Targeting the tumor microenvironment for pancreatic ductal adenocarcinoma therapy

Introduction

Pancreatic cancer is one of the most dangerous cancers, and it has a very low overall 5-year survival rate (8%) that has shown little improvement (1). There are 10 types of major neoplasms of the pancreas, and invasive ductal adenocarcinoma is common (2). Pancreatic ductal adenocarcinoma (PDAC) cells infiltrate into the tissue to make the tumor firm and metastasize to other organs (3).

Solid tumor cancer cells exhibit self-sufficiency in growth signals, which results in unlimited cell proliferation, the ability to obtain nutrients, resistance to apoptosis, insensitivity to the growth inhibitory pathway, and the capacity to invade and metastasize (4). PDAC has these characteristics as well. Through microscopy, PDAC cells were observed to have a close relationship with nerves, lymphatic spaces and small veins, indicating that PDAC is very likely to become a metastatic carcinoma. The tumor microenvironment (TME) of PDAC is highly immunosuppressive and has been associated with cell secretion disorders and interactions with nerves (5-7). The majority of the PDAC tumor volume is filled by stroma/desmoplastic reaction, and the heterogeneous stroma consists of fibroblasts, myofibroblasts, immune cells, pancreatic stellate cells (PaSCs), extracellular matrix (ECM), and soluble proteins, such as cytokines, growth factors and blood vessels (8,9). The interaction of the tumor cells with factors in TME suppresses the immune reaction of the body to escape immunocyte apoptosis. Due to the complex tumor microenvironment and high interstitial fluid pressure (IFP), PDAC has chemoresistance and radioresistance, which creates major challenges for therapy (10). The interactions between components in TME and the tumor cells results in tumor development, metastasis and immune escape. In addition to poor therapy effects, there is no early detection test for PDAC, and many patients lack recognizable symptoms or signs of the disease. Therefore, patients are continuously considered to be at high-risk after being diagnosed with PDAC.

Fibroblasts

Desmoplasia is an important feature of the PDAC tumor microenvironment but can be an obstacle for therapeutic agents; fibroblasts are considered to be “murderers” that result in desmoplasia. Cancer cells expend a large amount of energy to recruit, proliferate and activate fibroblasts; then, as a reward, the activated fibroblasts deposit ECM and secrete numerous factors that affect tumor development (11) (Figure 1). An important origin of cancer-associated fibroblasts (CAFs) is from the Notch signaling pathway that activates PaSCs (12).

Figure 1 The relationship between PDAC cells, fibroblasts and the ECM. PDAC cells expend energy to recruit fibroblasts during its early development stage by secreting factors such as SHH, SDF-1, TGF-β1 and PDGF. Fibroblasts deposit into the ECM and promote tumor cell proliferation, invasion and metastasis by secreting PGDF and SDF-1. The dense ECM promotes PDAC development through stimulation of the integrin/FAK signaling pathway in PDAC cells and contact-mediated lymphocyte trapping. PDAC, pancreatic ductal adenocarcinoma; ECM, extracellular matrix.

Several types of fibroblasts are deposited in the ECM, which may explain the poor therapy results of targeting CAFs. Two major types of PDAC FAP⁺ fibroblasts exist: periglandular αSMA^high myofibroblastic CAFs (myCAFs), which likely restrain tumor growth; and diffusely distributed αSMA^low interleukin-6 (IL-6)-positive inflammatory CAFs, which promote tumor growth by secreting ECM proteins and cytokines such as IL-6 and IL-11 (13,14). Based on the fibroblast subtype, inhibiting IL-6R to reduce STAT3 activation slows tumor development (15). Although there are two types of CAFs and their functions seem to be opposed, CAFs were demonstrated to be a promoting tumor factor in the TME. The PDAC cell interactions with stromal fibroblasts increase hyaluronic acid production, causing an obvious increase in the migration of PDAC cells (16). New research has reported that the Wnt-nonproducing subtype, a kind of PDAC cell, required Wnt from CAFs (17).

CAFs stop CD8⁺ and CD4⁺ T cells, NK cells and Tregs to juxtatumoral compartments to exert normal physiological function (18-20). CAFs secrete CXCL12 to keep CXCR4⁺ T cells away from tumors and secrete CCL5, CCL2 and CCL17 to recruit monocytes and Tregs, which results in immunosuppression (21). Due to the presence of fibroblasts and other stellate cells, tumors have high intratumor IFP and matricellular tension (MCT) (22), both of which promote tumor progression, reduce vasculature to cut off therapeutic agents and induces tissue hypoxia (23). Subsequently, through autophagy-mediated degradation and a reduction in protein synthesis in the PaSCs, hypoxia reduces the expression of lumican, an inhibitor of tumor progression that is located in the TME (24).

During tumor metastasis, the fibroblasts, along with other cells, affect the microenvironment of the target organ. During the early stage of PDAC liver metastasis, metastasis-associated macrophages (MAMs), a kind of inflammatory monocyte, secrete granulin and activate resident hepatic stellate cells that turn into myofibroblasts, which secrete periostin to result in a fibrotic microenvironment that promotes metastatic tumor growth (25). This finding may explain why myofibroblasts appear when metastases only comprise 6–7 cells in the cell population within a metastatic lesion (26). Research has shown that PDAC cells that are treated with CAF-conditioned media have an increased risk of metastasis; the reason for this increase is the loss of metastasis suppressor 1 (27).

Since the elimination of desmoplasia has been indicated be harmful to patients, new research is more focused on ECM reprogramming to find new effective therapeutic agents. Malik et al. reported that CAFs remodel cell-derived extracellular matrices (CDMs) by altering the underlying substrate stiffness and inhibit tumor growth in an extracellular signal-regulated kinase 2 (EKR2)-dependent manner (28). CAFs treated with physiological stiffness (~1.5 kPa) generate CDMs similar to normal fibroblasts, and the biomechanical manipulation generated on physiological stiffness CDMs leads to a decrease in the nuclear translocation of pERK1/2 in KRAS-mutated pancreatic cells.

The immune system

Immune cells may affect the composition of pancreatic stroma to affect the progression of PDAC (29). Recent research reports that prominent overall leukocyte infiltration is associated with increased survival (30).

The TME and immune system have a close relationship, and a number of interactions between the TME and immune system affect tumor development (Figure 2) (31-36). The T cell immunocytes in particular can recognize the non-self and then remove the invaders. The process of normal cells turning into cancer cells is an example of the self to non-self progression, and T cells can kill the cancer cells. Once the effector T cell activation reaches a threshold, Tregs become activated and release TGF-β and IL-10 immunosuppressive cytokines to negatively regulate T cell function (37). To develop and proliferate, the cancer cells break this balance between the inhibitors and activation level though abrogating coactivatory signals and augmenting coinhibitory signals, which ultimately heightens anergy and exhaustion (38).

Figure 2 Interactions of the immune system, TME factors and pancreatic cancer cells. γδ T cells promote tumor development and block CD8+ and CD4+ T cell infiltration to kill tumor cells. The fibroblasts recruit Bregs by secreting CXCL13, and Bregs secret IL-35, which results in tumor progression. However, fibroblasts block stromal fibroblasts, which then block Treg accumulation and PDAC progression. Pancreatic cancer cells drive macrophages into the TME with CCL2, and macrophages drive tumor growth. Fusobacterium in the PDAC microenvironment were reported to have a relationship with PDAC development, but its mechanism is not clear. TME, tumor microenvironment; PDAC, pancreatic ductal adenocarcinoma.

In a healthy body, the action of T cells through major histocompatibility complex (MHC) engagement is regulated by stimulating signals and inhibitory signals, which are referred to as immune checkpoints. To breakdown and benefit from this regulation, tumor cells expand the coinhibitory signal and abolish the coactivatory signal (38).

Research has focused on cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1), two factors that function on immune checkpoint inhibitors. The CLTA-4 pathway is activated by tumor cells to downregulate the CD28 costimulatory receptor, an important ligand for transmitting secondary signals for T cell activation and increasing the threshold of T cell activation, resulting in immunosuppression (39). Regulated by FUBP1, IRF1, cytokines, etc., the cancer cells upregulate PD-1, which binds to PD-1 receptors on T cells to cause a loss of T cell function, cell anergy, or cell death (40-43). In addition to CTLA-4 and PD-1, new targets have been identified. Bruton tyrosine kinase (BTK), a key B-cell and macrophage kinase, affects B-cell and macrophage-mediated T cell suppression in pancreatic adenocarcinomas, inhibits T cell antitumor function and improves the PDAC response to chemotherapy (44). The inhibitor of receptor tyrosine kinase Axl can be established as a preclinical mechanistic target to improve the overall survival rate (45). Dickkopf-3 (DKK3) is a potential therapeutic target as well (46).

Interleukins (ILs) are secreted by many kinds of cells that participate in immune regulation, hematopoiesis and inflammation. IL-6 is one of IL family members produced by pancreatic satellite cells (PSCs) and PDAC cells and is associated with tumor growth, survival, metastasis, chemoresistance, etc. (47,48). IL-6 can activate MDSCs via the JAK/STAT3 pathway, and a high MDSC level indicates a high risk of a poor patient outcome (49). The STAT3 pathway and NRF2 pathway, which are induced by STAT3, promote epithelial-mesenchymal transition (EMT) in PDAC (39,50). The IL-6/JAK/STAT3/NREF2 pathway benefits tumor development and thus targeting this pathway may inhibit tumor growth and metastasis to prolong patient survival. After EMT marker expression, IL-6 may be associated with a number of activation factors for the macrophage phenotype switch (51).

Neural invasion and neuroendocrinology

Neural invasion may depend on specific cancer cells and their interaction with the neural stroma (52). Neural invasion is a pathohistological hallmark of PDAC, and the observed incidence in PDAC is as high as 100% (53,54). The nerves that infiltrate the pancreatic tumor tissue interact with the TME to play an important role in carcinogenesis, tumor development and metastasis.

The complex tumor microenvironment has numerous factors that affect tumor development, and neurotransmitters must be included. For normal pancreatic beta-cells, dopamine increases proliferation, decreases apoptosis by extending intracellular cAMP content and prevents beta-cell dedifferentiation (55). However, in tumor tissue, dopamine receptors are activated and inhibit cancer cell growth and migration at both the cell level and tumor level in mice with xenografts (56). Some research reports that nitric oxide (NO) suppresses pancreatic cell apoptosis, and the overproduction of NO reduces insulin secretion (57,58). Pancreatic cancer cells express a high level of inducible NO synthase due to the overproduction of NO, and a high level of NO is significantly associated with poor survival (59). NO may be a therapeutic target and predictor of prognosis in the early stages.

Serotonin (5-HT) is a neurotransmitter in the central nervous system, and it also acts as a hormone in the peripheral region. 5-HT cannot cross the blood-brain barrier so 95% of 5-HT is directly produced in the periphery. In healthy bodies, 5-HT regulates pancreatic beta-cell autocrine function, promotes gluconeogenesis, suppresses glucose uptake, regulates adipose and affects other organs (60). In PDAC, 5-HT uptake promoted activation of the small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1), which is required for the transdifferentiation of acinar cells into acinar-to-ductal metaplasia (ADM), a key determinant in PDAC development (61). The level of 5-HT increases in PDAC tissue, and 5-HT promotes proliferation, prevents cell apoptosis, increases levels of metabolic enzymes involved in glycolysis, and increases participation in the phosphate pentose pathway and hexosamine biosynthesis pathway, activates PI3K-Akt-mTOR signaling and affects the Warburg effect by increasing protein levels of MYC and HIF1A (62). Gamma-aminobutyric acid (GABA) is a major neurotransmitter in the central nervous system and is widely distributed in the peripheral region; GABRP, a receptor of GABA, has been reported to have a close relationship with PDAC. GABRP interacts with macrophages. GABRP induces macrophages into PDAC tissue, and the deletion of macrophages mechanistically abrogates the oncogenic function of GABRP as GABRP interacts with KCNN4 to induce Ca²⁺ entry, which leads to activation of the NF-κB signaling pathway and ultimately facilitates macrophage infiltration by inducing CXCL5 and CCL20 expression (63).

Hormones adjust cell metabolism, growth, and differentiation, and these physiological process changes are hallmarks of cancer. Chronic psychological stress may promote tumor development, and norepinephrine (NE) is the main hormone in the response to stress. Researchers have reported that NE may promote the biological behavior of malignant PDAC through the Notch-1 pathway (64). Angiotensin has not been reported to directly affect PDAC cells; however, the expression of angiotensin-converting enzyme 2 (ACE2) decreases in PDAC tissue, and a reduction of ACE2 expression promotes pancreatic cancer cell proliferation (65). Furthermore, chronic systemic angiotensin inhibitor use in primary PDAC is associated with longer overall survival independent of chemotherapy due to the reduction of potentially malignant cancer cells and stimulation of the immune system (66). To break PDAC chemoresistance, research has attempted to use melatonin to inhibit the activation of NF-κB and increase melatonin expression by sorafenib to block the PDGFR-β/STAT3 pathway because the melatonin receptor mediates STAT3 (67,68).

P2RY2 is a purinergic receptor activated by ATP and belongs to the G-protein coupled receptor family. Investigations have revealed that inhibiting P2RY2 impaired cell growth and delayed tumor development because of its roles in reprogramming PDAC metabolism (69).

Insulin is an important hormone secreted by the pancreas. Increases in insulin and glucose concentration promote fibrosing response and cell proliferation in type 2 diabetes, which may be associated with PDAC (70). Apart from regulating glycometabolism, insulin with the insulin-like growth factor (IGF) family also promotes cell proliferation. Insulin/IGF signaling (IIS) drives cell proliferation via the Yorkie/YAP pathway, and YAP can be stimulated by insulin receptor and G protein-coupled receptor (GPCR) crosstalk through PI3K and PKD in PDAC (71,72). In addition to directly affecting PDAC cells, insulin also affects downstream factors, such as hormone-sensitive lipase, which regulates pancreatic cancer development (73).

Hormones and immunocytes can affect tumor development together. Pancreatic cancer cells secrete high levels of adrenomedullin (ADM), and CD11b⁺ myelomonocytic cells express all AMD receptor components, through which myelomonocytic cells enhance migration and invasion activities through the MAPK, PI3K/Akt and eNOS signaling pathways, as well as the expression and activity of MMP-2. Furthermore, AMD increases the expression of VCAM-1 and ICAM-1 to promote the adhesion and trans-endothelial migration of myelomonocytic cells in endothelial cells, and as we all know, myelomonocytic cells are related to tumor development (74).

Therapy

Because of the difficulties in diagnosing PDAC, only 15% to 20% of PDAC patients can be treated with resection procedures (75). Furthermore, the results of chemotherapy and radiotherapy are poor; therefore, new therapy options are necessary.

To treat PDAC, immunotherapy has been developed to target immune molecules, such as IL-2, to blocking immune checkpoints (76,77). The targeting of immune molecules (PD-1 and PD-L1) to disrupt immune checkpoints and treat tumors has been applied to metastatic melanoma, and this strategy is on track to replace chemotherapy and become a mainstream treatment (78,79). However, in PDAC, the treatment is not as straightforward as the treatment of melanoma. The CAFs that compose the ECM have suppressive immune functions and must be eliminated before a T cell response can ensue. CAFs secrete CCL2 and CXCL2 to recruit myeloid cells and synergistically suppress the immune response; clinical research has started targeting these chemokines and/or its receptors (80).

Vaccine immunotherapy for PDAC is currently being investigated, and this method has two steps that are designed to treat PDAC. The low number of lymphocytes and high number of immunosuppressive cells are responsible for the poor immune response of PDAC (81). The first step aims to accumulate lymphocytes, which secrete interferon gamma and other immune elements that induce tumor cells and immune cells to express a high level of PD-L1/PD-1 (82,83). The second step aims to inhibit the PD-L1/PD-1 signaling pathway to increase lymphocyte proliferation and function (41). The vaccine therapy method is expected to increase the number of immune cells and enhance their function to prevent tumor growth.

Because of the desmoplastic changes of the ECM, the treatment of PDAC has become a huge challenge; the components of the PDAC ECM, including CAFs, collagen, proteoglycan, and hyaluronan, are targeted for treatment. Some labs have begun trying to stop the detrimental collaboration of CAFs and tumor cells through drugs, and the use of 4-methylumbelliferone attempted to solve the hyaluronan problem (84,85). In this clinic, studies have tested the safety and efficacy of the Nab-paclitaxel-gemcitabine method, and because this method has low toxicity, it is recommended as a first-line therapy for patients with metastatic disease (86,87).

The TME stromal factors lead to PDAC being more complicated, immunosuppressive and resistant to drugs. Recent research has focused not only on the factors themselves but also on the downstream or upstream factors. KRAS, one of the most potent of all human oncogenes, regretfully cannot be effectively targeted as a drug because of its smooth surface (88,89). To target KRAS, microRNA was designed to decrease KRAS expression at the RNA level (90). The TGF-β, TGF-βR and TGF-β pathway activation protein SMAD commonly has mutations in pancreatic cancer. To target the TGF-β pathway, siRNA and negatively regular protein SnoN were tested for their ability to decrease cell proliferation as a possible targeted therapy for TGF-β (91,92). SHH binds to the PTCH1 receptor and regulates the Smoothened protein (SMO) and its downstream pathways; treating tumors with inhibitors of the SMO receptor in combination with gemcitabine decreases desmoplasia and collagen deposition in TME and enhances the gemcitabine concentration, resulting in increased overall survival in mice (93). However, the results of the SMO antagonist in clinical research disappointedly demonstrated no significant results (94).

Apart from therapy methods, many medicines and therapy methods have been investigated to conquer this deadly cancer. Tamoxifen, a candidate for PDAC therapy, remodels TME and reduces the tumor cell survival rate by hypoxia-inducible factor-1 alpha (95). A series of antibodies for immune cell inhibitors that target the immunosuppressive TME of PDAC have been tested (96). Urolithin A, which targets the PI3K/AKT/mTOR pathway, has been investigated as a potential therapeutic agent in recent research (97).

Discussion

Due to its complex microenvironment, PDAC suppresses the immune system and resists radiotherapy and chemotherapy, causing cognitive difficulty in PDAC patients. The soluble proteins and cells that surround the tumor cells stop drugs and immunocytes from reaching the cancer cells, which results in difficulties for therapy and a low overall 5-year survival rate. Breaking this obstacle has become the first step to treating pancreatic cancer, but recent clinical research has not found an effective way to approach and kill pancreatic cancer cells. The studies that targeted CAFs and PD-1 immune inhibitors have shown the effort applied in advancing pancreatic cancer therapy, but it is not enough. More factors and mechanisms need to be found to support the basic knowledge behind clinical work, and more drug and therapy methods need to be applied to identify basic scientific problems.

Acknowledgements

State Key Laboratory of Oncogenes and Related Genes, Ren Ji Hospital, Shanghai Jiao Tong University of Medicine, Shanghai Jiao Tong University.

Funding: This work was supported by the grants from National Natural Science Foundation of China (81871923, 81802890, 81872242, 81502382, 81500461 and 81761138045), State Key Laboratory of Oncogenes and Related Genes (No. 91-17-24) and Shanghai health and family planning commission foundation (201740105).

Footnote

Conflicts of Interest: The authors have no conflicts of interest to declare.

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