Role of the tumor microenvironment in pancreatic cancer

Abstract Pancreatic cancer remains a highly recalcitrant disease despite the development of systemic chemotherapies. New treatment options are thus urgently required. Dense stromal formation, so‐called “desmoplastic stroma,” plays controversial roles in terms of pancreatic cancer growth, invasion, and metastasis. Cells such as cancer‐associated fibroblasts, endothelial cells, and immune cells comprise the tumor microenvironment of pancreatic cancer. Pancreatic cancer is considered an immune‐quiescent disease, but activation of immunological response in pancreatic cancer may contribute to favorable outcomes. Herein, we review the role of the tumor microenvironment in pancreatic cancer, with a focus on immunological aspects.

matrix metalloproteinase (MMP), and secreted protein acidic and rich in cysteine (SPARC). 5 Under normal circumstances, the extracellular matrix conserves cellular polarity, proliferation, and migration while inhibiting dysplasia. 6 In contrast, dysregulated integrin subunits, seen in the basement membrane in pancreatic cancer tissue, contribute to cancer-cell survival and invasiveness. 7,8 Hyaluronan, a glycosaminoglycan, is deposited in high concentration in the extracellular matrix of pancreatic cancer. 9 Once hyaluronan binds to its receptor, CD44, subsequent interactions prolong cancer-cell survival and promote cancer cell growth.
Stromal cells in pancreatic cancer comprise cancer-associated fibroblasts (CAFs), endothelial cells, and immune cells. Pancreatic stellate cells are a subset of CAFs. 10 CAFs are a major component of pancreatic cancer stroma, derived from different kinds of progenitor cells such as fibroblasts, pancreatic stellate cells, and epithelial, endothelial, and mesenchymal stem cells. 11,12 CAFs express α-smooth muscle actin (α-SMA), a well-known and reliable marker of CAF, stromal cell-derived factor-1α, fibroblast activation protein, and fibroblast specific protein-1. 3,11 CAFs are activated by transforming growth factor β (TGFβ), sonic hedgehog, tumor necrosis factor α (TNFα), platelet-derived growth factor (PDGF), and interleukin (IL)-1, -6, and -10. 11,13 TGFβ regulates tumor growth, differentiation, and immune cell function. 14 TGFβ initially plays a tumor-suppressive role, but enhances tumor growth as cancer progresses. 6,15 TGF-β1 enhances the ability of CAFs to form abundant filopodia, which allows CAFs to migrate into cancer cell nests. 13 CAFs are stimulated by several types of mediators such as C-C motif chemokine ligand 2 (CCL2), hepatocyte growth factor (HGF), and fibroblast growth factor (FGF). 11 However, some growth factors including insulin-like growth factor (IGF), epidermal growth factor (EGF), and TGFβ are derived from CAFs. 6,14 CAFs induce desmoplasia through the secretion of collagen types I and III, fibronectin, proteoglycans, and glycosaminoglycans, leading to increased mechanical pressure in the extracellular matrix, which may promote cancer-cell migration and inhibit vascularization. 3 CAFs provide cancer cells with nourishment under low-glucose conditions. 11 CAFs also contribute to epithelial-to-mesenchymal transition (EMT), cancer invasion, angiogenesis, and metastasis. 16,17 F I G U R E 1 Schematic of the tumor microenvironment in pancreatic cancer. The tumor microenvironment in pancreatic cancer contributes to tumor progression in a multifaceted way. Cancer-associated fibroblasts (CAFs) and the extracellular matrix (ECM) comprise the desmoplastic stroma and enhance cancer growth, invasion, and metastasis in direct or indirect ways. In contrast, immune-suppressor cells such as regulatory T cells (Treg), myeloid-derived suppressor cells (MDSC), and tumor-associated macrophages (TAM) inhibit CD8 + T cells, which play a key role in the antitumor immune response, by establishing an immunosuppressive tumor microenvironment. Cytokines secreted by CAFs, immune cells, or other components mediate these processes. Antifibrotic therapy, immunotherapy, induction of immunomodulation, and bacterial therapy may improve the unfavorable tumor microenvironment associated with pancreatic cancer. CAR-T, chimeric antigen receptor T cell; CCR4, chemokine receptor type 4; EGF, epidermal growth factor; EMT, epithelial mesenchymal transition; IGF, insulin-like growth factor; IL, interleukin; MMP, matrix metalloproteinase; PD-1, programmed cell death protein 1; PDGF, plateletderived growth factor; PD-L1, programmed cell death ligand 1; PEGPH20, pegvorhyaluronidase alfa; SPARC, secreted protein acidic and rich in cysteine; TGFβ, transforming growth factor β; TIL, tumor-infiltrating lymphocyte; TNFα, tumor necrosis factor α

| Immune-suppressive tumor microenvironment in pancreatic cancer
Pancreatic cancer is thought to be immune-quiescent, as a variety of immune-suppressive mechanisms can inhibit antitumor immunity. 18  Transforming growth factor β excreted by pancreatic cancer cells or extracellular matrix also restrains immune cell function. 22,23 Cancer cell-derived indoleamine 2,3-dioxygenase, a tryptophanmetabolizing enzyme, results in effector T cells becoming deficient in tryptophan, inducing immunological tolerance. 24 In a mouse model, major immune-suppressor cell lines including regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs) have been seen in pancreatic tissue even in the early stages of carcinogenesis. 25 Tregs were identified as CD4 + CD25 + immune-suppressive cells in 1995. 26 The transcriptional factor Foxp3 was shown to be a master regulator of Treg function in 2003. 27 Tregs comprise 5%-10% of peripheral CD4 + T cells in healthy hosts, whereas higher concentrations of Tregs were reported in patients with cancers, including pancreatic cancer. 28,29 Tregs maintain immune cell homeostasis by controlling self-reactive T cells. The immune-suppressing mechanisms induced by Tregs are as follows: suppression of effector T cells by secreting immune-suppressive cytokines such as TGFβ or competing for IL-2; induction of effector T-cell apoptosis by cytotoxic enzymes such as granzyme B; and inhibition of dendritic cell maturation and function. 30 In pancreatic cancer tissue, abundant Tregs are present. 6 Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), which is constantly expressed on Tregs, plays a central role in suppressing antigen-presenting cells. MDSCs induce immune evasion by inhibiting both innate and adaptive antitumor immunity in pancreatic cancer. 21 Pancreatic-cancer patients with higher levels of circulating MDSCs correlated with poorer overall survival. 31 TAMs are macrophages that comprise a major component of immune cells in the tumor microenvironment. TAMs contribute to immune suppression as well as promoting angiogenesis. Cytokines such as CC chemokine, a protein belonging to the CXC chemokine family called stromal-derived factor 1, and vascular endothelial growth factor attract TAMs into the tumor microenvironment. 32 TAMs support cancer progression by secreting a variety of growth factors. 33 IL-10 secreted from TAMs contributes to establishment of the immunosuppressive tumor microenvironment by preventing dendritic cell-mediated antitumor immune responses. 34 Peranzoni et al 35 reported that macrophages inhibit CD8 + tumor-infiltrating lymphocytes (TIL).

| Tumor-infiltrating immune cells in pancreatic cancer
Cancer cells are surveyed by the host immune system, which eliminates cancer in the first phase. Cancer and immunity are then balanced in the next phase. In the last immune-evasion phase, cancer appears in the human body. This theory of immunoediting was developed only recently. 36 TILs are considered to reflect immunoediting. 37 TILs are observed in several tumor types, including colorectal cancer, gastric cancer, hepatocellular carcinoma, bile duct cancer, and pancreatic cancer, which are reported to have prognostic value. 38 Regarding pancreatic cancer, Fukunaga et al 39

| C AN CER S TROMA-TARG E TING THER APY FOR PAN CRE ATI C C AN CER
Depletion of the desmoplastic stroma has led to better chemotherapy delivery and drug response in preclinical models of pancreatic cancer. [53][54][55] Antifibrotic therapy therefore appears to represent a promising strategy in the treatment of pancreatic cancer.
Therapeutic strategies to target CAFs in pancreatic cancer include treatments that reduce the abundance of stroma in pancreatic cancer, such as nab-paclitaxel, pegvorhyaluronidase alfa (PEGPH20), pirfenidone, SOM230 and CD40 agonists, 53,56-59 and that reduce CAFs proliferation, including hedgehog pathway inhibitors, multi-MMP inhibitors, TGFβ inhibitor, retinoic acid 53,60-62 or vitamin D receptor activation to reprogram CAFs to a quiescent phenotype. 63 Chemotherapy combining nab-paclitaxel with gemcitabine has recently become the standard regimen for patients with metastatic pancreatic cancer, significantly prolonging overall survival in the MPACT trial, which was an international, multicenter, open-label, randomized phase III study. 64 Exploratory analysis was carried out to gain insight into the role of SPARC expression as a predictor of survival, because nab-paclitaxel was reported to decrease CAFs and increase microvessel density, attributed to increased drug concentration in tumors treated by nab-paclitaxel in preclinical models. [65][66][67] However, stromal and tumor levels of SPARC as mea- The first successful approach to reduce CAF proliferation that led to depletion of tumor stroma and better gemcitabine delivery and prolonging survival in initial preclinical studies, was achieved through inhibition of sonic hedgehog signaling. 54 The results of that study paved the way for clinical trials. Various hedgehog-pathway inhibitors were tested in a phase II trial in the setting of advanced solid tumors, including pancreatic cancer.
Unfortunately, this very promising approach in targeting the proliferation of CAFs using hedgehog-pathway inhibitors failed in phase II trials. 70 Other preclinical studies indicated that sonic hedgehog signaling inhibition resulted in tumor progression even though desmoplasia was decreased. 71,72 Moreover, clinical research using pancreatic cancer patient specimens demonstrated that high stromal density was associated with longer survival. 73 Given these results, part of the components of desmoplastic stroma work as tumor-restraining rather than as tumor-promoting. 74 A similar lesson has been learned from multi-MMP inhibitors, which did not improve survival among patients with PDAC in clinical trials, despite encouraging preclinical data. Recent data, however, have shown that some MMPs are protective against cancer and others are not, so non-selective inhibition also cancels the protective effects of some MMPs. Furthermore, initial clinical trials were faulty in that inhibitors were tested in late-stage cancers, whereas animal data were obtained during cancer initiation. Timing has to be taken into consideration, and entry criteria for clinical trials should be early-stage of cancer patients in order to match animal data. 60 In a mouse model, a TGFβ antagonist suppressed metastasis without any adverse effects. 75 In another report, TGFβ inhibition reduced pancreatic cancer stroma in an orthotopic pancreaticcancer mouse model, suggesting TGFβ inhibition as a potential treatment for controlling stroma density. 62 In a phase Ib clinical trial, the TGFβ inhibitor galunisertib was given in combination with gemcitabine to patients with advanced or metastatic pancreatic cancer. 76 The response rate with TGFβ inhibition therapy was 42.9% with acceptable safety. Tumor-infiltrating lymphocyte adoptive cell therapy has been developing since the 1980s. TILs extracted from resected specimens were stimulated and cultured in vitro, then transfused into patients.

| IMMUNOTHER APY FOR PAN CRE ATIC C AN CER
Rosenberg and Restifo 87 reported that the objective response rate for TIL adoptive cell therapy in melanoma patients ranged from 34% to 56%. Although the efficacy of TIL adoptive cell therapy for pancreatic cancer has not yet been reported, 88  Chimeric antigen receptor T-cell (CAR-T) therapy has shown high remission rate for patients with acute lymphoblastic leukemia. 90 Cultured T cells transferred with the CAR gene using a retroviral or lentiviral vector are reinjected into the host. CAR-T therapy directly stimulates cell-mediated immunity, and can thus induce stronger antitumor immune reaction than antibody therapy. 91 Several studies of CAR-T therapy for pancreatic cancer are under way. 88 Targeting immunosuppressive cells may be promising. Tumorinfiltrating Tregs in melanoma patients highly express chemokine receptor type 4 (CCR4), a potential target for Treg depletion.
CCR4 antibody has been shown to remove effector-type Treg both in vivo and in vitro. 92 Mogamulizumab, a humanized anti-CCR4 antibody therapy for solid tumors, is under clinical study. 93 Bacterial therapy may become a potential immunotherapy for pancreatic cancer. Salmonella typhimurium A1-R has been shown to be effective in patient-derived xenograft mouse models of pancreatic cancer. 94 In addition, S. typhimurium A1-R enhanced CD8 + TILs in a syngeneic pancreatic cancer mouse model, suggesting activation of host antitumor immunity. 95

| CON CLUS IONS
The tumor microenvironment in pancreatic cancer contributes to tumor growth, invasion, and metastasis in a multifaceted way, including immune evasion. New immunotherapies or cancer stromatargeting therapies have potential to induce a cure for pancreatic cancer.

D I SCLOS U R E
Conflicts of Interest: Authors declare no conflicts of interest for this article.