Abstract
Background
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.
Purpose
(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.
Methods
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.
Results
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.
Conclusions
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.
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References
Apte MV, Haber PS, Applegate TL et al (1998) Periacinar stellate shaped cells in rat pancreas: identification, isolation, and culture. Gut 43:128–133
Bachem MG, Schneider E, Gross H et al (1998) Identification, culture, and characterization of pancreatic stellate cells in rats and humans. Gastroenterology 115:421–432
Erkan M, Adler G, Apte MV et al. StellaTUM: current consensus and discussion on pancreatic stellate cell research. Gut 2011
Hwang RF, Moore T, Arumugam T et al (2008) Cancer-associated stromal fibroblasts promote pancreatic tumor progression. Cancer Res 68:918–926
Vonlaufen A, Joshi S, Qu C et al (2008) Pancreatic stellate cells: partners in crime with pancreatic cancer cells. Cancer Res 68:2085–2093
Vonlaufen A, Phillips PA, Xu Z et al (2008) Pancreatic stellate cells and pancreatic cancer cells: an unholy alliance. Cancer Res 68:7707–7710
Bailey JM, Swanson BJ, Hamada T et al (2008) Sonic hedgehog promotes desmoplasia in pancreatic cancer. Clin Cancer Res 14:5995–6004
Olive KP, Jacobetz MA, Davidson CJ et al (2009) Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer. Science 324:1457–1461
Sherman MH, Yu RT, Engle DD et al (2014) Vitamin d receptor-mediated stromal reprogramming suppresses pancreatitis and enhances pancreatic cancer therapy. Cell 159:80–93
Erkan M, Michalski CW, Rieder S et al (2008) The activated stroma index is a novel and independent prognostic marker in pancreatic ductal adenocarcinoma. Clin Gastroenterol Hepatol 6:1155–1161
Erkan M, Weis N, Pan Z et al (2010) Organ-, inflammation- and cancer specific transcriptional fingerprints of pancreatic and hepatic stellate cells. Mol Cancer 9:88
Vonlaufen A, Phillips PA, Yang L et al (2010) Isolation of quiescent human pancreatic stellate cells: a promising in vitro tool for studies of human pancreatic stellate cell biology. Pancreatology 10:434–443
Bachem MG, Schunemann M, Ramadani M et al (2005) Pancreatic carcinoma cells induce fibrosis by stimulating proliferation and matrix synthesis of stellate cells. Gastroenterology 128:907–921
Algul H, Treiber M, Lesina M, Schmid RM (2007) Mechanisms of disease: chronic inflammation and cancer in the pancreas—a potential role for pancreatic stellate cells? Nat Clin Pract Gastroenterol Hepatol 4:454–462
Arumugam T, Brandt W, Ramachandran V et al (2011) Trefoil factor 1 stimulates both pancreatic cancer and stellate cells and increases metastasis. Pancreas 40:815–822
Jesnowski R, Furst D, Ringel J et al (2005) Immortalization of pancreatic stellate cells as an in vitro model of pancreatic fibrosis: deactivation is induced by matrigel and N-acetylcysteine. Lab Invest 85:1276–1291
Mathison A, Liebl A, Bharucha J et al (2010) Pancreatic stellate cell models for transcriptional studies of desmoplasia-associated genes. Pancreatology 10:505–516
Paulo JA, Urrutia R, Banks PA et al (2011) Proteomic analysis of a rat pancreatic stellate cell line using liquid chromatography tandem mass spectrometry (LC-MS/MS). J Proteomics 75:708–717
Paulo JA, Urrutia R, Banks PA et al (2011) Proteomic analysis of an immortalized mouse pancreatic stellate cell line identifies differentially-expressed proteins in activated vs nonproliferating cell states. J Proteome Res 10:4835–4844
Satoh M, Masamune A, Sakai Y et al (2002) Establishment and characterization of a simian virus 40-immortalized rat pancreatic stellate cell line. Tohoku J Exp Med 198:55–69
Sparmann G, Hohenadl C, Tornoe J et al (2004) Generation and characterization of immortalized rat pancreatic stellate cells. Am J Physiol Gastrointest Liver Physiol 287:G211–G219
Apte MV, Park S, Phillips PA et al (2004) Desmoplastic reaction in pancreatic cancer: role of pancreatic stellate cells. Pancreas 29:179–187
Farrow B, Rowley D, Dang T, Berger DH (2009) Characterization of tumor-derived pancreatic stellate cells. J Surg Res 157:96–102
Acknowledgments
We want to thank Monika Meinhardt, Dariel Thereska, Xiao Yu, and Annette Heller for technical assistance and helpful discussions about experiments and data.
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O.S. was supported by a grant of the German Research Foundation (STR 690/3-1). N.D. was supported by a LGFG scholarship (Landesgraduiertenförderungsgesetz, Baden-Württemberg).
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All authors declare that they have no conflict of interest.
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All procedures performed in studies involving human samples were approved by and in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.
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Informed consent was obtained from all patients before tissue collection.
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Oliver Strobel and Nigora Dadabaeva contributed equally to this work.
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Strobel, O., Dadabaeva, N., Felix, K. et al. Isolation and culture of primary human pancreatic stellate cells that reflect the context of their tissue of origin. Langenbecks Arch Surg 401, 89–97 (2016). https://doi.org/10.1007/s00423-015-1343-6
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DOI: https://doi.org/10.1007/s00423-015-1343-6