Activation of JAK-STAT pathway is required for platelet-derived growth factor-induced proliferation of pancreatic stellate cells

AM: To clarify the role of Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway in platelet-derived growth factor (PDGF) induced proliferation in activated pancreatic stellate cells (PSCs). METHODS: PSCs were isolated from rat pancreas tissue, and used in their culture-activated, myofibroblast-like phenotype. STAT-specific binding activity was assessed by electrophoretic mobility shift assay. Activation of Src, JAK2, STAT1, STAT3, and ERK was determined by Western blotting using anti-phosphospecific antibodies. Cell proliferation was assessed by measuring the incorporation of 5-bromo-2'-deoxyuridine. RESULTS: PDGF-BB induced STAT-specific binding activity, and activation of Src, JAK2, STAT1, STAB, and ERK. Ethanol and acetaldehyde at clinically relevant concentrations decreased basal activation of JAK2 and STAT3. PDGF-induced activation of STAT1 and STAT3 was inhibited by a Src inhibitor PP1 and a JAK2 inhibitor AG490, whereas PDGF-induced activation of ERK was inhibited by PP1, and not by AG490. PDGF-induced proliferation was inhibited by PP1 and AG490 as well as by STAT3 antisense oligonucleotide. CONCLUSION: PDGF-BB activated JAK2-STAT pathway via Src-dependent mechanism. Activation of 3AK2-STAT3 pathway, in addition to ERK, may play a role in PDGF-induced proliferation of PSCs.

Endothelin-1 stimulates contraction and migration of rat pancreatic stellate cells

AIM： To examine the ability of FT-1 to affect the cell functions of PSCs and the underlying molecular mechanisms. METHODS： PSCs were isolated from the pancreas of male Wistar rats after perfusion with collagenase, and cells between passages two and five were used. Expression of ET-1 and FT receptors was assessed by reverse transcription-PCR and immunostaining. Phosphorylation of myosin regulatory light chain （MLC）, extracellular-signal regulated kinase （FRK）, and Akt was examined by Western blotting. Contraction of PSCs was assessed on hydrated collagen lattices. Cell migration was examined using modified Boyden chambers. Ceil proliferation was assessed by measuring the incorporation of 5-bromo-2＇- deoxyuridine. RESULTS： Culture-activated PSCs expressed ETA and ETB receptors, and ET-1. ET-1 induced phosphorylation of NLC and FRK, but not Akt. ET-1 induced contraction and migration, but did not alter proliferation of PSCs. FT-1-induced contraction was inhibited by an ETA receptor antagonist BQ-123 and an ETB receptor antagonist BQ-788, whereas migration was inhibited by BQ-788 but not by BQ-123. A Rho kinase inhibitor Y-27632 abolished both contraction and migration. CONCLUSION： ET-1 induced contraction and migration of PSCs through El receptors and activation of Rho-Rho kinase. ETA and FTB receptors play different roles in the regulation of these cellular functions in response to ET-1.

Green tea polyphenol epigallocatechin-3-gallate blocks PDGF-induced proliferation and migration of rat pancreatic stellate cells

AIM: To clarify the effects of epigallocatechin-3-gallate (EGCG) on the platelet-derived growth factor (PDGF)-BB-induced proliferation and migration of pancreatic stellate cells (PSCs). METHODS: PSCs were isolated from rat pancreas tissue and used in their culture-activated, myofibroblast-like phenotype. Cell proliferation was assessed by measuring the incorporation of 5-bromo-2'-deoxyuridine. Cell migration was assessed using modified Boyden chambers. Cyclin D1, p21waf1, and p27kip1 expression and phosphorylation of PDGF β-receptor, extracellular signal-regulated kinase, and Akt were examined by Western blotting. Activation of phospha-tidylinositol 3-kinase was examined by kinase assay using phosphatidylinositol as a substrate. Cell cycle was assessed by flow cytometry after staining with propidium iodide. RESULTS: EGCG at non-cytotoxic concentrations inhibited PDGF-induced proliferation and migration. This effect was associated with the inhibition of cell cycle progression beyond the G1 phase, decreased cyclin Dl and increased p27kip1 expression. EGCG inhibited tyrosine phosphorylation of PDGF p-receptor and downstream activation of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase/ Akt pathways. CONCLUSION: EGCG inhibited PDGFBB-induced proliferation and migration of PSCs through the inhibition of PDGFmediated signaling pathways.

YAP activates pancreatic stellate cells and enhances pancreatic fibrosis

Background:Pancreatic stellate cells(PSCs)foster the progression of pancreatic adenocarcinoma and chronic pancreatitis(CP)by producing a dense fibrotic stroma.However,the incomplete knowledge of PSCs biology hampers the exploration of antifibrotic therapies.Here,we explored the role of the Hippo pathway in the context of PSCs activation and experimental CP.Methods:CP model was created in rats with the tail vein injection of dibutyltin dichloride(DBTC).The expression of Yes-associated protein(YAP)in CP tissue was assessed.Primary and immortalized rats PSCs were treated with the YAP-inhibitor verteporfin.Furthermore,YAP siRNA was employed.Subsequently,DNA synthesis,cell survival,levels ofα-smooth muscle actin(α-SMA)protein,presence of lipid droplets and PSCs gene expression were evaluated.Upstream regulators of YAP signaling were studied by reporter gene assays.Results:In DBTC-induced CP,pronounced expression of YAP in areas of tubular structures and periduc-tal fibrosis was observed.Verteporfin diminished DNA replication in PSCs in a dose-dependent fash-ion.Knockdown of YAP reduced cell proliferation.Primary cultures of PSCs were characterized by a de-crease of lipid droplets and increased synthesis ofα-SMA protein.Both processes were not affected by verteporfin.At the non-cytotoxic concentration of 100 nmol/L,verteporfin significantly reduced mRNA levels of transforming growth factor-β1(Tgf-β1)and Ccn family member 1(Ccn1).YAP signaling was acti-vated by TGF-β1,but repressed by interferon-γ.Conclusions:Activated YAP enhanced PSCs proliferation.The antifibrotic potential of Hippo pathway in-hibitors warrants further investigation.

Molecular determinants of the profibrogenic effects of endothelin-1 in pancreatic stellate cells

AIM： To gain molecular insights into the expression and functions of endothelin-1 （ET-1） in pancreatic stellate cells （PSC）.METHODS： PSCs were isolated from rat pancreas tissue, cultured, and stimulated with ET-1 or other extracellular mediators. Cell proliferation was assessed by measuring the incorporation of 5-bromo-2＇-deoxyuridine into DNA and cell migration was studied in a transwell chamber assay. Gene expression at the level of mRNA was quantified by real-time Polymerase chain reaction. Expression and phosphorylation of proteins were monitored by immunoblotting, applying an infrared imaging technology. ET-1 levels in cell culture supernatants were determined by an enzyme immunometric assay. To study DNA binding of individual transcription factors, electrophoretic mobility shift assays were performed.RESULTS： Among several mediators tested, transforming growth factor-β1 and tumour necrosis factor-α displayed the strongest stimulatory effects on ET-1 secretion. The cytokines induced binding of Smad3 and NF-κB, respectively, to oligonucleotides derived from the ET-1 promoter, implicating both transcription factors in the induction of ET-1 gene expression. In accordance with previous studies, ET-1 was found to stimulate migration but not proliferation of PSC. Stimulation of ET-1 receptors led to the activation of two distinct rnitogen-activated protein kinases, p38 and extracellular signal-regulated kinases （ERK）1/2, as well as the transcription factor activator protein-1. At the mRNA level, enhanced expression of the PSC activation marker, α-smooth muscle actin and two proinflammatory cytokines, interleukin （IL）-1β and IL-6, was observed. CONCLUSION： This study provides novel lines of evidence for profibrogenic and proinflammatory actions of ET-1 in the pancreas, encouraging further studies with ET-1 inhibitors in chronic pancreatitis.

Modified methods for isolation of pancreatic stellate cells from human and rodent pancreas

Primary cultures of pancreatic stellate cells（PSCs） remain an important basis for in vitro study.However,effective methods for isolating abundant PSCs are currently lacking.We report on a novel approach to isolating PSCs from normal rat pancreases and human pancreatic ductal adenocarcinoma（PDAC） tissue.After anaesthesia and laparotomy of the rat,a blunt cannula was inserted into the pancreatic duct through the anti-mesentery side of the duodenum,and the pancreas was slowly infused with an enzyme solution until all lobules were fully dispersed.The pancreas was then pre-incubated,finely minced and incubated to procure a cell suspension.PSCs were obtained after the cell suspension was filtered,washed and subject to gradient centrifugation with Nycodenz solution.Fresh human PDAC tissue was finely minced into 1×1×l mm^3 cubes with sharp blades.Tissue blocks were placed at the bottom of a culture plate with fresh plasma（EDTA-anti-coagulated plasma from the same patient,mixed with CaCL） sprinkled around the sample.After culture for 5-10 days under appropriate conditions,activated PSCs were harvested.An intraductal perfusion of an enzyme solution simplified the procedure of isolation of rat PSCs,as compared with the multiple injections technique,and a modified outgrowth method significantly shortened the outgrowth time of the activated cells.Our modification in PSC isolation methods significantly increased the isolation efficiency and shortened the culture period,thus facilitating future PSC-related research.

Roles of Smad3 and Smad7 in rat pancreatic stellate cells activated by transforming growth factor-beta 1

BACKGROUND: Pancreatic stellate cells (PSCs) play a major role in promoting pancreatic fibrosis. Transforming growth factor beta 1 (TGF-beta 1) is a critical mediator of this process. This study aimed to determine the expression of the Smad3 and Smad7 genes in the process of PSC activation, and explore the mechanisms of chronic pancreatitis. METHODS: The expressions of Smad3 and Smad7 in PSCs before and after TGF-beta 1 treatment were detected by reverse transcription-polymerase chain reaction and Western blotting analysis. Smad3 expression was detected in PSCs after treatment with 5 ng/ml of TGF-beta 1 for 24 hours. RESULTS: Smad7 expression was decreased in TGF-beta 1 -activated PSCs (P<0.05) in a dose-dependent manner. When TGF-beta 1 concentration reached 10 ng/ml, the expression of p-Smad3, Smad3, and Smad7 was inhibited (P<0.05). CONCLUSIONS: TGF-beta 1 promotes the expression of Smad3 and inhibits the expression of Smad7 during the activation of PSCs. In contrast, high-dose TGF-beta 1 downregulates the expression of Smad3 in completely activated PSCs.

Pancreatic stellate cells promote proliferation and invasiveness of human pancreatic cancer cells via galectin-3

AIM: To investigate the role of pancreatic stellate cells (PSCs) and galectin-3 (GAL-3) in the proliferation and infiltration of pancreatic cancer cell line SW1990. METHODS: Human pancreatic cancer cell line SW1990 and PSCs were cultured in vitro . Supernatant fluid of cultured PSCs and SW1990 cells was collected. Expression of GAL-3 in SW1990 cells and PSCs was detected by ELISA, RT-PCR and Western blotting. Proliferation of cultured PSCs and SW1990 cells was measured by 3-(4, 5-methylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. Infiltration of SW1990 cells was detected by a cell infiltration kit. RESULTS: SW1990 cells expressed GAL-3 and this was up-regulated by the supernatant fluid of cultured PSCs. PSCs did not express GAL-3. SW1990 cells stimulated proliferation of PSCs via GAL-3. GAL-3 antibody inhibited SW1990 cell proliferation, while the supernatant fluid of PSCs stimulated proliferation of SW1990 cells through interaction with GAL-3 protein. The supernatant fluid of PSCs enhanced the invasiveness of SW1990 cells through interaction with GAL-3. CONCLUSION: GAL-3 and PSCs were involved in the proliferation and infiltration process of pancreatic cancer cells.

Effects of angiotensin Ⅱ receptor antagonist, Losartan on the apoptosis, proliferation and migration of the human pancreatic stellate cells

AIM： To investigate the effects of AT, （Type 1 angiotensin Ⅱ receptor） antagonist （Losartan） on the apoptosis, proliferation and migration of the human pancreatic stellate cells （hPSCs）. METHODS： hPSCs were isolated from pancreatic sample of patients with pancreatic carcinoma using radioimmunoassay （RIA） technique to detect the concentration of AngⅡ in culture media and cell homogenate. Immunocytochemistry （ICC） and in situ hybridization （ISH） methods were utilized to test AT1 expression in hPSCs. Effects of Losartan on hPSCs proliferation, apoptosis and migration were investigated using BrdU incorporation, TUNEL, flow cytometry （FCM）, and phase-contrast microscope separately when cells treated with Losartan. Immunofluorescence and Western blot were applied to quantify the expression of type Ⅰ collagen in hPSCs. RESULTS： There exists AT1 expression in hPSCs, while no AngⅡ was detected in culture media and cell homogenate. Losartan induces cell apoptosis in a doseand time-dependent manner （apparently at 10^-5 mol/L）, no pro-proliferative effect was observed in the same condition. Corresponding dosage of Losartan can also alleviate the motion capability and type Ⅰ collagen content of hPSCs compared with AngⅡ treatment and non-treatment control groups. CONCLUSION： These findings suggest that paracrine not autocrine functions of AngⅡ may have effects on hPSCs, which was mediated by AT1 expressed on cells, while Losartan may exert anti-fibrotic effects by inhibiting hPSCs motion and partly by inducing apoptosis.

Distinct antifibrogenic effects of erlotinib,sunitinib and sorafenib on rat pancreatic stellate cells

AIM: To study if three clinically available small molecule kinase inhibitors (SMI), erlotinib, sunitinib and sorafenib, exert antifibrogenic effects on pancreatic stellate cells (PSC) and analyze the basis of their action.

ROLE OF PANCREATIC STELLATE CELLS AND GALECTIN-3 ON PROLIFERATION AND INFILTRATION OF HUMAN PANCREATIC CANCER CELL LINE SW1990

Objective To investigate the role of pancreatic stellate cells (PSCs) and galectin-3 (GAL-3) on the proliferation and infiltration of pancreatic cancer cell line SW1990. Methods Human pancreatic cancer cell line SW1990 and PSCs were cultured in vitro. Supernatant of cultured PSCs and SW1990 cells was collected. Expressions of GAL-3 in SW1990 cells and PSCs were detected by ELISA, RT-PCR and Western blot. The proliferation of those cultured PSCs and SW1990 cells were measured by MTT assay and flowcytometry. Infiltration of SW1990 cells was detected by cell infiltration kit. Results SW1990 cells expressed GAL-3 and the expression was up-regulated by the supernatant fluid of cultured PSCs. PSCs did not express GAL-3. SW1990 cells could stimulate the proliferation of PSCs via GAL-3. GAL-3 antibody could inhibit SW1990 cells proliferation and infiltration, which indicated that supernatant of PSCs might stimulate the proliferation of SW1990 cells through the interaction with GAL-3 protein. The supernatant fluid of PSCs could enhance the invasiveness of SW1990 cells through the interaction with GAL-3. Conclusion GAL-3 and PSCs was involved in the proliferation and infiltration process of pancreatic cancer.

Thromboxane A2 receptor contributes to the activation of rat pancreatic stellate cells induced by 8-epi-prostaglandin F2α

Background:Pancreatic stellate cells(PSCs)activation plays a critical role in the development of chronic pancreatitis.Previous studies confirmed that thromboxane A2 receptor(TxA2r)was overexpressed in activated PSCs in rats.The purpose of this study was to investigate the role of TxA2r in the activation of PSCs induced by 8-epi-prostaglandin F2α(8-epi-PGF2α).Methods:TxA2r expression in both quiescent and activated PSCs was detected by immunocytochemistry and immunoblot assay.Isolated PSCs were treated with 8-epi-PGF2α(10^-6,10^-7,10^-8 mol/L)for 48 h,and SQ29548(10^-4,10^-6,and 10^-7 mol/L),a TxA2r-specific antagonist,for 48 h,respectively,to identify the drug concentration with the best biological effect and the least cytotoxicity.Then isolated PSCs were treated with SQ29548(10^-4 mol/L)for 2 h,followed by 10^-7 mol/L 8-epi-PGF2α for 48 h.Real-time polymerase chain reaction was performed to detect the messenger RNA(mRNA)levels of a-smooth muscle actin(a-SMA)and collagen I.Comparisons between the groups were performed using Student’s t test.Results:TxA2r was up-regulated in activated PSCs in vitro compared with quiescent PSCs(all P<0.001).Compared with the control group,different concentrations of 8-epi-PGF2α significantly increased mRNA levels of a-SMA(10^-6 mol/L:2.23±0.18 vs.1.00±0.07,t=10.70,P<0.001;10^-7 mol/L:2.91±0.29 vs.1.01±0.08,t=10.83,P<0.001;10^-8 mol/L,1.67±0.07 vs.1.00±0.08,t=11.40,P<0.001)and collagen I(10^-6 mol/L:2.68±0.09 vs.1.00±0.07,t=24.94,P<0.001;10^-7 mol/L:2.12±0.29 vs.1.01±0.12,t=6.08,P<0.001;10^-8 mol/L:1.46±0.15 vs.1.00±0.05,t=4.93,P=0.008).However,different concentrations of SQ29548 all significantly reduced the expression of collagen I(10^-4 mol/L:0.55±0.07 vs.1.00±0.07,t=10.47,P<0.001;10^-6 mol/L:0.56±0.10 vs.1.00±0.07,t=6.185,P<0.001;10^-7 mol/L:0.27±0.04 vs.1.00±0.07,t=15.41,P<0.001)and a-SMA(10^-4 mol/L:0.06±0.01 vs.1.00±0.11,t=15.17,P<0.001;10^-6 mol/L:0.28±0.03 vs.1.00±0.11,t=11.29,P<0.001;10^-7 mol/L:0.14±0.04 vs.1.00±0.11,t=12.86,P<0.001).After being treated with SQ29548(10^-4 mol/L)and then 8-epi-PGF2α(10^-7 mol/L),the mRNA levels of a-SMA(0.20±0.08 vs.1.00±0.00,t=17.46,P<0.001)and collagen I(0.69±0.13 vs.1.00±0.00,t=4.20,P=0.014)in PSCs were significantly lower than those of the control group.Conclusions:The results show that 8-epi-PGF2α promoted PSCs activation,while SQ29548 inhibited PSCs activation induced by 8-epi-PGF2α.The result indicated that TxA2r plays an important role during PSC activation and collagen synthesis induced by 8-epi-PGF2α in vitro.This receptor may provide a potential target for more effective antioxidant therapy for pancreatic fibrosis.

Inhibition of pancreatic stellate cell activity by adipose-derived stem cells

BACKGROUND：Pancreatic stellate cells（PSCs）play a critical role in the development of pancreatic fibrosis.In this study we used a novel method to isolate and culture rat PSCs and then investigated the inhibitory effects of adipose-derived stem cells（ADSCs）on activation and proliferation of PSCs.METHODS：Pancreatic tissue was obtained from SpragueDawley rats for PSCs isolation.Transwell cell cultures were adopted for co-culture of ADSCs and PSCs. PSCs prolifera- tion and apoptosis were determined using CCK-8 and flow cytometry, respectively, a-SMA expressions were analyzed using Western blotting. The levels of cytokines [nerve growth factor （NGF）, interleukin-10 （IL-10） and transforming growth factor-ill （TGF-[31）] in conditioned medium were detected by ELISA. Gene expression （MMP-2, MMP-9 and TIMP-1） was analyzed using qRT-PCR. RESULTS： This method produced 17.6_＋6.5 ~ 103 ceils per gram of the body weight with a purity of 90%-95% and a viability of 92%-97%. Co-culture of PSCs with ADSCs significantly inhib- ited PSCs proliferation and induced PSCs apoptosis. Moreover, a-SMA expression was significantly reduced in PSCs＋ADSCs compared with that in PSC-only cultures, while expression of fibrinolytic proteins （e.g., MMP-2 and MMP-9） was upregulated and anti-fibrinolytic protein （TIMP-1） was downregulated. In addition, NGF expression was up-regulated, but IL-10 and TGF-β1 expressions were down-regulated in the coculture conditioned medium compared with those in the PSC- only culture medium. CONCLUSIONS： This study provided an easy and reliable technique to isolate PSCs. The data demonstrated the inhibitory effects of ADSCs on the activation and proliferation of PSCs in vitro.

Key players in pancreatic cancer-stroma interaction: cancer-associated fibroblasts, endothelial and inflammatory cells

Pancreatic cancer(PC) is the most aggressive type of common cancers, and in 2014, nearly 40000 patients died from the disease in the United States. Pancreatic ductal adenocarcinoma, which accounts for the majority of PC cases, is characterized by an intense stromal desmoplastic reaction surrounding the cancer cells. Cancer-associated fibroblasts(CAFs) are the main effector cells in the desmoplastic reaction, and pancreatic stellate cells are the most important source of CAFs. However, other important components of the PC stroma are inflammatory cells and endothelial cells. The aim of this review is to describe the complex interplay between PC cells and the cellular and noncellular components of the tumour stroma. Published data have indicated that the desmoplastic stroma protects PC cells against chemotherapy and radiation therapy and that it might promote the proliferation and migration of PC cells. However, in animal studies, experimental depletion of the desmoplastic stroma and CAFs has led to more aggressive cancers. Hence, the precise role of the tumour stroma in PC remains to be elucidated. However, it is likely that a contextdependent therapeutic modification, rather than pure depletion, of the PC stroma holds potential for the development of new treatment strategies for PC patients.

Inhibitory effects of interferons on pancreatic stellate cell activation

AIM： To analyze and to compare the effects of interferon （IFN）-α, IFN-β, and IFN-γ on pancreatic stellate cell （PSC） activation/n vitro and to elucidate the molecular basis of IFN action. METHODS： PSCs were isolated from rat＇s pancreatic tissue, cultured and stimulated with recombinant rat IFNs. Cell proliferation and collagen synthesis were assessed by measuring the incorporation of 5-bromo-2＇ -deoxyuridine （BrdU） into DNA and [^3H]-proline into acetic acid-soluble proteins, respectively. Apoptotic ceils were determined by FACS analysis （sub-G1 peak method）. Exhibition of the myofibroblastic PSC phenotype was monitored by immunoblot analysis of （α-smooth muscle actin （α-SMA） expression. To assess the activation of signal transducer and activator of transcription （STAT）, Western blots using phospho- STAT-specific antibodies were performed. In studies on STAT1 function, expression of the protein was inhibited by siRNA. RESULTS： IFN-β and IFN-γ, but not IFN-α significantly diminished PSC proliferation and collagen synthesis. IFN-γ, was the only IFN that clearly inhibited α-SMA expression. Under the experimental conditions used, no enhanced rate of apoptotic cell death was observed in response to any IFN treatment. IFN-β and IFN-γ, induced a strong increase of STAT1 and STAT3 tyrosine phosphorylation, while the effect of IFN-α was much weaker. Inhibition of STAT1 expression with siRNA was associated with a significantly reduced growth-inhibitory effect of IFN-γ. CONCLUSION： IFN-β and particularly IFN-γ, display inhibitory effects on PSC activation in vitro and should be tested regarding their in vitro efficiency. Growth inhibition by IFN-γ action requires STAT1.

An immortalized rat pancreatic stellate cell line RP-2 as a new cell model for evaluating pancreatic fibrosis,inflammation and immunity

BACKGROUND： Pancreatic stellate cells （PSCs） play a critical role in the pathogenesis of pancreatic fibrosis and have emerging functions as progenitor cells, immune cells or intermediaries in pancreatic exocrine secretion. Increasing evidence has shown that desmin as an exclusive cytoskeleton marker of PSC is only expressed in part of these cells. This study was to establish a desmin-positive PSC cell line and evaluate its actions on pancreatic fibrosis, inflammation and immunity.

Pancreatic cancer stroma:understanding biology leads to new therapeutic strategies

Pancreatic ductal adenocarcinoma(PDA)is among the deadliest cancers in the United States and in the world.Late diagnosis,early metastasis and lack of effective therapy are among the reasons why only 6%of patients diagnosed with PDA survive past 5 years.Despite development of targeted therapy against other cancers,little progression has been made in the treatment of PDA.Therefore,there is an urgent need for the development of new treatments.However,in order to proceed with treatments,the complicated biology of PDA needs to be understood first.Interestingly,majority of the tumor volume is not made of malignant epithelial cells but of stroma.In recent years,it has become evident that there is an important interaction between the stromal compartment and the less prevalent malignant cells,leading to cancer progression.The stroma not only serves as a growth promoting source of signals but it is also a physical barrier to drug delivery.Understanding the tumor-stroma signaling leading to development of desmoplastic reaction and tumor progression can lead to the development of therapies to decrease stromal activity and improve drug delivery.In this review,we focus on how the current understanding of biology of the pancreatic tumor microenvironment can be translated into the development of targeted therapy.

Pancreatic cancer and its stroma: A conspiracy theory

Pancreatic cancer is characterised by a prominent desmoplastic/stromal reaction that has received little attention until recent times. Given that treatments focusing on pancreatic cancer cells alone have failed to significantly improve patient outcome over many decades, research efforts have now moved to understanding the pathophysiology of the stromal reaction and its role in cancer progression. In this regard, our Group was the first to identify the cells(pancreatic stellate cells, PSCs) that produced the collagenous stroma of pancreatic cancer and to demonstrate that these cells interacted closely with cancer cells to facilitate local tumour growth and distant metastasis. Evidence is accumulating to indicate that stromal PSCs may also mediate angiogenesis, immune evasion and the well known resistance of pancreatic cancer to chemotherapy and radiotherapy. This review will summarise current knowledge regarding the critical role of pancreatic stellate cells and the stroma in pancreatic cancer biologyand the therapeutic approaches being developed to target the stroma in a bid to improve the outcome of this devastating disease.

PSC-induced Galectin-1 Promotes the Malignant Behavior of Pancreatic Ductal Adenocarcinoma

Background Galectin-1 is aβ-galactoside-binding protein overexpressed in the pancreatic stellate cells(PSCs)of pancreatic ductal adenocarcinoma(PDAC),while its expression is typically low in pancreatic cancer cells(PCCs).The point at which galectin-1 expression in PCCs increases,and its association with PDAC progression,have been unclear.Methods Galectin-1 expression in PDAC and metastatic lymph nodes was investigated using an immunohistochemical assay.PANC-1 PCC cells were co-cultured with PSCs expressing different levels of galectin-1.Subsequently,galectin-1 was overexpressed in PANC-1 cells using recombinant lentiviruses,and their proliferation,invasion,anchorage-independent growth,and in vivo tumorigenicity were evaluated.Results There was intermediate galectin-1 expression in PCCs,and it was positively associated with galectin-1 expression in PSCs in the PDAC tissues.Galectin-1 was strongly expressed in the metastatic lymph nodes.In the co-culture,high galectin-1 expression in the PSCs increased the galectin-1 expression in the PANC-1 cells.The galectin-1 overexpression in the PANC-1 cells enhanced their clone formation ability,proliferation,and invasion,increased the expression of proliferating cell nuclear antigen(PCNA)and BCL-2,and decreased Bax expression,promoting the establishment and growth of tumors.Conclusion High galectin-1 expression in PSCs induces galectin-1 expression in PCCs and subsequently promotes the malignant biological behavior of PDAC.

Pancreatic cancer organotypics: High throughput, preclinical models for pharmacological agent evaluation

Pancreatic cancer carries a terrible prognosis,as the fourth most common cause of cancer death in the Western world.There is clearly a need for new therapies to treat this disease.One of the reasons no effective treatment has been developed in the past decade may in part,be explained by the diverse influences exerted by the tumour microenvironment.The tumour stroma cross-talk in pancreatic cancer can influence chemotherapy delivery and response rate.Thus,appropriate preclinical in vitro models which can bridge simple 2D in vitro cell based assays and complex in vivo models are required to understand the biology of pancreatic cancer.Here we discuss the evolution of 3D organotypic models,which recapitulare the morphological and functional features of pancreatic ductal adenocarcinoma(PDAC).Organotypic cultures are a valid high throughput preclinical in vitro model that maybe a useful tool to help establish new therapies for PDAC.A huge advantage of the organotypic model system is that any component of the model can be easily modulated in a short timeframe.This allows new therapies that can target the cancer,the stromal compartment or both to be tested in a model that mirrors the in vivo situation.A major challenge for the future is to expand the cellular composition of the organotypic model to further develop a system that mimics the PDAC environment more precisely.We discuss how this challenge is being met to increase our understanding of this terrible disease and develop novel therapies that can improve the prognosis for patients.