Kallikreins play an important role in tumour microenvironment and as cancer biomarkers in different cancer entities. Previous studies suggested an upregulation of KLK10 and KLK6 in pancreatic ductal adenocarcinoma (PDAC). Therefore, we evaluated the clinicopathological role of these kallikreins and their value as biomarkers in PDAC. Differential expression was validated by DNA-microarrays and immunohistochemistry in normal and malignant pancreatic tissues. Sera concentrations of both kallikreins were evaluated using ELISA. In silico analysis of possible protein interactions and gene silencing of KLK10 in vitro using siRNAs gave further insights in the pathomechanisms. Gene expression analysis and immunohistochemistry demonstrated a strong expression for KLK10 and KLK6 in PDAC. Statistical analysis showed that co-expression of these kallikreins correlated with an R1-resection status (P ¼ 0.017) and worse outcome for overall survival (P ¼ 0.031). Multivariate analysis proofed that co-expression is an independent prognostic factor for survival (P ¼ 0.043). Importantly, KLK10 knockdown in AsPC-1 cells significantly reduced cell migration, whereas computational analysis suggested interaction of KLK6 with angiogenetic factors as an important mechanism. Co-expression of KLK10 and KLK6 plays an unfavourable role in PDAC. Our results suggest that this effect is likely mediated by an interaction with the factors of the extracellular matrix and enhancement of cancer cell motility.

There is increasing demand for efficient and physiological in vitro cell culture systems suitable for testing new pharmaceutical drugs or for evaluating materials for tissue regeneration. In particular, co-cultures of two or more tissue-relevant cell types have the advantage to study the response of cells on diverse parameters in a more natural environment with respect to physiological complexity. We developed a direct bone cell co-culture system using human peripheral blood monocytes (hPBMC) and human bone marrow stromal cells (hBMSC) as osteoclast/osteoblast precursor cells, respectively, strictly avoiding external supplements for the induction of differentiation. The sophisticated direct hPBMC/hBMSC co-culture was characterized focusing on osteoclast function and was compared with two indirect approaches. Only in the direct co-culture, hPBMC were triggered by hBMSC into osteoclastogenesis and became active resorbing osteoclasts. Bisphosphonates and sulfated glycosaminoglycans were used to examine the suitability of the co-culture system for evaluating the influence of certain effectors on bone healing and bone regeneration and the contribution of each cell type thereby. The results show that the investigated substances had more pronounced effects on both osteoblasts and osteoclasts in the co-culture system than in respective monocultures.

© F e r r a t a S t o r t i F o u n d a t i o n Design and Methods Generation of human mesenchymal stem cellsThe study was approved by the local institutional review board. Bone marrow samples were collected from healthy donors after obtaining informed consent. Isolation and cultivation of MSCs were performed as described. 19 The characteristic phenotype of MSCs (CD44 + , CD73 + , CD90 + , CD105 + , CD166 + , CD14 -, CD34 -, CD45 -) was determined by flow cytometry. Furthermore, their ability to differentiate into osteocytes and adipocytes was analyzed. Immunomagnetic isolation of slanDCs and T cellsBlood samples were obtained with informed consent from healthy donors. Immunomagnetic isolation of slanDCs (purity: >90%) from peripheral blood mononuclear cells (PBMCs) was performed as previously described.15 CD4 + T cells, naïve CD45RA + CD4 + T cells and CD8 + T lymphocytes were purified from PBMCs by negative depletion using immunomagnetic separation (purity: >90%) according to the manufacturer´s instructions (Miltenyi Biotec, Bergisch-Gladbach, Germany). The culture medium used for all experiments was previously described. 17 Flow cytometric analysisExpression analysis of surface or intracytoplasmic molecules of MSCs, slanDCs, CD4 + and CD8 + T cells was performed as described 17 using the following monoclonal antibodies: FITC-conjugated anti-CD3, FITCconjugated anti-CD4, PE-conjugated anti-CD8, FITCconjugated anti-CD14, PE-conjugated anti-CD34, FITCconjugated anti-CD44, FITC-conjugated anti-CD45, PEconjugated anti-CD45RA, PE-conjugated anti-CD73, PE-conjugated anti-CD83, PE-conjugated anti-CD86, FITC-conjugated anti-CD90, FITC-conjugated anti-CD105, PE-conjugated anti-CD166, FITC-conjugated anti-human leukocyte antigen (HLA)-DR, PE-conjugated anti-intercellular adhesion molecule (ICAM)-1, FITCconjugated anti-interferon (IFN)-γ, PE-conjugated anti-IL-4, PE-conjugated anti-IL-6 and PE-conjugated isotype-specific anti-mouse antibodies (all BD Biosciences, San José, CA, USA), FITC-conjugated anti-immunoglobulin like transcript (ILT) 3 and FITC-conjugated anti-ILT4 (R&D Systems, Wiesbaden, Germany). Negative controls included labeled isotype-matched irrelevant antibodies (BD Biosciences). M-DC8 hybridoma supernatant was used as described. 17 Maturation and cytokine production of slanDCsFreshly isolated slanDCs (2×10 5 /well) were cultivated for 12 h in the presence or absence of MSCs (4×10 4 /well) and washed. Subsequently, expression levels of the maturation marker CD83, the costimulatory molecule CD86, HLA-DR and the adhesion molecule ICAM-1 at the surface of slanDCs were determined by flow cytometry. In additional experiments, expression levels of ILT3 and ILT4 on slanDCs were analyzed after 24 h by flow cytometry.To investigate cytokine release, slanDCs were plated in round-bottomed 96-well plates at 2×10 5 /well and incubated with MSCs at different MSC-DC ratios (1:2,5; 1:5; 1:10). After 6 h, 1 µg/mL lipopolysaccharide (LPS, SigmaAldrich, Taufkirchen, Germany) was added for an additional 18 h to stimulate cyto...