1 Pancreatic Stellate Cells in Health and Disease

Pancreatic stellate cell: physiologic role, role in fibrosis, and cancer

MV Apte, RC Pirola, JS Wilson

Pancreatic Research Group, South Western Sydney Clinical School, University of New South Wales, and Ingham Institute for Applied Medical Research, Sydney, Australia

Keywords : Pancreatic stellate cells, fibrogenesis, acute pancreatitis, chronic pancreatitis, pancreatic cancer, stromal-tumour interactions,

Corresponding Author:

Professor Minoti Apte OAM Director, Pancreatic Research Group SWS Clinical School, University of New South Wales Level 4, Ingham Institute for Applied Medical Research Liverpool, NSW 2170 AUSTRALIA

Ph: 61-2-87389029 Fax: 61-2-96029441 Email: [email protected]

Abstract

Ever since the first descriptions of methods to isolate pancreatic stellate cells (PSCs) from rodent and human pancreas 17 years ago, rapid advances have been made in our understanding of the biology of these cells and their functions in health and disease. PSCs are now well established as central players in pancreatic fibrogenesis, but are also increasingly acknowledged for their roles in the normal pancreas. This review summarises interesting new studies over the past 12 months, including improved methods of PSC immortalisation, factors causing PSC activation as well as those inducing quiescence, and translational research aimed at inhibiting the facilitatory effects of PSCs on disease progression in chronic pancreatitis as well as pancreatic cancer.

Introduction

Pancreatic stellate cells (PSCs) are resident cells of the pancreas, comprising 4-7% of all parenchymal cells in the gland [1], and are now well established as key players in pancreatic fibrogenesis. However, PSCs were only able to be isolated for biological studies about 17 years ago, and the advances made in the field in this relatively short period have been commendably rapid. Presently, while basic PSC biology continues to be explored, researchers in the field also appear to be turning to a more translational approach aiming to develop novel treatments for chronic pancreatitis and pancreatic cancer by targeting activated PSCs. This review touches upon current knowledge relating to PSC isolation and characterisation and the contribution of these cells in health and disease, with particular emphasis on findings reported over the past year, as relevant to the section under discussion.

Pancreatic Stellate Cells – Isolation and Characterisation

Quiescent PSCs can be isolated from normal rodent and human pancreas by density gradient centrifugation, while pre-activated PSCs can be isolated from diseased pancreas by the outgrowth method [2, 3]. Both methods have been crucial to studies assessing PSC physiology. The advantage of the first lies in its ability to yield quiescent PSCs thereby allowing studies of the transformation of the cells from their non-activated normal state to an activated state.

In view of the challenges in terms of labour, time and limited viability of primary cell cultures, researchers have attempted to develop immortalised PSC lines so as to make it easier to rapidly obtain cells in sufficiently large numbers for experimental use. However, unlike primary PSCs, early preparations of immortalised cell lines appeared to exhibit unusually high doubling rates, prompting a cautionary note from a panel of experts in the

3 field about the importance of validating any data obtained with these lines, in primary PSC cultures [4]. Indeed, Pomianowska et al [5] have recently reported differences in functional responses of immortalised and primary PSCs to prostaglandin E2, further emphasising the need to confirm findings in primary cells. Recently, Rosendahl et al [6] have reported the development of a method to generate conditionally immortalised ‘normal’ PSCs and tumour associated PSCs. The authors used tissue from a resected surgical specimen from a patient with pancreatic ductal adenocarcinoma. Neoplastic and non-neoplastic regions of the specimen were used to isolate tumour-derived and normal PSCs by the outgrowth method.

Primary cells were then infected with retrovirus-containing supernatants from a packaging cell line producing a construct containing a temperature-sensitive simian virus-40 large T antigen and human telomerase. The authors report that these transformed cells proliferate rapidly at 33°C but when transferred to normal culture conditions at 37°C, the SV40LT is switched off and the cells exhibit the phenotype and slower growth rate of cells in primary culture. The immortalised cells express PSC selective markers and exhibit some lipid droplets in their cytoplasm. The authors report differences between the normal versus tumour associated PSC lines with regard to basal and IGF-1 stimulated motility as well as their proteome profile. The above-described conditionally immortalised PSC lines are an improvement over previously reported cell lines. However, it must be noted that the ‘normal’

PSC lines were derived from pre-activated PSCs (via the outgrowth method) thus precluding any studies on the fully quiescent phenotype. Although the authors report the presence of lipid droplets in the cytoplasm of the normal PSC line, vitamin A content was not specifically measured (Oil Red-O staining non-specifically stains all lipids), neither was the response of the two types of cell lines to known activating factors such as platelet derived growth factor

(PDGF), TGFβ or other cytokines, compared to primary PSCs. Thus, while these cell lines

4 are potentially useful for ‘proof of principle’ studies, it is essential that the findings of such studies are validated using primary PSCs.

Islet Stellate Cells : A recent study by Zha et al [7] reports the isolation of stellate cells from pancreatic islets that were obtained from rat pancreas digested with collagenase. Upon culture of the isolated islet clusters, the authors noted outgrowth of cells from the edges of the explants. These cells exhibited PSC markers including GFAP and αSMA and also expressed the ECM proteins collagens I and III and fibronectin. The cells had fewer lipid droplets than standard PSCs, appeared to be more easily activated to αSMA expressing cells in culture, but exhibited lower rates of proliferation and migration compared to conventional PSCs. The authors propose that islet stellate cells may represent a subpopulation of PSCs, which may play a role in islet fibrosis and islet cell dysfunction.

Pancreatic Stellate Cells in Health

Although the earliest studies on PSC biology focussed on the role of these cells in pathological fibrosis, over the past decade, considerable attention has been paid to the possible functions of these cells in health [1]. This includes elucidation of 1) the ability of

PSCs to maintain normal ECM turnover in the gland by regulating synthesis as well as degradation (via matrix degrading enzymes) of ECM proteins; 2) the possible function of

PSCs as intermediary cells in cholecystokinin-mediated pancreatic exocrine secretion; 3) the capacity of PSCs for recognising pathogen associated molecular patterns (PAMPs) via toll like receptors expressed on the cell surface and the ability to phagocytose necrotic acinar cells and neutrophils, indicating a role in innate immunity (no role in acquired immunity has yet been demonstrated), 4) the expression of stem cell markers nestin, CD133, SOX9 and

GDF3 by PSCs and their capacity to function as progenitor cells. A detailed discussion of

5 previous work is not within the brief for this review and there have not been any reports in the literature in the past year to add to the above information.

Origin of PSCs

Pancreatic stellate cells are now known to be part of a wider retinoid-storing stellate cell system within the body. These retinoid-storing cells are located in the parenchyma and /or around blood vessels in the liver (hepatic stellate cells) as well as other extra-hepatic organs.

Transcriptomic and proteomic analyses have revealed significant similarities between hepatic and pancreatic stellate cells, although organ specific differences have also been noted [8].

Interestingly, both HSCs and PSCs were found to be very distinct from skin fibroblasts [8].

Since stellate cells express both mesenchymal and neurotrophic factors, their lineage was a matter of some debate in the field. However, lineage tracing studies have firmly established that hepatic stellate cells are of mesenchymal origin [9]. Similar studies for PSCs are awaited.

Pancreatic Stellate Cells in Disease

As noted earlier, the central role of PSCs in the fibrotic processes in chronic pancreatitis and pancreatic cancer have been studied in some depth over the past 15 years. A major step in this process is the activation of PSCs from their quiescent to their myofibroblast-like phenotype.

Activation of PSCs

As would be reasonably expected, studies aimed at identifying the factors that could activate

PSCs in diseased states were based on knowledge of the mediators that are upregulated during pancreatic necroinflammation and of agents known to cause pancreatic injury such as alcohol. The indices of activation routinely assessed in these studies included loss of vitamin

A lipid droplets, αSMA expression, proliferation, migration, and production of ECM

6 proteins. A large variety of growth factors, cytokines and chemokines have thus been shown to activate PSCs in vitro. Other known activators include alcohol and its metabolites, endotoxin, oxidant stress, hyperglycaemia, hypoxia, angiogenic factors, proteases and an ever increasing list of factors pertinent to pancreatic injury (see review[10]). A recent study reports on the effects of one particular chemokine CX3CL1 on PSC activation. CX3CL1 levels are increased in the serum of patients with alcoholic chronic pancreatitis [11].

Expression of this chemokine has also been reported to correlate with severity of pain in chronic pancreatitis. Uchida and colleagues [12] had reported in 2013 that cultured rat PSCs secreted CX3CLI and that this secretion was significantly induced by exposure to ethanol. In their recent follow up study they have shown that PSCs also express the receptor (CX3CR1) for this chemokine and that CX3CL1 increases PSC proliferation in vitro [11]. Thus, during pancreatic necroinflammation both paracrine and autocrine effects of CX3CL1 on PSCs may be envisaged resulting in activation of the cells, which then contribute to further injury.

Another recently described activating factor is pancreatic parathyroid hormone-related protein (PTHrP) that is produced by islet cells in normal pancreas, but has been reported to be upregulated in the exocrine pancreas in a mouse model of caerulein-induced pancreatitis.

Bhatia and colleagues [13] used repeated caerulein injections to produce a model of chronic pancreatitis in wild-type mice and in mice with acinar cell-specific targeted disruption of the

PTHrP gene. They found that compared to wild type mice, caerulein-induced inflammation and fibrosis were significantly decreased in the mice with deficient acinar PTHrP production, suggesting an important role for PTHrP in chronic pancreatitis. Using acinar cells and PSCs isolated from wild type and PTHrP deficient mice, the authors showed that PSCs express

PTH1R, the receptor for PTHrP, and that upon exposure to the ligand, PSCs exhibit increased procollagen mRNA expression.

Signalling Pathways in PSCs

Several intracellular signalling pathways that regulate specific PSC functions have now been identified (see Table 1). Most recently, Hu et al [14] have reported that the canonical Wnt signalling pathway (which regulates cell development and cell fate) plays a crucial role in the activation of PSCs in culture since incubation of PSCs with the Wnt antagonist Dkk

(Dickkopf protein) significantly reduced PSC proliferation, αSMA expression and cytokine production. In a mouse model of chronic pancreatitis, the authors detected increased expression of Wnt and its second messenger β catenin, but found it difficult to detect the Wnt antagonist Dkk. The authors postulate that an imbalance between the Wnt pathway and its antagonist may allow persistent activation of PSCs and thus facilitate fibrogenesis in CP. For a majority of the pathways (noted in Table 1), which are stimulated by the binding of relevant ligands to their receptors, the common downstream event is most likely intracellular calcium modulation.

MicroRNAs (small non-coding RNAs) have been attracting increasing attention in cell biology in general, and are now implicated in the regulation of a range of cellular functions such as differentiation, proliferation, apoptosis, protein synthesis etc. It has been reported that miR-15b and miR-16 influence apoptosis in rat PSCs by targeting the anti-apoptotic factor

Bcl-2 [15]. Masamune et al [16] have detected upregulation of 42 microRNAs and downregulation of 42 others in activated PSCs compared to quiescent PSCs. The differentially expressed microRNAs are implicated in cell development, cell growth and proliferation, cell movement, cell death and cell survival. Most recently, Charrier et al [17] have reported the existence of a positive feedback loop between connective tissue growth factor (CCN2, known to activate PSCs) and miR-21. Increased production of CCN2 by activated PSCs is accompanied by increased miR-21 expression. Interestingly, PSC derived

8 exosomes were found to contain both miR-21 and CCN2 mRNA, and these exosomes were taken up by other PSCs in culture, prompting the authors to postulate that PSCs can stimulate other PSCs in the vicinity via an exosome-mediated paracrine pathway. It may be reasonably predicted that the PSC microRNA field will be an active area of research in the coming years and will increasingly elucidate the role of specific microRNAs in PSC functions.

As would be expected, inhibition of the pathways mediating PSC activation noted above can facilitate PSC quiescence. Factors known to induce PSC quiescence include retinol or its metabolites, curcumin, the anthraquinone derivative rhein and more recently identified agents such as, bone morphogenic protein (BMP) [18] and the tyrosine kinase inhibitors sorafenib and sunitinib [19]; the above factors act via inhibition of MAPK, AP-1, Sonic Hedgehog,

TGFβ/Sma2 signalling and PI3K/AKT respectively. Melatonin, (an indole produced by the pineal gland) when used in pharmacological doses has also been recently shown to inhibit

PSC proliferation and to induce cell death possibly via mobilisation of calcium and activation of apoptotic pathways [20].

PSCs in Acute Pancreatitis

PSC proliferation occurs as an early event in pancreatic necroinflammation and PSCs are now acknowledged to play a role in the repair / regeneration of the pancreas from an acute episode by : i) providing an ECM scaffold for restitution of acinar cells through interactions of ECM proteins with integrin receptors on acinar cells; ii) removing excessive ECM via secretion of matrix degrading enzymes; iii) effecting restitution of normal PSC numbers via loss of activated cells through either apoptosis, senescence or reversion to quiescence although the relative contributions of these 3 in the removal of activated PSCs after pancreatic injury remain to be clarified.

PSCs in Chronic Pancreatitis

Evidence accumulated from in vitro and in vivo studies over the past 3 decades has unequivocally demonstrated the central role of activated PSCs in the fibrosis of chronic pancreatitis. While the majority of proliferating PSCs in CP are sourced from resident cells in the gland, a small fraction (5-18%) is thought to be derived from circulating bone marrow cells (see review [21]). Using a single haematopoietic stem cell isolated from transgenic mice expressing enhanced green fluorescent protein (EGFP), Ino et al [22] have reported that the lineage of a subset of PSCs in the pancreas of CCL4-treated mice could be traced back to terminally differentiated monocytes that were recruited to the injured pancreas under the influence of monocyte chemotactic protein 1 (MCP-1) in the gland. However, the mechanisms by which monocytes acquired PSC features in the injured pancreas remain to be determined. Nonetheless, it is generally agreed that the PSCs in chronic pancreatitis are mainly derived from the resident cells with some contribution from bone marrow-derived pluripotent cells.

Up until a few years ago, most of the research interest regarding the role of PSCs in CP was directed towards the exocrine pancreas. However, in recent times, the possible role of these cells in the endocrine dysfunction of chronic pancreatitis has attracted some attention.

Increased PSC numbers have been detected in fibrotic areas around and within the islets of

Langerhans in the pancreas of Goto-Kakizaki rats (a model of type 2 diabetes) and in vitro work has shown that PSCs inhibit insulin secretion by beta cells as well as causing apoptosis of those cells. Recent studies have reported that hyperglycaemia aggravates the detrimental effects of PSCs on beta cell function [23], and that in hyperglycaemic mice, caerulein- induced chronic pancreatitis is significantly aggravated when compared to normoglycaemic mice [24]. The above observation suggest a positive feedback loop between PSCs and islet

10 cell function, whereby PSCs cause beta cell dysfunction with the resultant hyperglycaemia further aggravating the detrimental effects of PSCs on beta cells while at the same time causing enhanced PSC activation and fibrosis.

Reversal of Pancreatic Fibrosis :

An improved understanding of PSC biology during pancreatic injury, particularly in chronic pancreatitis, was the driver for many subsequent studies aimed at developing novel therapeutic approaches to minimise or reverse the fibrosis. These treatments which have mostly been applied in experimental models of pancreatic fibrosis, include i) inhibition of profibrogenic growth factors TGFβ and TNFα, ii) anti-oxidants, iii) protease inhibitors, iv) modulation of signalling molecules (eg troglitazone binding to the peroxisome proliferator receptor gamma, PPARγ); v) inhibition of collagen synthesis by targeted treatment of PSCs with collagen siRNA; vi) an anthraquinone derivative Rhein; and vii) in case of alcoholic pancreatitis, withdrawal of alcohol administration. In the past 12 months, two new treatments have been reported including i) a prostacyclin analogue ONO-1301 which suppressed fibrosis in the dibutyl tin (DBTC)-induced chronic pancreatic model, by inhibiting the production of pro-inflammatory and profibrogenic cytokines by monocytes [25]; ii) analogues of apigenin, a flavonoid, which significantly reduced fibrosis in a mouse model of chronic pancreatitis produced by repeated caerulein injections [26]. The above studies are encouraging from the point of view of identifying potentially useful therapies for pancreatic fibrosis. However, the challenge lies in translating these pre-clinical findings to the clinical situation.

Pancreatic Stellate Cells in Pancreatic Cancer

The central role of pancreatic stellate cells in producing the collagenous stroma in pancreatic cancer is now well accepted. Activated PSCs are found not only in fully developed PDACs

11 but also around pancreatic intraepithelial neoplasms (PanINs, early pre-malignant lesions).

With regard to the influence of PSCs on cancer progression, the weight of experimental evidence to date (in vitro studies and in vivo subcutaneous, orthotopic and transgenic models) supports the notion that PSCs promote local tumour growth and metastasis. However, a recent report by Ozdemir and colleagues [27] has raised questions about the facilitatory influence of cancer-associated fibroblasts in pancreatic cancer. Using a mouse model of pancreatic cancer with conditionally depleted αSMA+ve myofibroblasts, the authors reported increased epithelial mesenchymal transition (EMT) of cancer cells in pancreatic tumours, increased hypoxia within tumours and decreased survival. While intriguing, these findings in mice are difficult to reconcile with the breadth of previous work using human PSCs and human cancer cells, that has consistently demonstrated a tumour permissive role for PSCs. It is possible that the influence of PSCs on cancer behaviour is a dynamic and stage-dependent process, such that in the earliest stages of carcinogenesis, PSC-produced stroma may represent an attempt to restrict/cordon off tumour cells, while in later stages the ‘protective’ effect is overwhelmed by the ability of cancer cells to subvert PSCs into cancer-permissive cells. Obviously, this is an area that requires further study.

Interactions between PSCs and cancer cells have been very well defined in recent years, with each cell type stimulating proliferation, migration and survival of the other. PSCs may also have a role in the well-known resistance of cancer cells to the first line chemotherapeutic agent Gemcitabine. Zhang et al [28] have recently reported that conditioned medium from human PSCs inhibited the cytotoxic and apoptotic effects of Gem on the human pancreatic cancer cell line Panc-1. This effect was regulated by the stromal derived factor 1 α (SDF-1α, produced by PSCs) – CXCR4 (receptor for SDF on cancer cells) axis, which in turn induced phosphorylation of several downstream signalling pathways including MAPK and PI3K in

12 the cancer cells, resulting in increased IL6 production by PANC-1 cells. IL6 then exerted an autocrine effect on cancer cells to protect them from Gem-induced apoptosis. The possible facilitation of chemoresistance by PSCs is supported by the findings of another recent study involving the analysis of human PSCs obtained from fine needle aspirates of pancreatic cancer tissue from patients before and after treatment with Gemcitabine (a first line chemotherapeutic agent) and concurrent radiation [29]. The authors found that PSCs survived the chemoradiation treatment and exhibited an even more activated phenotype post-treatment, supporting the concept that PSCs may play a key role in chemoresistance and/or recurrence of pancreatic cancer.

Interactions between PSCs and other cells in the stroma (endothelial cells, immune cells and neural cells) are now being increasingly studied. In the past year, our Group has demonstrated that PSCs stimulate endothelial cell proliferation and tube formation (a measure of angiogenesis), effects that are mediated via the hepatocyte growth factor (HGF, secreted by

PSCs) / c-MET (receptor for HGF expressed on endothelial cells) pathway, leading to the induction of downstream signalling pathways PI3 kinase and p38 kinase [30]. PSCs may also contribute to the acknowledged ‘immune evasion’ phenomenon in pancreatic cancer. In this regard, PSCs have been shown to i) sequester CD8+ T cells in the stroma, preventing them from invading the peri-tumoral regions to exert their anti-cancer effects [31] and to induce apoptosis of T-cells via secretion of the β-galactoside binding protein, galectin-1 [32]; ii) induce migration of myeloid derived suppressor cells into the stroma [33]; iii) induce degranulation of mast cells leading to the release of tryptase and IL13 which cause PSC and cancer cell proliferation [34]; iv) induce cytokine production by macrophages leading to further activation of PSCs [35]. In terms of neural elements within the stroma, a recent report has demonstrated that neurite growth towards cancer cells as well as the ability of cancer

13 cells to invade neurons are both significantly enhanced in the presence of PSCs, an effect that is [36] likely mediated via activation of PSCs by sonic hedgehog paracrine signalling.

However, whether PSCs interact directly with stromal neuronal cells is yet to be determined.

In view of the now widely accepted role of the stroma in pancreatic cancer progression, most of the research in recent times has been devoted to translational studies aiming to identify novel strategies to interrupt stromal-tumour interactions in pancreatic cancer. The majority of these have used pre-clinical models, with a few progressing to early phase clinical trails.

Successful outcomes (reduced tumour growth and metastasis and/or increased survival) have been reported in experimental models with i) the antifibrotic agent pirfenidone [37]; ii) all trans retinoic acid (ATRA) which induces PSC quiescence [38]; and most recently, iii) calcipotriol, a vitamin D receptor ligand also shown to induce PSC quiescence [39].

Interestingly, a recent study has exploited the ability of PSCs to express gamma glutamyltransferase (γGT) to maximise therapeutic delivery to endothelial cells in pancreatic cancer, by using a glutathione-S conjugate of a trivalent arsenical compound, which is converted to its active form upon cleavage of the γ glutamyl residue by γGT from PSCs [40].

It may be reasonable to postulate that glutathione S conjugates of other drugs could be similarly delivered to specific target cells in pancreatic cancer.

Treatments targeting the stroma (see review [10] that have entered clinical trials after encouraging results in pre-clinical models include i) Pegylated human recombinant hyaluronidase (which degrades hyaluronan in ECM; ii) Nanoparticle albumin complexed paclitaxel with and without gemcitabine; iii) CD40 agonist monoclonal antibody to activate macrophages; iv) Hedgehog pathway inhibitors; and iv) Renin-angiotensin inhibitors such as losartan and olmasartan. It must be acknowledged here that the above have mostly only

14 shown modest beneficial effects in patients. However, given that treatments focussed on cancer cells alone have not improved patient outcome for decades, and that accumulating evidence supports a major role for the stromal reaction in pancreatic cancer, it is reasonable to turn to stromal reprogramming (via a multipronged approach targeting PSCs, endothelial cells and immune cells in the stroma), as an important addition to the existing armamentarium of chemotherapies for pancreatic cancer.

Summary

The field of pancreatic stellate cell biology in health and disease has seen a significant expansion over the past decade (Figure 1). Our understanding of the molecular processes underpinning PSC quiescence and activation and the consequent influence of these cells on pancreatic pathophysiology is constantly improving. Armed with this knowledge researchers are now turning to translational studies, ultimately aimed at developing novel treatment strategies to modulate PSC function so as to improve clinical outcomes in patients with two of the notoriously hard to treat diseases of the pancreas – chronic pancreatitis and pancreatic cancer.

Acknowledgments

The authors acknowledge the work and support of the members of the Pancreatic Research

Group.

Financial Contribution : The authors have received grant support from the National Health and Medical Research Council and the Cancer Council of New South Wales.

Conflicts of Interest : MA has a signed Materials Transfer Agreement with Amgen Inc.

References and Recommended Reading :

Papers of particular interest, highlighted within the annual period of review have been highlighted as : n of special interest; nn of outstanding interest

Salient Points

1. PSC immortalisation is an issue under active investigation to facilitate research with

human PSCs.

2. New evidence is emerging regarding the role of PSCs in endocrine cell function,

islet fibrosis and diabetes

3. New evidence available regarding the putative importance of specific microRNAs

in regulating PSC functions

4. Translational studies assessing the effects of therapeutically targeting activated

PSCs in chronic pancreatitis and pancreatic cancer are gaining momentum.

Table 1: Pancreatic Stellate Cells – Signalling Pathways

Signalling Pathways PSC Functions

Mitogen activated protein kinase (MAPKP αSMA expression, proliferation, migration ECM protein synthesis

Phosphatidylinositol 3 kinase (PI3K) Migration, proliferation, ECM protein synthesis

Protein kinase C (PKC) ECM protein synthesis

Hedgehog Migration

JAK-STAT Proliferation

Smads ECM protein synthesis

Rho, Rho kinase Actin cytoskeleton, stress fibre formation

Transcription factors (AP-1, NFκB, Gli-1) Activation, migration, proliferation, ECM protein synthesis

Peroxisome proliferator activated receptor αSMA expression, proliferation, gamma (PPARγ) phagocytosis

Wnt/β-catenin Proliferation, ECM production

MicroRNAs (miR-15, 16B, 21 and others) Apoptosis, cell activation

Figure Legend

Figure 1 Pancreatic Stellate Cells in Health and Disease. The Figure depicts the functions of quiescent (vitamin A containing) cells in health and their conversion to a myofibroblast-like phenotype by activating factors pertinent to pancreatic disease. Quiescent cells serve i) to maintain normal ECM turnover; ii) to mediate innate immunity; iii) as intermediary cells in cholecystokinin (CCK)-mediated pancreatic exocrine secretion and iv) as progenitor cells.

Activated PSCs have the following functions i) In acute pancreatitis – repair and regeneration; ii) In chronic pancreatitis – excessive ECM production, islet cell dysfunction; iii) In pancreatic cancer – production of collagenous stroma; ii) cross-talk with cancer cells, endothelial cells, immune cells, and neural elements; iii) providing a growth permissive environment for local growth and metastasis of pancreatic tumours.

References

1. Apte MV, Pirola RC, Wilson JS. Pancreatic stellate cells: a starring role in normal and diseased pancreas. Front Physiol. 2012;3:344. 2. Apte MV, Haber PS, Applegate TL, et al. Periacinar Stellate Shaped Cells in Rat Pancreas - Identification, Isolation, and Culture. Gut. 1998;43:128-33. 3. Bachem MG, Schneider E, Gross H, et al. Identification, Culture, and Characterization of Pancreatic Stellate Cells in Rats and Humans. Gastroenterology. 1998;115:421-32. 4. Erkan M, Adler G, Apte MV, et al. StellaTUM: current consensus and discussion on pancreatic stellate cell research. Gut. 2012;61:172-8. 5. Pomianowska E, Sandnes D, Grzyb K, et al. Inhibitory effects of prostaglandin E2 on collagen synthesis and cell proliferation in human stellate cells from pancreatic head adenocarcinoma. BMC cancer. 2014;14:413. 6. Rosendahl AH, Gundewar C, Said Hilmersson K, et al. Conditionally immortalized human pancreatic stellate cell lines demonstrate enhanced proliferation and migration in response to IGF-I. Experimental cell research. 2015;330:300-10. 7. Zha M, Li F, Xu W, et al. Isolation and characterization of islet stellate cells in rat. Islets. 2014;6:e28701. 8. Buchholz M, Kestler HA, Holzmann K, et al. Transcriptome analysis of human hepatic and pancreatic stellate cells: organ-specific variations of a common transcriptional phenotype. J Mol Med (Berl). 2005;83:795-805. 9. Cassiman D, Barlow A, Vander Borght S, et al. Hepatic stellate cells do not derive from the neural crest. J Hepatol. 2006;44:1098-104. 10. Apte MV, Pirola RC, Wilson JS. Stellate Cells in Nonhepatic Organs In: Gandhi CS, Pinzani M, editors. Stellate Cells in Health and Disease. 1. United Kingdom: Elsevier; 2015. p. 271-99. 11. Uchida M, Ito T, Nakamura T, et al. Pancreatic stellate cells and CX3CR1: occurrence in normal pancreas and acute and chronic pancreatitis and effect of their activation by a CX3CR1 agonist. Pancreas. 2014;43:708-19. 12. Uchida M, Ito T, Nakamura T, et al. ERK pathway and sheddases play an essential role in ethanol-induced CX3CL1 release in pancreatic stellate cells. Lab Invest. 2013;93:41- 53. 13. Bhatia V, Rastellini C, Han S, et al. Acinar cell-specific knockout of the PTHrP gene decreases the proinflammatory and profibrotic responses in pancreatitis. Am J Physiol Gastrointest Liver Physiol. 2014;307:G533-49. 14. Hu Y, Wan R, Yu G, et al. Imbalance of Wnt/Dkk negative feedback promotes persistent activation of pancreatic stellate cells in chronic pancreatitis. PLoS One. 2014;9:e95145. 15. Shen J, Wan R, Hu G, et al. miR-15b and miR-16 induce the apoptosis of rat activated pancreatic stellate cells by targeting Bcl-2 in vitro. Pancreatology. 2012;12:91-9. 16. Masamune A, Nakano E, Hamada S, et al. Alteration of the microRNA expression profile during the activation of pancreatic stellate cells. Scand J Gastroenterol. 2013;49:323- 31. 17. Charrier A, Chen R, Chen L, et al. Connective tissue growth factor (CCN2) and microRNA-21 are components of a positive feedback loop in pancreatic stellate cells (PSC) during chronic pancreatitis and are exported in PSC-derived exosomes. Journal of cell communication and signaling. 2014;8:147-56. 18. Gao X, Cao Y, Staloch DA, et al. Bone morphogenetic protein signaling protects against cerulein-induced pancreatic fibrosis. PLoS One. 2014;9:e89114.

19. Elsner A, Lange F, Fitzner B, et al. Distinct antifibrogenic effects of erlotinib, sunitinib and sorafenib on rat pancreatic stellate cells. World J Gastroenterol. 2014;20:7914- 25. 20. Santofimia-Castano P, Garcia-Sanchez L, Ruy DC, et al. Melatonin induces calcium mobilization and influences cell proliferation independently of MT1/MT2 receptor activation in rat pancreatic stellate cells. Cell biology and toxicology. 2015. 21. Apte MV, Wilson JS, Lugea A, Pandol SJ. A starring role for stellate cells in the pancreatic cancer microenvironment. Gastroenterology. 2013;144:1210-9. 22. Ino K, Masuya M, Tawara I, et al. Monocytes infiltrate the pancreas via the MCP- 1/CCR2 pathway and differentiate into stellate cells. PLoS One. 2014;9:e84889. 23. Zha M, Xu W, Zhai Q, et al. High glucose aggravates the detrimental effects of pancreatic stellate cells on Beta-cell function. International journal of endocrinology. 2014;2014:165612. 24. Zechner D, Knapp N, Bobrowski A, et al. Diabetes increases pancreatic fibrosis during chronic inflammation. Experimental biology and medicine (Maywood, NJ). 2014;239:670-6. 25. Niina Y, Ito T, Oono T, et al. A sustained prostacyclin analog, ONO-1301, attenuates pancreatic fibrosis in experimental chronic pancreatitis induced by dibutyltin dichloride in rats. Pancreatology. 2014;14:201-10. 26. Chen H, Mrazek AA, Wang X, et al. Design, synthesis, and characterization of novel apigenin analogues that suppress pancreatic stellate cell proliferation in vitro and associated pancreatic fibrosis in vivo. Bioorganic & medicinal chemistry. 2014;22:3393-404. 27. Ozdemir BC, Pentcheva-Hoang T, Carstens JL, et al. Depletion of carcinoma- associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer with reduced survival. Cancer cell. 2014;25:719-34. 28. Zhang H, Wu H, Guan J, et al. Paracrine SDF-1alpha signaling mediates the effects of PSCs on GEM chemoresistance through an IL-6 autocrine loop in pancreatic cancer cells. Oncotarget. 2015;6:3085-97. 29. Cabrera MC, Tilahun E, Nakles R, et al. Human Pancreatic Cancer-Associated Stellate Cells Remain Activated after in vivo Chemoradiation. Frontiers in oncology. 2014;4:102. 30. Patel MB, Pothula SP, Xu Z, et al. The role of the hepatocyte growth factor/c-MET pathway in pancreatic stellate cell-endothelial cell interactions: antiangiogenic implications in pancreatic cancer. Carcinogenesis. 2014;35:1891-900. 31. Ene-Obong A, Clear AJ, Watt J, et al. Activated Pancreatic Stellate Cells Sequester CD8+ T-Cells to Reduce Their Infiltration of the Juxtatumoral Compartment of Pancreatic Ductal Adenocarcinoma. Gastroenterology. 2013. 32. Tang D, Gao J, Wang S, et al. Apoptosis and anergy of T cell induced by pancreatic stellate cells-derived galectin-1 in pancreatic cancer. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine. 2015. 33. Mace TA, Bloomston M, Lesinski GB. Pancreatic cancer-associated stellate cells: A viable target for reducing immunosuppression in the tumor microenvironment. Oncoimmunology. 2013;2:e24891. 34. Ma Y, Hwang RF, Logsdon CD, Ullrich SE. Dynamic mast cell-stromal cell interactions promote growth of pancreatic cancer. Cancer Res. 2013;73:3927-37. 35. Shi C, Washington MK, Chaturvedi R, et al. Fibrogenesis in pancreatic cancer is a dynamic process regulated by macrophage-stellate cell interaction. Lab Invest. 2014;94:409- 21. 36. Li X, Wang Z, Ma Q, et al. Sonic hedgehog paracrine signaling activates stromal cells to promote perineural invasion in pancreatic cancer. Clin Cancer Res. 2014;20:4326-38.

37. Kozono S, Ohuchida K, Eguchi D, et al. Pirfenidone inhibits pancreatic cancer desmoplasia by regulating stellate cells. Cancer Res. 2013;73:2345-56. 38. Froeling FE, Feig C, Chelala C, et al. Retinoic acid-induced pancreatic stellate cell quiescence reduces paracrine Wnt-beta-catenin signaling to slow tumor progression. Gastroenterology. 2011;141:1486-97, 97 e1-14. 39. Sherman MH, Yu RT, Engle DD, et al. Vitamin D receptor-mediated stromal reprogramming suppresses pancreatitis and enhances pancreatic cancer therapy. Cell. 2014;159:80-93. 40. Ramsay EE, Decollogne S, Joshi S, et al. Employing pancreatic tumor gamma- glutamyltransferase for therapeutic delivery. Molecular pharmaceutics. 2014;11:1500-11.