Introduction
Pancreatic ductal adenocarcinoma (PDAC) remains the most malignant type of cancer with a 5-year survival rate of only 11%.1 It is projected that PDAC will become the second leading cause of mortality in the United States by 2030.2 Radical surgery and adjuvant chemotherapy can gain long-term survival, but most patients have already lost the opportunity for surgery when they are first diagnosed.3,4 For most patients with locally advanced or metastatic disease, the standard treatment involves a combination of cytotoxic chemotherapy and supportive care. Unfortunately, such combination therapies come at the cost of significant side effects, while overall response rates are limited.5,6 Therefore, it is important to explore new therapeutic approaches to effectively treat PDAC.
Immunotherapy (i.e. immune checkpoint inhibitors) has achieved great success for an impressive breadth of solid tumors; however, PDAC is an exceptional case due to its distinctive histopathological features.7,8 The tumor mutational burden of pancreatic cancer is generally lower compared to other types of cancer, which in turn leads to minor antigenicity.9 The unique characteristics of the tumor microenvironment (TME) in PDAC can pose challenges for effective immune response. The dense extracellular matrix and poorly formed vascular system can limit the infiltration of immune cells. Even if a small number of immune cells do manage to infiltrate, they may not be able to function properly due to the local environment. These factors contribute to the “cold” or “immune-excluded” landscape of the TME, which is not capable of initiating a strong immune response on its own.10 It is important to continue exploring innovative approaches to address these challenges and develop effective treatments for pancreatic cancer.
Immunogenic cell death (ICD) is a variant of programmed cell death that emits damage-associated molecular patterns (DAMPs) and other signals, thereby activating the immune reaction. Mechanically, ICD provides plenty of corpses which can be identified as TNA/TSA to compensate for the low immunogenicity of pancreatic cancer. On the other hand, danger signals emitted by ICD can mobilize members of the immune system and thereby remodulate the so-called “immune-excluded” TME of pancreatic cancer to an immune-reactive state, ultimately initiating the antitumor immune response and leading to tumor regression. Indeed, part of traditional PDAC therapy has been demonstrated to exert antitumor effects through ICD. Understanding ICD also leads to the exploitation of considerable novel agents for the immunotherapy of PDAC. Hence, inducing immunogenic cell death appears to be a feasible strategy and may shed light on immunochemotherapy for PDAC.11,12
We searched MEDLINE and PubMed databases for relevant articles and reviews published from January 1, 2005, to April 31, 2023, with combinations of the following keywords: (“immunogenic cell death” OR “ICD”) AND (“PDAC” OR “pancreatic ductal adenocarcinoma” OR “pancreatic” OR “pancreas”). We also considered the reference lists of all included articles and of previous related reviews.
Paradigm of ICD
In the past decades, researchers have classified cell death into two different types: apoptosis is thought to be physiological and immuno-tolerogenic, whereas necrosis is pathological and immunogenic. The notion of ICD was first proposed by Casares et al. in 2005, identifying ICD as a variant of apoptosis that can generate cancer vaccines and stimulate antineoplastic immune responses in vivo.13 With deeper investigation, ICD has been recognized as an independent form of programmed cell death. In 2018, the Nomenclature Committee on Cell Death defined ICD as ‘a functionally peculiar form of RCD that is sufficient to activate an adaptive immune response specific for endogenous (cellular) or exogenous (viral) antigens expressed by dying cells’,14 emphasizing the immunogenicity of ICD.
A range of factors can trigger the initiation of ICD, such as viral infection, stress, therapeutic drugs (e.g. oxaliplatin, irinotecan), specific forms of radiation therapy, and some physical therapies. Certain inducers may trigger endoplasmic reticulum stress (ERS), which can raise the production of reactive oxygen species (ROS),15 while others may initially cause DNA or chromatin damage, followed by ERS. ERS subsequently generates host-derived, immune-activating molecules, such as DAMPs.16 DAMPs act as a signal of danger that can be detected by pattern recognition receptors (PRRs) present on the surface of innate immune cells. They cooperate with the new antigen presented or exposed from the cell corpse and ultimately cause an adaptive immune response. Eventually, the matured dendritic cells (DCs) recruit effective T cells, leading to long-term immunologic memory (Fig. 1).17 Indeed, the actual event of ICD can vary widely from the ideal pattern. However, the basic principles apply to any situation.
Prerequisites of ICD: antigenicity and adjuvanticity
Immunogenic cell death requires antigenicity and adjuvanticity.18 The premise for mature T cells to recognize antigens is that they must be correctly presented through MHC-II molecules and different from those normally presented on the MHC-I. Tumor cells naturally harbor tumor-associated antigens (TAA) and tumor neoantigens (TNA) that are different from normal peptides.19,20 The release or presentation of TAA and TNA when the structure of dying cells collapses (e.g. treated with certain chemotherapies) provides the antigenicity required for ICD. However, the complexity and arbitrary nature of peptide chain structures leads to complexity in antigenicity. In contrast, adjuvanticity is completely defined by DAMPs, greatly simplifying its molecular characterization.21 Early release of ATP during ICD binds to P2RY2 (purinergic P2Y receptor 2) on the surface of DCs or macrophages, emitting a “find me” signal, followed by flipping of calreticulin (CALR) to the cell surface, emitting an “eat me” signal. In addition, high-mobility group box 1 (HMGB1) ultimately promotes DC maturation and antigen presentation. The temporal and spatial release of these DAMPs is orchestrated, ultimately leading to the recruitment of effector T cells.22 Memory T cells are subsequently produced and rapidly respond upon the second activation. Compared to complex peptide chain molecules, the adjuvanticity characterized by these clear immune small molecules is more manipulable and druggable.16
DAMPs of ICD
Molecules that are released by cells in response to various stresses are known as DAMPs. These molecules act as signals to activate the immune system, which then targets and eliminates damaged cells. DAMPs are recognized by innate immune cells through a variety of PRRs, such as Toll-like receptors (TLRs) and NOD-like receptors (NLRs). This recognition leads to the activation of downstream signaling pathways, including the NF-κB and MAPK pathways, which trigger the immune response. The three canonical DAMPs are CALR, extracellular ATP, and HMGB1, and they are thought to be biological markers for ICD.23 As research deepens, more and more DAMPs are gradually being discovered. This has improved our understanding of ICD and provided more targets for manipulating ICD.
CALR
During the early stages of ICD, CALR, an endoplasmic reticulum (ER) chaperone, is translocated from the lumen of the ER to the plasma membrane. This exposure of CALR acts as an “eat-me” signal, aiding in the engulfment of dying cells or their remnants by DCs or their precursors. This process provides these cells with a plentiful supply of antigenic material.24,25 The process of CALR translocation involves a series of events. Firstly, eIF2α is phosphorylated, leading to the inhibition of protein translation. Secondly, caspase 8 is activated, which triggers the cleavage of BCAP31. This results in the aggregation of pro-apoptotic Bcl-2 family members, BAX and BAK1, at the outer mitochondrial membrane. Lastly, the anterograde transport of CALR to the Golgi apparatus is facilitated by VAMP1 and SNAP25.26 CALR usually translocates to the cell surface along with ERp57, which is a member of the protein disulfide isomerase family A. This occurs in most cases of ICD.27
CALR functions by binding to LDL-receptor-related protein 1, also known as CD91, which is the PRR expressed by antigen-presenting cells such as DCs and serves as the main ER chaperone sensor.28,29 A mechanism that relies on BTK is involved in CD91 ligation-induced engulfment of cellular corpses.30,31 The ability of dying cancer cells to establish protective immunity is dependent on the key role of CALR in the immunogenicity of RCD. This can be disrupted through RNAi-mediated knockdown, CRISPR/Cas9-mediated deletion, or neutralizing antibody-mediated blockade of CALR. Conversely, exogenous recombinant CALR can enable the other variant of RCD to provoke protective immunity.30,32 Recently, there have been studies demonstrating that CALR can also enhance innate immunity by improving interleukin (IL)15 trans-presentation to natural killer (NK) cells. This broadens the understanding of CALR’s immunogenicity.33,34
HMGB1
HMGB1 is a protein that is abundant in the nucleus and does not belong to the histone family. During ICD in dying cells, HMGB1 is released. This release happens when both the nuclear lamina and the plasma membrane become permeable.35,36 The precise molecular mechanisms still require further clarification. HMGB1 exerts strong pro-inflammatory effects through its interaction with TLR2, TLR4, and AGER (also known as RAGE), leading to its activation.37 The binding of HGMB1 to TLR4 and RAGE receptors on DCs promotes antigen presentation and induces DC maturation.38,39 Preclinical in vivo models have shown that when cancer cells are deprived of HMGB1 or when the host is deficient in TLR4, cell death is not considered immunogenic according.40,41 It is worth mentioning that the immunogenic characteristics of HMGB1 rely on its redox condition. Studies have demonstrated that the immunostimulatory function of HMGB1 is limited by the caspase-induced generation of ROS, which leads to the oxidation of HMGB1.42 It is possible that the bidirectionality of HMGB1 could contribute to the observed paradoxical effect of HMGB1 on nucleic acid-mediated antitumor activity in mice. However, further research is needed to fully understand the mechanisms behind this phenomenon.43
ATP
ATP is released via pannexin channels in an autophagy-dependent manner through active exocytosis of ATP-containing vesicles during ICD.44,45 By binding to the purinergic receptor P2Y2 (P2RY2), extracellular ATP functions as a significant “find-me” signal for DC precursors and macrophages. This process helps in the recruitment of myeloid cells to the locations where there is active ICD.46,47 Extracellular ATP triggers pro-inflammatory effects by activating the NLRP3 inflammasome through CASP1, leading to the secretion of mature IL1B and IL18. IL1β is best known for its role in inflammation.48,49 Activation of CD8+ T cells and IL17-producing γδ T cells is the result of the effects originating from the P2X7 receptor, which is an inotropic receptor known as purinergic receptor P2X7 (P2RX7).48 The immunogenicity of cell death is dependent on events related to immune responses driven by ICD. This includes the accumulation of ATP in the microenvironment of dying cancer cells and the presence of P2RX7 or P2RY2 in the myeloid compartment of the host.
However, the immunogenicity of cell death can be reduced if ATP fails to accumulate or if P2RX7 or P2RY2 are absent. CD39 and CD73, which are the ectoenzymes that degrade ATP, can also exert immunosuppressive effects when overexpressed in malignant tissues.50–52 In breast cancer patients who receive anthracycline-based chemotherapy, loss-of-function polymorphisms in P2RX7 have been linked to unfavorable clinical outcomes.53 It is notable that the release of ATP during cell death is similar to the release of HGMB1 and can also occur during non-immunogenic forms of cell death. However, in vitro studies have shown that normalizing extracellular ATP levels to the percentage of dead cells or to the ATP plateau that can be achieved with a detergent can help distinguish between immunogenic and non-immunogenic forms of cell death.
Other DAMPs of ICD
Interferon (IFN)-1 is a typical DAMP molecule. Type I IFN production is a result of cells undergoing ICD, which is triggered by the detection of endogenous RNA and DNA.53–55 The immunogenicity of ICD is significantly reduced through the enzymatic degradation of extracellular nucleic acids, in line with this idea.43,56 The former is recognized by the endosomal TLR3,5,57 whereas the latter binds with cytosolic cyclic GMP-AMP synthase (cGAS), which subsequently initiates the cGAS-STING signaling pathway.58–60 The importance of cGAS-STING signaling in the immunogenicity of anthracyclines and radiation therapy has been demonstrated through studies on mice. Defects in key components of the cGAS-STING signaling pathway or cotreatment with IFNAR1-blocking antibodies have been shown to decrease therapeutic efficacy.61,62 The broad immunostimulatory effects of IFN-1 are due to its ability to interact with immune cells expressing IFNAR1.63 It has been reported that IFN-I can boost the cytotoxic capabilities of CD8+ T cells and NK cells,64 as well as facilitate cross-priming through DCs.65,66 In addition, macrophages can be stimulated to secrete pro-inflammatory mediators by type I IFN, while also inhibiting the immunosuppressive functions of regulatory T cells expressing CD4+CD25+FOXP3+.67 Malignant cells have been found to express a range of IFN-stimulated genes (ISGs) upon activation by IFN-I. This includes the expression of C-X-C motif chemokine ligand 10 (CXCL10), which acts as a chemoattractant for T cells.5
When cells undergo ICD they release various biomolecules that can trigger a pro-inflammatory response when they interact with DCs. One such biomolecule is heat shock protein, which can bind to CD91 and promote the maturation of DCs and uptake of dying cells, as previously discussed in the research on CALR.68 ANXA1 is a regulatory protein that plays a crucial role in maintaining homeostasis. It facilitates the uptake of tumor antigens by the TME when it is released by dying cells. This function is essential for the effective response to anthracycline-based chemotherapy in vivo, as malignant cells lacking ANXA1 exhibit limited sensitivity to this treatment.69 In a recent study, histones were identified as DAMPs in PDAC that are targeted by diaciclib.70 The supernatant of dinaciclib-treated KPC cell culture directly induced an increase in the expression of CD80 or CD86 on the DC cell surface, and this effect could be blocked by histone-neutralizing antibodies rather than HMGB1 antibodies.
Inducing ICD in PDAC
ICD can be induced in various manners, mainly including three categories in PDAC: 1) therapeutic drugs: including chemotherapeutics, targeted drugs, and immunomodulatory drugs; 2) biological molecules: oncolytic viruses, oncolytic peptides,71 and some biological immune regulatory factors; 3) physical therapy: stereotactic body radiotherapy (SBRT)72 and photodynamic therapy (PDT) (Table 1).73 Most of these inducers have shown profound effects on the tumor immune microenvironment in preclinical PDAC tumor models and have been confirmed to have a killing effect on tumors in various ways. There has been a growing trend in recent years towards the use of combination therapy regimens, which involve not only the combination of different ICD inducers, but also the use of ICD inducers in conjunction with other medications.74,75 The development of nanocarrier technology has enabled the use of multidrug combinations and precise pharmacokinetic control. In fact, many of the combination therapies mentioned below have used nanocarrier platforms. Combination strategies often offer more promising prospects compared to monotherapy. This leads to the following insight: in the immuno-incompetent microenvironment of pancreatic cancer, coordinated activation of multiple adaptive immune nodes and the accumulation of potential immune driving forces may be required to ultimately surpass the critical threshold to reach the trigger point (Fig. 2).
Table 1Emerging ICD inducers and their combination in PDAC
| Agent | Mechanism | Ref. |
|---|
| Silicasome-carried platinum or OXA | Increase the release of CALR and HMGB1 | 77 |
| Irinotecan + PDL1 | Neutralize acidic pH values, trigger autophagy, enhance the release perforin and granzyme B | 78 |
| Irinotecan + 3M-052 | Mediating dendritic cell activation by activate toll-like receptor (TLR) 7/8 | 79 |
| Diaciclib | Inhabit CDK1/2/5,release the histone H3/4 as DAMPs | 70 |
| ACUPA/TPP-I3A | Induce the release of DAMPs, upregulate CD80 and CD86 expression on DCs | 90 |
| OV OBP-702 | Enhance secretion of extracellular ATP and HMGB1 by inducing p53-mediated apoptosis and autophagy | 80 |
| Oncolytic peptide LTX-315 | Inhibit PD-L1 expression and enhanced lymphocyte infiltration | 81 |
| TT856-1313 | Promote the lymph node–like structures close contact with pancreatic tumors, enhance CD4 T cells infiltration | 82 |
| oHSV + CD40L | Increase the infiltration of DC and CD8 T cells and decrease the infiltration of Treg cells | 83 |
| Radiotherapy/SBRT | Local release of cytokines and danger signals by dying cells, alter cytokine secretion by surviving radioresistant cells | 84,86 |
| SBRT + R848 | Activate Toll-like receptor 7/8, increase tumor antigen-specific CD8+ T cells, decrease regulatory T cells, and enhance antigen-presenting cells maturation, as well as increase interferon gamma, granzyme B, and CCL5 | 85 |
| SBRT + anti-CD40 | Promote DC maturation by activating CD40, augment T cell priming, induce memory CD8 T cell responses | 72 |
| PTT + nanoplatform IMQ@IONs/ICG | Thermal ablation of the tumor, induce infiltration of CD8 T cells | 73,87 |
| IRE | Direct killing effect by IRE, promotes M1 macrophage polarization | 88 |
| IRE + anti-PD1 | Direct killing effect by IRE, activates dendritic cells, and alleviates stroma-induced immunosuppression without depleting tumor-restraining collagen | 89 |
Therapeutic drugs
The current first-line chemotherapy regimen for PDAC involves a quadruple regimen called mFOLFIRINOX, which has shown significant extension of survival in clinical trials. Oxaliplatin has been identified as a classic ICD inducer.76 Using mesoporous silica nanoparticles as a platform to carry oxaliplatin plus an indoleamine 2,3-dioxygenase (IDO) inhibitor indoleamine 2,3-dioxygenase (IND), oxaliplatin can target dead tumor cell corpses, as well as simultaneously inhibit the IDO pathway to facilitate ICD, with smaller doses and less bone marrow toxicity.77 The same strategy is also used for irinotecan (IRIN)-encapsulation, in which silicon can deliver IRIN to the lysosomes of cancer cells, neutralize their acidic pH values, and then trigger autophagy inhibition and ER stress response, ultimately leading to “type II” ICD and excessive expression of programmed death-ligand 1 (PD-L1), while enhancing the expression of PD-L1 and antagonizing the response of PD-1.78 The team then added 3M-052, a TLR7/8 agonist, into the IRIN silicon capsule, which further strengthened the immunogenic cell death response induced by IRIN by mediating DC activation, reducing adverse pharmacokinetics and off-target systemic inflammatory effects.79
In addition to exploring the potential of traditional chemotherapy drugs for inducing ICD, some small molecule drugs that can potentially lead to ICD have also been screened and shown to have strong multiple immune effects. Diaciclib, a CDK1/2/5 inhibitor, not only overwhelms adaptive immune resistance mediated by IFN in PDAC, but also induces ICD through histone release.70 Ingenol-3-mebutate (I3A), the main active ingredient in the sap of the Euphorbia plant, can be used as an alternative immune adjuvant to upregulate CD80 and CD86 expression on DCs. Recently, a small-sized sequential receptor nanocarrier has further developed the immunogenic potential of I3A, inducing ICD and reshaping the microenvironment in PDAC.
Biological molecules
Oncolytic viruses/peptides are a promising tumor immunotherapy that uses the concept of “tumor vaccines” to destroy cancer cells. These agents trigger an antitumor immune response by releasing high-quality vaccine-like neoantigens, ultimately leading to tumor-targeted cell cytotoxic T lymphocyte-dependent immune attack. Oncolytic viruses and peptides that have been tested in multiple PDAC models include OBP-702, a p53-expressing telomerase-specific oncolytic adenovirus that significantly enhances ICD through p53-mediated cell apoptosis and autophagy;80 LTX-315, an oncolytic peptide derived from bovine lactoferrin that induces antitumor immunity by targeting the ATP11B-PD-L1 axis;81 and the use of attenuated Listeria monocytogenes to selectively deliver immunogenic tetanus toxin protein (TT856-1313).82 Insights into tumor immunity have also led to the development of other immune-positive biomolecules aimed at enhancing immune responses by increasing adjuvanticity rather than immunogenicity. For example, adding CD40L to oHSV in oncolytic viruses can significantly promote of DC maturation, followed by more ICD markers, stronger antigen presentation, and more CD8+ T cell infiltration, ultimately leading to tumor regression.83
Physical therapy
Although PDAC is considered insensitive to radiotherapy, the development of new radiotherapy techniques is challenging this notion. In theory, the therapeutic effect of radiotherapy is to kill cancer cells that are irradiated, as well as reshape the TME, ultimately initiating an immune response targeting the tumor.84 However, in the immunologically “cold” TME of PDAC, simple radiotherapy alone does not seem to be enough to overcome this barrier. To further enhance immune reactivity, the use of TLR 7/8 ligand R848hu85 or anti-CD4072 is employed to allow immunogenic cell death during SBRT, with the key point being the exogenous provision of additional “danger signals” that initiate DAMPs and downstream ICD. A recent study analyzed tumor specimens from patients receiving SBRT using mIHC, RNA-seq, and TCR-seq, further promoting the clinical efficacy of SBRT combined with immunotherapy in PDAC.86
Photothermal therapy (PTT) is a medical treatment that uses thermal energy to generate heat in targeted tissues or cells. Absorption of photon energy produces significant thermal damage in target cells, especially tumor cells that are sensitive to heat-induced damage, followed by IRS and immunogenic cell death. PTT can supply enough antigenicity, but additional adjuvants can still be added to enhance its immunogenic effect in PDAC.73,87 In addition, the oxygen consumption and microvascular damage driven by PDT will further exacerbate hypoxia and lactate formation. A recent study used a nanodrug delivery platform to reverse hypoxia and downstream lactate metabolism by carrying other precursor drugs, eliminating this side effect. Another non-thermal ablation technique that can induce ICD, irreversible electroporation (IRE), differs from PTT in that it not only kills pancreatic cancer cells to provide immunogenicity but also promotes M1 macrophage polarization88 or reprograms the matrix89 to provide adjuvanticity, making it more self-contained.
Conclusions
At present, a novel therapeutic strategy is needed for PDAC. ICD, which is known as a special form of RCD that can augment immunogenicity, is a promising entity for initiating an immune response. Here, we reviewed the basis of ICD, highlighting its adjuvanticity and utility. A large amount of research demonstrates the success of exogenous provision of DAMPs and a combined regimen targeting multiple immune checkpoints, allowing for “conditional” ICD. This can transform “cold” tumors into “hot” tumors and lead to tumor regression. Meanwhile, an increasing number of new adjuvant molecules for ICD are being discovered, which lays the foundation for exploring new druggable targets. Although there are still shortcomings in the current evaluation and testing models for ICD, new technologies such as RNA-seq and TCR-seq are emerging in trial therapies for ICD, which may bring new breakthroughs in evaluating ICD.
Abbreviations
- ANXA1:
annexin A1
- ATP:
adenosine triphosphate
- CALR:
calreticulin
- cGAS:
cyclic GMP-AMP synthase
- DAMPs:
damage-associated molecular patterns
- DCs:
dendritic cells
- ER:
endoplasmic reticulum
- ERS:
endoplasmic reticulum stress
- HMGB1:
high-mobility group box 1
- ICD:
immunogenic cell death
- IDO:
indoleamine 2,3-dioxygenase
- IFN:
interferon
- IL:
interleukin
- IRE:
irreversible electroporation.
- IRIN:
irinotecan
- NK cell:
natural killer cell
- NLR:
NOD-like receptor
- OXA:
oxaliplatin
- P2RX7:
purinergic receptor P2X7
- P2RY2:
purinergic P2Y receptor 2
- PDAC:
pancreatic ductal adenocarcinoma
- PD-L1:
programmed death-ligand 1
- PDT:
photodynamic therapy
- PRR:
pattern recognition receptor
- PTT:
photothermal therapy
- RAGE:
receptor for advanced glycation end products
- ROS:
reactive oxygen species
- SBRT:
stereotactic body radiotherapy
- STING:
signaling pathway and stimulator of interferon genes
- TAA:
tumor-associated antigen
- TLR:
toll-like receptor
- TME:
tumor microenvironment
- TNA:
tumor neoantigen
Declarations
Funding
The work was supported by a grant from the National Natural Science Foundation of China (Grant no. 81871974 to BS).
Conflict of interest
One of the authors, Prof. Bei Sun has been an associate editor of Cancer Screening and Prevention since March 2022. The authors have no other conflict of interests related to this publication.
Authors’ contributions
Contributed to study concept and design (CH and BS), acquisition of the data (CZ), drafting of the manuscript (CH, XLW and YL), critical revision of the manuscript (YL), and supervision (BS).