Immortalization of pancreatic stellate cells as an in vitro model of pancreatic fibrosis: deactivation is induced by matrigel and N-acetylcysteine

Jesnowski, Ralf; Fürst, Daniel E.; Ringel, Jörg; Chen, Ying; Schrödel, Andrea; Kleeff, Jörg; Kolb, Armin; Schareck, W.; Löhr, Matthias

doi:10.1038/labinvest.3700329

Cited by 132 publications

(141 citation statements)

References 64 publications

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“…Slides were analysed using the Axioplan 2 imaging microscope (Carl Zeiss Light Microscope, Göttingen, Germany). For immunocytochemistry, immortalised primary human pancreatic stellate cells (IPSCs) (Jesnowski et al, 2005) and Panc-1 cells were seeded on SuperFrost microscope slides (Menzel GmbH & Co KG, Braunschweig, Germany) overnight till adherent, and fixed with 3.5% paraformaldehyde for 25 min, and quenched with 30 mM glycine/PBS for 5 min. Permeabilisation of the cell membrane was carried out with 0.1% Triton X-100 for 5 min at room temperature.…”

Section: Immunohistochemistry and Immunocytochemistrymentioning

confidence: 99%

Regulation and functional role of the Runt-related transcription factor-2 in pancreatic cancer

et al. 2007

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Recent evidence suggests that Runt-related transcription factors play a role in different human tumours. In the present study, the localisation of the Runt-related transcription factor-2 (Runx2), its transcriptional activity, as well as its regulation of expression was analysed in human pancreatic ductal adenocarcinoma (PDAC). Quantitative real-time PCR and immunohistochemistry were used for Runx2 expression and localisation analysis. Runt-related transcription factor-2 expression was silenced using specific siRNA oligonucleotides in pancreatic cancer cells (Panc-1) and immortalised pancreatic stellate cells (IPSCs). Overexpression of Runx2 was achieved using a full-length expression vector. TGF-b1, BMP2, and other cytokines were assessed for their potential to regulate Runx2 expression. There was a 6.1-fold increase in median Runx2 mRNA levels in PDAC tissues compared to normal pancreatic tissues (Po0.0001). Runt-related transcription factor-2 was localised in pancreatic cancer cells, tubular complexes, and PanIN lesions of PDAC tissues as well as in tumour-associated fibroblasts/stellate cells. Coculture of IPSCs and Panc-1 cells, as well as treatment with TGF-b1 and BMP2, led to increased Runx2 expression in Panc-1 cells. Runt-related transcription factor-2 overexpression was associated with decreased MMP1 release as well as decreased growth and invasion of Panc-1 cells. These effects were reversed by Runx2 silencing. In conclusion, Runx2 is overexpressed in PDAC, where it is regulated by certain cytokines such as TGF-b1 and BMP2 in an auto-and paracrine manner. In addition, Runx2 has the potential to regulate the transcription of extracellular matrix modulators such as SPARC and MMP1, thereby influencing the tumour microenvironment.

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Section: Immunohistochemistry and Immunocytochemistrymentioning

confidence: 99%

Regulation and functional role of the Runt-related transcription factor-2 in pancreatic cancer

et al. 2007

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“…L3.6p cells were provided by Dr Isaiah Fidler (MD Anderson) (16). RLT-PSC human pancreatic stellate cells (PSCs) were provided by Dr Ralf Jesenofsky (University of Heidelberg) (17); HPSC-T human PSCs were provided by Dr Rosa Hwang (MD Anderson) (18); LTC-14 rat PSCs were provided by Dr Gisele Sparman (University Hospital of Rostock) (19) and imPSC mouse PSCs were provided by Dr Raul Urrutia (Mayo Clinic) (20). Both human and murine PSCs were obtained directly from the labs that isolated the cells and were functionally validated by their expression patterns in the indicated studies.…”

Section: Methodsmentioning

confidence: 99%

TGF-β-induced stromal CYR61 promotes resistance to gemcitabine in pancreatic ductal adenocarcinoma through downregulation of the nucleoside transporters hENT1 and hCNT3

Hesler¹,

Huang²,

Starr³

et al. 2016

CARCIN

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Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer in part due to inherent resistance to chemotherapy, including the first-line drug gemcitabine. Although low expression of the nucleoside transporters hENT1 and hCNT3 that mediate cellular uptake of gemcitabine has been linked to gemcitabine resistance, the mechanisms regulating their expression in the PDAC tumor microenvironment are largely unknown. Here, we report that the matricellular protein cysteine-rich angiogenic inducer 61 (CYR61) negatively regulates the nucleoside transporters hENT1 and hCNT3. CRISPR/Cas9-mediated knockout of CYR61 increased expression of hENT1 and hCNT3, increased cellular uptake of gemcitabine and sensitized PDAC cells to gemcitabine-induced apoptosis. In PDAC patient samples, expression of hENT1 and hCNT3 negatively correlates with expression of CYR61. We demonstrate that stromal pancreatic stellate cells (PSCs) are a source of CYR61 within the PDAC tumor microenvironment. Transforming growth factor-β (TGF-β) induces the expression of CYR61 in PSCs through canonical TGF-β-ALK5-Smad2/3 signaling. Activation of TGF-β signaling or expression of CYR61 in PSCs promotes resistance to gemcitabine in PDAC cells in an in vitro co-culture assay. Our results identify CYR61 as a TGF-β-induced stromal-derived factor that regulates gemcitabine sensitivity in PDAC and suggest that targeting CYR61 may improve chemotherapy response in PDAC patients.

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“…Limitations in the availability of pancreatic surgical specimens gave rise to the immortalization of PSCs. 23 Transformed PSC lines provided the additional benefit of eliminating potential confounders, such as variability between PSC isolation and culture practices. However, immortalization has been met with skepticism in the most recent PSC consensus conference.…”

Section: Discussionmentioning

confidence: 99%

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

Han

Delitto

Zhang

et al. 2015

Laboratory Investigation

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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. Evidence continues to accumulate implicating the intense desmoplastic reaction common to both chronic pancreatitis (CP) and pancreatic cancer (PC) is not simply a bystander process from a dynamic inflammatory state, but rather represents a complex interplay within the microenvironment responsible for the initiation and maintenance of key pathologic feedback systems. 1 Pancreatic stellate cells (PSCs) appear to represent a critical determinant in the development and maintenance of this desmoplastic microenvironment in pancreatic disease states. 2-4 However, recent investigations have questioned the role of PSCs in cancer. Specifically, depletion of activated PSCs in genetically engineered mouse models of PC has paradoxically demonstrated pro-tumor effects. 5,6 While disparate from prior observations showing the tumor promoting abilities of activated PSCs, 3,7-9 these investigations still implicate an interplay between PSCs, other cells within the tumor microenvironment, and the cancer. 5,6 Given these disparate findings, further investigation into the biology of PSCs in various disease states is essential and necessitates a high degree of experimental standardization. To this effect, an expert panel recently published guidelines regarding PSC isolation, identification, and activation. 10 Human PSCs are most typically isolated via the outgrowth method, whereby direct culture of minced pancreatic s...

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Immortalization of pancreatic stellate cells as an in vitro model of pancreatic fibrosis: deactivation is induced by matrigel and N-acetylcysteine

Cited by 132 publications

References 64 publications

Regulation and functional role of the Runt-related transcription factor-2 in pancreatic cancer

Regulation and functional role of the Runt-related transcription factor-2 in pancreatic cancer

TGF-β-induced stromal CYR61 promotes resistance to gemcitabine in pancreatic ductal adenocarcinoma through downregulation of the nucleoside transporters hENT1 and hCNT3

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

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