Immune microenvironment and immunotherapy in pancreatic cancer: a narrative review

Introduction

Pancreatic cancer is a malignancy of the digestive system, whose incidence is on the rise, while the prognosis of it is almost the worst of all types of solid tumors (1). In the United States, for example, pancreatic cancer is the third leading cause of cancer death in the country, and among all kinds of solid tumors, it has the lowest 5-year survival rate, with about 11% (2). Global cancer statistics in 2020 show that pancreatic cancer is the seventh leading cause of cancer death in both sexes, and the median survival of pancreatic cancer patients is less than 12 months (3). Although a variety of treatments, including surgery, radiation therapy, chemotherapy, and immunotherapy, have been applied to the treatment of pancreatic cancer, the effectiveness is still very limited, and the prognosis is still very poor. Surgical resection combined with adjuvant therapy is the only sensible option at present (4,5). However, due to the occlusion of pancreatic cancer, the difficulty of early detection, and its rapid progression, most patients have no chance for surgery when the diagnosis is confirmed (6). Because traditional therapies struggle to provide patients with better outcomes, many emerging options, including immunotherapy, are receiving increasing attention. Many immunotherapies have achieved gratifying results in the experimental phase, but in clinical applications, immunotherapy has not yet provided significant benefits to patients, and some side effects have also been reported (7). With the deepening of related research, the immunosuppressive microenvironment of pancreatic cancer has gradually been discovered, which is mostly why immunotherapy has little effect (8). Many scholars have conducted detailed studies on the composition of the immunosuppressive microenvironment of pancreatic cancer and its role in tumor progression. Their findings will set the foundation for future advances in immunotherapy. There has been much literature reviewing the tumor microenvironment (TME) of pancreatic cancer as a whole. However, few reviews that explain the relationship between the microenvironment and pancreatic cancer treatment from an immunological perspective. This paper not only describes the components of the microenvironment of pancreatic cancer and their mechanism of action from an immunological point of view but also describes the principle and progress of related immunotherapy, and a future perspective was elaborated in the end. Our goal is to enable researchers to understand the immune microenvironment of pancreatic cancer better, generate more ideas, and ultimately make more remarkable progress through this review. We present the following article in accordance with the Narrative Review reporting checklist (available at https://pcm.amegroups.com/article/view/10.21037/pcm-22-55/rc).

Methods

This is a narrative review. The PubMed database was searched using the keywords “Immune microenvironment”, “Pancreatic cancer”, “Tumor-associated macrophages”, “MDSCs”, “Tregs”, “Tregs and pancreatic cancer”, “Tregs and TME”, “Pancreatic stellate cells”, “Cancer-associated fibroblasts”, “PSCs and pancreatic cancer”, “and pancreatic cancer”, “Immunotherapy”, “Immune checkpoint inhibitors”, “PD-1/PD-L1”, “CTLA-4”, “Oncolytic virus”, “Tumor vaccines”, “Monoclonal antibody”, “Monoclonal antibody and pancreatic cancer”. The date of the literature was limited from January 1st, 1990 to July 1st, 2022. We reviewed and summarized the current literature about the immune microenvironment of pancreatic cancer and related immunotherapies (Table 1). We excluded articles not published in English.

Immune microenvironment of pancreatic cancer

As elaborated above, the immune microenvironment of pancreatic cancer is gradually entering the field of scholars. The specific immune microenvironment is composed of immune cells, including myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), dendritic cells (DCs), regulatory T-cells (Tregs), and cell matrix components related to tumor immunity, including pancreatic stellate cells (PSCs), cancer-associated fibroblasts (CAFs), extracellular matrix (ECM), I will explain their role in the development of pancreatic cancer below.

Immune cells

MDSCs

MDSCs are a group of cells derived from bone marrow hematopoietic stem cells (HSCs). In pathological conditions such as chronic inflammation and cancer, HSCs perform a large amount of bone marrow hematopoiesis (9). The same pathological conditions can lead to upregulation of the expression of multiple inflammatory factors in the body. Under the stimulation of inflammation-related signals, immature myeloid cells do not differentiate into monocytes and granulocytes as usual but deviate from the original differentiation path and differentiate into a group of cells with immature phenotype and morphology and weak phagocytic function. This is what we currently call MDSCs (10-12). As early as 2011, Condamine proposed a 2-phase model to describe the proliferation of MDSCs (13). Subsequently, in 2020, Karin supplemented and revised this model, proposing a 4-phase model (steps 1–4: myelopoiesis, mobilization in the blood, homing to the tumor site, retention at the tumor site), which is close to a perfect one (14). MDSCs are not present in normal pancreatic tissue. In the state of pancreatic intraepithelial neoplasia (PIN), a small number of MDSCs can be found in the tissues surrounding the pancreas lesion and in the spleen. As pancreatic lesions progress, when PIN progress to pancreatic cancer, the number of MDSCs in pancreatic tissues and spleen increases significantly (15), which suggests that MDSCs may not play a significant role in the relatively early stage of tumor immune microenvironment. Some in vitro experiments have found that MDSCs can inhibit specific anti-tumor T cell responses (16-18), and this effect has also been supported to some extent in in vivo experiments (19-21). Regardless of the animals’ age or the degree of tumor burden, there was a negative correlation between the number of MDSCs and CD8⁺ T cells (15). This negative correlation echoes the immunosuppressive effects of MDSCs.

With the continuous development of studies, multiple mechanisms of the immunosuppressive effects of MDSCs have also been reported. Currently, there are four main immunosuppressive mechanisms of MDSCs: (I) arginase (ARG). Although studies of arginine metabolism in the process of tumorigenesis and development were noted decades ago, attention has increased in just recent years. ARG has two main types, one (ARG1) is present in the cytoplasm, and the other (ARG2) is present in the mitochondria (22). The primary role of ARG is to break down L-arginine into L-ornithine and urea, which affects the tumor from two aspects. On the one hand, L-ornithine, the decomposition product of L-arginine, is an important substrate for the generation of polyamines, which are fully proven to be one of the essential factors in promoting tumor growth (23). On the other hand, L-arginine can promote the proliferation of T cells. Once it is depleted, it will reduce the expression of cyclins D3 and CDK4 in T cells (24), thus inhibiting the proliferation of T cells. (II) Inducible nitric oxide synthase (iNOS). iNOS is a member of the NOS family (25), and has been found to have a high expression level in MDSCs. Many cytokines such as vascular endothelial growth factor (VEGF), granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin (IL)-6, etc., can induce high expression of iNOS in the bone marrow (26-29). The main function of iNOS is to produce NO, thus inhibiting the function of T cells in many ways. On the one hand, when T cells in the mitotic phase are exposed to a relatively high concentration of NO, the JAK3 and STAT5 pathways within the cells are activated, which causes T cells to be stopped in the G0/G1 phase of the mitosis (30,31). On the other hand, NO can inhibit the body’s immunity against tumors by inhibiting the expression of major histocompatibility complex (MHC) II molecules (32) and promoting T cell apoptosis (33). (III) Reactive oxygen species (ROS). The production of ROS is also considered an important mechanism for MDSCs to suppress the immune response. Various substances, including tumor growth factor β (TGF-β) and IL-6, can induce MDSCs to produce ROS (34). Studies have reported that under in vitro experimental conditions, selective inhibition of ROS production in MDSCs isolated from tumor mice will lead to the disappearance of the immunosuppressive effect of MDSCs (35,36). (IV) Peroxynitrite. It has been found that high expression of peroxynitrite is a crucial feature of MDSCs in the immune microenvironment. This feature strongly correlates with the high expression of ARG and iNOS in MDSCs (37). The peroxynitrite on the surface of MDSCs directly contacts the surface of the T cells, causing the T cell receptor (TCR) and CD8 molecules to react with the nitro group. This process that hinders their binding to the antigen peptide, which subsequently leads to the result that the T cells lose their response to specific tumor antigens (38,39). In addition to the above four mechanisms, MDSCs secrete IL-10 under the influence of interferon γ (IFN-γ) to inhibit T cell function (40,41), and changes in the expression of substances such as programmed cell death 1 ligand 1 (PD-L1), L-selectin (42) are all mechanisms of the immunosuppressive effect of MDSCs.

TAMs

TAMs are important immune cells in tumor tissue, and these cells play a vital role in the genesis, development, metastasis, and immune escape of pancreatic cancer (43). The sources of TAMs are not the same as most macrophages in the common tissue. Traditionally, most macrophages come from yolk cyst cells, while TAMs are derived from monocytes in the bone marrow (44). Under the action of complex cytokine networks, TAMs can mainly differentiate into classically activated M1 macrophages and alternatively activated M2 macrophages (45,46). The process of macrophage differentiation is often referred to as polarization. In the traditional view, M1 and M2 macrophages are often seen as two separate populations, but some studies have found that they may transform into each other under certain conditions (47,48). In the immune microenvironment of tumors, M1 macrophages produce substances such as tumor necrosis factor α (TNF-α), IL-12, IL-23, reactive nitrogen intermediate (RNI), etc., which are manifested as promoting inflammation and anti-tumor effects (49,50). In contrast, M2 macrophages, activated by glucocorticoids, IL-4, and IL-13, secrete IL-10 and TGF-β, which promote the proliferation of Th2 cells, thereby exhibiting inhibition of inflammation and pro-tumor effects (49-52). Existing studies have shown that M2 are much more numerous and valuable in pancreatic ductal adenocarcinoma (PDAC) than M1 macrophages (53). Moreover, the number of M2 macrophages was negatively correlated with the prognosis and survival of patients and positively correlated with the metastasis and immune evasion of tumors (54). At present, there are many studies on TAMs, and their role in pancreatic cancer is gradually becoming clear (55-57). First, TAMs play an important role in pancreatic cancer. In the development of pancreatic cancer, some acinar cells with high plasticity in normal pancreatic tissue, which are stimulated by some inflammation will have the characteristics of ductal cells. This process is called acinar duct metaplasia (ADM). If this adverse stimulation persists, ADM will progress to PIN, which further progresses to PDAC. In the process of pancreatic lesion progression, mutations of the KRAS gene can significantly increase the rate of progression of lesions (53). Since TAMs are the earliest cells that play an immune role in pancreatic inflammation (58) compared to MDSCs (15), their infiltration can amplify inflammatory stimuli. Not only that, TAMs mainly consist of M2 macrophages that can promote mutations in the KRAS gene of pancreatic cells, and the mutated KRAS gene can also promote more macrophages to polarize to M2 (59). This creates a positive-feedback pathway that accelerates the progression of the tumor. IL-6 and IL-10 secreted by TAMs can activate the STAT3 cell pathway in pancreatic cells, improving their sensitivity to inflammatory stimuli (47). Recent studies have also shown that some growth factors, C-C motif chemokine ligand (CCL)5 and TNF-α, etc., can synergistically activate the necrosis factor κB (NF-κB) and phosphatidylinositol 3-kinase (PI3K) pathways within M2 cells and other cells to induce ADM (60,61). TAMS can also promote the combination of heparin and endothelial growth factor receptor (EGFR), which also plays a role in inducing ADM (47) (Figure 1). Second, TAMs are involved in constructing the immunosuppressive microenvironment of pancreatic cancer and helping tumors spread and immune escape. TAMs in the PDAC immune microenvironment can secrete substances such as IL-10, TGF-β, CCL17, CCL12, prostaglandin E2 (PGE2), ARG1, etc., which can downregulate CD8⁺ T cell activity (62-64). TAMs can upregulate signaling of immune checkpoints at tumor cell, including increased expression of cytotoxic T-lymphocyte associated protein 4 (CTLA-4) and PD-L1, blocking T cell immune recognition and losing response to tumor cells (64,65). TAMs are also involved in the invasion and progression of tumors, and an important mechanism is the bilateral promotion of VEGF and TAMs. Studies have shown that TAMs can be enriched in the hypoxic and no-vascular areas of tumors and upregulate the expression of VEGF in this region (66). VEGF can promote blood vessel formation and facilitate tumor spread. Further studies confirmed that VEGF could also recruit TAMs to infiltrate tumor tissue (67). The formation of this positive feedback pathway is exceptionally conducive to tumor invasion and spread and provides a possible theoretical basis for early hematogenous metastasis.

Figure 1 The role of M2 cells in the immune microenvironment of pancreatic cancer (by Figdraw). Monocytes from the bone marrow are polarized in different directions under the influence of Th1 and Th2 cells. Th1 cells secrete cytokines such as TNF-α and IL-23, which promote the polarization of monocytes to the M1 phenotype, while Th2 cells secrete substances such as IL-4 and IL-13 to promote its polarization to the M2 phenotype. M1 and M2 macrophages also activate Th cells and upregulate their function by secreting cytokines. Also, M2 can also chemotaxis more M2 cells’ infiltration through the CCL2/CCR2 axis. M1 mainly plays an anti-tumor effect by secreting anti-tumor cytokines, while M2 can play a pro-tumor effect in a variety of ways. In the immune microenvironment of pancreatic cancer, M2 macrophages account for the vast majority of TAMs, and they can play a pro-tumor effect in the following ways: (I) M2 cells promote KRAS gene mutations in pancreatic acinar cells, which in turn promote the polarization of monocytes to the M2 phenotype, thereby forming a positive feedback pathway. (II) M2 cells are usually enriched in non-vascular areas, and they can promote tumor angiogenesis by upregulating the expression of VEGF, which in turn recruits more M2 infiltration into the tumor. (III) M2 can secrete many immunosuppressive cytokines, such as TGF-β and IL-10, which can inhibit CD8⁺ T cell activation and promote Tregs activation. In addition, in M2 cells, there are complex signaling pathways. IL-6 and IL-10 can promote cell proliferation through the JAK-STAT3 pathway, while TNF-α and growth factor-like substances play the same role respectively through the NF-κB pathway and the PI3K-Akt pathway. CCL, C-C motif chemokine ligand; CCR2, C-C motif chemokine receptor 2; CXCL12, C-X-C motif chemokine ligand 12; CXCR4, C-X-C motif chemokine receptor 4; IL, interleukin; NF-κB, nuclear factor κB; PI3K, phosphatidylinositol 3-kinase; TAMs, tumor-associated macrophages; TGF-β, transforming growth factor β; TNF-α, tumor necrosis factor α; VEGF, vascular endothelial growth factor.

Tregs

In the TME of pancreatic cancer, infiltration of Tregs is an important feature. Since Tregs’ primary function is to regulate immune system function, its role in the tumor immune microenvironment is also considered to negatively regulate immune function (68). Several studies have confirmed Tregs’ role in tumors’ immunosuppressive microenvironment. For example, the increase of Tregs in cancer tissue and peripheral blood is directly related to poor prognosis (69). Some treatments targeting at Tregs which aim to weaken Tregs’ function have also been proven effective (70,71). Tregs are habitually divided into two groups. One is the natural Tregs (nTreg), which persistently express forkhead box P3 (FOXP3) at a high level and play an immunomodulatory role through direct contact with other cells (72). Another group is called induced Tregs (iTreg). They can be activated by T cell antigen recognition receptors and secrete cytokines includes mainly TGF-β, which act as an immunosuppressor. However, some scholars prefer the new naming method according to Tregs’ origin (73). Tregs derived from the thymus gland are called tTreg, while those from the periphery are called pTreg. This nomenclature has certain benefits, but its main problem is the lack of specific markers to distinguish them. At present, in addition to NR1 and Helios, which have been studied to a certain extent (74-76), other specific markers that can be used to distinguish the two are still limited. Tregs can also be divided into different phenotypes according to the expression of substances such as CTLA-4, FOXP3, CD69, programmed cell death 1 (PD-1), etc. For example, Yadav divided Tregs into the following three types: activated Treg for CD45RA⁻FOXP3⁺, resting Treg for CD45RA⁺FOXP3⁻, and non-Treg for CD45RA⁻FOXP3⁻ (77).

There are many studies on Treg’s involvement in the immune microenvironment, but the following two conclusions are clear. (I) Tregs participate in the immune microenvironment by secreting immunosuppressive cytokines. TGF-β and IL-10 are representatives (78,79). These cytokines can act directly on natural killer T (NKT) cells, reducing their activity (80). They can also act on DCs (81,82), then affect the proliferation of cytotoxic T lymphocyte (CTL) and effector T cells. (II) Tregs participate in the process of tumor immunity by upregulating the expression of immune checkpoint molecules such as CTLA-1 and PD-1/PD-L1. Upregulation of CTLA-4 expression is an important mechanism for Treg’s involvement in immune microenvironment. In normal tissues, once a potent combination of MHC and TCR occurs, the expression of CTLA-4 rises, inhibiting T cell activation, which is a potential protective mechanism to protect the body from severe immune damage (83). During the activation of normal T cells, the combination of CD28 and CD80/86, which provides a co-stimulating signal, is essential. CTLA-4 is homologous with CD28, it can also bind to CD80/86, but this kind of binding cannot provide a co-stimulation signal, however, it will hinder the binding of CD28 and CD80/86 through the mechanism of competition and then lead to the obstruction of T cell activation (84,85). The role of PD-1/PD-L1 is similar to that of CTLA-4. If the binding of MHC to TCR and the binding of PD-1 to PD-L1 occur simultaneously on the surface of T cells, the activity of such T cells will be inhibited, and the secretion of TNF-α and IFN-γ will also be reduced (86,87). Nevertheless PD-1/PD-L1 also differs from CTLA-4. CTLA-4 is mainly expressed intracellularly and will be transferred to the cell surface when T cells are stimulated (83). Therefore, the CTLA-4-mediated immunosuppressive response mainly occurs before the effect phase. While PD-1/PD-L1 is on the surface of activated T cells, so the immunosuppressive response it mediates occurs during the effector phase (88).

Of course, Tregs can interact with many components in the immune microenvironment, and the complete action network is still undiscovered. For example, it is reported that ectonucleotidases and CD39/73, a molecule that is rare in normal people but can be detected in patients with head and neck tumors (89), synergistically use exogenous adenosine triphosphate (ATP) to produce immunosuppressive adenosine, which may have a relation with the suppressive activity of Tregs (90). Shockingly, some new studies have found that Treg cells may have somehow inhibited tumorigenesis (75,91,92), which is more pronounced in some malignant tumors of the airway and digestive tract. Because such organs and tissues are often exposed to a mixture of air, secretions, and microbe for a long time, such an environment is often more likely to lead to inflammation, which is the most likely origin of many tumors. It is related to the DNA instability and pro-angiogenesis effect brought by inflammation to cells. Some report notes that the immune tolerance induced by Tregs in inflammation may potentially protect the body from tumor distress (93-96).

Other cells

In the complex immune microenvironment of pancreatic cancer, there are not only the above three immune cells but a variety of cells, including DCs, B cells, monocytes, and neutrophils, are also involved in the microenvironment. Jang’s research noted that DCs in the TME of pancreatic cancer patients could express indoleamine 2,3-dioxygenase (IDO), calming the intense immune response of T cells down (97). γδT cells can inhibit the activation of anti-tumor αβT cells through PD-1/PD-L1 mechanisms that are similar to Tregs (98). The immune microenvironment of pancreatic cancer is a complex whole in which immune cells interact to form a network, and the complex connections have yet to be explored.

Cell matrix components

PSC and CAF

PSCs play a non-negligible role in the progression of pancreatic cancer, which not only promotes pancreatic cancer to infiltrate surrounding areas and metastasize to distant places but also participates in the composition of the immune microenvironment (99) (Figure 2). PSCs can interact with immune cells, secrete cytokines, and convert to CAFs (100). In normal pancreatic tissue, PSCs are inactive, producing many ECM components or degrading them by secreting enzymes (101). Therefore, PSCs can maintain the ECM in homeostasis in normal pancreatic tissue. However, once PSCs are stimulated by inflammation, they are activated, and these PSCs are more inclined to produce ECM components than to degrade them. When PSCs are activated, the lipid droplets full of vitamin A in their cells disappear, and PSCs express a considerable amount of alpha smooth muscle actin (α-SMA) (102). If this inflammatory stimulus is chronic and persistent, then PSCs will be continually affected by activation signals and cannot accept the apoptosis signal, which is why the apoptosis process of PSCs is not occurring. Continuously activated PSCs secrete large amounts of ECM components, which are mainly fibrous components (103). It is thought to be the reason why PDAC tissue is highly fibrous. Other studies have pointed out that PSCs not only accept stimulus signals from other cells or molecules, but once the activation signal appears, PSCs will promote self-activation and proliferation through autocrine. Validated autocrine components include IL-1, IL-6, IL-8, TNF-α, TGF-β, etc. (104,105). In addition, the relationship between PSCs and some immune cells has been elucidated. Studies have shown that PSCs can prevent CD8⁺ T cells from migrating to tumor sites (106), which may be related to the contact effect of the fiber components with T cells. It has also been shown that PSCs can recruit MDSCs to infiltrate the tumor site and can, enhance the activity of MDSCs, and promote their differentiation (107). Some studies point out that PSCs play a positive role in helping MDSCs infiltrate the tumor site, and increase in the activity of MDSCs and the acceleration of MDSCs differentiation have also been confirmed to be related to PSCs (107-109).

Figure 2 The role of PSCs and CAFs in the immune microenvironment of pancreatic cancer (by Figdraw). Normally, PSCs play a role in maintaining ECM stability in pancreatic tissue, and they can not only produce ECM components but degrade them as well. Normal PSCs are activated under the stimulation of cytokines and reactive oxygen species produced by inflammation or tumors. Activated PSCs can amplify this kind of activation effect by autocrine TGF-β and IL-6. When stimulation of inflammation or tumor persists, intracellular vitamin A lipid drops of activated PSCs decrease, and the expression of α-SMA increases, followed by the completion of the transition to CAFs. CAFs express large amounts of matrix proteins, which is the reason why the pancreatic cancer microenvironment is highly fibrotic. Activated PSCs and CAFs can promote the formation of the immunosuppressive microenvironment through a variety of mechanisms. Activated PSCs can promote infiltration of MDSCs. Activated PSCs can inhibit the activity of CD8⁺ T cells, while CAFs can facilitate CD8⁺ T cell migration out of the tumor microenvironment through the CXCL12/CXCR4 pathway. CAFs can induce the conversion of CD4⁺ T cells to Tregs by expressing only MHC class II molecules without generating co-stimulatory signals. CAFs can also promote Treg infiltration from the periphery to the tumor site by secreting chemokines including CCL2. α-SMA, alpha-smooth muscle actin; CAFs, cancer-associated fibroblasts; CCL, C-C motif chemokine ligand; CXCL12, C-X-C motif chemokine ligand 12; CXCR4, C-X-C motif chemokine receptor 4; ECM, extracellular matrix; IL, interleukin; MDSCs, myeloid-derived suppressor cells; PSCs, pancreatic stellate cells; ROS, reactive oxygen species; TGF-β, transforming growth factor β; TME, tumor microenvironment; Tregs, regulatory T-cells.

CAFs also influence the immune microenvironment of pancreatic cancer (Figure 2). Currently known studies have identified its association with the fibrotic matrix component of pancreatic cancer and its resistance to chemotherapy drugs, including gemcitabine (110). CAFs are a class of cells that are not proliferative and do not belong to the category of endothelial cells or immune cells but can produce ECM components (111). In pancreatic cancer’s TME, the main source of CAFs is activated PSCs. Although CAFs are matrix components, they can still be involved in forming inhibitory immune microenvironments in various ways. First, CAFs can affect the chemotaxis of effector T cells through the CXCL12/CXCR4 axis, preventing them from infiltrating the tumor site (112). Second, CAFs can also mediate monocytes’ and Tregs’ infiltration by secreting chemokines including CCL5 (112). Moreover, monocytes are an essential source of MDSCs. Some CAFs can express MHC-II molecules but cannot express co-stimulating molecules, leading to the transformation from CD4⁺ T cells to Tregs (113). There is much more research on CAFs-mediated immunosuppressive microenvironments, and treatments that target CAFs have had some effect in animal models, which may be related to the increased activity of CTL after CAFs reduction (114).

Other components

Among the cellular matrix components of pancreatic cancer, there are many components that can mediate the formation of inhibitory immune microenvironments to varying degrees through many different mechanisms. For example, stromal proteins can physically block the contact between anti-tumor immune cells and tumor cells (115); hyaluronic acid can bind water molecules in large quantities to construct an environment of high fluid pressure which act as a barrier from substances such as chemotherapy drugs elsewhere (116). The epithelial to mesenchymal transformation (EMT) process, which is highly representative in pancreatic cancer TME, can also promote tumor proliferation and metastasis (117). In general, the cellular matrix composition of pancreatic cancer TME is very complex. It is still a long way from fully understanding it and applying its principles to clinical practice.

Progress in immunotherapy for pancreatic cancer

As mentioned earlier, traditional therapies still do not provide satisfactory results for patients, especially for metastatic lesions, and we lack effective treatments. This is the background to the rise of immunotherapy. Therapies like tumor vaccines, chimeric antigen receptor T-cell (CAR-T) immunotherapy, immune checkpoint inhibitors, monoclonal antibody therapies, oncolytic viruses, and therapies targeting the stromal components of TME cells are being increasingly studied, and these immunotherapies are also seen as potential means of conquering pancreatic cancer. In animal experiments, many immunotherapies have achieved gratifying results, and some drugs have entered phase II clinical trials. However, immunotherapy also has some problems, the most important of which are severe autoimmune responses and even patient death. Despite all this, it is believed that shortly, immunotherapy will receive more attention, the problem will be solved, and more drugs will be put into clinical application, which will undoubtedly benefit more patients.

Tumor vaccines

Tumor vaccines are an emerging therapy among the currently known immunotherapies, and its effects have been initially affirmed. We can use DNA or peptides as antigens to activate immune cells, traditionally known as the vaccine principle. We can also directly utilize activated DCs or complete tumor cells as components of vaccines. An ideal vaccine target should be a class of substances expressed only in tumor cells and not in normal cells. Unfortunately, no such proteins have been found in pancreatic cancer tissues, so it is a more reasonable choice to develop a vaccine by taking advantage of differences in the expression levels of certain substances in tumor cells and normal cells. Research on carcinoembryonic antigen (CEA), human epidermal growth factor receptor 2 (HER2), mucin 1 (MUC1), P53, etc., as vaccine targets has been reported (118-121). Traditional vaccines tend to use autologous cells treated with radiation. However, in the treatment of pancreatic cancer, not many patients have the chance to undergo surgery, and even if a small number of people are able to do so, they will eventually be unable to gain enough autologous tumor cells for various reasons (122). Therefore, using allogeneic tumor cells in vitro culture as a stable source of tumor cells has become the primary method of preparing pancreatic cancer tumor vaccines (123). Today, the pancreatic cancer tumor vaccine that has been fully studied is mainly the GVAX vaccine. It uses genetic engineering methods to reconstruct pancreatic cancer cells and make them highly express GM-CSF, thus activating the immune system. There is a study showing that GM-CSF rises at the tumor site a few days after GVAX vaccination, which increases the body’s immune response (124). A phase-1 clinical trial showed that 3 of the 8 patients receiving two doses of the GVAX vaccine achieved a disease-free survival effect of 15 years (125). In another phase-2 clinical trial, patients receiving the GVAX vaccine also showed promising clinical efficacy (126). However, some phase-2 clinical trials show that GVAX combined with ipilimumab did not provide survival benefits for patients with pancreatic cancer compared with combination chemotherapy (127). These contradictory results may be related to how the GVAX vaccine is used, including dosage, frequency, and other therapies in combination.

Another emerging vaccine is the Algenpantucel-L vaccine. It consists of two different radiation-treated human pancreatic cancer cell lines that mainly express α-1,3-galactosyltransferase, which can catalyze the formation of α-galactosylated epitopes on cell membranes. A phase-2 clinical trial showed an 81 percent improvement in 1-year disease-free survival for patients using the vaccine and a 96 percent improvement in 1-year overall survival (128). Another phase-2 clinical trial showed that this vaccine is more effective in combination with neoadjuvant chemotherapy (129). In addition, tumor vaccines based on telomerase, mutated KRAS gene, and heat shock protein (HSP) peptide complexes are being studied.

Immune checkpoint inhibitors

Immune checkpoints are defined as programmed death receptors and their ligands. Tumor cells inhibit immune cell activity by expressing immune checkpoint molecules, thereby influencing the host’s immune response to the tumor. At present, the immune checkpoints that have been fully studied are mainly CTLA-4 and PD-1/PD-L1. The antitumor effects of CTLA-4 inhibitors can be traced back to 1996 when a study reported that CTLA-4 antibodies could cause mice to exhibit rejection of transplanted tumors (130). Subsequently, several clinical trials have been carried out (131-133). Among those, one phase-3 clinical trial demonstrated that using ipilimumab, a CTLA-4 antibody, is beneficial for patients with melanoma (134). Although many scholars have noted the anti-tumor effects of CTLA-4 inhibitors, and there are many related drugs in development, the use of CTLA-4 still has some problems and controversies. Studies have shown that the using ipilimumab and nivolumab can lead to a probability of adverse therapeutic reaction, mainly autoimmune diseases. If the two are combined, this probability rises to 60% (135). However, some scholars believe that we can avoid similar adverse reactions by improving drugs. In other words, severe adverse reactions may not be due to their inhibition of CTLA-4. The study reported that among the many CTLA-4 antibodies, the drug that was the most effective produced the least number of antibodies targeting DNA (136). Another study confirmed this from a pathological point of view (137). Du and his colleagues point out that CTLA-4 inhibitors may not appear to be a sufficient condition for tumor treatment, which is subversive in the traditional view of CTLA-4. It was found that CTLA-4 inhibitors did not block that many CTLA-4 and CD80/86 bindings at the cell surface, and even though CTLA-4 antibodies successfully blocked the binding of the two, CTLA-4-mediated physiological responses were observed, while the body’s immunity to tumors was enhanced (138). This finding may suggest the existence of another mechanism related to tumor immunity related to CTLA-4. CTLA-4 inhibitors have a more limited range of use than PD-1/PD-L1 inhibitors. However, scholars are still keen to study the antitumor effects of CTLA-4 inhibitors because many studies have shown that the use of CTLA-4 inhibitors can improve the efficacy of PD-1 inhibitors (139-141).

PD-1/PD-L1 is another target of the immune checkpoint inhibitors. In treating non-small cell lung cancer (NSCLC), U.S. Food and Drug Administration (FDA) has recommended anti-PD-L1 therapy for patients with immunohistochemical PD-L1 positive (142,143). In the treatment of colorectal cancer, the treatment of PD-L1 antibodies combined with PD-L1 secretion inhibitors also yielded comparable results (144). At present, the combination therapy of PD-1/PD-L1 inhibitors with other anti-tumor drugs has entered the field of scholars, which mainly includes the combination of PD-1/PD-L1 inhibitors with CTLA-4 inhibitors, chemotherapy drugs, and targeted drugs. A phase-3 clinical trial of a combination of PD-1 and CTLA-4 antibodies showed significant benefits in patients with melanoma (145), but this also meant more substantial side effects (146). In patients with NSCLC, regimens of PD-1 antibodies in combination with platinum-based chemotherapy are better than those of single agents (147). Several trials in patients with renal cell cancer have also shown that PD-1 antibodies combined with axitinib, a VEGF receptor (VEGFR) inhibitor, and other chemotherapy drugs are more effective than monotherapy (148,149).

Adoptive immunotherapy (CAR-T therapy)

CAR-T therapy is an immunotherapy for the treatment of tumors. T cells are activated and can specifically identify tumor cells by cell designing and engineering, to achieve the effect of killing tumor cells and treating tumors. CAR-T therapy has shown promising efficacy in some hematologic tumors, and a representative example is its use of it in acute B-lymphoblastic leukemia (150). Regrettably, however, CAR-T therapy has not been able to show satisfactory results in treating solid tumors. One reason for this is that many tumors, including pancreatic cancer, are in an immunosuppressive microenvironment, which limits the function of T cells. Therefore, some scholars are studying the combined effects of CAR-T therapy with TME targeting therapies. A more important reason is that solid tumors lack specific targets for designing and engineering T cells. In pancreatic cancer, markers such as mesothelin (MSLN) and MUC1 tend to be overexpressed, and the elevation is inversely correlated with the prognosis of patients (151), providing us with potential targets. Nevertheless, many targets are tumor-associated antigens (TAA) rather than tumor-specific antigens (TSA) (120), which means they exist in normal cells, and this will lead to the “on-target, off-tumor” effect. The so-called “on-target, off-tumor” effect is that CAR-T cells recognize normal cells as tumor cells and attack them, which leads to a variety of autoimmune reactions, including colitis. It even leads to deaths of patients (152,153). Although CAR-T therapy for solid tumors is still not perfect, it is fortunate that in animal experiments, its efficacy of it for pancreatic cancer has been confirmed (154-156), and clinical trial results have also been reported (157). Among the many targets reported at present, CAR-T therapy for MSLN has achieved the best efficacy, while its side effects, mainly consisting of autoimmune reactions, are relatively few (158). The results are satisfying. All pancreatic cancer cells express MSLN, while normal pancreatic cells hardly express this substance, which provides a theoretical basis for avoiding the “on-target, off-tumor” effect (159). There have been some studies reporting the role of MSLN in many types of solid tumors (160-162). However, to fully reveal its principles for tumorigenesis and progression and put them into clinical practice, we still have a long way to go. Studies have shown that in PDAC, specific T-cell responses against MSLN have been observed (163,164). Zheng’s research suggests that MSLN may regulate the growth and apoptosis of tumor cells through p53-mediated pathways (165). Another study has found that the interaction between MSLN and CA125 may be related to the adhesion of tumor cells (166). Related clinical trials are also underway. A small Phase-1 clinical trial shows that CAR-T therapy to MSLN in patients with recurrent and(or) metastatic pancreatic cancer leads to prolonged survival (167).

Immunotherapy targeting components of pancreatic cancer TME

As described previously, pancreatic cancer has a complex immune microenvironment that plays a crucial role in the progression of pancreatic cancer. Therefore, the treatment aimed at the immune microenvironment of pancreatic cancer has been in people’s field of vision. The immune microenvironment of pancreatic cancer consists of cells and matrix components, and accordingly, the treatment begins in these two aspects. (I) Treatments targeted at immune cells. The main targets are TAMs and MDSCs. Some studies have shown that CD40 activation is key to macrophage transition to an anti-tumor phenotype (168), which provides us with a potential therapeutic basis. In addition, some therapies that target cytokines and chemokines are also progressing. The CCL2/C-C motif chemokine receptor 2 (CCR2) axis is the main driving force for TAM chemotaxis and infiltration into the tumor (169). A phase-1 clinical trial showed that CCR2 inhibitors combined with FOLFIRINOX chemotherapy yielded clinical benefits in at least 50 percent of patients (170). The differentiation of TAMs and MDSCs is modulated by colony stimulating factor (CSF)-1/CSF-1R, while the prospect of CSF-1R blockers has been shown. The effect of CSF-1R blockers in the treatment of tumors in a separate combination with CD40 agonists and immune checkpoint inhibitors has been reported (171,172). Activation of the CXCL2/CXCR2 axis can facilitate MDSCs chemotactic to tumors. Accordingly, trials blocking both CXCR-2 and CSF-1R have been published, which shows that this therapy can achieve anti-tumor purposes by improving immune cell infiltration (173-175). (II) Treatments targeted the cell matrix components. One approach is to deplete the tumor matrix, and a representative drug is the hyaluronidase. A study shows that IDO inhibitors combined with hyaluronidase significantly increased the infiltration of antitumor T cells (176). However, other studies have shown that treatment for matrix depletion may lead to pancreatic cancer being more likely to metastasize, possibly because of the loss of the restrictive effect of the dense matrix around tumor cells (177). Another approach is stromal modulation therapy targeting vitamin D and vitamin D receptors. Patients with vitamin D deficiency have been reported to have a relatively poor prognosis (178). Vitamin D receptors are expressed on the surface of PSCs, meaning that vitamin D-associated metabolism is part of matrix metabolism in the TME. Studies have shown that activation of vitamin D receptors can cause a decrease in fibrosis levels in the TME, leading to an improved response of tumors to various chemotherapy drugs, including gemcitabine (179). In addition to vitamin D and vitamin D receptors, fibroblast activated proteins (FAPs) are also associated with highly fibrotic matrix in pancreatic cancer (180), and therapies targeting FAPs are also being studied (181).

Monoclonal antibody therapy

Monoclonal antibody therapy is well-known immunotherapy which can specifically bind to some cell surface receptors or cytokines, block the conduction of signals, weaken their physiological effects, and finally achieve the effect of inhibiting tumor growth and even eradicating tumors. Currently, cetuximab is a common monoclonal antibody that has a very high affinity with the epidermal growth factor receptor (EGFR) but has no physiological activity of EGFR ligands. It can competitively inhibit the activation of EGFR, thereby blocking intracellular downstream signaling pathways and inhibiting tumor cell proliferation. Some clinical trials about the possibility of using cetuximab in pancreatic cancer have shown results. In a phase-2 clinical trial, the combined use of cetuximab and gemcitabine was confirmed to be possible in patients with advanced pancreatic cancer (182). Another retrospective study confirmed that conventional chemotherapy combined with cetuximab and bevacizumab had better efficacy than chemotherapy alone (183). However, the use of monoclonal antibody therapy in pancreatic cancer is controversial. Clinical trials have also shown that adding cetuximab to gemcitabine and cisplatin does not improve survival in patients (184). Besides cetuximab, a number of monoclonal antibodies have also been put into clinical trials, such as FG-3019, which targets connective tissue growth factor (CTGF), and H2Mab-19, which targets HER2.

Oncolytic virus therapy

Oncolytic virus therapy is emerging immunotherapy that achieves anti-tumor effects by attacking tumor cells with particular viruses. Part of the virus’s anti-tumor effect comes from virus-mediated lysis of tumor cells. However, more importantly, the lysis of tumor cells can expose some tumor antigens that are difficult to identify using traditional methods, thus achieving the effect of activating patients’ anti-tumor immunity. At present, some viruses have been studied as ideal sources of oncolytic viruses, including adenoviruses, measles viruses, and herpes simplex viruses (HSV), etc.. On adenovirus, there is still not so much research. Moreover, very few of them can be put into clinical trials and applications. An adenovirus with a defective E1B-55kD gene can proliferate in cells with deficient in the p53 gene to replicate only in pancreatic cancer cells (185). In phase-1 clinical trials, the virus showed excellent safety, with an elevated immune response in patients proved by increased T cell responses and elevated antibody levels (186). However, the virus did not show good clinical efficacy in subsequent clinical trials. Another reported adenovirus-derived oncolytic virus is adenovirus with two defective genes of E1ACR and E1B19K, whose main role is inhibiting cell autophagy. Studies have pointed out that the combined use of gemcitabine and this kind of virus has shown gratifying results in animal models (187), but reliable clinical research data are still lacking. HSV is also considered to be a possible source of oncolytic viruses. An oncolytic virus named FusOn-H2 can replicate in cells where the Ras signaling pathway is activated, making it possible to kill tumor cells accurately (188). Another oncolytic virus, L1BR1, comes from the HSV-2 virus, while the US3 gene is not activatable. The US3 gene can inhibit cell apoptosis. In a small range study, patients with pancreatic cancer benefited from the combination of chemotherapy and the L1BR1 virus (189). Measles virus is also an ideal resource for an oncolytic virus, which enters cells through the CD46 molecule, a cell surface molecule that many tumor cells, including pancreatic cancer cells, highly express. Currently, two kinds of measles virus with MV-NIS gene and prodrug-convertase gene have shown good effect in animal experiments (190,191), but few reports of clinical trials are published. Some representative oncolytic viruses are listed below (Table 2).

Perspective

At present, although the focus of many studies is on the immune microenvironment and immunotherapy of pancreatic cancer, and related research has also drawn considerable attention and make much progress, we still know very little about the microenvironment of pancreatic cancer, with the unsatisfactory progress and clinical effect of immunotherapy. The immune microenvironment of pancreatic cancer is an extremely complex network, and every tiny impact on any point, such as immune cells, non-immune cells and matrix components, can affect the occurrence, progression and treatment effect of pancreatic cancer. In this complex network, the main influence on the immune microenvironment is immune cells and related cytokines, which interact with one another to form an immunosuppressive microenvironment thus promote the occurrence and development of pancreatic cancer. But the immunotherapy for pancreatic cancer is also in a huge gap between ideal and reality. Some immunotherapies, such as oncolytic virus therapy, CAR-T therapy and immune checkpoint inhibitor therapy, can specifically kill tumor cells, which is theoretically highly possible to stop tumor progression, but many clinical trial results prove that we are naive and ignorant to a certain extent. The treatment of pancreatic cancer will remain a huge challenge for some time. The immune microenvironment of pancreatic cancer and related immunotherapies are still frontier in the field. For solid tumors, especially pancreatic cancer, the therapeutic effect of surgery is very limited. Therefore, fully understanding the immune microenvironment of pancreatic cancer, using patients’ own anti-tumor immune response and various types of immunotherapy principles to specifically kill tumor cells will bring a glimmer of hope to overcome pancreatic tumors. It is hoped that with the development of science and technology, pancreatic cancer will be less and less harmful to humans until we can completely overcome this problem.

Conclusions

Pancreatic cancer is a malignant tumor of the digestive system with a very poor prognosis, a high case fatality rate, and a low 5-year survival rate, and R0 resection combined with chemotherapy as a standardized therapy still cannot achieve satisfactory results currently. With the study of the immune microenvironment of pancreatic cancer, it has been found that the inhibitory immune microenvironment of pancreatic cancer is related to the immune incapacitation of patients and related-drug resistance, and it is also an important reason why pancreatic cancer is so difficult to treat. In this paper, we review research in related fields and systematically describe the various components and their interactions in the immune microenvironment of pancreatic cancer. It has become a wide consensus that the stimulation of pancreatic tissue inflammation to pancreatic acinar cells is one of the important reasons for the occurrence of pancreatic cancer. In fact, from the occurrence of inflammation, the formation process of pancreatic cancer immune microenvironment has begun. The first cells to initiate the immune response are TAMs, which can differentiate in 2 directions, but in the pancreatic cancer TME, most TAMs will differentiate into M2 macrophages that exhibit pro-tumor effects. M2 can participate in the formation of the immune microenvironment by promoting gene mutations in acinar cells and secreting immunosuppressive cytokines. MDSCs respond relatively slowly to tumors. They originate in the bone marrow, circulate through the bloodstream and proliferate in the microenvironment. Such cells can either produce immunosuppressive effects mediated by a variety of substances (including ARG, iNOS, ROS, etc.) or induce T cells to differentiate into Tregs by direct contact with effector T cells, hindering their cytotoxic effect on tumors. Some therapies targeting TAMs and MDSCs are being studied, such as CD40 monoclonal antibodies, CCR2 inhibitors, and CSF-1R blockers. Tregs are also an important component of the immunosuppressive microenvironment. The role of Tregs in secreting inhibitory cytokines has been well understood, and another important immunosuppressive mechanism is the upregulation of immune checkpoint molecular expression, for which immune checkpoint inhibitor therapy has made some breakthroughs. In the immune microenvironment, there are not only immune cells but also cell matrix components, mainly composed of PSCs, CAFs, and ECMs. PSCs are activated by inflammation or tumor stimulation, highly express the ECM component, and convert to CAFs. PSCs and CAFs are the leading causes of high fibrosis of pancreatic cancer tissue, which greatly limits the role of exogenous therapeutic drugs and peripherally derived immune cells. Therapies for matrix components have also been reported, mainly matrix depletion therapy with hyaluronidase as the core and matrix modulation therapy with vitamin D receptor agonists as the core. The efficacy and feasibility of the latter are better than the former. Since surgery combined with chemotherapy is not effective enough in the treatment of pancreatic cancer, immunotherapy is considered a promising treatment. In addition to some of the therapies mentioned above that target the components of the immune microenvironment of pancreatic cancer, there are also breakthroughs in immunotherapy (oncolytic virus therapy, CAR-T therapy, tumor vaccines, and monoclonal antibody therapy), and many clinical trials are underway. The breakthrough of immunotherapy has brought a glimmer of light to pancreatic cancer patients, and it is hoped that in the near future, this light will become a burst of shining sunshine, helping us overcome pancreatic cancer and help more patients regain their health. Although the narrative of this review is nearing its end, and its discussion of the immune microenvironment of pancreatic cancer and related immunotherapies is clear, it is undeniable that this review still has its limitations for various reasons. First, due to the single database used to search the literature (PubMed only), some of the relevant studies and literature have not been included in this review. Second, because the authors focused only on the immune microenvironment of pancreatic cancer rather than the TME of pancreatic cancer as a whole, the literature cited and the conclusions drawn are not so comprehensive. Third, although immunotherapy is the hope of pancreatic cancer treatment, certainly, that traditional therapies cannot be abandoned. The treatment of pancreatic cancer should be a combination of multiple therapies rather than a victory of monotherapy. Perhaps in the future, the progress of immunotherapy for pancreatic cancer will reach unprecedented heights, but there is still a great possibility that other therapies will be retained in clinical practice.

Acknowledgments

The authors thank Drs. Xianjun Yu and Si Shi from Fudan University for their participation in the writing of this article and especially their support in the creation of the iconography.

Funding: This study was jointly supported by the National Natural Science Foundation of China (No. U21A20374), Shanghai Municipal Science and Technology Major Project (No. 21JC1401500), Scientific Innovation Project of Shanghai Education Committee (No. 2019-01-07-00-07-E00057), Clinical Research Plan of Shanghai Hospital Development Center (No. SHDC2020CR1006A), and Xuhui District Artificial Intelligence Medical Hospital Cooperation Project (No. 2021-011).

Footnote

Reporting Checklist: The authors have completed the Narrative Review reporting checklist. Available at https://pcm.amegroups.com/article/view/10.21037/pcm-22-55/rc

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://pcm.amegroups.com/article/view/10.21037/pcm-22-55/coif) and report supports from the National Natural Science Foundation of China (No. U21A20374), Shanghai Municipal Science and Technology Major Project (No. 21JC1401500), Scientific Innovation Project of Shanghai Education Committee (No. 2019-01-07-00-07-E00057), Clinical Research Plan of Shanghai Hospital Development Center (No. SHDC2020CR1006A), and Xuhui District Artificial Intelligence Medical Hospital Cooperation Project (No. 2021-011). The authors have no other conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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