3D pancreatic carcinoma spheroids induce a matrix-rich, chemoresistant phenotype offering a better model for drug testing.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer related death. It is lethal in nearly all patients, due to an almost complete chemoresistance. Most if not all drugs that pass preclinical tests successfully, fail miserably in the patient. This raises the question whether traditional 2D cell culture is the correct tool for drug screening. The objective of this study is to develop a simple, high-throughput 3D model of human PDAC cell lines, and to explore mechanisms underlying the transition from 2D to 3D that might be responsible for chemoresistance. METHODS: Several established human PDAC and a KPC mouse cell lines were tested, whereby Panc-1 was studied in more detail. 3D spheroid formation was facilitated with methylcellulose. Spheroids were studied morphologically, electron microscopically and by qRT-PCR for selected matrix genes, related factors and miRNA. Metabolic studies were performed, and a panel of novel drugs was tested against gemcitabine. RESULTS: Comparing 3D to 2D cell culture, matrix proteins were significantly increased as were lumican, SNED1, DARP32, and miR-146a. Cell metabolism in 3D was shifted towards glycolysis. All drugs tested were less effective in 3D, except for allicin, MT100 and AX, which demonstrated effect. CONCLUSIONS: We developed a high-throughput 3D cell culture drug screening system for pancreatic cancer, which displays a strongly increased chemoresistance. Features associated to the 3D cell model are increased expression of matrix proteins and miRNA as well as stromal markers such as PPP1R1B and SNED1. This is supporting the concept of cell adhesion mediated drug resistance.

Stabilin-1 and stabilin-2 are both directed into the early endocytic pathway in hepatic sinusoidal endothelium via interactions with clathrin/AP-2, independent of ligand binding.

Liver sinusoidal endothelial cells (LSECs) mediate clearance of hyaluronan (HA) and scavenger receptor ligands, for example, advanced glycation end product (AGE)-modified proteins and oxidized lipids from the circulation. We recently cloned stabilin-1 and -2, two members of a novel family of transmembrane proteins expressed in LSECs. By using primary LSECs and HEK293 cells separately expressing either stabilin, we have investigated their roles in the early events of endocytosis with respect to localization, ligand-binding properties, and associations with clathrin and adaptor protein (AP)-2. Both stabilins were present at the cell surface, although surface levels of stabilin-1 were limited. In addition, stabilins were present in early endosomal antigen (EEA)-1+ organelles colocalizing with endocytosed AGE-modified bovine serum albumin (BSA). Treating cells with monensin further pronounced this distribution. Recombinant stabilin-2, but not recombinant stabilin-1, bound HA and the scavenger receptor ligands AGE-modified BSA, formaldehyde-treated BSA, and collagen N-terminal propeptides. In LSECs, both stabilins were associated with clathrin and AP-2, but not with each other. These interactions did not change upon addition of exogenous HA, suggesting that stabilins are constitutively internalized. In conclusion, hepatic stabilins are both present in the early endocytic pathway, associating with clathrin/AP-2, but whereas stabilin-2 has a clear scavenging profile, stabilin-1 does not recognize these ligands.

Functional regulation of semaphorin receptors by proprotein convertases.

PLEXIN genes encode receptors for secreted and membrane-bound semaphorins. It was proposed that the extracellular domain of plexins acts as an inhibitory moiety, preventing receptor activation. Here we show that plexin-B1 and plexin-B2 undergo proteolytic processing in their extracellular portion, thereby converting single-chain precursors into non-disulfide-linked, heterodimeric receptors. We demonstrate that plexin processing is mediated by subtilisin-like proprotein convertases, by inhibition with alpha1-antitrypsin Portland, and by mutagenesis of the substrate-cleavage sites. We provide evidence indicating that proprotein convertases cleave plexins in a post-Golgi compartment and, likely, at the cell surface. In addition, we find that both cell surface targeting and proteolytic processing of plexin-B1 depend on protein-protein interaction motifs in the cytoplasmic domain of the receptor. We then show that proteolytic conversion of plexin-B1 into a heterodimeric receptor greatly increases the binding and the functional response to its specific ligand semaphorin 4D/CD100. Thus, we conclude that cleavage by proprotein convertases is a novel regulatory step for semaphorin receptors localized at the cell surface.

Stabilin-1 and -2 constitute a novel family of fasciclin-like hyaluronan receptor homologues.

MS-1, a high-molecular-mass protein expressed by non-continuous and angiogenic endothelial cells and by alternatively activated macrophages (Mphi2), and the hepatic sinusoidal endothelial hyaluronan clearance receptor are similar with respect to tissue distribution and biochemical characteristics. In the present study we purified these proteins by immuno- and hyaluronan-affinity chromatography respectively, sequenced tryptic peptides and generated full-length cDNA sequences in both mouse and human. The novel genes, i.e. stabilin-1 and stabilin-2, code for homologous transmembrane proteins featuring seven fasciclin-like adhesion domains, 18-20 epidermal-growth-factor domains, one X-link domain and three to six B-(X(7))-B hyaluronan-binding motifs. Northern-blotting experiments revealed the presence of both stabilins in organs with predominant endothelial sinuses such as liver, spleen and lymph node: stabilin-1 mRNA was also detected in organs with predominant Mphi2 cells, such as placenta, and in interleukin-4/glucocorticoid-stimulated Mphi2 cells in vitro. A polyclonal antibody made against human recombinant stabilin-1 confirmed the expression of stabilin-1 protein in splenic sinus endothelial cells in vivo and in Mphi2 in vitro. On the basis of high similarity at the protein level and the unique domain composition, which differs from that of all other known fasciclin-like proteins and hyaluronan receptors, stabilin-1 and stabilin-2 define a novel family of fasciclin-like hyaluronan receptor homologues that might play a role in cell-cell and cell-matrix interactions in vascular function and inflammatory processes.