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Bacterial infections of bones remain a serious complication of endoprosthetic surgery. These infections are difficult to treat, because many bacterial species form biofilms on implants, which are relatively resistant towards antibiotics. Bacterial biofilms elicit a progressive local inflammatory response, resulting in tissue damage and bone degradation. In the majority of patients, replacement of the prosthesis is required. To address the question of how the local inflammatory response is linked to bone degradation, tissue samples were taken during surgery and gene expression of the macrophage inflammatory proteins MIP1α (CCL3) and MIP2α (CXCL2) was assessed by quantitative RT-PCR. MIPs were expressed predominantly at osteolytic sites, in close correlation with CD14 which was used as marker for monocytes/macrophages. Colocalisation of MIPs with monocytic cells could be confirmed by histology. In vitro experiments revealed that, aside from monocytic cells, also osteoblasts were capable of MIP production when stimulated with bacteria; moreover, CCL3 induced the differentiation of monocytes to osteoclasts. In conclusion, the multifunctional chemokines CCL3 and CXCL2 are produced locally in response to bacterial infection of bones. In addition to their well described chemokine activity, these cytokines can induce generation of bone resorbing osteoclasts, thus providing a link between bacterial infection and osteolysis.

Background & Aims: Cancer cells often lose contact inhibition to undergo anchorage-independent proliferation and become resistant to apoptosis by inactivating the Hippo signaling pathway, resulting in activation of the transcriptional co-activator yes-associated protein (YAP). However, the oncogenic mechanisms of YAP activity are unclear., Methods: By using cross-species analysis of expression data, the Notch ligand Jagged-1 (Jag-1) was identified as a downstream target of YAP in hepatocytes and hepatocellular carcinoma (HCC) cells. We analyzed the functions of YAP in HCC cells via overexpression and RNA silencing experiments. We used transgenic mice that overexpressed a constitutively activated form of YAP (YAP(S127A)), and measured protein levels in HCC, colorectal and pancreatic tumor samples from patients., Results: Human HCC cell lines and mouse hepatocytes that overexpress YAP(S127A) up-regulated Jag-1, leading to activation of the Notch pathway and increased proliferation. Induction of Jag-1, activation of Notch, and cell proliferation required binding of YAP to its transcriptional partner TEA domain family member 4 (TEAD4); TEAD4 binding required the Mst1/2 but not β-catenin signaling. Levels of YAP correlated with Jag-1 expression and Notch signaling in human tumor samples and correlated with shorter survival times of patients with HCC or colorectal cancer., Conclusions: The transcriptional regulator YAP up-regulates Jag-1 to activate Notch signaling in HCC cells and mouse hepatocytes. YAP-dependent activity of Jag-1 and Notch correlate in human HCC and colorectal tumor samples with patient survival times, suggesting the use of YAP and Notch inhibitors as therapeutics for gastrointestinal cancer. Transcript profiling: microarray information was deposited at the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=jxepvsumwosqkve&acc=GSE35004)., (Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Introduction: Stroma reaction leading to fibrosis is the most characteristic histopathological feature of both pancreatic carcinoma and chronic pancreatitis with increased fibrosis compared with healthy pancreatic tissue and further increased fibrosis during radiochemotherapy. Recent studies using intravoxel incoherent motion-derived parameters did not show differences for structural diffusion constant D between these 2 diseases. The aim of this study was to verify the hypothesis that D correlates with the histopathological grade of fibrosis in pancreatic lesions., Materials and Methods: We included 15 patients with histopathologically proven pancreatic carcinoma and 9 patients with histopathologically proven focal chronic pancreatitis. Diffusion-weighted magnetic resonance imaging was performed using 10 b values between 25 and 800 s/mm² before surgery. We calculated the apparent diffusion coefficient and the intravoxel incoherent motion-derived parameters D and f within tumors and focal chronic pancreatitis. The resected tissue was evaluated with regard to the grade of fibrosis., Results: Fourteen patients were found to have moderate fibrosis and 10 patients had severe fibrosis. The difference between the D values for the moderate and severe fibrosis was significant with mean (SD) D value of 1.02 × 10⁻³ (0.48 × 10⁻³ mm/s) and mean (SD) D of 1.22 × 10⁻³ (0.76 × 10⁻³) mm²/s. There were no significant differences for the f and ADC values., Conclusions: Contrary to our hypothesis, D rises from moderate to severe fibrosis. It seems that cellular complexes surrounded by fibrosis provide more structural limitations than does fibrosis alone. Our data suggest that D is not intuitively related to the degree of fibrosis. Compared with healthy tissue, D is reduced in moderate fibrosis but increases when severe fibrosis is present.

Pancreatic ductal adenocarcinoma (PDAC) presenting with a micropapillary growth pattern is frequently associated with a prominent neutrophil infiltration into the tumor. The relevance of neutrophil infiltrates for tumor progression, however, is still debated. To gain insight into the role of polymorphonuclear neutrophils (PMNs) in PDAC, we assessed their effect on pancreatic tumor cells grown in vitro as monolayers. Time-lapse video microscopy showed a PMN-induced dyshesion of the tumor cells, and subsequent experiments revealed that this dyshesion was due to PMN elastase-mediated degradation of E-cadherin, an adhesion molecule that mediates the intercellular contact of the tumor cells. E-cadherin degradation by elastase or--(for comparison) down-modulation by specific siRNA, significantly increased the migratory capacity of the pancreatic tumor cells, leading to the hypothesis that PMNs could contribute to the invasive tumor growth. To address this issue, biopsies of patients with PDAC (n = 112) were analyzed. We found that E-cadherin expression correlated negatively with PMN infiltration, compatible with the notion that E-cadherin is cleaved by PMN-derived elastase, which in turn could result in the dispersal of the tumor cells, enhanced migratory capacity and thus invasive tumor growth., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Galectin-3 influences neoangiogenesis, tumor cell adhesion, and tumor-immune-escape mechanisms. Hence, the expression of galectin-3 in pancreatic ductal adenocarcinoma (PDAC) was evaluated. Galectin-3 expression in PDAC cell lines was proven by the presence of intracellular protein and by release into the supernatant. Furthermore, galectin-3 was found in the majority of human tissue samples. Serum concentrations of galectin-3 in PDAC patients did not differ significantly from healthy donors and did not correlate with established tumor markers. In conclusion, galectin-3 is expressed in PDAC tissues suggesting a role in tumor development; however, no relationship between expression and clinical findings could be established.

Major histocompatibility complex class II antigens (MHC class II) are constitutively expressed by professional antigen presenting cells and present antigenic peptides to specific CD4+ T lymphocytes. MHC class II expression, however, can also be induced on epithelial cells and in a variety of solid tumors. We tested MHC class II expression on tissue samples derived from patients with pancreatic ductal adenocarcinoma (PDAC) and pancreatic endocrine tumors (PET). Immunohistochemistry revealed MHC class II expression in 86 of 112 (76.8%) PDAC samples and in 30 of 43 (70.0%) PET samples. In PDAC and PET, MHC class II expression correlated significantly with severity and activity of intratumoral inflammation, as well as with the infiltration of CD4+ T lymphocytes. High MHC class II expression significantly correlated with a better histological grade of differentiation in PDAC. In vitro MHC class II expression could be induced on PDAC tumor cell lines by interferon-γ. These cells were then able to present the staphylococci enterotoxin B superantigen to T lymphocytes, which resulted in T cell proliferation. Our findings suggest that MHC class II expression on pancreatic tumor cells is induced by the intratumoral inflammatory reaction in pancreatic tumors.

Standard cancer cell lines do not model the intratumoural heterogeneity situation sufficiently. Clonal selection leads to a homogeneous population of cells by genetic drift. Heterogeneity of tumour cells, however, is particularly critical for therapeutically relevant studies, since it is a prerequisite for acquiring drug resistance and reoccurrence of tumours. Here, we report the isolation of a highly tumourigenic primary pancreatic cancer cell line, called JoPaca-1 and its detailed characterization at multiple levels. Implantation of as few as 100 JoPaca-1 cells into immunodeficient mice gave rise to tumours that were histologically very similar to the primary tumour. The high heterogeneity of JoPaca-1 was reflected by diverse cell morphology and a substantial number of chromosomal aberrations. Comparative whole-genome sequencing of JoPaca-1 and BxPC-3 revealed mutations in genes frequently altered in pancreatic cancer. Exceptionally high expression of cancer stem cell markers and a high clonogenic potential in vitro and in vivo was observed. All of these attributes make this cell line an extremely valuable model to study the biology of and pharmaceutical effects on pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is frequently associated with fibrosis and a prominent inflammatory infiltrate in the desmoplastic stroma. Moreover, in PDAC, an epithelial-to-mesenchymal transition (EMT) is observed. To explore a possible connection between the infiltrating cells, particularly the polymorphonuclear neutrophils (PMN) and the tumor cell transition, biopsies of patients with PDAC (n = 115) were analysed with regard to PMN infiltration and nuclear expression of β-catenin and of ZEB1, well-established indicators of EMT. In biopsies with a dense PMN infiltrate, a nuclear accumulation of β-catenin and of ZEB1 was observed. To address the question whether PMN could induce EMT, they were isolated from healthy donors and were cocultivated with pancreatic tumor cells grown as monolayers. Rapid dyshesion of the tumor cells was seen, most likely due to an elastase-mediated degradation of E-cadherin. In parallel, the transcription factor TWIST was upregulated, β-catenin translocated into the nucleus, ZEB1 appeared in the nucleus, and keratins were downregulated. EMT was also induced when the tumor cells were grown under conditions preventing attachment to the culture plates. Here, also in the absence of elastase, E-cadherin was downmodulated. PMN as well as prevention of adhesion induced EMT also in liver cancer cell line. In conclusion, PMN via elastase induce EMT in vitro, most likely due to the loss of cell-to-cell contact. Because in pancreatic cancers the transition to a mesenchymal phenotype coincides with the PMN infiltrate, a contribution of the inflammatory response to the induction of EMT and-by implication-to tumor progression is possible.

In the recent years, the participation of the animal lectin galectin (gal)-3 in inflammation and in host defence mechanisms was extensively studied. In vivo studies implied - among others - a role of gal-3 in the recruitment of polymorphonuclear neutrophils (PMN) to sites of bacterial infection. In that context, we asked the question whether gal-3 was chemotactic for PMN. Functional assays revealed that gal-3 was not chemotactic for PMN, but that it inhibited the spontaneous migration and the chemotaxis of PMN towards complement C5a, interleukin (IL)-8, or ATP. Moreover, gal-3 inhibited the shape change and the actin polymerisation of PMN that occurs in response to C5a or IL-8. By use of FITC-labelled gal-3, we found that it attached rapidly to the PMN membrane in a lactose-sensitive manner. In response to gal-3 the MAP kinase p38 was phosphorylated. This kinase is crucial for the migration of PMN towards end-target chemokines, such as C5a, and is activated in response to C5a or IL-8. When PMN were preincubated with gal-3, the C5a-induced p38 phosphorylation was transiently enhanced, but eventually down-modulated. We conclude that by interfering with the chemokine-induced p38 phosphorylation gal-3 inhibits chemotaxis of PMN., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Solid-pseudopapillary neoplasms of the pancreas are tumors of low malignant potential whose histogenesis has been discussed controversially. In the series of 15 solid-pseudopapillary neoplasms presented here, we demonstrate that discovered on gastrointestinal stromal tumor 1 is expressed significantly by these tumors (diffuse expression in 8 cases, focal expression in 4 cases, and scarce expression in 3 cases). Similar to the high expression of CD117, this finding parallels the immunohistochemical findings in gastrointestinal stromal tumors. Using double immunohistochemistry and immunofluorescence, we furthermore show that centroacinar cells express discovered on gastrointestinal stromal tumors 1. Thus, our findings suggest that, similarly to CD10 or vimentin, the expression of discovered on gastrointestinal stromal tumors 1 may serve as a novel marker for centroacinar cells and for solid-pseudopapillary neoplasms, which is suggestive of a centroacinar origin of these neoplasms., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background: Anaplastic pancreatic cancers are rare neoplasms. The available data are focused on pathologic and molecular features, and little is known about the clinical presentation and management. The outcome of operative exploration and resection is unknown., Methods: From a prospective database, all consecutive operations for anaplastic pancreatic cancer performed at our institution were identified. The clinicopathologic details were analyzed and the outcome was compared with a matched group of typical pancreatic ductal adenocarcinomas (nested case-control study)., Results: Eighteen patients with anaplastic pancreatic cancer were identified. The patients had a median age of 64 years. The tumors were large (median diameter, 4 cm) and showed peripheral contrast enhancement in radiologic imaging. Fifteen (83%) patients underwent resection, a palliative bypass procedure was performed in 1 (6%) patient, and 2 patients underwent exploration with biopsy only. Perioperative morbidity was 39% and mortality was 6%. The median survival rate in patients with anaplastic pancreatic cancer was 5.7 months and was less than in the control group of patients with pancreatic ductal adenocarcinoma (15.7 months). In anaplastic pancreatic cancer, the median duration of survival was significantly greater after R0/R1 resection, as compared with palliative surgery (7.1 vs 2.3 months). The duration of survival was significantly greater in neoplasms with osteoclast-like giant cells. In 3 (17%) patients, long-term survival of 33, 49, and 161 months was observed., Conclusion: Anaplastic pancreatic cancer is an aggressive type of pancreatic cancer with a short median survival; however, because of the observation of prolonged survival after resection, resection should be performed whenever possible. The presence of osteoclast-like giant cells is associated with a favorable prognosis., (Copyright © 2011 Mosby, Inc. All rights reserved.)

Aims: Treatment of cachexia requires pharmacological intervention which, in turn, requires knowledge of the mediators and processes. Cachexia markers that are specifically expressed in pancreatic cancer and secreted into the blood circulation have yet to be identified. The aim of our study was to investigate the serum protein profiles and protein alterations associated with cachexia and to identify potential disease protein biomarkers indicative for this syndrome., Main Methods: Serum samples from cachectic and non-cachectic patients undergoing pancreatic cancer (PaCa) surgery and controls were investigated by Surface Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI-TOF-MS). The identity of detected discriminatory markers was determined by a combination of protein fractionation, chromatographic purification steps, gel electrophoresis, and mass spectrometry., Key Findings: Using Cu-IMAC array and CM-10 array based SELDI-TOF-MS. we identified eleven up- and four down-regulated proteins associated with cachexia. CiphergenExpress analysis revealed four disease-associated protein features (38559Da, 9138Da, 8925Da and 3358Da) that were elevated by a factor of 2.3, 1.7, 1.4 and 1.4, respectively. Zinc-α2-glycoprotein (ZAG), apolipoproteins apo C-II and apo C-III and glucagon-like peptide-1 (GLP-1) were identified as markers for PaCa-associated cachexia syndrome. ZAG levels were additionally evaluated in serum and tissue samples by ELISA and immunohistochemistry and the obtained data confirmed the SELDI-TOF-MS results., Significance: The identified proteins could be routinely and reliably measured in the serum of patients and provide an elegant non-invasive approach for early diagnosis of cachectic pancreatic cancer patients. Controlling ZAG and GLP-1 activity could be beneficial in the management of cancers and cachexia-induced conditions., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The protease ADAM10 influences progression and metastasis of cancer cells and is overexpressed in various malignancies. Therefore, the aim of our study was to evaluate the expression and potential function of ADAM10 in the pathophysiology of pancreatic cancer (PDAC). ADAM10 expression in normal pancreatic (NP), chronic pancreatitis (CP), PDAC tissues, as well as PDAC cell lines was determined. To evaluate whether rhADAM10 or ADAM10 silencing influences cancer cell viability, MTT assay was used. Matrigel invasion and wound healing assays were performed to observe influence on invasion and migration. ADAM10 mRNA was expressed in all samples of NP, CP and PDAC tissue and cell lines. Western blotting and immunohistochemistry revealed stronger ADAM10 expression in PDAC than in NP. ADAM10 silencing or rhADAM10 had no effect on cell viability. ADAM10 silencing markedly reduced invasiveness and migration of cancer cells. These findings establish ADAM10 as a contributing factor in PDAC invasion and metastasis.

Aims: Undifferentiated (anaplastic) pancreatic cancer and undifferentiated pancreatic carcinoma with osteoclast-like giant cells (giant cell tumour) are rare variants of pancreatic ductal adenocarcinoma. Representing biologically highly aggressive neoplasms, they are frequently diagnosed at an advanced stage. The response to established chemo- or radiochemotherapeutic treatment regimens is poor, and undifferentiated pancreatic cancer generally has a dismal prognosis. As additional therapeutic options have not yet been investigated in undifferentiated pancreatic cancer, the aim was to analyse the expression of putative therapeutic targets that have shown promising results in various other neoplasms., Methods and Results: Fifteen cases of undifferentiated pancreatic cancer (seven containing osteoclast-like giant cells) were investigated clinicopathologically and immunohistochemically for putative therapeutic targets. Whereas L1CAM, cyclooxygenase (COX)-2 and epidermal growth factor receptor (EGFR) were found to be significantly expressed in 80%, 93% and 87% of the investigated tumours, respectively, there was no substantial expression of c-kit (CD117) and there was no detectable expression of Her2/neu., Conclusions: The expression of L1CAM, COX-2 and EGFR in the majority of undifferentiated pancreatic carcinomas suggests that they might represent targets for adjuvant therapy in anaplastic pancreatic cancer. On the other hand, c-kit and Her2/neu seem to have no relevance for the therapy of these tumours.

The chemokine receptor CXCR6 has been described on lymphoid cells and is thought to participate in the homing of activated T-cells to non-lymphoid tissue. We now provide evidence that the chemokine receptor CXCR6 is also expressed by activated polymorphonuclear neutrophils (PMN) in vivo: Examination of biopsies derived from patients with pancreatic carcinoma by confocal laser scan microscopy revealed a massive infiltration of PMN that expressed CXCR6, while PMN of the peripheral blood of these patients did not. To answer the question whether CXCR6 expression is a property of infiltrated and activated PMN, leucocytes were collected from patients with localized soft tissue infections in the course of the wound debridement. By cytofluorometry, the majority of these cells were identified as PMN. Up to 50% of these PMN were also positive for CXCR6. Again, PMN from the peripheral blood of these patients were nearly negative for CXCR6, as were PMN of healthy donors. In a series of in vitro experiments, up-regulation of CXCR6 on PMN of healthy donors by a variety of cytokines was tested. So far, a minor, although reproducible, effect of tumour necrosis factor (TNFalpha) was seen: brief exposure with low-dose TNFalpha induced expression of CXCR6 on the surface of PMN. Furthermore, we could show an increased migration of PMN induced by the axis CXCL16 and CXCR6. In summary, our data provide evidence that CXCR6 is not constitutively expressed on PMN, but is up-regulated under inflammatory conditions and mediates migration of CXCR6-positive PMN.

We investigated the expression pattern and clinical significance of the costimulatory ligands B7-1, B7-2, B7-H1, and B7-DC, and their counter-receptors CTLA-4 and PD-1 in pancreatic cancer. Gene expression of all examined costimulatory molecules was significantly upregulated in pancreatic cancer tissues. B7-1, B7-2, B7-H1, and B7-DC protein was detectable in pancreatic cancer cells. Only the expression of B7-H1 significantly correlated with postoperative survival (p<0.0001). B7-H1 was inducible in cultured pancreatic cancer cells by IFN-gamma and significantly correlated with the level of IFN-gamma expression in human pancreatic cancer tissues (Spearman rho=0.4536,p=0.0029). B7-H1 positive tumors showed an increased prevalence of tumor-infiltrating regulatory T cells (Tregs) compared to B7-H1 negative tumors. Among the investigated costimulatory molecules only tumor-associated B7-H1 seems to be of prognostic relevance in pancreatic cancer. B7-H1 might, therefore, be involved in the downregulation of antitumor responses through regulation of Tregs in pancreatic cancer. Our findings also suggest a dual role of IFN-gamma in antitumor response. Through induction of B7-H1 in pancreatic cancer cells IFN-gamma might contribute to the evasion of antitumor immunity.

CXC chemokines have a major influence on the angiogenesis, growth and metastatic potential of pancreatic ductal adenocarcinoma. CXCL16 is a unique transmembrane CXC chemokine, which is shed by members of the disintegrins and metalloproteases (ADAMs), in particular by ADAM10 and ADAM17. In our study, we evaluated expression and potential function of CXCL16 and its receptor CXCR6. CXCL16 and the receptor CXCR6 are upregulated in pancreatic ductal adenocarcinoma (PDAC) and chronic pancreatitis tissues in contrast to normal pancreatic tissues at the mRNA and protein levels. In 85 and 100% of the investigated samples, tumor cells showed positive immuno-staining for CXCL16 and CXCR6, respectively; furthermore, tubular complexes of chronic pancreatitis and the invasive front of PDAC were immunopositive for CXCL16 and CXCR6. Stimulation of PDAC cells with proinflammatory cytokines increased CXCL16 protein levels, whereas silencing of ADAM10 with siRNA transfection led to a decrease in CXCL16 protein levels in cell culture supernatants. No effects on cell viability were notable after incubation of cancer cells with CXCL16. However, CXCL16 markedly increased invasiveness of PDAC cells. Clinically, 82.5% of PDAC patients had higher CXCL16 serum values than the highest value seen in healthy donors. SELDI-TOF-MS analysis confirmed the upregulation of CXCL16 in sera of PDAC patients. In conclusion, CXCL16 in both transmembrane and soluble forms, and its receptor CXCR6, seem to play an important role in the pathobiology of pancreatic cancer and might be potential markers for pancreatic cancer diagnosis and a target for multimodal therapy concepts in the future.

CXC chemokines influence angiogenesis, growth, and metastatic potential of pancreatic cancer. Therefore, the expression and potential function of CXCL14, a recently described CXC chemokine, was evaluated. CXCL14 is upregulated in pancreatic cancer tissues compared to chronic pancreatitis and normal pancreas. Immunolocalization revealed a distinct expression of CXCL14 in tubular complexes in chronic pancreatitis and in particular at the invasive front of pancreatic cancer tissues. Stimulation of pancreatic cancer cells with CXCL14 showed no effects on cell viability and on chemosensitivity. However, CXCL14 clearly increased invasiveness of pancreatic cancer cells without affecting MMP-2 and VEGF secretion, whereas CXCL14 influenced NFkB p65 levels. In conclusion, CXCL14 might play a pivotal role in the pathobiology of pancreatic cancer, probably by regulating cancer invasion.