Targeting the tumor microenvironment in pancreatic ductal adenocarcinoma

Abstract

Introduction:

The dismally slow improvement in patient survival over the years for pancreatic cancer patients is mainly due to two factors: the late diagnosis, at which point the disease is spread to distant organs; and the fact that tumor cells are surrounded by a dense, highly immunosuppressive microenvironment. The tumor microenvironment not only shields pancreatic cancer cells from chemotherapy but also leaves it unsusceptible to various immunotherapeutic strategies that have been proven successful in other types of cancer.

Areas covered:

This review highlights the main components of the pancreatic tumor microenvironment, how they cross-talk with each other to generate stroma and promote tumor growth. Additionally, we discuss the most promising treatment targets in the microenvironment whose modulation can be robustly tested in combination with standard of care chemotherapy. Currently active clinical trials for pancreatic cancer involving components of the microenvironment are also listed.

Expert opinion:

Although immunotherapeutic approaches involving checkpoint inhibition are being pursued enthusiastically, there is still more work to be done with several other emerging immune targets that could provide therapeutic benefit.

1. Introduction

Unlike other cancer types where significant advances have been made, therapies for the treatment of pancreatic cancer have largely failed to move through the clinical trials. After the approval of Gemcitabine by the Food and Drug Agency (FDA) in 1997, the most significant clinical benefit has been shown by FOLFIRINOX, which is a combination of oxaliplatin, irinotecan, leucovorin and 5-fluorouracil, showing a median survival of 11.1 months as compared to 6.8 month for gemcitabine [1]. A combination of Gemcitabine and nanoparticle albumin-bound Paclitaxel (Nab-Paclitaxel) is also used as an alternative frontline therapy [2]. However, when compared to FOLFIRINOX in a metastatic pancreatic cancer study, Gemcitabine plus Nab-Paclitaxel showed only a marginal advantage [3]. The study revealed that Gemcitabine plus Nab-Paclitaxel was better suited for older patients because of lower toxicity and ease of administration. Addition of immunotherapy to this regimen is the next hope towards increasing patient survival. The microenvironment in Pancreatic Ductal Adenocarcinoma (PDAC) constitutes up to 80% of the total tumor mass and as a major component, plays a vital role in creating an immunosuppressive environment, promoting disease progression and metastasis [4]. This stromal microenvironment, composed of cellular and acellular components, forms a physical barrier around the tumor cells, shielding them from immune surveillance. Chemoresistance of PDAC is also thought to be largely due to the robust stroma. Water retention capability of some extracellular matrix proteins discussed later, causes interstitial fluid pressure that makes it difficult to deliver anti-tumor drugs to the area.

Over the past years, there have been many efforts exploring different ways in which to manipulate the stromal microenvironment to achieve a more immunogenic environment, better drug delivery and ultimately improve patient survival in the clinical studies. In this review, we will highlight the major components of the pancreatic TME, their interaction with tumor cells and strategies to target TME that have clinical potential.

2. Major players in pancreatic tumor microenvironment

Over the past decade, studies have brought into light the profound importance of the pancreatic tumor microenvironment (TME) as a driving force in tumor development and progression. Not only does the desmoplastic stroma create a protective shield from therapeutics [5], it also aids in the process of epithelial to mesenchymal transition (EMT) and causes tumor cell dissemination into surrounding tissue. This process involves constant remodelling of the stroma, and activation of different signalling pathways in the TME [6,7]. The following sections enlist and discuss some of the major components of the TME and how they interact amongst themselves and the tumor cells to promote tumorigenesis. Figure 1 depicts the pancreatic tumor microenvironment and summarizes the cross-talk amongst different components.

Figure 1: Pancreatic tumor microenvironment and cross-talk within its components.

Schematic depicting pancreatic acinar cells progressing through precancerous lesion types to PDAC and their interaction with different stromal cell types. Activated stellate cells and transformed acinar cells undergoing acinar to ductal metaplasia (ADM), release cytokines that recruit M1 macrophages, generating an inflammatory environment. Fibroblasts and PanIN cells release factors that promote polarization of M1 macrophages to pro-tumor M2 macrophages. Activated stellate cells and fibroblasts also release proteins that make up a dense and fibrous extracellular matrix, a hallmark of pancreatic cancer. Tumor cells release HIF-1α that can attract MDSCs. MDSCs along with regulatory T cells, immunosuppressive factors released by TAMs/M2 macrophages and suppressive signals from tumor cells cause the suppression of effector T cells.

2.1. Pancreatic stellate cells (PSCs)

The lineage mapping of PSCs remains to be a topic of ongoing investigation, however some results from hepatic stellate cell studies support a mesodermal origin based on functional similarities [7,8]. Other studies suggest that they can originate from more than one source. In the context of pancreatic cancer and chronic pancreatitis, a fraction of stellate cells is recruited from the bone-marrow [9,10]. Infiltrating monocytes can also differentiate to PSCs under the influence of MCP-1 [11]. Under physiological conditions, stellate cells are known to play a role in the maintenance of the basement membrane [12]. They also synthesize matrix proteins, matrix metalloproteinases (MMP-2, MMP-9, MMP-13) and MMP inhibitors that regulate extracellular matrix (ECM) turnover [13,14]. On the other hand, under pathological conditions, PSCs can proliferate, get activated and exhibit increased production of ECM proteins (α-SMA, collagen) and MMPs [15]. Factors that can activate quiescent stellate cells range from inflammatory cytokines to platelet-derived growth factor (PDGF) and transforming growth factors TGF-α, TGF-β [16,17] [18,19]. Sources of these factors can be pancreatic acinar cells and other cell types found in the inflammatory microenvironment, as discussed later [20]. Importantly, tumor cells are also shown to induce stellate cell proliferation and matrix production [21]. Apart from receiving activating signals from other sources in a paracrine manner, PSCs themselves express factors like TGF-β1 and PDGF to perpetuate their own proliferation and activation [22]. Once activated, PSCs can secrete chemoattractant molecules such as IL-8, macrophage chemoattractant protein-1 (MCP-1), and RANTES to contribute to the inflammatory nature of the microenvironment [23].

2.2. Cancer associated fibroblasts (CAFs)

Primary function of fibroblasts is to facilitate tissue regeneration in the face of injury. Under physiological conditions, they are present in organ tissues in a quiescent state and become activated only upon receiving signals from surrounding damaged tissue. Once activated, they proliferate, generate growth factors and produce extracellular matrix (ECM) proteins to aid the healing process [24]. Activated fibroblasts that arise in the TME are called CAFs and are one of the most predominant cell types found in the stroma with several functional subtypes [25]. This heterogeneity in the CAF population is dependent on their interaction with cancer cells and on the local signals they receive from their immediate surroundings [26]. Activated CAF populations in turn have been shown to facilitate the growth and proliferation of tumor cells [27], and a recently published study also reported that Laminin-332 and α3β1 Integrin expression by CAFs can facilitate cell invasion [28]. CAFs can also carry out their effector functions via secreted cytokines. Amongst those, the granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-6 (IL-6) have recently emerged to be important in enhancing monocyte differentiation to pro-tumor macrophages [29] that further contribute to tumor progression.

2.3. Myeloid derived suppressor cells (MDSCs)

MDSCs are a group of bone-marrow derived cells, immature in nature, whose primary function is immuno-regulation and tissue repair. Owing to their ability to curtail immune responses, in the context of TME, MDSCs create an immunosuppressive environment. Pancreatic cancer cells can induce mobilization of MDSCs out of the bone marrow and into systemic circulation before they get recruited into the TME [30]. In pancreatic cancer patients, the number of circulating MDSCs has been shown to correlate with the stage of the cancer and metastasis [31]. As the primary tumor grows and a hypoxic environment is created, hypoxia inducible factors (HIF) such as HIF1α are upregulated, and serve as key mediators of MDSC recruitment to the pancreatic TME [32]. As mentioned above, the main role MDSCs play in the TME is that of immunosuppression. They can release reactive oxygen species (ROS) that can induce stress in other immune cells present in the TME [33]. Oxidative stress in T cells suppresses the expression of CD3 ζ chain, which hampers their proliferation [34]. Another important mechanism by which MDSCs induce immunosuppression in the TME in response to IL-10 secreted by T cells is by the upregulation of the programmed death-ligand 1 (PD-L1) [35]. Once the programmed death protein 1 (PD-1) present on activated T cells is engaged by the upregulated PD-L1 on MDSCs, T cells go into a cell cycle arrest, rendering the lymphocytes inactive.

Since MDSCs are a mixed population of immature immune cells that are not fully differentiated, they exhibit a certain amount of plasticity. There is evidence that MDSCs in a hypoxic environment can differentiate into macrophages through an upregulation of CD45 phosphatase and downregulation of STAT3 [36]. Tumor-associated macrophages are another major component of the TME, as discussed ahead.

2.4. Tumor-associated macrophages (TAMs)

TAMs found in the microenvironment arise from three main sources. 1) They originate from the tissue-resident population that arises from the embryonic yolk-sac [37], 2) they are attracted into the pancreas by various chemoattractants present in the tumor stroma such as IL-4, IL-13, intercellular adhesion molecule-1 (ICAM-1) and colony stimulating factor-1 (CSF-1) [38,39] and 3) they are generated by a polarization switch from inflammatory M1 macrophages to a tumor promoting M2-like phenotype [40,41]. While M1 macrophages are more predominant in the early stages of pancreatic lesion formation creating a fibrotic and inflammatory environment, M2-like macrophages are found in later stages of the progression and are referred to as TAMs. In humans, abundance of M2 macrophages in tumors is correlated to early metastasis, tumor recurrence and ultimately reduced overall survival [42,43]. These cells are known to secrete factors like IL-10 and TGF-β and promote tumor progression by creating a fibrotic and immunosuppressive environment [44,45]. TGF-β is known to have a paradoxical effect in pancreatic TME, where it inhibits cell growth in early stages but is known to have an immunosuppressive role as the disease progresses [41,46]. In turn, pancreatic cancer cells can also modulate polarization of macrophages to a tumor promoting M2 phenotype [41,47]. Tumor cells have been shown to produce IL-13, a polarization factor for M2 macrophages [41], and to enhance the differentiation of macrophages to an M2-like phenotype under hyperglycaemic conditions [48]. Various studies have shown that TAMs can propagate the disease by playing a significant role in tumor cell invasion and metastasis [49–52].

3. Targeting the tumor microenvironment

The dense desmoplasia surrounding the tumor cells is a huge obstacle for drug delivery, and studies that combined stromal targeting with standard of care chemotherapy showed an increase in efficacy [2]. However, more recent studies suggest that completely depleting tumor stroma may in fact, have a more detrimental prognosis indicating that the tumor stroma, in addition to having largely tumor promoting characteristics, stifles tumor growth to some extent. For example, Ozdemir et al showed that complete depletion of tumor stroma by targeting CAFs accelerated the progression of PDAC with reduced overall survival [53]. Such studies underscore the highly complex nature of tumor stroma and that targeting the pancreatic tumor stroma doesn’t simply require complete ablation but in fact needs to be careful modulated. The key components of the stroma, as discussed above, are stellate cells, CAFs, MDSCs and the TAMs, that cross-talk with each other and the tumor cells and create an immunosuppressive environment, refractive to therapy. But the acellular component of the TME, comprised of ECM proteins such as collagen I, III, IV, hyaluronic acid, fibronectin, laminin etc. are equally important. These ECM proteins can provide a scaffold for cytokines and growth factors, interact with tumor cells directly to enhance growth, and create a physical barrier for chemotherapeutics and immune cells. Therefore, it is imperative for any therapeutic strategy against pancreatic cancer, that a combination of drugs simultaneously targeting the stromal components and the tumor cells be used. Table 1 summarizes some of the currently active clinical trials for pancreatic cancer that are using agents targeting the pancreatic TME alone or in combination with chemotherapeutics after surgical removal of the tumors. Some of the most promising TMA targeting strategies are discussed in detail as follows. Figure 2 broadly summarizes the main targeting strategies in pancreatic TME, some of which are discussed in detail as follows.

Table 1.

Currently active and/or recruiting clinical trials for pancreatic cancer, testing drugs that target different components of the tumor microenvironment, alone or in combination with standard of care chemotherapy or other therapies. Clinical trials currently investigating TME targeting strategies

Clinical Trial Identifier	Study drugs/ Biologicals/ Other interventions	Phase
Checkpoint inhibition
NCT03331562	Pembrolizumab, Paricalcitol	Phase II
NCT03727880	Pembrolizumab, Defactinib	Phase II
NCT02646748	Pembrolizumab, Itacitinib, INCB050465	Phase I
NCT03681951	GSK3145095, Pembrolizumab	Phase I, Phase II
NCT03184870	BMS-813160, Nivolumab, Nab-paclitaxel, Gemcitabine, 5-fluorouracil, Leucovorin, Irinotecan	Phase I, Phase II
NCT02734160	Galunisertib, Durvalumab	Phase I
NCT03481920	PEGPH20, Avelumab	Phase I
NCT02311361	Durvalumab, Tremelimumab	Phase I, Phase II
NCT02777710	Pexidartinib, Durvalumab	Phase I
NCT02826486	BL-8040, Pembrolizumab	Phase II
NCT02648282	Cyclophosphamide, GVAX, Pembrolizumab	Phase II
NCT02930902	Pembrolizumab, Paricalcitol, Gemcitabine, Nab-paclitaxel, surgical resection	Phase I
NCT03153410	Cyclophosphamide, GVAX, Pembrolizumab, IMC-CS4	Phase I
NCT02713529	AMG820, Pembrolizumab	Phase I, Phase II
NCT02451982	Cyclophosphamide, GVAX, Nivolumab, Urelumab	Phase I, Phase II
NCT03190265	Cyclophosphamide, Nivolumab, Ipilimumab, CRS-207, GVAX	Phase II
NCT03161379	Cyclophosphamide, Nivolumab, GVAX	Phase II
NCT02243371	CRS-207, GVAX, Nivolumab,	Phase II
NCT03519308	Nivolumab, Paricalcito, Gemcitabine, Nab-paclitaxel	Phase I
NCT02868632	MEDI4736, Tremelimumab	Phase I
NCT01473940	Ipilimumab, Gemcitabine	Phase I
NCT02588443	RO70097890, Gemcitabine, Nab-paclitaxel	Phase I
NCT02807844	MCS110, PDR001	Phase I, Phase II
Targeting CAF mediated immunosuppression
NCT03277209	Plerixafor	Phase I
NCT02826486	BL-8040, Pembrolizumab	Phase II
Targeting MDSC recruitment
NCT02345408	CCX872-B, FOLFIRINOX	Phase I
NCT03681951	GSK3145095, Pembrolizumab	Phase I, Phase II
Targeting stromal depletion
NCT02715804	PEGPH20, Gemcitabine, Nab-paclitaxel	Phase III
NCT02910882	PEGPH20, Gemcitabine, Radiation	Phase II
NCT03481920	PEGPH20, Avelumab	Phase I
GVAX
NCT02451982	Cyclophosphamide, GVAX, Nivolumab, Urelumab	Phase I, Phase II
NCT02648282	Cyclophosphamide, GVAX, Pembrolizumab	Phase II
NCT02243371	CRS-207, GVAX, Nivolumab	Phase II
NCT03153410	Cyclophosphamide, GVAX, Pembrolizumab, IMC-CS4	Phase I
NCT03161379	Cyclophosphamide, Nivolumab, GVAX	Phase II
NCT03190265	Cyclophosphamide, Nivolumab, Ipilimumab, CRS-207, GVAX	Phase II
Others
NCT02030860	Paricalcitol, Gemcitabine, Abraxane	N/A
NCT02929797	CD8+NKG2D+ AKT Cell	Phase I
NCT02562898	Ibrutinib, Paclitaxel, Gemcitabine	Phase I, Phase II
NCT02923921	AM0010, FOLFOX	Phase III
NCT02550327	Anakinra, Gemcitabine, Nab-paclitaxel, Cisplatin	Phase I
NCT02559674	ALT-803, Gemcitabine, Nab-paclitaxel	Phase I

Figure 2: Therapeutic targets in the tumor microenvironment.

Several components outside and within the microenvironment contribute to effector T cell suppression. Dendritic cells in the regional lymph nodes expressing CD80/86 on the surface, bind to CTLA-4 present on the T cells blocking appropriate priming for T cells. This can be targeted by anti-CTLA-4 antibodies. Tumor cells express the ligand PD-L1, which can also render the effector T cells inhibited and unable to perform effector functions once bound to the PD1 receptor on T cells. This can be targeted by anti-PD1 and anti-PD-L1 antibodies. Regulatory T cells (Tregs), whose primary function is to curtail effector T cell function, are heavily recruited in the microenvironment by GM-CSF. GVAX in combination with chemotherapeutics leads to reduced Treg recruitment and enrichment of effector T cells. T cell suppression can also be reduced by targeting MDSCs via COX-2 inhibition, targeting the CSF-1 receptor or by using triterpenoids that are shown to reduce MDSCs in the tumors by downregulating ROS production. Alternatively activated or M2 macrophages are one of the major orchestrators of the immunosuppressive environment, making them an important target. M2 polarization can be caused by IL-13, making IL-13 neutralizing antibodies a potential therapeutic. Pomalidomide treatment can also reduce M1 to M2 polarization. Lastly, the dense stroma in the tumor poses an enormous challenge in PDAC therapy and its depletion is essential. Targeting the stromal proteins by MMP inhibition and hyaluronic acid depletion are promising strategies for better delivery of any therapy including chemotherapeutic drugs.

3.1. Immunotherapy, targeting the cellular component of the TME

3.1.1. Enhancing T cell-mediated immunity

Unlike other solid tumors, pancreatic cancer is unique in having a strongly immunosuppressive microenvironment, adding to the complexity of immunotherapeutic targeting. T cells are the main effectors that can potentially target tumor cells through cytotoxic activity. But their cytotoxic function is preceded by a complex process involving priming, antigen presentation by dendritic cells and tumor infiltration. To evade T cell cytotoxicity, tumors develop mechanisms that inhibit various steps in this process. Increased recruitment of regulatory T cells, expression of PD-L1 (ligand for PD1 expressed on T cells) being some of them. Therefore, employing strategies to overcome these inhibitory mechanisms is essential for successful immunotherapy in PDAC. Enhancing T cell-mediated immunity, especially using the chimeric antigen receptor (CAR) T cell therapy has been successful in the treatment of patients with lymphoma and acute myeloid leukemia [54], but is still under investigation as a treatment for pancreatic cancer [55]. Mesothelin is an attractive target for CAR-T cell therapy since it is expressed in 80% of pancreatic cancers and is correlated with an unfavourable patient outcome [56]. Beatty et al showed that adoptive transfer of mesothelin specific mRNA CAR-T (CARTmeso) cells was safe in patients with minimal off-target effects and infiltrated primary and metastatic sites [57,58]. Apart from mesothelin, some of the other antigens used as a target for CAR-T cell therapy include prostrate stem cell antigen (PSCA), Muc-1, carcinoembryonic antigen (CEA), or fibroblast activation protein (FAP) [59].

Checkpoint inhibition in pancreatic cancer, even in combination with chemotherapy has not shown any significant improvement in therapy [60,61]. However, alteration of the TME prior to checkpoint inhibition such that it becomes more immunogenic may result in better outcomes. Treatment with the GM-CSF vaccine (GVAX) in combination with chemotherapy was shown to deplete regulatory T cells from pancreatic tumors and form lymphoid aggregates within the tumor, making the microenvironment less immunosuppressive [62]. When used in combination with Ipilimumab (checkpoint inhibitor-monoclonal antibody targeting CTLA-4), the overall survival was better than Ipilimumab alone (3.6 months for Ipilimumab; 5.7 months for combination treatment) [63]. Focal adhesion kinase (FAK) has recently been identified as being upregulated in PDAC and is correlated with poor CD8+ T cell infiltration. A study targeting FAK in neoplastic tumor cells showed that when used alone, FAK inhibitor showed limited tumor progression, lesser fibrosis and fewer immunosuppressive cells in KPC mouse tumors [64]. The study also showed that after FAK inhibitor treatment, KPC tumors which were not susceptible to checkpoint inhibition became more responsive to PD-1 antagonists. FAK inhibitors are being tested in the clinic for other solid tumor malignancies ( NCT00787033) and are an attractive drug candidate for being tested in pancreatic cancer patients.

Another promising strategy for enhancing T cell mediated immunity is by using CD40 agonist antibodies [65,66]. CD40 is a molecule expressed mainly on antigen presenting cells and some fibroblasts and endothelial cells. Engagement of the CD40 receptor with the CD40 ligand, which is expressed on T cells, macrophages and smooth muscle cells [67], is known to enhance the activation of antigen presenting cells, priming them for cytotoxic T cell responses [68]. In pancreatic cancer, CD40 stimulation has been evaluated in combination with Gemcitabine treatment with the understanding that the chemotherapeutic aids the release of tumor antigens, followed by antigen presentation by APCs that are then “licenced” with the help of a CD40 agonist. This leads to the stimulation of T cells that are cytotoxic against the cells expressing tumor antigens [69].

Other ways of enhancing T cell mediated immunotherapy include MAP kinase pathway inhibition. It is widely known that more than 90% of the pancreatic cancer patients harbour a KRAS mutation. Combination of the KRAS/MAP kinase pathway inhibition and PD-L1 checkpoint blockade showed a synergistic effect and tumor regression [70].

Due to the highly fibrotic nature of the pancreatic TME that poses a physical barrier for immune cell entry, design of any therapeutic regimen should incorporate a strategy to reprogram the immunosuppressive microenvironment.

3.1.2. Reprograming macrophage polarization

Macrophages are inarguably one of the key players in the entire tumor development process. Recruitment of inflammatory macrophages (M1 polarized) into the pancreas is mediated by several chemokines, cytokines and growth factors such as CCL2, CCL5, GM-CSF, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and ICAM-1 [39,71–74]. Other cytokines present in the inflamed pancreatic milieu cause polarization of the M1 macrophages to an alternative active form referred to as M2 macrophages. A recent study showed that the expression of IL-13 at the PanIN lesions correlated with the presence of M2 macrophages and that it can cause M1 to M2 polarization [41]. These M2 macrophages thrive in and foster an environment that is fibrotic and immunosuppressive in nature, perpetuates tumor growth, facilitates metastasis, and ultimately leads to full-blown carcinoma. Therefore, they are also referred to as tumor-associated macrophages (TAMs). Presence of these macrophages in pancreatic cancer patients correlates with poor prognosis [75]. Given the critical role played by these cells in tumor biology, it stands to reason that fine-tuning the polarization of these macrophages in combination with adjuvant interventions presents a potential therapeutic opportunity. A recently published study shows that treatment of pancreatic tumors with an agent known as Pomalidomide led to reduced M2 macrophages in the microenvironment through downregulation of interferon regulatory factor 4 (IRF4) [45]. Other studies have shown that repolarization of M2 macrophages to an M1-like phenotype can be achieved by targeting nuclear factor-κB (NF-κB) signalling and can also result in tumoricidal activity [76]. Interestingly, both studies also indicated that targeting TAMs, not only induces tumoricidal activity, but also enhances T cell mediated cytotoxic effects, making the TME more immunogenic in nature. Another study delineated the role of tumor necrosis factor (TNF) in modulating macrophage polarization [77]. The study showed that TNF can do so in two different ways. It can negatively regulate M2 macrophage associated gene expression and can downregulate IL-13 production from eosinophils in the environment, which as discussed earlier, is a key regulator of M1 to M2 polarization. Other factors that can potentially repolarize M2 macrophages to an M1 phenotype include TLR4 neutralizing antibodies, PPAR-gamma agonists or Trabectadin [78].

Studies highlighted above, underscore the enormous potential of macrophages in reshaping the pancreatic TME. R programing of TAMs to an M1 phenotype modulates the TME such that it also helps improve immunosurveillance from other immune cells as well as improve T cell mediated immunity [79].

3.1.3. Targeting MDSCs

Given their enormous role in causing an immunosuppressive TME, MDSCs are considered a potential target for therapy. Use of an agent called as Triterpenoid in pre-clinical models of lung and colon carcinoma showed reduced MDSC function through downregulation of reactive oxygen species (ROS) and inhibition of STAT3 [80]. The same study showed that Triterpenoid treatment was well tolerated in patients. However, when used in patients with unresectable tumors in combination with Gemcitabine, Triterpenoid had no significant effects on MDSCs in peripheral blood, but increase T cell response. MDSCs express CSF-1R on their surface, which is a receptor for the growth factor CSF-1. One study showed that blocking this interaction led to depletion of MDSCs in the tumors along with some macrophages. It also showed that when used in combination with checkpoint inhibition, there was significant pancreatic tumor regression [81]. Downstream of growth factor receptors, the JAK-STAT signalling pathway is known to be essential in MDSC function. However, limited data is available to evaluate the potential of STAT inhibition in reducing MDSC function in pancreatic cancer. Rosiglatizone, an FDA approved drug, which suppresses JAK-STAT signalling, in combination with Gemcitabine showed reduced MDSC accumulation in the tumor and extended overall survival in a pre-clinical pancreatic cancer model [82]. LTP-1, an anti-mitotic agent and STAT3 inhibitor was shown to inhibit pancreatic tumor cell growth in vitro and tumor formation in vivo, but its mechanism of action on MDSCs was not explored [83]. Another agent, Sildenafil (phosphodiesterase-5 inhibitor) is known to inhibit MDSC function by downregulating arginase-1, IL4Ra and ROS leading to NK cell mediated cytotoxicity in the tumor [84]. In addition to these agents, cyclooxygenase-2 (COX-2) inhibition was also shown to inhibit recruitment and immunosuppressive function of MDSCs by targeting arginase-1 levels in a model of lung carcinoma [85]. These studies highlight several avenues that could potentially prove beneficial in the treatment of pancreatic cancer as adjuvant therapies to standard of care chemotherapeutics. In fact, Gemcitabine was discovered to target the splenic MDSC population years after it was approved for use in pancreatic cancer patients [86]. Also, gemcitabine treatment after tumor resection was shown to enhance NK cell mediated cytotoxic effects [87]. Therefore, at least a part of the effects seen by gemcitabine could be through its role in depleting MDSCs.

3.2. Targeting the non-cellular components of the TME

The non-cellular components of the TME mainly comprise of ECM proteins and matrix metalloproteases, both of which can be good therapeutic targets. ECM proteins are secreted into the stroma mainly by the fibroblasts and PSCs in the TME. These proteins provide structural integrity and are directly involved in promoting tumor cell proliferation and migration [88]. They are generally found to be upregulated in pancreatic cancer. Fibronectin, which is secreted by fibroblasts, can bind integrin molecules on cell surfaces and regulate cell survival, adhesion and migration, but abrogation of this interaction has not produced good results in the clinic despite promising pre-clinical results [89]. Some of the other potential strategies for targeting the non-cellular components of the TME are discussed as follows.

3.1.2. Matrix metalloproteinase (MMP) inhibition

MMPs are proteolytic enzymes and are known for their wide ranged role in cancer initiation, growth and metastasis [90]. Unfortunately, clinical studies on pancreatic cancer patients have only shown modest success involving MMP inhibitors. A phase-1 study with the drug Marmistat (multifamily MMP inhibitor) on pancreatic cancer patients with unresectable tumors showed an overall survival equivalent to that with gemcitabine but failed to show any synergy with gemcitabine when used in combination [91]. As we learn more about the variety of MMPs found in pancreatic stroma and the diverse roles they play, it is becoming clear that efforts should be more targeted towards specific MMPs. Studies have shown that MMPs such as MMP-9 are upregulated in pancreatic cancer and are essential in mediating cell invasion [92]. In a phase III clinical trial with advanced and metastatic PDAC patients, Tanomastat, which inhibits MMP-9 along with MMP-2, MMP-3 and MMP-13 had minimal success [93]. Lack of success with MMP inhibitors is partly due to structural similarities between different MMPs resulting in off-target effects. Over the past few years, numerous miRNAs have been identified as post-transcriptional MMP regulators and can target MMP molecules with relatively greater specificity [94]. Also, miRNAs can target more than one MMP and may be beneficial in targeting a network of pathways. MMPs can also be regulated differently by more than one miRNAs. For example, miR-143 was shown to decrease MMP-2 and MMP-9 expression in pancreatic cancer cells [95], whereas miR-21, miR-221 and miR-222 led to their upregulation [96,97]. From these studies it becomes clear that even though MMPs are attractive therapeutic targets, a closer examination of their varied roles in pancreatic TME and their regulation via miRNAs needs to be carried out before they can be further tested in clinical trials.

3.2.2. Hyaluronic acid depletion

Extracellular matrix (ECM) proteins found in the vicinity of tumor cells and the cellular component of the TME, are extremely important in conferring physical properties to the stroma. Apart from collagen, which is the most abundant ECM protein, hyaluronic acid (HA) can also provide support and structure to the stroma and more importantly, it is targetable by therapeutics. HA is a glycosaminoglycan secreted by tumor cells and forms a gel-like elastic matrix between collagen fibres that helps retain growth factors and cytokines in the stroma. It can also bind to surface receptors on tumor cells and promote proliferation, migration and invasion [98]. HA’s capacity to absorb and retain water causes an increase in the interstitial fluid pressure (IFP) that hinders drug delivery to the tumor cells [99,100]. Given the multifaceted role played by HA in pancreatic stroma, HA targeting has been of great interest and has shown to cause tumor depletion in several preclinical tumor models [99,101]. Targeting HA was shown to enhance drug delivery by relieving the IFP [102]. A recent study showed that pancreatic tumors treated with Halofuginone, an anti-fibrotic agent, targeted PSCs and led to reduced HA in the tumors and showed overall reduced ECM production [54]. PEGylated recombinant hyaluronidase (PEGPH20) in combination with gemcitabine showed an improvement in overall survival and is emerging as an attractive adjuvant therapy [103]. Currently, patients are being recruited for a phase I clinical trial where PEGPH20 will be given in combination with a checkpoint inhibitor Avelumab to assess overall response rate in patients with metastatic or locally advanced pancreatic cancer ( NCT03481920).

4. Conclusion

Numerous studies over the past decade have made clear that the tumor microenvironment has a major contribution in promoting the hallmarks of pancreatic cancer. Not only do stromal cells promote the proliferation of cancer cells, they also provide a protective shell to decrease efficacy of chemotherapeutic drugs. Therefore, a combination therapy approach targeting stroma and cancer cells could be most efficient. However, studies have suggested that complete abrogation of the stroma can have unwanted detrimental effects on tumor growth and metastasis. This is due to the complexity of the TME which consists of a plethora of target cells and molecules present in the milieu of tumor cells (Figure 1). Therefore, rather than total depletion of one or more components of the TME, therapies should be aimed at modulating the composition of the tumor stroma to achieve the correct alignment of different mechanisms in the microenvironment with an overall goal to promote efficient targeting of tumor cells.

5. Expert opinion

New advances in the field show that despite the low immunogenicity of the PDAC microenvironment, careful modulation can make pancreatic tumors amenable to immunotherapy [104]. To improve chemotherapeutic responses and patient survival, strategic multipronged targeting of interdependent mechanisms in the TME that orchestrate disease progression will be key for advancement of therapy. Currently, there is a huge emphasis on checkpoint inhibitors for PDAC therapy as indicated by the clinical trials listed in Table 1, but it is important to note that T cells may not be the ultimate targetable factor in the goal of developing anti-tumor immunity. There are several mechanisms in play that provide an opportunity for intervention, some of which have been discussed in this review. Repolarization of TAMs into inflammatory/M1 macrophages poses an attractive target, as not only do M1 macrophages possess phagocytic capability to destroy tumor cells [105], but can also release tumor antigens in doing so. Released tumor antigens can then boost antigen presentation and stimulate T cell responses. However, translating macrophage-oriented therapies into clinic requires further investigation into identifying the most effective therapeutic targets and identifying any compensatory mechanisms that the tumors might develop in response to depleting TAMs from the TME. Further research is also required to determine how the depletion of acellular TME components effects tumor growth, resistance to chemotherapy and metastasis. Disappointing clinical studies involving MMP inhibitors, discussed earlier, underscore the need for intricate evaluation of the cost and benefit of depleting tumor stroma. Although there is limited success with current clinical trials, they still help to gain insight into the previously obscure nature of various TME components and how they interact with each other. Based on the new knowledge in this ever-advancing field of study, we will be able to further characterize the known therapeutic targets and perhaps discover better ones in future work.

Highlights.

• Desmoplasia in the stromal microenvironment makes up to ~80% of total tumor mass in PDAC, creating a physical barrier and conferring chemoresistance

• Pancreatic TME is largely non-immunogenic and tumor promoting in nature

• PSCs, CAFs, MDSCs and TAMs are major cellular components of the TME, that cross-talk amongst each other and with tumor cells to activate signaling pathways beneficial to tumor growth and metastasis

• Remodeling of the stroma to enhance anti-tumor immunity requires simultaneous targeting of TME and tumor cells

• Majority of ongoing clinical trials for PDAC are utilizing small molecule inhibitors and monoclonal antibodies targeting TME along with frontline chemotherapy