Human Pancreatic Stellate Cells Research Articles

Kindlin-2 in pancreatic stellate cells promotes the progression of pancreatic cancer

Pancreatic stellate cells (PSCs) play a pivotal role in pancreatic fibrosis associated with pancreatic ductal adenocarcinoma (PDAC). Kindlin-2 is a focal adhesion protein that regulates the activation of integrins. This study aimed to clarify the role of kindlin-2 in PSCs in pancreatic cancer. Kindlin-2 expression in 79 resected pancreatic cancer tissues was examined by immunohistochemical staining. Kindlin-2-knockdown immortalized human PSCs were established using small interfering RNA. Pancreatic cancer cells were treated with conditioned media of PSCs, and the cell proliferation and migration were examined. SUIT-2 pancreatic cancer cells were subcutaneously injected into nude mice alone or with PSCs and the size of the tumors was monitored. Kindlin-2 expression was observed in PDAC and the peritumoral stroma. Stromal kindlin-2 expression was associated with shorter recurrence-free survival time after R0 resection. Knockdown of kindlin-2 resulted in decreased proliferation, migration, and cytokine expression in PSCs. The PSC-induced proliferation and migration of pancreatic cancer cells were suppressed by kindlin-2 knockdown in PSCs. In vivo, co-injection of PSCs increased the size of the tumors, but this effect was abolished by kindlin-2 knockdown in PSCs. In conclusion, kindlin-2 in PSCs promoted the progression of pancreatic cancer.

Abstract A21: Chemokine directed migration of activated stellate cells in pancreatic ductal adenocarcinoma

Abstract Multiple studies have shown that the extreme desmoplasia observed in pancreatic tumors modulates the effectiveness of chemotherapeutic agents. Despite this, the specific mechanisms by which stroma develops in pancreatic tumors are unknown and the role that it plays in pathogenesis of the disease is unsettled. Chemokines are a family of nearly 50 different secreted proteins playing a key role in the recruitment of a wide variety of cell types in health and disease. While we and others have extensively explored the role of the chemokine CXCL12 in pancreatic cancer, the expression and potential role of other chemokines, or their cognate receptors, in pancreatic cancer remains an area of active exploration. To this end, we profiled the expression of the chemokine family in pancreatic cancer. RT-PCR of a battery of established human pancreatic cancer cell lines revealed elevated transcript levels of the chemokine ligands CCL28 and CXCL16, as well as their cognate receptors CCR10 and CXCR6, respectively. These results were mirrored in cell lines derived from transgenic murine models of pancreatic cancer. Immunohistochemical analysis of both diseased and normal human pancreatic tissue showed that the expression of CCL28, CXCL16, CCR10, and CXCR6 proteins were upregulated in pancreatic adenocarcinoma compared with normal exocrine ductal epithelium. Interestingly, CCR10 and CXCR6 were also expressed in pancreatic tumor fibroblasts, while their cognate ligands, CCL28 and CXCL16 were absent in the stromal compartment. Based on those data we hypothesized that expression of chemokines in cancer cells with the concomitant expression of their cognate receptors by activated fibroblasts facilitates recruitment of those cancer-associated fibroblasts into the tumor microenvironment. Analysis of tissue from patients with pancreatitis revealed that CCL28/CCR10, but not CXCL16/CXCR6, proteins were upregulated. Consistent with its known regulation by inflammatory mediators, we found that interferon-gamma stimulated significant increases in CCL28 secretion by human pancreatic cancer cell lines. In contrast, interferon-gamma had little if any effect on CCL28 secretion by an immortalized activated human pancreatic stellate cell (HPSC) line. Stimulation with exogenous CCL28 elicited the prototypical dose dependent calcium signaling expected of a ligand activated chemokine receptor. CCL28 stimulated the dose dependent directional migration of HPSC without altering cell viability. These data are consistent with HPSC being a functional target for the chemokine. Finally, in a 2-D co-culture model, CCL28 secreted from human pancreatic cancer cells induced HPSC chemotactic migration. Pretreatment with neutralizing antibody to the chemokine blocked HPSC chemotaxis toward the cancer cells. Together, these data suggest that the CCL28-CCR10 chemokine axis plays a role in the inflammation-mediated recruitment of cancer-associated fibroblasts into the stromal compartment of pancreatic tumors. More broadly, we have demonstrated a new role for chemokines in the pancreatic tumor microenvironment, suggestive of their potential role in stromal remodeling in pancreatic cancer. This is the first study to establish the differential expression and functional role for the CCL28 chemokine in pancreatic disease. Citation Format: Ishan Roy, Kathleen A. Boyle, A Craig Mackinnon, Rosa F. Hwang, Susan Tsai, Douglas B. Evans, Michael B. Dwinell.{Authors}. Chemokine directed migration of activated stellate cells in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A21.

Abstract A18: The integrin αvβ6 regulates PDAC cell growth and stromal cell behavior

Abstract Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death in the UK. Five-year survival rates are less than 3% and haven’t improved over the last 40 years. Thus novel approaches are required urgently. We have found that over 90% of PDAC express αvβ6 and high expression of the integrin β6 gene (ITGB6) correlates with a significant reduction (Hazard Ratio 2.07; Logrank p=3.17x10-8) in overall survival from PDAC. Thus we have examined the biological functions mediated by αvβ6 in PDAC cell lines. A panel of human PDAC cell lines was incubated with αvβ6-blocking antibodies in order to define the role of αvβ6 in vitro. Some cell lines were driven into G2/M cell cycle arrest (Panc0403), whilst others died through apoptosis (CfPac1). Moreover, adhesion, migration and invasion through Matrigel were suppressed by blockade of αvβ6. Treatment of subcutaneous tumors composed of human αvβ6-positive cell lines in combination with human pancreatic stellate cells, with an αvβ6-blocking antibody resulted in significantly reduced tumor volume and growth rate in vivo, via modulation of the stromal compartment. In combination, these data suggested that targeting αvβ6 may be an effective therapeutic strategy in PDAC. To investigate the biological roles and molecular bases of αvβ6 in PDAC, cell lines were generated from PDAC tumors of a novel PDAC transgenic mouse. Two primary cell populations from PDAC tumors, abbreviated as KVC1 and KPCD3, demonstrated both epithelial and mesenchymal characteristics. Thus, cells were subsequently sorted by FACS to obtain high-αvβ6 expressing monoclonal cell lines. Resultant monoclonal cell lines expressed higher levels of αvβ6 than the parental line and expressed only epithelial markers. Transduction with lentivirus particles containing shRNA to itgb6 under a tetracycline-inducible promoter (SMARTvector, Dharmacon) was optimized for four high-αvβ6 expressing monoclonal cell lines, and doxycycline-induced knock-down of β6 protein was 70-80%, confirmed using FACS and western blotting. In vitro functional assays demonstrated that knock-down of itgb6 reduced invasion of Matrigel. To assess the role of αvβ6 in an assay that better reflected the PDAC environment, we developed 3D organotypic assays with inducible knockout PDAC combined with mouse embryo fibroblasts or mouse pancreatic stellate cells. Doxycycline treatment reduced PDAC tumor cell growth and invasion, but also reduced the contraction of the collagen-Matrigel gel matrix. Since αvβ6 activates TGFβ, and as TGFβ promotes fibroblast and stellate cell contractile propensity, current studies will determine whether this is the mechanism of how αvβ6 regulates stromal cell behavior. Citation Format: Claire S. Reader, Jennifer P. Morton, Owen J. Sansom, John F. Marshall.{Authors}. The integrin αvβ6 regulates PDAC cell growth and stromal cell behavior. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A18.

Exosomes Derived From Pancreatic Stellate Cells: MicroRNA Signature and Effects on Pancreatic Cancer Cells.

Pancreatic stellate cells (PSCs) interact with pancreatic cancer cells in the tumor microenvironment. Cell constituents including microRNAs may be exported from cells within membranous nanovesicles termed exosomes. Exosomes might play a pivotal role in intercellular communication. This study aimed to clarify the microRNA signature of PSC-derived exosomes and their effects on pancreatic cancer cells. Exosomes were prepared from the conditioned medium of immortalized human PSCs. MicroRNAs were prepared from the exosomes and their source PSCs, and the microRNA expression profiles were compared by microarray. The effects of PSC-derived exosomes on proliferation, migration, and the mRNA expression profiles were examined in pancreatic cancer cells. Pancreatic stellate cell-derived exosomes contained a variety of microRNAs including miR-21-5p. Several microRNAs such as miR-451a were enriched in exosomes compared to their source PSCs. Pancreatic stellate cell-derived exosomes stimulated the proliferation, migration and expression of mRNAs for chemokine (C - X - C motif) ligands 1 and 2 in pancreatic cancer cells. The stimulation of proliferation, migration, and chemokine gene expression by the conditioned medium of PSCs was suppressed by GW4869, an exosome inhibitor. We clarified the microRNA expression profile in PSC-derived exosomes. Pancreatic stellate cell-derived exosomes might play a role in the interactions between PSCs and pancreatic cancer cells.

Human pancreatic stellate cells modulate 3D collagen alignment to promote the migration of pancreatic ductal adenocarcinoma cells.

A hallmark of pancreatic ductal adenocarcinoma (PDAC) is the ability for cancer cells to aggressively infiltrate and navigate through a dense stroma during the metastatic process. Key features of the PDAC stroma include an abundant population of activated pancreatic stellate cells (PSCs) and highly aligned collagen fibers; however, important questions remain regarding how collagen becomes aligned and what the biological manifestations are. To better understand how PSCs, aligned collagen, and PDAC cells might cooperate during the transition to invasion, we utilized a microchannel-based in vitro tumor model and advanced imaging technologies to recreate and examine in vivo-like heterotypic interactions. We found that PSCs participate in a collaborative process with cancer cells by orchestrating the alignment of collagen fibers that, in turn, are permissive to enhanced cell migration. Additionally, direct contact between PSCs, collagen, and PDAC cells is critical to invasion and co-migration of both cell types. This suggests PSCs may accompany and assist in navigating PDAC cells through the stromal terrain. Together, our data provides a new role for PSCs in stimulating the metastatic process and underscores the importance of collagen alignment in cancer progression.

Epithelial to Stromal Re-Distribution of Primary Cilia during Pancreatic Carcinogenesis.

BackgroundThe Hedgehog (HH) pathway is a mediator in pancreatic ductal adenocarcinoma (PDAC). Surprisingly, previous studies suggested that primary cilia (PC), the essential organelles for HH signal transduction, were lost in PDAC. The aim of this study was to determine the presence of PC in human normal pancreas, chronic pancreatitis, and during carcinogenesis to PDAC with focus on both epithelia and stroma.MethodsPC were analyzed in paraffin sections from normal pancreas, chronic pancreatitis, intraductal papillary-mucinous neoplasia, and PDAC, as well as in primary human pancreatic stellate cells (PSC) and pancreatic cancer cell lines by double immunofluorescence staining for acetylated α-tubuline and γ-tubuline. Co-staining for the HH receptors PTCH1, PTCH2 and SMO was also performed.ResultsPC are gradually lost during pancreatic carcinogenesis in the epithelium: the fraction of cells with PC gradually and significantly decreased from 32% in ducts of normal pancreas, to 21% in ducts of chronic pancreatitis, to 18% in PanIN1a, 6% in PanIN2, 3% in PanIN3 and to 1.2% in invasive PDAC. However, this loss of PC in the neoplastic epithelium is accompanied by a gain of PC in the surrounding stroma. The fraction of stromal cells with PC significantly increased from 13% around normal ducts to about 30% around PanIN and PDAC. HH-receptors were detected in tumor stroma but not in epithelial cells. PC are also present in PSC and pancreatic cancer cell lines.ConclusionPC are not lost during pancreatic carcinogenesis but re-distributed from the epithelium to the stroma. This redistribution may explain the re-direction of HH signaling towards the stroma during pancreatic carcinogenesis.

Insulin promotes proliferation and fibrosing responses in activated pancreatic stellate cells.

Epidemiological studies support strong links between obesity, diabetes, and pancreatic disorders including pancreatitis and pancreatic adenocarcinoma (PDAC). Type 2 diabetes (T2DM) is associated with insulin resistance, hyperglycemia, and hyperinsulinemia, the latter due to increased insulin secretion by pancreatic beta-cells. We reported that high-fat diet-induced PDAC progression in mice is associated with hyperglycemia, hyperinsulinemia, and activation of pancreatic stellate cells (PaSC). We investigated here the effects of high concentrations of insulin and glucose on mouse and human PaSC growth and fibrosing responses. We found that compared with normal, pancreata from T2DM patients displayed extensive collagen deposition and activated PaSC in islet and peri-islet exocrine pancreas. Mice fed a high-fat diet for up to 12 mo similarly displayed increasing peri-islet fibrosis compared with mice fed control diet. Both quiescent and activated PaSC coexpress insulin (IR; mainly A type) and IGF (IGF-1R) receptors, and both insulin and glucose modulate receptor expression. In cultured PaSC, insulin induced rapid tyrosine autophosphorylation of IR/IGF-1R at specific kinase domain activation loop sites, activated Akt/mTOR/p70S6K signaling, and inactivated FoxO1, a transcription factor that restrains cell growth. Insulin did not promote activation of quiescent PaSC in either 5 mM or 25 mM glucose containing media. However, in activated PaSC, insulin enhanced cell proliferation and augmented production of extracellular matrix proteins, and these effects were abolished by specific inhibition of mTORC1 and mTORC2. In conclusion, our data support the concept that increased local glucose and insulin concentrations associated with obesity and T2DM promote PaSC growth and fibrosing responses.

The TGFβ-SMAD3 pathway inhibits IL-1α induced interactions between human pancreatic stellate cells and pancreatic carcinoma cells and restricts cancer cell migration.

BackgroundThe most abundant cells in the extensive desmoplastic stroma of pancreatic adenocarcinomas are the pancreatic stellate cells, which interact with the carcinoma cells and strongly influence the progression of the cancer. Tumor stroma interactions induced by IL-1α/IL-1R1 signaling have been shown to be involved in pancreatic cancer cell migration. TGFβ and its receptors are overexpressed in pancreatic adenocarcinomas. We aimed at exploring TGFβ and IL-1α signaling and cross-talk in the stellate cell cancer cell interactions regulating pancreatic adenocarcinoma cell migration.MethodsHuman pancreatic stellate cells were isolated from surgically resected pancreatic adenocarcinomas and cultured in the presence of TGFβ or pancreatic adenocarcinoma cell lines. The effects of TGFβ were blocked by inhibitors or amplified by silencing the endogenous inhibitor of SMAD signaling, SMAD7. Pancreatic stellate cell responses to IL-1α or to IL-1α-expressing pancreatic adenocarcinoma cells (BxPC-3) were characterized by their ability to stimulate migration of cancer cells in a 2D migration model.ResultsIn pancreatic stellate cells, IL-1R1 expression was found to be down-regulated by TGFβ and blocking of TGFβ signaling re-established the expression. Endogenous inhibition of TGFβ signaling by SMAD7 was found to correlate with the levels of IL-1R1, indicating a regulatory role of SMAD7 in IL-1R1 expression. Pancreatic stellate cells cultured in the presence of IL-1α or in co-cultures with BxPC-3 cells enhanced the migration of cancer cells. This effect was blocked after treatment of the pancreatic stellate cells with TGFβ. Silencing of stellate cell expression of SMAD7 was found to suppress the levels of IL-1R1 and reduce the stimulatory effects of IL-1α, thus inhibiting the capacity of pancreatic stellate cells to induce cancer cell migration.ConclusionsTGFβ signaling suppressed IL-1α mediated pancreatic stellate cell induced carcinoma cell migration. Depletion of SMAD7 upregulated the effects of TGFβ and reduced the expression of IL-1R1, leading to inhibition of IL-1α induced stellate cell enhancement of carcinoma cell migration. SMAD7 might represent a target for inhibition of IL-1α induced tumor stroma interactions.

Abstract B22: Necuparanib inhibits pancreatic cancer progression and invasion in a 3D tumor and stromal cell co-culture system

Abstract Pancreatic cancer is one of the most aggressive types of cancer, with only about 5% of patients surviving 5 years past the initial diagnosis. Despite advances with new chemotherapy combinations, overall survival outcomes are still dismal due to the cancer's complex pathology. Novel multi-targeted agents or therapeutic combinations able to disrupt the aggressive fibrotic microenvironment and restrict tumor proliferation are a current focus in the field. Necuparanib (formerly M402) is a heparan sulfate-like molecule that binds and inhibits multiple heparin-binding growth factors, chemokines, and adhesion molecules in cancer progression and metastasis and as such has been shown to modulate multiple pathways in tumors. In order to further explore how necuparanib affects the tumor and its microenvironment, a 3-dimensional (3D) culture system mimicking pancreatic cancer was developed containing a co-culture of human pancreatic tumor cells and pancreatic stellate cells, the most abundant stromal cell in the pancreas. Morphological and histological assessment of the 3D tumor spheroids demonstrated that necuparanib inhibits a) pancreatic tumor cell growth, b) pancreatic tumor cell invasion, and c) pancreatic stellate cell growth. Necuparanib's activity in these assays was shown to be dose-dependent. Moreover, using a negative control for necuparanib engineered to lack the ability to bind growth factors, we established that necuparanib's activity is growth factor binding dependent. The ability to inhibit both tumor and stromal cells demonstrates that necuparanib is a very promising multi-targeting agent for difficult-to-treat cancers such as desmoplastic pancreatic tumors. These studies aid in our understanding of the key biological targets and pathways responsible for necuparanib's anti-tumor effects and support the current Phase 2 safety and efficacy study being performed in patients with metastatic pancreatic cancer. Citation Format: Amanda MacDonald, Michelle Priess, Jennifer Curran, Silva Krause. Necuparanib inhibits pancreatic cancer progression and invasion in a 3D tumor and stromal cell co-culture system. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B22.

Abstract 1318: Pancreatic cancer therapy using polyion micelles co-loaded with cyclopamine and paclitaxel

Abstract Sonic hedgehog (SHH) signaling pathway is an important regulator during the initiation and progression of pancreatic cancer, and sustained activation of this pathway contributes to excessive deposition of tumor stroma and maintenance of tumor-initiating cells. Cyclopamine, a potent inhibitor of the SHH pathway, has shown promising antitumor efficacy. However, cyclopamine is insoluble in water and is highly toxic, and cyclopamine monotherapy did not effectively inhibit tumor growth in a range of preclinical pancreatic cancer models. We hypothesized that polyion complex polymeric micelles that simultaneously deliver cyclopamine and paclitaxel (M-CPA/PTX) would display greater efficacy against pancreatic tumor than micelles loaded with cyclopamine alone (M-CPA) or paclitaxel alone (M-PTX). We tested this hypothesis in cell cultures and in an orthotopic human pancreatic cancer Miapaca-2 xenograft model and a genetically engineered mouse model of pancreatic ductal carcinoma (PDAC), the KPC model. Our results showed that M-CPA interrupted the SHH pathway by antagonizing the smoothened receptor, reduced expression of Gli-1 transcription factor, and inhibited proliferation of Miapaca-2 cells and stroma-producing human pancreatic stellate cells. In Miapaca-2 cells, M-CPA decreased the fraction of CD133+ cells (cancer stem cells) and decreased tumorsphere formation, whereas M-TXL increased the fraction of CD133+ cells and tumorsphere formation. M-CPA negated the stimulation of human pancreatic stellate cells by exogenous SHH ligands. In vivo, M-CPA/PTX reduced tumor cell proliferation, depleted tumor stroma, and increased tumor vasculature density, leading to effective inhibition of tumor growth in Miapaca-2 xenografts. In the KPC model, M-CPA/PTX significantly prolonged media survival of mice with PDAC compared to non-treatment control mice. In conclusion, M-CPA/PTX was an effective strategy to treat pancreatic cancer. (supported in part by the Viragh Family Foundation and the John S. Dunn Foundation) Citation Format: Jun Zhao, Chunhui Wu, Xiaoxia Wen, Junjie Li, Qian Huang, Ying Ma, Stephen Ullrich, Huamin Wang, Jason Fleming, Chun Li. Pancreatic cancer therapy using polyion micelles co-loaded with cyclopamine and paclitaxel. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1318.

Profile of MMP and TIMP Expression in Human Pancreatic Stellate Cells: Regulation by IL-1α and TGFβ and Implications for Migration of Pancreatic Cancer Cells

Pancreatic ductal adenocarcinoma is characterized by a prominent fibroinflammatory stroma with both tumor-promoting and tumor-suppressive functions. The pancreatic stellate cell (PSC) is the major cellular stromal component and the main producer of extracellular matrix proteins, including collagens, which are degraded by metalloproteinases (MMPs). PSCs interact with cancer cells through various factors, including transforming growth factor (TGF)β and interleukin (IL)-1α. The role of TGFβ in the dual nature of tumor stroma, i.e., protumorigenic or tumor suppressive, is not clear. We aimed to investigate the roles of TGFβ and IL-1α in the regulation of MMP profiles in PSCs and the subsequent effects on cancer cell migration. Human PSCs isolated from surgically resected specimens were cultured in the presence of pancreatic cancer cell lines, as well as IL-1α or TGFβ. MMP production and activities in PSCs were quantified by gene array transcripts, mRNA measurements, fluorescence resonance energy transfer–based activity assay, and zymography. PSC-conditioned media and pancreatic cancer cells were included in a collagen matrix cell migration model. We found that production of IL-1α by pancreatic cancer cells induced alterations in MMP and tissue inhibitors of matrix metalloproteinase (TIMP) profiles and activities in PSCs, upregulated expression and activation of MMP1 and MMP3, and enhanced migration of pancreatic cancer cells in the collagen matrix model. TGFβ counteracted the effects of IL-1α on PSCs, reestablished PSC MMP and TIMP profiles and activities, and inhibited migration of cancer cells. This suggests that tumor TGFβ has a role as a suppressor of stromal promotion of tumor progression through alterations in PSC MMP profiles with subsequent inhibition of pancreatic cancer cell migration.

The radiosensitizing effects of Nelfinavir on pancreatic cancer with and without pancreatic stellate cells

AimsWe have previously shown in a phase I trial that nelfinavir (NFV) is safe with chemoradiation in PDAC with good signs for efficacy. Reverse translationally, we aimed to test the influence of PSCs on nelfinavir mediated radiosensitization to PDAC preclinically, because PDAC is very rich in desmoplasia and PSCs are known to mediate radioresistance. MethodsIn a direct co-culture model of several PDAC cell lines with PSC we performed clonogenic assays +/− nelfinavir. This was repeated exposing cells to hypoxic conditions. In xenograft PDAC tumors we tested radiation +/− nelfinavir +/− PSC. ResultsNFV sensitized both, PDAC only and PDAC cocultured with PSC (PDAC: Panc-1, MiaPaCa-2, PSN-1). In Panc-1 and PSN-1 this effect was larger +PSC compared to −PSC. Human pancreatic stellate cells (hPSC) were also sensitized by NFV which reduced p-FAK levels in hPSC, an effect that we previously found to sensitize specifically PDAC/PSC coculture. Contrarily, LY294002 reduced p-Akt in PSC (hPSC and LTC-14) but had no impact on PSC radiation survival. In vitro, nelfinavir sensitized Panc-1 and PSN-1 under normoxic and hypoxic conditions. In PSN-1 xenografts, +PSC led to faster tumor regrowth after radiation vs −PSC. The regrowth delay effect of nelfinavir after radiation was dramatically larger +PSC vs −PSC (time to reach 250mm3 183% vs 22%). ConclusionNFV mediated radiosensitization in PDAC with stroma is partly mediated by p-FAK inhibition (Chen et al., 2013). In vitro, NFV sensitizes both normoxic and hypoxic PDAC +/− PSC to a roughly similar extent. The dramatic increased effect of xenograft regrowth inhibition by nelfinavir in tumors with PSC is attributed to vascular normalization (Brunner et al., 2014) rather than direct modification of hypoxia as shown by the tumor regrowth after gemcitabine with NFV.

MicroRNA-199a and -214 as potential therapeutic targets in pancreatic stellate cells in pancreatic tumor.

Pancreatic stellate cells (PSCs) are the key precursor cells for cancer-associated fibroblasts (CAFs) in pancreatic tumor stroma. In this study, we explored miRNA as therapeutic targets in tumor stroma and found miR-199a-3p and miR-214-3p induced in patient-derived pancreatic CAFs and TGF-β-activated human PSCs (hPSCs). Inhibition of miR-199a/-214 using hairpin inhibitors significantly inhibited TGFβ-induced differentiation markers (e.g. α-SMA, collagen, PDGFβR), migration and proliferation. Furthermore, heterospheroids of Panc-1 and hPSCs attained smaller size with hPSCs transfected with anti-miR-199a/-214 compared to control anti-miR. The conditioned medium obtained from TGFβ-activated hPSCs induced tumor cell growth and endothelial cell tube formation. Interestingly, these inductions were abrogated in hPSCs transfected with anti-miR-199a or miR-214. Moreover, IPA analyses revealed signaling pathways related to miR-199a (TP53, mTOR, Smad1) and miR-214 (PTEN, Bax, ING4). Taken together, this study reveals miR-199a-3p and miR-214-3p as major regulators of PSC activation and PSC-induced pro-tumoral effects, representing them as key therapeutic targets in pancreatic cancer.

IL-6/STAT3 Plays a Regulatory Role in the Interaction Between Pancreatic Stellate Cells and Cancer Cells.

Pancreatic stellate cells (PSCs) play a pivotal role in pancreatic fibrosis, a characteristic feature of pancreatic cancer. Although it is still controversial, previous studies have suggested that PSCs promote the progression of pancreatic cancer by regulating the cell functions of cancer cells. PSCs produce large amounts of IL-6, which promotes the accumulation of myeloid-derived suppressor cells via a signal transducers and activator of transcription 3 (STAT3)-dependent mechanism. But the role of IL-6/STAT3 pathway in the interaction between PSCs and pancreatic cancer cells remains largely unknown. To clarify the role of IL-6/STAT3 in the interaction between PSCs and cancer cells. Human pancreatic cancer cells (Panc-1 and SUIT-2 cells) were treated with conditioned medium of immortalized human PSCs (PSC-CM). The effects of PSC-CM and IL-6 neutralization on the mRNA expression profiles were examined using Agilent's microarray. Activation of STAT3 was assessed by Western blotting using an anti-phospho-specific antibody. Cellular migration was examined by a two-chamber assay. The expression of markers related to epithelial-mesenchymal transition (EMT) was assessed by real-time reverse transcription PCR. PSC-CM induced the activation of STAT3 in pancreatic cancer cells. Neutralization of IL-6 suppressed the PSC-CM-induced upregulation of genes including complement factor B, lipocalin, and chemokine (C-C motif) ligand 20. Inhibition of IL-6/STAT3 pathway by anti-IL-6 antibody or a STAT3 inhibitor (NSC74859) inhibited the PSC-CM-induced migration and the expression of EMT-related markers (Snail and cadherin-2) in pancreatic cancer cells. IL-6/STAT3 pathway regulates the PSC-induced EMT and alterations in gene expression in pancreatic cancer cells.

The role and related mechanism of YAP on invasion and desmoplastic reaction in pancreatic cancer

Pancreatic cancer (PC), a hypovascular tumor, thrives under hypoxic conditions. Pancreatic stellate cells (PSCs) promote PC progression by secreting soluble factors, but their functions in hypoxia are poorly understood. This study aimed to clarify the effects of hypoxic conditions on the interaction between PC cells and PSCs.We isolated human PSCs from fresh pancreatic ductal adenocarcinomas and analyzed functional differences in PSCs between normoxia (21% O2) and hypoxia (1% O2), including expression of various factors related to tumor–stromal interactions. We particularly analyzed effects on PC invasiveness of an overexpressed molecule—connective tissue growth factor (CTGF)—in PSCs under hypoxic conditions, using RNA interference techniques.Conditioned media from hypoxic PSCs enhanced PC cell invasiveness more intensely than that from normoxic PSCs (P < 0.01). When co-cultured with PSCs, PC cell invasion was more enhanced under hypoxia than under normoxia (P < 0.05). Among various soluble factors, which were related to invasiveness, CTGF was one of the overexpressed molecules in hypoxic PSCs. A higher level of CTGF expression was also found in supernatant of hypoxic PSCs than in supernatant of normoxic PSCs. PC cell invasiveness was reduced by CTGF knockdown in hypoxic PSCs co-cultured with PC cells (P < 0.05).Hypoxia induces PSCs' secretion of CTGF, leading to enhancement of PC invasiveness. CTGF derived from hypoxia-stimulated PSCs may be a new therapeutic target for pancreatic cancer.

Isolation and culture of primary human pancreatic stellate cells that reflect the context of their tissue of origin.

Pancreatic stellate cells (PSCs) play a critical role in pancreatic ductal adenocarcinoma (PDAC). Activated PSCs are the main source of fibrosis in chronic pancreatitis and of desmoplasia in PDAC. The majority of studies on PSC are based on in vitro experiments relying on immortalized cell lines derived from diseased human pancreas or from animal models. These PSCs are usually activated and may not represent the biological context of their tissue of origin. (1) To isolate and culture primary human PSC from different disease contexts with minimal impact on their state of activation. (2) To perform a comparative analysis of phenotypes of PSC derived from different contexts. PSCs were isolated from normal pancreas, chronic pancreatitis, and PDAC using a hybrid method of digestion and outgrowth. To minimize activation by serum compounds, cells were cultured in a low-serum environment (2.5 % fetal bovine serum (FBS)). Expression patterns of commonly used markers for PSC phenotype and activity were compared between primary PSC lines derived from different contexts and correlated to expression in their original tissues. Isolation was successful from 14 of 17 tissues (82 %). Isolated PSC displayed stable viability and phenotype in low-serum environment. Expression profiles of isolated PSC and matched original tissues were closely correlated. PDAC-derived PSC tended to have a higher status of activation if compared to PSC derived from non-cancerous tissues. Primary human PSCs isolated from different contexts and cultured in a low-serum environment maintain a phenotype that reflects the stromal activity present in their tissue of origin.

Anti-IL-6 and PD-L1 antibody combination therapy reduces tumor progression in murine models of pancreatic cancer

Meeting abstracts Checkpoint inhibition has gained traction as an immunotherapeutic approach. However, limited efficacy has been observed in patients with pancreatic ductal adenocarcinoma (PDAC). Prior studies by our group have indicated that human pancreatic stellate cells (PSC), a major component

Isolation and characterization of human islet stellate cells

Background and aimsWe have previously demonstrated that islet stellate cells (ISCs) exhibiting a similar phenotype to classical pancreatic stellate cells (PSCs) could be isolated from rat islets, where they may contribute to islet fibrosis in type 2 diabetes mellitus (T2DM). This study was designed to determine whether human islets also contain ISC. Materials and methodsUsing standard explants techniques, human ISCs were enriched from freshly isolated human islets. Immunofluorescence visualization of markers for PSCs(α-smooth muscle actin;α-SMA), desmin, vimentin, glial fibrillary acidic protein (GFAP) was used to characterize the human ISC. Cell counting kit-8 (CCK-8) was used to assess the proliferation of ISC. The wound-healing assay and the transwell migration were used to assess the migration capacity of ISC. Immunofluorescence against collagen typesI (col-I), collagen typesIII (col-III) and fibronectin (FN) was performed to identify extracellular matrix (ECM) component synthesized by ISC. Adipogenic and osteogenic differentiation were tried to detected stem cell potential. ResultsIn culture, ISC with triangular shape grow out from human islets. The passaged ISC expressed α-SMA, desmin, vimentin, GFAP and was positive for col-I, col-III and FN. The proliferation and migration ability of ISC was significantly slower than those of PSC. And both the human PSC and ISC were able to differentiate in vitro into adipocyte- and osteoblast-like cells. ConclusionSimilar to our previous rat experiment, the current study shows that human islets also contain ISC which is phenotypically similar but not identical to human PSC.

Primary outgrowth cultures are a reliable source of human pancreatic stellate cells

Recent advances demonstrate a critical yet poorly understood role for the pancreatic stellate cell (PSC) in the pathogenesis of chronic pancreatitis (CP) and pancreatic cancer (PC). Progress in this area has been hampered by the availability, fidelity, and/or reliability of in vitro models of PSCs. We examined whether outgrowth cultures from human surgical specimens exhibited reproducible phenotypic and functional characteristics of PSCs. PSCs were cultured from surgical specimens of healthy pancreas, CP and PC. Growth dynamics, phenotypic characteristics, soluble mediator secretion profiles and co-culture with PC cells both in vitro and in vivo were assessed. Forty-seven primary cultures were established from 52 attempts, demonstrating universal α-smooth muscle actin and glial fibrillary acidic protein but negligible epithelial surface antigen expression. Modification of culture conditions consistently led to cytoplasmic lipid accumulation, suggesting induction of a quiescent phenotype. Secretion of growth factors, chemokines and cytokines did not significantly differ between donor pathologies, but did evolve over time in culture. Co-culture of PSCs with established PC cell lines resulted in significant changes in levels of multiple secreted mediators. Primary PSCs co-inoculated with PC cells in a xenograft model led to augmented tumor growth and metastasis. Therefore, regardless of donor pathology, outgrowth cultures produce PSCs that demonstrate consistent growth and protein secretion properties. Primary cultures from pancreatic surgical specimens, including malignancies, may represent a reliable source of human PSCs.

Alcohol and cigarette smoke components activate human pancreatic stellate cells: implications for the progression of chronic pancreatitis.

Chronic pancreatitis, a known complication of alcohol abuse, is characterized histopathologically by prominent fibrosis. Pancreatic stellate cells (PSCs) are responsible for producing this fibrous tissue in chronic pancreatitis and are activated by alcohol. Progression of alcoholic chronic pancreatitis (as assessed by calcification and fibrosis) is thought to be facilitated by concurrent smoking, but the mechanisms are unknown. This study aimed to (a) determine whether human PSCs (hPSCs) and rat PSCs express nicotinic acetylcholine receptors (nAChRs), which are known to bind 2 important components of cigarette smoke, namely nicotine and nicotine-derived nitrosamine ketone (NNK), and (b) examine the effects of cigarette smoke components in the presence and absence of alcohol on PSC activation in vitro. Western blotting was used to detect the presence of nAChRs in primary cultures of PSCs. Clinically relevant concentrations of cigarette smoke components (either cigarette smoke extract [CSE], NNK, or nicotine) ± ethanol (EtOH) were used to treat primary cultures of PSCs, and stellate cell activation was assessed by cell migration, proliferation, collagen production, and apoptosis. We demonstrate, for the first time, that PSCs express nAChRs (isoforms α3, α7, β, ε) and that the expression of the α7 isoform in hPSCs is induced by CSE + EtOH. We also provide novel findings that PSCs are activated by CSE and NNK (both alone and in combination with EtOH) as evidenced by an increase in cell migration and/or proliferation. Further, we demonstrate that activation of PSCs by CSE + EtOH and NNK + EtOH may be mediated via nAChRs on the cells. PSCs are activated by clinically relevant concentrations of cigarette smoke components (CSE and NNK), alone and in combination with EtOH. Thus, in alcoholics who smoke, progression of pancreatic fibrosis may be facilitated by the combined effects of alcohol and cigarette smoke components on hPSC behavior.