Cells free fulltext establishment and characterization of paired. Hpastec are responsible for the synthesis and the degradation of the extracellular matrix proteins that promote tissue repair. Interactions of pancreatic stellate cells with neighbou. This chapter will detail the methodology for isolation of quiescent pancreatic stellate cells from both rat and human pancreas. The stellate cells are a prime contributor to stromal fibrosis formation in pancreatic cancer 12, and key regulators in fibrogenesis during necroinflammation in chronic pancreatitis 14 16. For example, mmp9 is secreted as a zymogen and maintained in the latent form presumably by the interaction of a conserved cysteine in its nh 2terminal prodomain with the. These cells promote the malignant process and block immune surveillance. Vitamin d receptormediated stromal reprogramming suppresses pancreatitis and enhances pancreatic cancer therapy. The human pdac cell line capan2 and the immortalized human pancreatic stellate cell psc. In recent years, enormous scientific effort has been devoted to understanding their roles in pancreatic cancer, which continues. As part of it, pancreatic stellate cells pscs provide a fibrotic niche.
Impact of the secretome of activated pancreatic stellate. Extracellular matrix composition significantly influences. Hpsc, primary human pancreatic stellate cells from a patient with cancer. Pancreatic stellate cells pscs of the tumor stroma are known to interact with pancreatic cancer cells pccs and influence the progression of pdac through a complex network of signaling. However, whether and how rsv influences the biological properties of. To this end, we were first to profile the proteome of secreted proteins from resected pdac, dcca, adjacent normal pancreatic tissues, and cancer cell lines to identify novel tumor protein biomarkers. Pdf secretion of fibronectin by human pancreatic stellate. After pancreas damage and in the fibrotic stroma, quiescent pasc become activated and differentiate into a myofibroblast phenotype that synthesizes and secretes large amounts of.
Mass spectrometrybased quantitative proteomic profiling of. Vitamin d receptormediated stromal reprogramming suppresses. Coculture of rat pancreatic stellate cell line sips and panc1. Activation of hepatic stellate cells hscs and subsequent uncontrolled accumulation of altered extracellular matrix ecm underpin liver fibrosis, a wound healing response to chronic injury, which can lead to organ failure and death. This study was aimed at investigating molecular changes at the level of the proteome that are associated with the activation of pancreatic stellate cells by proinflammatory factors, namely tnf. In response to profibrogenic stimuli such as cytokines, growth factors and ethanol metabolites, psc undergo a phenotypic transition from a quiescent to an activated, myofibroblastlike stage 1. Pancreatic fibrosis, a prominent feature of chronic pancreatitis cp, induces persistent and permanent damage in the pancreas. Pancreatic stellate cells pscs are important players in pancreatic fibrosis and are major contributors to the extracellular matrix proteins observed with the stromal response characteristic of pancreatic ductal adenocarcinoma pdac. Multiplexed quantitative proteomics for highthroughput comprehensive proteome comparisons of human cell lines. Psc, pancreatic stellate cell full size image to evaluate the effect of pscs secreted soluble factors on cancer cell chemosensitivity, pccs were incubated with psccm for 24 h and analyzed for the response to the cytotoxic effects of gemcitabine using the mtt assay fig.
The images were analyzed by the scion image software version 4. Pancreatic stellate cell conditioned medium psccm protein. Isolation of quiescent pancreatic stellate cells from rat. Resveratrol rsv, a natural polyphenol, exhibits potent antioxidant and anticancer effects. The silap standard was prepared from proteins secreted by human pancreatic cell lines in culture, allowing for the relative quantification of a panel of pancreas specific protein biomarkers in serum. Periostin, secreted from stromal cells, has biphasic. Pascs are mediated by paracrine and autocrine stimuli and share similarities with the hepatic stellate cell. Pancreatic stellate cells pscs play a pivotal role in pancreatic fibrosis, a characteristic feature of pancreatic cancer. A total of 796 different proteins homo sapiens were identified, after the protein grouping performed by maxquant software. The pancreatic islets or islets of langerhans are the regions of the pancreas that contain its endocrine hormoneproducing cells, discovered in 1869 by german pathological anatomist paul langerhans. Pancreatic stellate cells pasc are emerging as key mediators in chronic pancreatitis and pancreatic cancer pathogenesis. Analysis of the human pancreatic stellate cell secreted proteome article in pancreas 404. Imagej software was used to calculate the area of the scratch.
Pancreatic stellate cells pascs are myofibroblastlike cells found in exocrine areas of the pancreas, and they play an important role in the pathogenesis of pancreatitis and pancreatic cancer 1,2,3. Mascot search results are combined using inhousedeveloped software. Liquid chromatographymass spectrometry author manuscript nih. Retinoic acidinduced pancreatic stellate cell quiescence. Pancreatic stellate cells demonstrate different proteomic and functional. Pancreatic stellate cells enhance stem cell like phenotypes in pancreatic cancer cells. Pancreatic stellate cell secreted il6 stimulates stat3. It remains difficult to characterize proteins secreted by a single cell type to the exclusion of all others in vivo. Termed stable isotope labeled proteome silap, the current study represents a novel implementation of this approach. The densitometric analysis was performed using labworks software uvp, cambridge, uk. Proteome variations in pancreatic stellate cells upon stimulation. Activated pancreatic stellate cells pasc are the main cell type in the stroma of chronic pancreatitis and pancreatic cancer and participate in the progression of these disorders 1, 2.
Pancreatic ductal adenocarcinoma pdac exists in a complex desmoplastic microenvironment. The migration capability of panc1 and pancco cells was. They are found adjacent to pancreatic acinar cells and around small pancreatic ducts and blood vessels. Upon achieving 90% confluence on the order of 10 7 cells, the growth media was aspirated and the cells were washed 3 times with icecold pbs. However, other important components of the pc stroma are inflammatory cells and endothelial cells. Proteomic analysis of extracellular matrix from the. The stellate cell is the major cell type involved in liver fibrosis, which is the formation of scar tissue in response to liver damage. Negative regulation of pancreatic stellate cell proliferation antibodies antibodies for proteins involved in negative regulation of pancreatic stellate cell proliferation pathways. Proteomic analysis of a rat pancreatic stellate cell line.
Mar 28, 2019 pancreatic ductal adenocarcinoma pdac exists in a complex desmoplastic microenvironment. Il6stat3 plays a regulatory role in the interaction between. Pancreatic tumor cells have been shown to express and secrete. All 4 stellate cell derivatives fs, primary normal human stellate cells. Isolation of quiescent pancreatic stellate cells from rat and. Stellate cells represent 58% of the total number of liver cells and 4% of pancreas cells. Pancreatic stellate cell research remains an untapped source of potential biomarker proteins. Relative quantification of serum proteins from pancreatic. Analysis of the human pancreatic stellate cell secreted proteome. Secretion of fibronectin by human pancreatic stellate cells. Pancreatic stellate cells pscs are important players in pancreatic fibrosis and. The liver is an immunologic organ consisting of parenchymal cells i. Pscs produce large amounts of il6, which promotes the accumulation of myeloidderived suppressor.
With the developing knowledge of this important cell type, we are at the cusp of developing effective therapies for the above diseases based upon targeting the psc and modulating its function. The pancreas serves two functions, endocrine and exocrine. Expression levels of proteins secreted by pccs and pscs were comparable to those. Mass spectrometrybased quantitative proteomic profiling. Dec 21, 2011 proteins secreted from activated pasc may be pivotal to the development of chronic pancreatitis. We have previously reported the identification of a pancreata mitoxantroneresistant cell population which expressed the abcg2 transporter with a pancreatic stellate cells phenotype pasc and ability of secreting insulin after inducing their differentiation. Pancreatic stellate cells demonstrate different proteomic and. Microfluidic coculture of pancreatic tumor spheroids with pscs may serve as a useful model for studying emt and drug resistance in a clinically relevant manner. The pancreatic acini are clusters of cells that produce digestive enzymes and secretions and make up the bulk of the pancreas.
Corticotropinreleasing factor receptors and stressrelated alterations of gut motor function yvette tache et al. Biology of pancreatic stellate cellsmore than just. Impact of the secretome of activated pancreatic stellate cells. Fibrosis is a major feature of chronic pancreatitis and desmoplasia, a stromal reaction characteristic of pancreatic ductal carcinoma cancer pdac. Proteome variations in pancreatic stellate cells upon. Our products were used in recent research regarding pancreatic stellate cells, a caf that is gaining considerable interest for new treatment options through advanced understanding of cancer biology. Activation of pancreatic stellate cells pscs initiates pancreatic fibrosis in chronic pancreatitis and furnishes a niche that enhances the malignancy of pancreatic cancer cells pccs in pancreatic ductal adenocarcinoma pdac. Pancreatic stellate cells pscs provide a major source of extracellular matrix ecm deposition during pancreatic injury, and persistent activation of pscs plays a vital role in the progression of pancreatic fibrosis.
Pancreatic stellate cells hpastec are the main fibroblastic cells of the pancreas. Pancreatic stellate cell secreted il6 stimulates stat3 dependent invasiveness of pancreatic intraepithelial neoplasia and cancer cells. Pancreatic stellate cell psc conditioned media cm activates stat3. As the existing in vitro experimental models offer only a limited resemblance to human pdac, there is a strong need for additional research tools to better understand pdac tumor biology, particularly the impact of the tumor stroma. The microarrays were produced as described previously in detail 29, 30. Il6stat3 plays a regulatory role in the interaction.
Psc secreted il6 activated stat3 signaling in noninvasive. Pancreatic stellate cells pscs, a major component of the tumor microenvironment in pancreatic cancer, play roles in cancer progression as well as drug resistance. It is now commonly accepted that these cells play a pivotal role in the desmoplastic reaction present in severe pancreatic disorders. Extracellular matrix composition significantly influences pancreatic stellate cell gene expression pattern. Liraglutide suppresses the metastasis of panc1 cocultured. The pancreatic islets constitute 1 to 2% of the pancreas volume and receive 1015% of its blood flow. Here, i focus on changes in the global proteome of cultured pancreatic cells when subjected to nicotine treatment. Conophylline cnp is a vinca alkaloid extracted from the leaves of the tropical plant, ervatamia microphylla, and has been shown to promote cell differentiation in pancreatic precursor cells. Pancreatic stellate cells pascs are classified as myofibroblastlike cells that are located in exocrine regions of the pancreas. Pancreatic stellate cell secreted il6 stimulates stat3 dependent invasiveness of pancreatic intraepithelial neoplasia and cancer cells nagaraj nagathihalli, jason a. We sought to catalogue the components of fibrotic liver ecm to obtain insights into disease etiology and aid identification of new biomarkers.
The diffuse stellate cell system is composed of star shaped cells woven into various mammalian organs including, but not limited to, the liver, pancreas or kidney 26, 32, 6. Culturing various cells in microfluidic microchannel devices has proven to be a useful in studying cellular interactions and drug sensitivity. Van saun, xizi dai, mahogany ambrose, qiaozhi guo, yanhua xiong, nipun merchant. Hepatic stellate cells, also known as ito cells, are pericytes found in the perisinusoidal space a small area between the sinusoids and hepatocytes of the liver. The exocrine pancreas normally harbors pscs in the preacinar and interlobular space as quiescent cells. Retinoic acid ameliorates pancreatic fibrosis and inhibits. Cells free fulltext establishment and characterization. Pancreatic stellate cells support tumour metabolism. Pscs produce large amounts of il6, which promotes the. The vitamin d receptor transcriptionally reprograms activated pancreatic stellate cells to a quiescent state to suppress pancreatitis as well as sensitize the stroma to conventional chemotherapy. Periostin, secreted from stromal cells, has biphasic effect on cell migration and correlates with the epithelial to mesenchymal transition of human pancreatic cancer cells. Pancreatic stellate cells support tumour metabolism through. An immortalized pancreatic stellate cell line from rat rattus norvegicus origin was propagated in dmem supplemented with 10% fetal bovine serum fbs. Activated pancreatic stellate cells pascs are the key cellular source of cancerassociated fibroblasts in the pancreatic stroma of patients with pancreatic ductal adenocarcinoma pdac, however, the activation mechanism of pascs is not yet known.
Regulation of pancreatic stellate cell activation by notch3. Isolation and culture of rat pancreatic stellate cells. Cafs are thus new drug targets for pancreatic and other cancer therapies. Pancreatic stellate cells, normally quiescent, are capable of remarkable transition into their activated myofibroblastlike phenotype. Microfluidic coculture of pancreatic tumor spheroids with. I investigate two padc cell lines, first a normal epithelial cell line, hpne human pancreatic nestinexpressing, and second a wellstudied pancreatic cancer cell line, panc1 pancreatic cancer 1. Proteins secreted from activated pasc may be pivotal to the development of chronic pancreatitis. Analysis of senescence markers in rodent pancreatic stellate. As part of it, pancreatic stellate cells pscs provide a fibrotic niche, stimulated by a dynamic.
Secretion of fibronectin by human pancreatic stellate cells promotes. Pancreatic ductal adenocarcinoma pdac is characterized by an extremely poor prognosis, and its treatment remains a challenge. While knowledge of the intracellular proteome is important, it is the secreted proteome, or secretome, that is of particular interest with regards to understanding intercellular interactions, for example with tumor cells. Pancreatic stellate cells enhance stem celllike phenotypes in pancreatic cancer cells. Cellular and molecular functions of hepatic stellate cells. Periostin, secreted from stromal cells, has biphasic effect. Immunoblots were quantified using imagej and analysis was performed with prism software graphpad software inc.
Jan 12, 2018 microfluidic coculture of pancreatic tumor spheroids with pscs may serve as a useful model for studying emt and drug resistance in a clinically relevant manner. Pancreatic stellate cell secreted il6 stimulates stat3 dependent. We explore the proteome of human hepatic stellate cells hhsc and human pancreatic stellate cells hpasc using mass spectrometry msbased quantitative proteomics to investigate pathophysiologic mechanisms. Pancreatic adenocarcinoma cells drive autophagy in tumour microenvironmentassociated stellate cells, which release alanine that is used by the cancer cells as a carbon source for a variety of. Proteomic analysis of extracellular matrix from the hepatic. However, it is also recognised that the results of studies of psc activation may be influenced by many different factors including stellate cell purity, culture media used and age of animals harvested or for humans donor age and disease normal pancreas, chronic pancreatitis, pancreatic cancer. As part of it, pancreatic stellate cells pscs provide a fibrotic niche, stimulated by a dynamic communication between activated pscs and tumour cells. Evidence suggests that fibrosis in both organs is partially regulated by organspecific stellate cells.
Periostin, secreted from stromal cells, has biphasic effect on cell migration and correlates with the epithelial to mesenchymal transition of human pancreatic cancer cells atsushi kanno division of gastroenterology, tohoku university graduate school of medicine, sendai city, miyagi, japan. Baseline stat3 level was obtained by immunoblotting total cell lysates from human left panel psc hpscs, pancreatic ductal cells h6c7, and pdac cells lines bxpc3, panc1, as well as mouse right panel psc mpscs, panin, pda and liver metastasis lmp cell lines. 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. Oncogenic kras regulates tumor cell signaling via stromal.
Proteins regulating the biomolecular pathways involved in the conversion of quiescent to activated pasc may have a significant influence on the development of chronic pancreatitis. Conophylline suppresses pancreatic cancer desmoplasia and. Analysis of senescence markers in rodent pancreatic. Although gain of oncogene functions and loss of tumor suppressor functions are driving forces in tumor development, the tumor microenvironment, comprising the extracellular matrix, surrounding stroma, signaling molecules and infiltrating immune and other cell populations, is now also recognized as crucial to tumor development and metastasis. Secretion of fibronectin by human pancreatic stellate. Sips1, spontaneously immortalized rat stellate cell line showed a reproducible change in d cell size and e counts as well as f a shift to the g. Venn diagram of secreted proteins identified from pancreatic stellate cells in. The characterization of the secretome of this cell population by twodimensional electrophoresis 2d coupled with mass spectrometry. Nicotine alters the proteome of two human pancreatic duct. Impact of the secretome of activated pancreatic stellate cells on. Analysis of the human pancreatic stellate cell secreted. Cancerassociated fibroblasts cafs are the main effector cells in the desmoplastic reaction, and pancreatic stellate cells are the most important source of cafs. Investigation of the roles of pasc in pancreatic disease may lead to a better understanding of the mechanism of fibrosis associated with chronic pancreatitis.
The characterization of the secretome of this cell population by twodimensional electrophoresis 2d coupled with. Resveratrol inhibits rospromoted activation and glycolysis. Analogously, pancreatic stellate cells pascs are located in the periacinar space of. Aug 10, 2016 pancreatic adenocarcinoma cells drive autophagy in tumour microenvironmentassociated stellate cells, which release alanine that is used by the cancer cells as a carbon source for a variety of. Activated pancreatic stellate cells pascs are the key cellular source of. Proteomics characterization of the secretome from rat. Regulation of pancreatic stellate cell activation by notch3 bmc.
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