Slit and Robo proteins are evolutionarily conserved molecules whose interaction underlies axon guidance and neuronal precursor cell migration. During development secreted Slit proteins mediate chemorepulsive signals on cells expressing Robo receptors. Because similar molecular mechanisms may be utilized in glioma cell invasion and neuroblast migration, we studied the expression of Slit2 and its transmembrane receptor Robo1 as well as their functional role in migration in glioma cells. qRT-PCR and immunohistochemistry of human specimens revealed that Slit2 was distinctly expressed by non-neoplastic neurons, but at only very low levels in fibrillary astrocytoma and glioblastoma. Robo1 also was mainly restricted to neurons in the normal brain, whereas astrocytic tumor cells in situ as well as glioblastoma cell lines overexpressed Robo1 at mRNA and protein levels. Recombinant human Slit2 in a concentration of 0.45 nM was repulsive for glioma cell lines in a modified Boyden chamber assay. RNAi-mediated knockdown of Robo1 in glioma cell lines neutralized the repulsive effect of Slit2, demonstrating that Robo1 served as the major Slit2 receptor. Our findings suggest that a chemorepulsive effect mediated by interaction of Slit2 and Robo1 participates in glioma cell guidance in the brain.

BackgroundRecent studies have demonstrated that a molecular subtype of glioblastoma is characterized by overexpression of extracellular matrix (ECM)/mesenchymal components and shorter survival. Specifically, gene expression profiling studies revealed that matrix gla protein (MGP), whose function has traditionally been linked to inhibition of calcification of arteries and cartilage, is overexpressed in glioblastomas and associated with worse outcome.MethodsIn order to analyze the role of MGP in glioblastomas, we performed expression, migration and proliferation studies.ResultsReal-time PCR and ELISA assays confirmed overexpression of MGP in glioblastoma biopsy specimens and cell lines at mRNA and protein levels as compared to normal brain tissue. Immunohistochemistry verified positivity of glial tumor cells for MGP. RNAi-mediated knockdown of MGP in three glioma cell lines (U343MG, U373MG, H4) led to marked reduction of migration, as demonstrated by wound healing and transwell assays, while no effect on proliferation was seen.ConclusionOur data suggest that upregulation of MGP (and possibly other ECM-related components as well) results in unfavorable prognosis via increased migration.

The poor prognosis of glioblastoma patients is related to diffuse brain invasion and interaction of tumor cells with extracellular matrices (ECM). We describe expression and function of the FACIT-collagen XVI in glioblastomas. We found upregulation of collagen XVI mRNA as well as protein in glioblastomas as compared to normal cortex. SiRNA knockdown resulted in decreased cell adhesion whereas increased adhesion was observed on surfaces coated with collagen XVI. The migration of glioblastoma cells on this substrate remained unchanged. Our results demonstrate de-novo expression of collagen XVI in glioblastomas as part of the tumor specific remodeling of the ECM. Structured summary:MINT-6743179: Collagen IV (uniprotkb:P02462-1) and Collagen XVI

The pathogenesis of choroid plexus papillomas, intraventricular papillary neoplasms most often occurring sporadically in children and young adults, remains poorly understood. To identify pathways operative in the development of choroid plexus papillomas, gene expression profiles obtained from laser-microdissected human choroid plexus papilloma cells (n = 7) were compared with that of normal choroid plexus epithelial cells laser microdissected from autopsy tissue (n = 8). On DNA microarray data analysis, 53 probe sets were differentially expressed in choroid plexus papilloma tumor cells (>7-fold). Up-regulation of TWIST1, WIF1, TRPM3, BCLAF1, and AJAP1, as well as down-regulation of IL6ST was confirmed using quantitative reverse transcription-PCR. Knockdown of Twist1 gene expression in the rat choroid plexus epithelial cell line Z310 significantly reduced proliferation as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and cell invasion in a Matrigel assay, whereas cell migration was not affected. Screening for expressional changes of cancer-related genes upon Twist1 knockdown revealed up-regulation of Cdkn1a, Cflar, and Serpinb2 and down-regulation of Figf. To conclude, using gene expression profiling, several genes differentially expressed in human choroid plexus papillomas could be identified. Among those, TWIST1 is highly expressed in choroid plexus papillomas and promotes proliferation and invasion.

Dry eye disease (DED) is a multifactorial disease characterized by a disrupted tear film homeostasis and inflammation leading to visual impairments and pain in patients. Aqueous-deficient dry eye (ADDE) causes the most severe progressions and depends mainly on the loss of functional lacrimal gland (LG) tissue. Despite a high prevalence, therapies remain palliative. Therefore, it is of great interest to develop new approaches to curatively treat ADDE. Mesenchymal stem/stromal cells (MSC) have been shown to induce tissue regeneration and cease inflammation. Moreover, an increasing amount of MSC was found in the regenerating LG of mice. Therefore, this study investigated the therapeutic effect of MSC transplantation on damaged LGs using duct ligation induced ADDE in mice. Due to the transplantation of sex-mismatched and eGFP-expressing MSC, MSC could be identified and detected until day 21. MSC transplantation significantly improved LG regeneration, as the amount of vital acinar structures was significantly increased above the intrinsic regeneration capacity of control. Additionally, MSC transplantation modulated the immune reaction as macrophage infiltration was delayed and TNFα expression decreased, accompanied by an increased IL-6 expression. Thus, the application of MSC appears to be a promising therapeutic approach to induce LG regeneration in patients suffering from severe DED/ADDE.

Lacrimal gland (LG) insufficiency is a main cause for severe dry eye leading to pain, visual impairment, and eventually loss of sight. Engineering of transplantable LG tissue with secretory capacity is a desirable goal. In this study, a three-dimensional decellularized LG (DC-LG) scaffold with preserved LG morphology was generated by treatment with 1% sodium deoxycholate and DNase solution using porcine LG tissue. To address clinical applicability, the primary in vitro culture of secretory active LG cells from a small tissue biopsy of 1.5 mm diameter was introduced and compared with an established isolation method by enzymatic digestion. Cells from both isolation methods depicted an epithelial phenotype, maintained their secretory capacity for up to 30 days, and exhibited progenitor cell capacity as measured by aldehyde dehydrogenase-1 activity, side population assay, and colony-forming units. Cells from passage 0 were reseeded into the DC-LG and secretory active cells migrated into the tissue. The cells resembled an LG-like morphology and the constructs showed secretory activity. These results demonstrate the possibility of engineering a secretory competent, three-dimensional LG construct using LG cells expanded from a small tissue biopsy and DC-LG as a matrix that provides the native structure and physiological niche for these cells.

No abstract

No abstract