StellaTUM: current consensus and discussion on pancreatic stellate cell research

The field of pancreatic stellate cell (PSC) biology is very young, as the essential in-vitro tools to study these cells (ie, methods to isolate and culture PSC) were only developed as recently as in 1998. Nonetheless, there has been an exponential increase in research output in this field over the past decade, with numerous research groups around the world focusing their energies into elucidating the biology and function of these cells. It is now well established that PSC are responsible for producing the stromal reaction (fibrosis) of two major diseases of the pancreas—chronic pancreatitis and pancreatic cancer. Despite exponentially increasing data, the methods for studying PSC remain variable. Although within individual laboratories methods are consistent, different methodologies used by various research groups make it difficult to compare results and conclusions. This article is not a review article on the functions of PSC. Instead, members of the Pancreatic Star Alliance (http://www.pancreaticstaralliance.com) discuss here and consolidate current knowledge, to outline and delineate areas of consensus or otherwise (eg, with regard to methodological approaches) and, more importantly, to identify essential directions for future research

Interaction of Stellate Cells with Pancreatic Carcinoma Cells

cancer

Transforming growth factors (TGFα and TGFβH) stimulate chondroitin sulfate and hyaluronate synthesis in cultured rat liver fat storing cells

AbstractThe synthesis of total sulfated glycosaminoglycans (GAG) was stimulated by transforming growth factors (TGFα 1.4-fold at 5 ngml, and TGFβ1 2.05-fold at 2.5 ngml) in primary cultures of rat liver fat storing cells (FSC). The combination of both TGFs resulted in an additively stimulated synthesis of total sulfated GAG (more than 3-fold), chondroitin sulfate (more than 15-fold) and hyaluronate (3.8-fold), respectively, whereas the formation of dermatan sulfate was unchanged and that of heparan sulfate was slightly reduced. In summary, TGFs were identified as important mediators of stimulated GAG synthesis in those cells of the liver (FSC), which are the primary site of matrix glycoconjugate production.Fat storing cell; Fibrogenesis; Glycosaminoglycans; Chondroitin sulfate; Hyaluronic acid; Transforming growth facto

Hexon modification to improve the activity of oncolytic adenovirus vectors against neoplastic and stromal cells in pancreatic cancer.

Primary pancreatic carcinoma has an unfavourable prognosis and standard treatment strategies mostly fail in advanced cases. Virotherapy might overcome this resistance to current treatment modalities. However, data from clinical studies with oncolytic viruses, including replicating adenoviral (Ad) vectors, have shown only limited activity against pancreatic cancer and other carcinomas. Since pancreatic carcinomas have a complex tumor architecture and frequently a strong stromal compartment consisting of non-neoplastic cell types (mainly pancreatic stellate cells = hPSCs) and extracellular matrix, it is not surprising that Ad vectors replicating in neoplastic cells will likely fail to eradicate this aggressive tumor type. Because the TGFβ receptor (TGFBR) is expressed on both neoplastic cells and hPSCs we inserted the TGFBR targeting peptide CKS17 into the hypervariable region 5 (HVR5) of the capsid protein hexon with the aim to generate a replicating Ad vector with improved activity in complex tumors. We demonstrated increased transduction of both pancreatic cancer cell lines and of hPSCs and enhanced cytotoxicity in co-cultures of both cell types. Surface plasmon resonance analysis demonstrated decreased binding of coagulation factor X to CKS17-modified Ad particles and in vivo biodistribution studies performed in mice indicated decreased transduction of hepatocytes. Thus, to increase activity of replicating Ad vectors we propose to relax tumor cell selectivity by genetic hexon-mediated targeting to the TGFBR (or other receptors present on both neoplastic and non-neoplastic cells within the tumor) to enable replication also in the stromal cell compartment of tumors, while abolishing hepatocyte transduction, and thereby increasing safety

Pancreatitis-associated protein inhibits human pancreatic stellate cell MMP-1 and -2, TIMP-1 and -2 secretion and RECK expression

BACKGROUND/AIMS: Pancreatic stellate cells (PSCs) play a key role in fibrogenesis associated with acute and chronic pancreatitis. Pancreatitis-associated protein (PAP), an acute-phase protein, is dramatically upregulated during acute and chronic pancreatitis. Assuming a protective role of PAP, we investigated its effects on human PSCs. METHODS: PSCs were obtained by outgrowth from fibrotic human pancreas tissue. PAP was expressed in the yeast Pichia pastoris. PAP was added at 10 ng/ml to cultured PSCs. Cell proliferation was determined by bromodeoxyuridine incorporation. PSC migration was assessed by a wound healing assay. Collagen types I and III, fibronectin, matrix metalloproteinases (MMPs), tissue inhibitors of MMPs (TIMPs) and reversion-inducing cysteine-rich protein with Kazal motifs (RECK) were demonstrated on protein and mRNA level. RESULTS: PAP had no significant effect on PSC proliferation and migration. Cell-associated fibrillar collagen types I and III and fibronectin increased after addition of PAP to PSCs. PAP diminished the expression of MMP-1 and -2 and TIMP-1 and -2 and their concentrations in PSC supernatants. RECK was detected on the surface of PSCs and its expression was reduced after PAP application. CONCLUSIONS: Our data offer new insights into the biological functions of PAP, which may play an important role in wound healing response and cell-matrix interactions

Cellular uptake inhibition experiments with soluble fiber knob protein and TGFBRII-specific antibody.

<p>(<b>A</b>) Lysates from pancreatic hPSCs and tumor cells were analyzed by SDS-PAGE and immunoblotting. TGFBRII was detected by an anti-TGFBRII-specific polyclonal antibody from rabbit (sc-1700). A549 cells were pre-incubated with soluble fiber knob protein, TGFBRII-specific antibody or rabbit serum (serving as isotype control), respectively, and transduced with the replication-deficient vectors AdGFPhCKS17 and AdGFPhWt (control) at a particle MOI of 100. After two hours the medium was replaced with fresh medium and incubated for additional 2 and 24 hours to (<b>B</b>) analyze GFP expression by flow cytometry or to (<b>C</b>) determine relative Ad genome levels by qPCR using total DNA isolated from cells. * <i>P</i> < 0.05, ** <i>P</i> < 0.01, n = 3.</p

Influence of hexon modification on biodistribution.

<p>For Kupffer cell depletion, 200 μl of clodronate liposomes were injected into the tail vein of BALB/c mice. After 24 hours, 3x10¹⁰ viral particles of AdGFPhWt or AdGFPhCKS17 were injected intravenously into the tail vein of mice (n = 4 or 5), and organs were collected 45 minutes or 72 hours later. (<b>A</b>) Relative Ad genome levels were determined from total DNA isolated from liver obtained 45 minutes or 72 hours after infection. (<b>B</b>) GFP expression was measured in liver lysates obtained 72 hours after infection by fluorimetry. (<b>C</b>) Analysis of relative Ad genome levels from total DNA isolated from spleen obtained 45 minutes or 72 hours after infection. * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.005, n = 4–5.</p

DNA replication and virus release of hexon-modified Ad vector in pancreatic cells.

<p>1 x 10⁶ hPSCs or 2.5x10⁶ A549, Panc1 or UlaPaCa cells were seeded. On the next day, replicating Ad vectors (AdhWt or AdhCKS17) were added at an infectious MOI of 20. Six hours post infection cells were washed with PBS to remove unbound virus, and fresh medium was added to the cells. The supernatants and the cell lysates were collected during the course of infection (at 48 and 72 hours post infection). (<b>A</b>) To determine the number of physical particles found in the supernatant at the indicated time points, viral DNA was isolated and subjected to quantitative PCR to analyse the Ad genome number. (<b>B</b>) The numbers of infectious particles per cell (viral release) within the supernatants or the cell lysates (indicated by “-”or “+”) were assessed by plaque assay. * <i>P</i> < 0.05, ** <i>P</i> < 0.01, n = 2.</p

Limitations of Ad vector-mediated transduction of early passage pancreatic cancer cell lines and primary hPSCs.

<p>(<b>A</b>) 2x10⁴ hPSCs or 2x10⁵ tumor cells were transduced with ΔE1 Ad1stGFP at a MOI of 5. To determine the Ad transduction rate the cells were subjected both to flow cytometry for detection of GFP expression (24 h.p.i.) expressed as the percentage of GFP-positive cells and to slot blot analysis to determine their relative Ad genome content (2 h.p.i.). (<b>B</b>) CAR levels on different cell lines and hPSCs were determined by flow cytometry as detailed in the Materials and Methods section. To analyze Ad replication rates and production of progeny virions in different cells, respectively, wildtype Ad5 (Ad5Wt) was used for infection of different cell lines at a slot blot adjusted actual MOI of 1. (<b>C</b>) Cells were harvested 2 and 48 hours p.i., genomic DNA was isolated and subjected to slot blot analysis using an Ad5 fiber-specific probe. Ad replication is expressed as the ratio of Ad genomes 48 h.p.i./ 2 h.p.i. in comparison to A549 cells (set to 100). (<b>D</b>) Forty-eight hours after infection cells harvested, lysed by repeated freezing and thawing, and treated with Benzonase. Two and 10 microliters of the lysate were used to reinfect A549 cells. The number of infectious particles was determined by DNA slot blot analysis [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117254#pone.0117254.ref040" target="_blank">40</a>] using a Ad5 fiber-specific probe and the number of infectious particles per tumor cell was calculated. * P < 0.05, ** P < 0.01 *** P < 0.005, n = 2–4.</p