Dkk-3 expression in the tumor endothelium: a novel prognostic marker of pancreatic adenocarcinomas.

Dkk-3 is proposed to be a new specific marker for tumor endothelial cells. Here we analyzed the clinical relevance of Dkk-3 expression in pancreas adenocarcinomas and determined its role on endothelial cell growth in vitro. Microvessel density in tumor samples was immunohistochemically determined using Dkk-3 and CD31 as endothelial cell markers, respectively. Based on the median microvessel density as a cut-off point, patients were categorized into high and low microvessel density groups and a correlation with survival and clinical parameters was assessed. Moreover, the role of Dkk-3 expression on chemosensitivity of endothelial cells was analyzed. In contrast to CD31 staining, Dkk-3-positive vessels were found only in tumor tissue and Dkk-3 microvessel density significantly correlated negative with tumor grading. In survival analysis the median survival time was 7 months for patients with Dkk-3 low, and 15 months for Dkk-3 high microvessel density (P = 0.0013). Subset analysis of patients receiving gemcitabine therapy showed that overall survival was significantly decreased in Dkk-3 low tumors than in high tumors (P = 0.009). In Cox regression Dkk-3 emerged as a significant independent parameter (P = 0.024). Dkk-3 overexpression in endothelial cells resulted in significantly enhanced growth inhibition after 5-fluorouracil or gemcitabine treatment compared to control endothelial cells and cancer cell lines. Dkk-3 low microvessel density was associated with tumor progression and worse clinical outcome. Overexpression of Dkk-3 enhanced endothelial cell growth inhibition to chemotherapeutic drugs. Therefore, we suggest that Dkk-3 high microvessel density may help to select patients who may benefit from chemotherapy.

Rotational transport of islets: the best way for islets to get around?

Islet transplantation is a valid treatment option for patients suffering from type 1 diabetes mellitus. To assure optimal islet cell quality, specialized islet isolation facilities have been developed. Utilization of such facilities necessitates transportation of islet cells to distant institutions for transplantation. Despite its importance, a clinically feasible solution for the transport of islets has still not been established. We here compare the functionality of isolated islets from C57BL/6 mice directly after the isolation procedure as well as after two simulated transport conditions, static versus rotation. Islet cell quality was assessed using real-time live confocal microscopy. In vivo islet function after syngeneic transplantation was determined by weight and blood sugar measurements as well as by intraperitoneal glucose tolerance tests. Vascularization of islets was documented by fluorescence microscopy and immunohistochemistry. All viability parameters documented comparable cell viability in the rotary group and the group transplanted immediately after isolation. Functional parameters assessed in vivo displayed no significant difference between these two groups. Moreover, vascularization of islets was similar in both groups. In conclusion, rotary culture conditions allows the maintenance of highest islet quality for at least 15 h, which is comparable to that of freshly isolated islets.

Keep on rolling: optimizing human islet transport conditions using a perfused rotary system.

The setup of an islet isolation facility designed along the rules of good manufacturing practice (GMP) is a technically challenging, cost and time intensive process. ( 1) Consequently, several institutions have decided to perform transplantation of islets isolated at another center with an already standing expertise. Such a solution includes the necessity to transport the isolated islets from the isolation to the transplantation facility. In spite of its importance, an ideal solution for the transport of the isolated human islets has still not been established.   Here, we present an islet transport device suited to transport human islet cells under reproducible conditions and minimized cell stress. The transport simulation of the human islets was performed in a transfused "rotary transport system for islets" termed "ROTi." Besides measuring standard metabolic (LDH, lactate, glucose) and physical parameters (pH, dissolved oxygen and temperature), we used five different live stains in combination with real time live confocal microscopy to document islet quality parameters. As live stains we added tetramethylrhodamine methyl ester, cell permeant acetoxymethylester, propidium iodide, annexin-fitc and fluorescent wheat germ agglutinin, and assessed mitochondrial membrane potentials, calcium levels, cell death, apoptosis or cell morphology, respectively. We compared the viability of human islets after 24 h incubation in the ROTi device to an incubation simulating "standard" shipment of islets in 50 ml tubes. All cell viability parameters studied (mitochondrial membrane potentials, calcium content, apoptosis, cell death as well as cell morphology) documented a significantly better cell viability in the ROTi fraction compared with the simulated "standard" shipment fraction. Besides maintaining islet cell viability, the ROTi bears the advantage of a better reproducibility of islet transport conditions.