Jan Sperveslage

The impact of agrin on the formation of orthogonal arrays of particles in cultured astrocytes from wild-type and agrin-null mice.

Astrocytic endfeet membranes are studded with aquaporin-4 (AQP4) containing orthogonal arrays of particles (OAP) which can be visualized exclusively by the freeze-fracturing method. They are predominantly expressed where the astroglial membrane is in contact with the superficial and perivascular basal lamina. This polarity seems to be essential for the integrity of the blood-brain barrier (BBB). The basal lamina containing many extracellular matrix (ECM) components such as collagen, laminin and heparansulfate proteoglycans like agrin is thought to influence this OAP-related polarity of astrocytes. Recently, we have shown that agrin, in particular the neuronal isoform A4B8, is capable of influencing the formation of OAPs in astrocytes when cultured in the presence of agrin-conditioned media. In this paper we wanted to investigate whether coating with exogenous agrin compared to coating with other ECM components would induce OAP formation in astrocytes of the agrin-null mouse. For this purpose, we cultured astrocytes from agrin-null and wild-type mice on agrin- or ECM-coated surfaces. Immunofluorescent cytochemical staining of AQP4 indicated a higher AQP4 expression level in cultures with agrin- or ECM-coated than in cultures with uncoated surfaces, whereas western blot analyses and PCR showed no differences. α-Dystroglycan is thought to be a potential receptor of agrin and was immunostained in wild-type as well as in agrin-null astrocytes. In freeze-fracture replicas, we observed an increase in OAP density in astrocytes when growing on agrin- and ECM-coatings. These results concurred with other experiments in which changes in volume were measured following hypotonic stress, which supported the positive influence of exogenous agrin on AQP4 insertion into the membrane, on OAP formation and on water transport.

Longitudinal Expression Analysis of αv Integrins in Human Gliomas Reveals Upregulation of Integrin αvβ3 as a Negative Prognostic Factor

Integrin inhibitors targeting αv series integrins are being tested for their therapeutic potential in patients with brain tumors, but pathologic studies have been limited by lack of antibodies suitable for immunohistochemistry (IHC) on formalin-fixed, paraffin-embedded specimens. We compared the expression of αv integrins by IHC in brain tumor and normal human brain samples with gene expression data in a public database using new rabbit monoclonal antibodies against αvβ3, αvβ5, αvβ6, and αvβ8 complexes using both manual and automated microscopy analyses. Glial tumors usually shared an αvβ3-positive/αvβ5-positive/αvβ8-positive/αvβ6-negative phenotype. In 94 WHO (World Health Organization) grade II astrocytomas, 85 anaplastic astrocytomas WHO grade III, and 324 glioblastomas from archival sources, expression of integrins generally increased with grade of malignancy. Integrins αvβ3 and αvβ5 were expressed in many glioma vessels; the intensity of vascular expression of αvβ3 increased with grade of malignancy, whereas αvβ8 was absent. Analysis of gene expression in an independent cohort showed a similar increase in integrin expression with tumor grade, particularly of ITGB3 and ITGB8; ITGB6 was not expressed, consistent with the IHC data. Parenchymal αvβ3 expression and ITGB3 gene overexpression in glioblastomas were associated with a poor prognosis, as revealed by survival analysis (Kaplan-Meier logrank, p = 0.016). Together, these data strengthen the rationale for anti-integrin treatment of glial tumors.

RelA regulates CXCL1/CXCR2-dependent oncogene-induced senescence in murine Kras-driven pancreatic carcinogenesis

Tumor suppression that is mediated by oncogene-induced senescence (OIS) is considered to function as a safeguard during development of pancreatic ductal adenocarcinoma (PDAC). However, the mechanisms that regulate OIS in PDAC are poorly understood. Here, we have determined that nuclear RelA reinforces OIS to inhibit carcinogenesis in the Kras mouse model of PDAC. Inactivation of RelA accelerated pancreatic lesion formation in Kras mice by abrogating the senescence-associated secretory phenotype (SASP) gene transcription signature. Using genetic and pharmacological tools, we determined that RelA activation promotes OIS via elevation of the SASP factor CXCL1 (also known as KC), which activates CXCR2, during pancreatic carcinogenesis. In Kras mice, pancreas-specific inactivation of CXCR2 prevented OIS and was correlated with increased tumor proliferation and decreased survival. Moreover, reductions in CXCR2 levels were associated with advanced neoplastic lesions in tissue from human pancreatic specimens. Genetically disabling OIS in Kras mice caused RelA to promote tumor proliferation, suggesting a dual role for RelA signaling in pancreatic carcinogenesis. Taken together, our data suggest a pivotal role for RelA in regulating OIS in preneoplastic lesions and implicate the RelA/CXCL1/CXCR2 axis as an essential mechanism of tumor surveillance in PDAC.

VE1 immunohistochemistry in pituitary adenomas is not associated with BRAF V600E mutation

(6 %) and 1 strong case (1 %); supplemental Figure S1). Twelve of the positive cases were HGH-producing adenomas, two were mixed mammosomatotroph adenomas and the four remaining adenomas expressed ACTH (including the case with score 3). To rule out non-specific enzymatic activity of the avidin–biotin method, the VE1 diaminobenzidine (DAB) staining results were repeated using two different polymer detection systems (Ventana OptiView and Leica BondMax), as recommended by Andrulis et al. [1] to reduce unspecific background staining, and reproducibly confirmed the positive reactions (Fig. 1b). The signal was even stronger than in the initial DAB staining of the same case (Fig. 1a). The cases were also stained with synaptophysin and CD68; possible occasional cross-reactivity was excluded [3]. Ten VE1 positive cases (one score 3, three score 2 and six score 1) were analyzed by clamped probe assay; they showed wild-type BRAF curves (Fig. 1c; for experimental methods see supplemental data). Sanger sequencing of BRAF exon 15 in the same cases confirmed the absence of V600E mutation (Fig. 1d). We minimized sequencing failure because we used only samples with 80 % tumor content and additionally performed clamped probe assays that are able to detect mutations even if the mutation/wild-type ratio is as low as 1 % [9]. Due to their highsequence homology with the BRAF activation segment, we also analyzed the activation segments of ARAF and CRAF by sequencing to rule out VE1 cross-reactivity with a putative homolog ARAF V453E or CRAF V492E mutation, again revealing wild-type sequences for all cases (Fig. 1d; for experimental procedures, see supplemental data). In contrast to many previously investigated cancers, our results indicate that even strong positive immunostaining of VE1 in pituitary adenomas does not indicate the presence of a BRAF V600E mutation, especially if it is restricted to the cells’ membranes. While V600E mutations Activating BRAF mutations are observed in malignant melanomas, pleomorphic xanthoastrocytomas, hairy cell leukemia, papillary thyroid carcinomas, colorectal cancer, ovarian cancer and ganglioglioma [1, 10, 11]. Generation of a monoclonal antibody specific for the BRAF V600E mutation (VE1) was published recently [3]. This antibody has been successfully validated for melanomas [5], lung adenocarcinomas [8] and thyroid carcinomas [2]. Although pituitary adenomas account for up to 10 % of all intracranial tumors [4], there is still a lack of data on VE1 immunostains in these tumors. In three studies in which BRAF in pituitary adenomas was examined by sequencing, a single V600E mutation was found in 145 cases [6, 7, 10]. We screened 78 pituitary adenomas (epidemiological data in supplemental Table 1) with VE1 antibody. Staining was observed in 18 cases (12 weak (15 %); 5 moderate

Lack of CCR7 expression is rate limiting for lymphatic spread of pancreatic ductal adenocarcinoma

CCR7 expression on tumor cells promotes lymphatic spread in several malignant tumors. However, a comprehensive characterization of the CCL19/CCL21–CCR7 axis in pancreatic ductal adenocarcinoma (PDAC), which is known for its high rates of lymph‐node metastases, is still lacking. CCR7 mRNA and CCR7 protein were found to be expressed in spheroid cultures of all six examined PDAC cell lines. In migration assays, CCR7 expressing PDAC cells showed enhanced migration toward CCL19 and CCL21, the two ligands of CCR7. In an orthotopic nude mouse model, CCR7‐transfected PT45P1 cells gave rise to significantly larger tumors and showed a higher frequency of lymph vessel invasion and lymph‐node metastases than mock‐transfected cells. In an analysis using quantitative real‐time PCR, CCR7 showed fourfold overexpression in microdissected PDAC cells compared to normal duct cells. Moderate‐to‐strong immunohistochemical CCR7 expression, found in 58 of 121 well‐characterized human PDACs, correlated with high rates of lymph vessel invasion. Conversely, PDACs completely lacking CCR7 expression showed only low rates of lymph vessel invasion and lymph‐node metastases. The evaluation of CCL21 expression by immunofluorescence staining revealed a significant upregulation of CCL21 in peritumoral and intratumoral lymph vessels compared to lymph vessels in disease‐free pancreata. In conclusion, our study revealed strong evidence that lack of CCR7 impairs the metastatic potential of PDAC. Lymph vessel invasion by CCR7 expressing PDAC cells may be additionally enhanced by upregulation of CCL21 in tumor‐associated lymph vessels, representing a previously unknown factor of lymphatic spread.

Loss of Chromosome 18 in Neuroendocrine Tumors of the Small Intestine: The Enigma Remains

Background/Aims: Neuroendocrine tumors of the small intestine (SI-NETs) exhibit an increasing incidence and high mortality rate. Until now, no fundamental molecular event has been linked to the tumorigenesis and progression of these tumors. Only the loss of chromosome 18 (Chr18) has been shown in up to two thirds of SI-NETs, whereby the significance of this alteration is still not understood. We therefore performed the first comprehensive study to identify Chr18-related events at the genetic, epigenetic and gene/protein expression levels. Methods: We did expression analysis of all seven putative Chr18-related tumor suppressors by quantitative real-time PCR (qRT-PCR), Western blot and immunohistochemistry. Next-generation exome sequencing and SNP array analysis were performed with five SI-NETs with (partial) loss of Chr18. Finally, we analyzed all microRNAs (miRNAs) located on Chr18 by qRT-PCR, comparing Chr18+/- and Chr18+/+ SI-NETs. Results: Only DCC (deleted in colorectal cancer) revealed loss of/greatly reduced expression in 6/21 cases (29%). No relevant loss of SMAD2, SMAD4, elongin A3 and CABLES was detected. PMAIP1 and maspin were absent at the protein level. Next-generation sequencing did not reveal relevant recurrent somatic mutations on Chr18 either in an exploratory cohort of five SI-NETs, or in a validation cohort (n = 30). SNP array analysis showed no additional losses. The quantitative analysis of all 27 Chr18-related miRNAs revealed no difference in expression between Chr18+/- and Chr18+/+ SI-NETs. Conclusion: DCC seems to be the only Chr18-related tumor suppressor affected by the monoallelic loss of Chr18 resulting in a loss of DCC protein expression in one third of SI-NETs. No additional genetic or epigenetic alterations were present on Chr18.

Establishment and Characterization of Primary Cell Lines of Squamous Cell Carcinoma of the Penis and its Metastasis

PURPOSE We established cell lines from penile squamous cell carcinoma and its lymph node metastasis, and investigated the role of chemokines, chemokine receptors and podoplanin in cancer progression. MATERIALS AND METHODS Tumor specimen of primary tumors, and lymph node and distant metastases were cultured in vitro and xenotransplanted in SCID beige mice. Specimens were analyzed by hematoxylin and eosin staining, and immunohistochemistry. Comparative screening for chemokines, chemokine receptors and podoplanin was done by polymerase chain reaction, fluorescence activated cell sorting and enzyme-linked immunosorbent assay. RESULTS We established 2 cell lines from a primary tumor and its corresponding lymph node metastasis, respectively. Heterotopic xenotransplantation revealed reliable tumor growth in vivo. Morphological and immunohistological analysis showed comparable features for human tumors, cell lines in vitro and xenotransplanted tumors in mice regarding the primary tumor and metastasis. Comprehensive analysis of chemokines and chemokine receptors in the metastasis derived cell line and in the cell line originating from the primary tumor revealed the most pronounced changes for CXCL14. This pattern was confirmed on the protein level. Comparative analysis of podoplanin showed marked down-regulation in the metastatic variant on the mRNA and protein levels. CONCLUSIONS To our knowledge we established the first pair of cell lines of a human primary penile tumor and the corresponding lymph node metastasis. These cell lines offer unique possibilities for further comparative functional investigations in in vitro and in vivo settings. They enable studies of new potential therapeutic agents and other assays to better understand the molecular mechanisms of penile cancer progression.

Findet die molekulare Diagnostik Einzug in die Pankreaspathologie

Genetic alterations of solid and cystic tumors of the pancreas have been increasingly more characterized over the last few years. Pancreatic ductal adenocarcinoma (PDAC) carries numerous point mutations and, to a lesser extent, deletions and amplifications of genes that are associated with at least 13 tumor relevant signalling pathways and processes. Besides the four common driver mutations in the KRAS, p53, CDKN2a and SMAD4 genes there are a number of low frequency driver mutations. The classification of PDAC subtypes has benefited from recent analyses of transcriptional profiles that revealed a classical KRAS driven and a KRAS independent quasi-mesenchymal subtype. The analyses of mRNA and miRNA expression profiles of fine needle aspirates serve as a basis for reliable preoperative diagnosis of pancreatic masses.The four most common cystic pancreatic tumors bear tumor-specific genetic alterations, such as GNAS mutations in intraductal papillary mucinous neoplasms, β-catenin mutations in solid pseudopapillary neoplasms and VHL mutations or loss of heterozygosity in serous cystadenoma. Recovery of DNA from aspirates of cyst fluids enables an improved preoperative management of cystic pancreatic tumors by mutational analysis. In addition to the analysis of DNA there are promising approaches in distinguishing benign and premalignant/malignant cystic tumors by evaluating miRNA profiles.In recent years much progress has been made in molecular genetic characterization and preoperative evaluation of pancreatic tumors. Hopefully these results will contribute to prognostic and therapeutic stratification of PDAC and to a reliable preoperative diagnostics of benign cystic pancreatic tumors in the future.