Epithelial to Stromal Re-Distribution of Primary Cilia during Pancreatic Carcinogenesis

Background The Hedgehog (HH) pathway is a mediator in pancreatic ductal adenocarcinoma (PDAC). Surprisingly, previous studies suggested that primary cilia (PC), the essential organelles for HH signal transduction, were lost in PDAC. The aim of this study was to determine the presence of PC in human normal pancreas, chronic pancreatitis, and during carcinogenesis to PDAC with focus on both epithelia and stroma. Methods PC were analyzed in paraffin sections from normal pancreas, chronic pancreatitis, intraductal papillary-mucinous neoplasia, and PDAC, as well as in primary human pancreatic stellate cells (PSC) and pancreatic cancer cell lines by double immunofluorescence staining for acetylated alpha-tubuline and gamma-tubuline. Co-staining for the HH receptors PTCH1, PTCH2 and SMO was also performed. Results PC are gradually lost during pancreatic carcinogenesis in the epithelium: the fraction of cells with PC gradually and significantly decreased from 32% in ducts of normal pancreas, to 21% in ducts of chronic pancreatitis, to 18% in PanIN1a, 6% in PanIN2, 3% in PanIN3 and to 1.2% in invasive PDAC. However, this loss of PC in the neoplastic epithelium is accompanied by a gain of PC in the surrounding stroma. The fraction of stromal cells with PC significantly increased from 13% around normal ducts to about 30% around PanIN and PDAC. HH-receptors were detected in tumor stroma but not in epithelial cells. PC are also present in PSC and pancreatic cancer cell lines. Conclusion PC are not lost during pancreatic carcinogenesis but re-distributed from the epithelium to the stroma. This redistribution may explain the re-direction of HH signaling towards the stroma during pancreatic carcinogenesis

Simultaneous and sequential lineups: decision processes of accurate and inaccurate eyewitnesses

The cognitive processes and decision-making strategies of eyewitnesses were tested for their predictive qualities in determining the accuracy of identifications from lineups. The sequential lineup presentation was compared with the traditionally employed simultaneous lineup under culprit (target) present and culprit absent conditions. Consistent with previous research the sequential presentation resulted in an equivalent number of correct identifications compared to the simultaneous lineup but reduced false identification rates. Although sequential lineups were found to be associated with the use of absolute strategies, those shown a simultaneous lineup reported the use of both relative and absolute strategies. Accurate identifications and rejections were found to be associated with the use of absolute strategies, irrespective of lineup presentation or presence of target. Also accurate identifications, at least with a sequential lineup, were generally made faster than inaccurate identifications. These results are compared to previous studies with respect to the effect that mode of processing (relative versus absolute judgements) has on a witness's decision making and identification accuracy

Epithelial to Stromal Re-Distribution of Primary Cilia during Pancreatic Carcinogenesis.

The Hedgehog (HH) pathway is a mediator in pancreatic ductal adenocarcinoma (PDAC). Surprisingly, previous studies suggested that primary cilia (PC), the essential organelles for HH signal transduction, were lost in PDAC. The aim of this study was to determine the presence of PC in human normal pancreas, chronic pancreatitis, and during carcinogenesis to PDAC with focus on both epithelia and stroma.PC were analyzed in paraffin sections from normal pancreas, chronic pancreatitis, intraductal papillary-mucinous neoplasia, and PDAC, as well as in primary human pancreatic stellate cells (PSC) and pancreatic cancer cell lines by double immunofluorescence staining for acetylated α-tubuline and γ-tubuline. Co-staining for the HH receptors PTCH1, PTCH2 and SMO was also performed.PC are gradually lost during pancreatic carcinogenesis in the epithelium: the fraction of cells with PC gradually and significantly decreased from 32% in ducts of normal pancreas, to 21% in ducts of chronic pancreatitis, to 18% in PanIN1a, 6% in PanIN2, 3% in PanIN3 and to 1.2% in invasive PDAC. However, this loss of PC in the neoplastic epithelium is accompanied by a gain of PC in the surrounding stroma. The fraction of stromal cells with PC significantly increased from 13% around normal ducts to about 30% around PanIN and PDAC. HH-receptors were detected in tumor stroma but not in epithelial cells. PC are also present in PSC and pancreatic cancer cell lines.PC are not lost during pancreatic carcinogenesis but re-distributed from the epithelium to the stroma. This redistribution may explain the re-direction of HH signaling towards the stroma during pancreatic carcinogenesis

Schematical structure and visualization of primary cilia (PC).

<p><b>(A)</b> Cross section of primary cilia, consisting of 9 microtubule duplets containing α and β tubline. <b>(B)</b> Structure of PC. Cilia are fixed by basal bodies on the cell membrane. <b>(C)</b> Immunofluorescent visualization of PC in Panc1 cells. The axonem is stained in red (acetylated α-tubline), the basal body is stained green (γ-tubline). <b>(D)</b> Immunofluorescent visualization of PC and the spindle apparatus in the BxPx3 cancer cell line.</p

Primary cilia (PC) in pancreatic intra-epithelial neoplasia (PanIN) and pancreatic cancer cells (PDAC).

<p><b>(A)</b> Comparison between PanIN 1a (*) and PanIN 1B (#), the latter showing papillary epithelium and reduced number of cilia. <b>(B)</b> PanIN 3 lesion. Epithelial cells do not carry cilia while PC are present in stromal cells (*). <b>(C)</b> Loss of epithelial PC in PDAC (indicated by arrows), while in the stromal cells there is a noticeable increase of both the length of PC and the number of PC carrying cells (*). <b>(D)</b> Length of epithelial PC is decreased in PanIN lesions in comparison to normal pancreas (donor). <b>(E)</b> Gradual loss of epithelial PC in PanIN lesions. In PDAC, almost no epithelial PC were detected. <b>(F)</b> In stromal tissue around PanIN lesions and PDAC (G1/G2), an increased length of PC and <b>(G)</b> increased fraction of cilia carrying cells was evident compared to normal pancreas (donor). Kruskal-Wallis test: <i>p</i> < 0.0001, post-hoc Dunns test: ***<i>p</i> < 0.001 vs. donor, **<i>p</i> < 0.01 vs donor, *<i>p</i> < 0.01 vs donor, ###<i>p</i> < 0. 001 vs PanIN 1A, ##<i>p</i> < 0. 01 vs PanIN 1A, +++<i>p</i> < 0. 001 vs PanIN 1B, +<i>p</i> < 0. 05 vs PanIN 1B, acetylated α-tubuline: red, γ-tubuline: green, DAPI: blue. Mean ± SEM.</p

Primary cilia (PC) in intraductal papillary-mucinous neoplasia (IPMN).

<p><b>A/B)</b> Two benign and <b>(C)</b> one malignant IPMN without PC. <b>(D)</b> In stromal tissue surrounding IPMN lesions, an increased fraction of cilia carrying cells (<b>E</b>, Kruskal-Wallis test: <i>p</i> < 0. 01) and an increased length of PC (<b>F</b>, Kruskal-Wallis test: <i>p</i> < 0. 001) were detected compared to normal pancreas (donor). Post-hoc Dunns test: *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0. 001, acetylated α-tubuline: red, γ-tubuline: green, DAPI: blue. Mean ± SEM.</p

PC in pancreatic stellate cells (PSC) and pancreatic cancer cell lines.

<p><b>(A)</b> MiaPaCa2 cells developing sporadic PC. <b>(B)</b> In these starving Panc1 cells, PC are expressed more frequently. <b>(C)</b> Multiple mitosis can be seen in Capan1 cells. The spindle apparatus never appears together with PC, inferring it is reabsorbed during mitosis. <b>(D)</b> PC are frequently present even in isolated PSC.</p

Re-Distribution of primary cilia (PC) from epithelial to stromal cells in pancreatic carcinogenesis.

<p>The fraction of primary cilia (PC) carrying cells decreases in epithelia, while there is a simultaneous increase of ciliated cells in stromal tissue during pancreatic carcinogenesis (from PanIN 1A to pancreatic G1/G2 ductal adenocarcinomas (PDAC). Mean ± SEM.</p

Engineering pro-angiogenic peptides using stable disulfide-rich cyclic scaffolds

Fragments from the extracellular matrix proteins laminin and osteopontin and a sequence from VEGF have potent proangiogenic activity despite their small size (< 10 residues). However, these linear peptides have limited potential as drug candidates for therapeutic angiogenesis because of their poor stability. In the present study, we show that the therapeutic potential of these peptides can be significantly improved by "grafting" them into cyclic peptide scaffolds. Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II) and sunflower trypsin inhibitor-1 (SFTI-1), naturally occurring, plant-derived cyclic peptides of 34 and 14 residues, respectively, were used as scaffolds in this study. Using this approach, we have designed a peptide that, in contrast to the small peptide fragments, is stable in human serum and at nanomolar concentration induces angiogenesis in vivo. This is the first report of using these scaffolds to improve the activity and stability of angiogenic peptide sequences and is a promising approach for promoting angiogenesis for therapeutic uses. (Blood. 2011;118(25):6709-6717