotherapy, value level kinetics of soluble high mobility group box 1 (HMGB1), soluble receptor for advanced glycation end products (sRAGE) as well as the established breast cancer biomarkers CA 15–3 and carcinoembryonic antigen (CEA) were investigated and correlated with therapy response objectified by pathological staging at surgery. In addition, biomarkers were measured in sera of 30 healthy controls (HC), 13 patients with benign breast diseases, and 28 metastatic breast cancer (MBC) patients. Pretherapeutic levels of soluble HMGB1 were decreased in MBC, while sRAGE was already decreased in LBC. In contrast, CA 15–3 and CEA were strongly elevated in MBC, but not in LBC. Combination of sRAGE and CA 15–3 enabled best discrimination of LBC from HC (AUC 78.2 %; sens 58 % at 95 % spec), while CA15-3 and CEA discriminated best between MBC and all controls (AUC 90.9 %; sens 70 % at 95 % spec). In LBC patients undergoing neoadjuvant chemotherapy, nine patients achieved complete remission (CR), 29 achieved partial remission (PR), while 13 had no change of disease (NC). NC patients tended to have higher HMGB1 and lower sRAGE levels before therapy onset (p?=?0.056 and p?=?0.054), while CA 15–3 and CEA did not predict therapeutic outcome. Furthermore, kinetics of HMGB1 during therapy correlated with efficacy of the treatment (p?=?0.053). Markers of immunogenic cell death are valuable for the diagnosis of MBC and early estimation of response to neoadjuvant therapy in LBC patients.     

Chemotherapy‐induced immunogenic modulation of tumor cells enhances killing by cytotoxic T lymphocytes and is distinct from immunogenic cell death

Certain chemotherapeutic regimens trigger cancer cell death while inducing dendritic cell maturation and subsequent immune responses. However, chemotherapy‐induced immunogenic cell death (ICD) has thus far been restricted to select agents. In contrast, several chemotherapeutic drugs modulate antitumor immune responses, despite not inducing classic ICD. In addition, in many cases tumor cells do not die after treatment. Here, using docetaxel, one of the most widely used cancer chemotherapeutic agents, as a model, we examined phenotypic and functional consequences of tumor cells that do not die from ICD. Docetaxel treatment of tumor cells did not induce ATP or high‐mobility group box 1 (HMGB1) secretion, or cell death. However, calreticulin (CRT) exposure was observed in all cell lines examined after chemotherapy treatment. Killing by carcinoembryonic antigen (CEA), MUC‐1, or PSA‐specific CD8⁺ CTLs was significantly enhanced after docetaxel treatment. This killing was associated with increases in components of antigen‐processing machinery, and mediated largely by CRT membrane translocation, as determined by functional knockdown of CRT, PERK, or CRT‐blocking peptide. A docetaxel‐resistant cell line was selected (MDR‐1⁺, CD133⁺) by continuous exposure to docetaxel. These cells, while resistant to direct cytostatic effects of docetaxel, were not resistant to the chemomodulatory effects that resulted in enhancement of CTL killing. Here, we provide an operational definition of “immunogenic modulation,” where exposure of tumor cells to nonlethal/sublethal doses of chemotherapy alters tumor phenotype to render the tumor more sensitive to CTL killing. These observations are distinct and complementary to ICD and highlight a mechanism whereby chemotherapy can be used in combination with immunotherapy.     

Is cell death a critical end point for anticancer therapies or is cytostasis sufficient?

Since the discovery of conventional chemotherapy and the development of new target-based agents, the importance of cytostasis in anticancer activity has been debated. This review examines the relative importance of both cytostasis and cytotoxicity based on both preclinical data and clinical reports. Several limitations of our basic and clinical methods to evaluate cytostasis and cytotoxicity will be highlighted. Molecular mechanisms of cytostasis will be analyzed, including interference with the cell cycle as well as putative links with necrosis and autophagy. Finally, we will cite evidence that most older and newer compounds are both cytostatic and cytotoxic. The relative role of cytostasis and cytotoxicity on future drug screening and clinical development will be explored.     

The bovine dialysable leukocyte extract IMMUNEPOTENT CRP induces immunogenic cell death in breast cancer cells leading to long-term antitumour memory

Background Cancer recurrence is a serious problem in breast cancer (BC) patients, and immunogenic cell death (ICD) has been proposed as a strategy to overcome this recurrence. IMMUNEPOTENT CRP (ICRP) acts as an immunomodulator and can be cytotoxic to cancer cells. Thus, we evaluated if ICRP induces ICD in BC cells.Methods Immunogenicity of ICRP-induced cell death was evaluated in vitro, analysing the principal biochemical characteristics of ICD in MCF-7, MDA-MB-231 and 4T1 cells. Ex vivo, we assessed the ability of killed cancer cells (KCC) obtained from ICRP-treated 4T1 cells (ICRP-KCC) to induce DC maturation, T-cell priming and T-cell-mediated cancer cytotoxicity. In vivo, we evaluated tumour establishment and antitumour immune memory after prophylactic ICRP-KCC vaccination in BALB/c mice.Results ICRP induced caspase-independent, ROS-dependent cell death, autophagosome formation, P-eIF2α, chaperone protein exposure, CD47 loss, ATP and HMBG1 release in BC cells. Additionally, ICRP-KCC promoted DC maturation, which triggered T-cell priming and cancer cytotoxicity. Prophylactic vaccination with ICRP-KCC prevented tumour establishment and induced long-term antitumour memory in BALB/c mice, involving DC maturation in lymph nodes, CD8+ T-cell augmentation in lymph nodes, peripheral blood and tumour site and ex vivo tumour-specific cytotoxicity by splenocytes.Conclusions ICRP induces ICD in BC cells, leading to long-term antitumour memory.Subject terms: Cancer immunotherapy, Breast cancer     

Emerging new cell therapies/immune therapies in B-cell non-Hodgkin's lymphoma

Abstract:Non-Hodgkin's lymphomas (NHL) are a heterogeneous group of lymphoproliferative disorders originating in B-lymphocytes, T-lymphocytes, or natural killer (NK) lymphocytes. First line therapy is well established and generally a combination of steroids and chemotherapy. Treatment of relapsed/refractory (R/R) NHL however remains a challenge with rapidly evolving new therapies. Many of these new therapies focus on manipulating the body’s natural immune mechanisms to identify and eradicate tumor cells. There has been much success with using checkpoint inhibitors in Hodgkin’s Lymphoma (HL). However, results have been modest in NHL, prompting further studies of immunomodulatory strategies to target NHL. In this article, we review the emerging immune and cell therapies for R/R B-NHL including checkpoint inhibitors, bispecific antibodies, chimeric antigen receptor (CAR) T-cell therapy, and small molecule inhibitors both alone and in combination.