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Sydney, Australia

Vonlaufen A.,Pancreatic Research Group | Vonlaufen A.,University of New South Wales | Vonlaufen A.,University of Geneva | Phillips P.A.,Pancreatic Research Group | And 13 more authors.
Pancreatology | Year: 2010

Background: Pancreatic stellate cells (PSCs) play a critical role in pancreatic fibrosis. To date, human PSC biology has been studied using cancer- or inflammation-associated (pre-activated) PSCs, but an in vitro model of quiescent normal human PSCs (NhPSCs) has been lacking. Aims: To (i) isolate and characterize quiescent NhPSCs, and (ii) evaluate the response of culture-activated NhPSCs to cytokines and LPS. Methods: Quiescent NhPSCs were isolated from normal pancreatic tissue using density gradient centrifugation. PSC markers, glial fibrillary acidic protein (GFAP), desmin, α-smooth muscle actin (αSMA) and the lipopolysaccharide (LPS) receptors TLR4 and CD14 were identified by immunoblotting and immunocytochemistry. The effect of platelet-derived growth factor (PDGF), transforming growth factor β (TGFβ) and LPS on NhPSC activation was also assessed. Results: Freshly isolated NhPSCs displayed a polygonal appearance with refringent cytoplasmic lipid droplets. Culture-activated NhPSCs expressed GFAP, desmin, αSMA, TLR4 and CD14, and were responsive to PDGF, TGFβ and LPS. Conclusion: Isolated NhPSCs expressed the same markers as rat PSCs and human cancer-associated PSCs and responded to PDGF and TGFβ similarly to rat PSCs. NhPSC preparations provide a useful in vitro tool to study the biology of PSCs in their physiological, non-activated state. © 2010 S. Karger AG, Basel and IAP.

Xu Z.,Pancreatic Research Group | Xu Z.,University of New South Wales | Vonlaufen A.,Pancreatic Research Group | Vonlaufen A.,University of New South Wales | And 16 more authors.
American Journal of Pathology | Year: 2010

Pancreatic stellate cells (PSCs) produce the stromal reaction in pancreatic cancer (PC), and their interaction with cancer cells facilitates cancer progression. This study investigated the role of human PSCs (hPSCs) in the metastatic process and tumor angiogenesis using both in vivo (orthotopic model) and in vitro (cultured PSC and PC cells) approaches. A sex mismatch study (injection of male hPSCs plus female PC cells into the pancreas of female mice) was conducted to determine whether hPSCs accompany cancer cells to metastatic sites. Metastatic nodules were examined by fluorescent in situ hybridization for the presence of the Y chromosome. Angiogenesis was assessed by i) immunostaining tumors for CD31, an endothelial cell marker; and ii) quantifying human microvascular endothelial cell (HMEC-1) tube formation in vitro on exposure to conditioned media from hPSCs. Transendothelial migration was assessed in vitro by examining the movement of fluorescently labeled hPSCs through an endothelial cell monolayer. Human PSCs i) were found in multiple metastatic sites in each mouse injected with male hPSCs plus female PC cells; ii) increased CD31 expression in primary tumors from mice injected with MiaPaCa-2 and hPSCs and stimulated tube formation by HMEC-1 in vitro; and iii) exhibited transendothelial migration that was stimulated by cancer cells. Human PSCs accompany cancer cells to metastatic sites, stimulate angiogenesis, and are able to intravasate/extravasate to and from blood vessels. Copyright © American Society for Investigative Pathology.

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