Molecular signature of stem-like glioma cells (SLGCs) from human glioblastoma and gliosarcoma.

Glioblastoma multiforme (GBM) and the GBM variant gliosarcoma (GS) are among the tumors with the highest morbidity and mortality, providing only palliation. Stem-like glioma cells (SLGCs) are involved in tumor initiation, progression, therapy resistance, and relapse. The identification of general features of SLGCs could contribute to the development of more efficient therapies. Commercially available protein arrays were used to determine the cell surface signature of eight SLGC lines from GBMs, one SLGC line obtained from a xenotransplanted GBM-derived SLGC line, and three SLGC lines from GSs. By means of non-negative matrix factorization expression metaprofiles were calculated. Using the cophenetic correlation coefficient (CCC) five metaprofiles (MPs) were identified, which are characterized by specific combinations of 7-12 factors. Furthermore, the expression of several factors, that are associated with GBM prognosis, GBM subtypes, SLGC differentiation stages, or neural identity was evaluated. The investigation encompassed 24 distinct SLGC lines, four of which were derived from xenotransplanted SLGCs, and included the SLGC lines characterized by the metaprofiles. It turned out that all SLGC lines expressed the epidermal growth factor EGFR and EGFR ligands, often in the presence of additional receptor tyrosine kinases. Moreover, all SLGC lines displayed a neural signature and the IDH1 wildtype, but differed in their p53 and PTEN status. Pearson Correlation analysis identified a positive association between the pluripotency factor Sox2 and the expression of FABP7, Musashi, CD133, GFAP, but not with MGMT or Hif1α. Spherical growth, however, was positively correlated with high levels of Hif1α, CDK4, PTEN, and PDGFRß, whereas correlations with stemness factors or MGMT (MGMT expression and promoter methylation) were low or missing. Factors highly expressed by all SLGC lines, irrespective of their degree of stemness and growth behavior, are Cathepsin-D, CD99, EMMPRIN/CD147, Intß1, the Galectins 3 and 3b, and N-Cadherin.

The EGFR phosphatase RPTPγ is a redox-regulated suppressor of promigratory signaling.

Spatially organized reaction dynamics between proto-oncogenic epidermal growth factor receptor (EGFR) and protein tyrosine phosphatases determine EGFR phosphorylation dynamics in response to growth factors and thereby cellular behavior within developing tissues. We show that the reaction dynamics of mutual inhibition between RPTPγ phosphatase and autocatalytic ligandless EGFR phosphorylation enable highly sensitive promigratory EGFR signaling responses to subnanomolar EGF levels, when < 5% receptors are occupied by EGF. EGF thereby triggers an autocatalytic phospho-EGFR reaction by the initial production of small amounts of phospho-EGFR through transient, asymmetric EGF-EGFR2 dimers. Single cell RPTPγ oxidation imaging revealed that phospho-EGFR induces activation of NADPH oxidase, which in turn inhibits RPTPγ-mediated dephosphorylation of EGFR, tilting the autocatalytic RPTPγ/EGFR toggle switch reaction towards ligandless phosphorylated EGFR. Reversibility of this reaction to EGF is maintained by the constitutive phosphatase activity of endoplasmic reticulum-associated TCPTP. This RPTPγ/EGFR reaction at the plasma membrane causes promigratory signaling that is separated from proliferative signaling induced by accumulated, liganded, phosphorylated EGF-EGFR in endosomes. Accordingly, loss of RPTPγ results in constitutive promigratory signaling from phosphorylated EGFR monomers. RPTPγ is thus a suppressor of promigratory oncogenic but not of proliferative EGFR signaling.

Primary URECs: a source to better understand the pathology of renal tubular epithelia in pediatric hereditary cystic kidney diseases.

BACKGROUND: In pediatric hereditary cystic kidney diseases, epithelial cell defects mostly result from rare, autosomal recessively inherited pathogenic variants in genes encoding proteins of the cilia-centrosome complex. Consequences of individual gene variants on epithelial function are often difficult to predict and can furthermore depend on the patient's genetic background. Here, we studied urine-derived renal tubular epithelial cells (URECs) from genetically determined, pediatric cohorts of different hereditary cystic kidney diseases, comprising autosomal recessive polycystic kidney disease, nephronophthisis (NPH) and the Bardet Biedl syndrome (BBS). UREC characteristics and behavior in epithelial function-related 3D cell culture were compared in order to identify gene and variant-specific properties and to determine aspects of epithelial (cell) dysfunction. RESULTS: UREC preparations from patients (19) and healthy controls (39) were studied in a qualitative and quantitative manner using primary cells cultured for up-to 21 days. In patients with biallelic pathogenic variants in PKHD1 or NPHP genes, we were able to receive satisfactory amounts of URECs of reproducible quality. In BBS patients, UREC yield was lower and more dependent on the individual genotype. In contrast, in UREC preparations derived from healthy controls, no predictable and satisfactory outcome could be established. Considering cell proliferation, tubular origin and epithelial properties in 2D/3D culture conditions, we observed distinct and reproducible epithelial properties of URECs. In particular, the cells from patients carrying PKHD1 variants were characterized by a high incidence of defective morphogenesis of monolayered spheroids-a property proposed to be suitable for corrective intervention. Furthermore, we explored different ways to generate reference cell lines for both-patients and healthy controls-in order to eliminate restrictions in cell number and availability of primary URECs. CONCLUSIONS: Ex vivo 3D cell culture of primary URECs represents a valuable, non-invasive source to evaluate epithelial cell function in kidney diseases and as such helps to elucidate the functional consequences of rare genetic disorders. In combination with genetically defined control cell lines to be generated in the future, the cultivation of primary URECs could become a relevant tool for testing personalized treatment of epithelial dysfunction in patients with hereditary cystic kidney disease.

Fibrocystin Is Essential to Cellular Control of Adhesion and Epithelial Morphogenesis.

Mutations of the Pkhd1 gene cause autosomal recessive polycystic kidney disease (ARPKD). Pkhd1 encodes fibrocystin/polyductin (FPC), a ciliary type I membrane protein of largely unknown function, suggested to affect adhesion signaling of cells. Contributions of epithelial cell adhesion and contractility to the disease process are elusive. Here, we link loss of FPC to defective epithelial morphogenesis in 3D cell culture and altered cell contact formation. We study Pkhd1-silenced Madin-Darby Canine Kidney II (MDCKII) cells using an epithelial morphogenesis assay based on micropatterned glass coverslips. The assay allows analysis of cell adhesion, polarity and lumen formation of epithelial spheroids. Pkhd1 silencing critically affects the initial phase of the morphogenesis assay, leading to a reduction of correctly polarized spheroids by two thirds. Defects are characterized by altered cell adhesion and centrosome positioning of FPC-deficient cells in their 1-/2-cell stages. When myosin II inhibitor is applied to reduce cellular tension during the critical early phase of the assay, Pkhd1 silencing no longer inhibits formation of correctly polarized epithelia. We propose that altered sensing and cell interaction of FPC-deficient epithelial cells promote progressive epithelial defects in ARPKD.

Application and Comparison of Supervised Learning Strategies to Classify Polarity of Epithelial Cell Spheroids in 3D Culture.

Three-dimensional culture systems that allow generation of monolayered epithelial cell spheroids are widely used to study epithelial function in vitro. Epithelial spheroid formation is applied to address cellular consequences of (mono)-genetic disorders, that is, ciliopathies, in toxicity testing, or to develop treatment options aimed to restore proper epithelial cell characteristics and function. With the potential of a high-throughput method, the main obstacle to efficient application of the spheroid formation assay so far is the laborious, time-consuming, and bias-prone analysis of spheroid images by individuals. Hundredths of multidimensional fluorescence images are blinded, rated by three persons, and subsequently, differences in ratings are compared and discussed. Here, we apply supervised learning and compare strategies based on machine learning versus deep learning. While deep learning approaches can directly process raw image data, machine learning requires transformed data of features extracted from fluorescence images. We verify the accuracy of both strategies on a validation data set, analyse an experimental data set, and observe that different strategies can be very accurate. Deep learning, however, is less sensitive to overfitting and experimental batch-to-batch variations, thus providing a rather powerful and easily adjustable classification tool.

Fibroblast growth factor 23 is induced by an activated renin-angiotensin-aldosterone system in cardiac myocytes and promotes the pro-fibrotic crosstalk between cardiac myocytes and fibroblasts.

Background: Fibroblast growth factor 23 (FGF23) is discussed as a new biomarker of cardiac hypertrophy and mortality in patients with and without chronic kidney disease (CKD). We previously demonstrated that FGF23 is expressed by cardiac myocytes, enhanced in CKD and induces cardiac hypertrophy via activation of FGF receptor 4 independent of its co-receptor klotho. The impact of FGF23 on cardiac fibrosis is largely unknown. Methods: By conducting a retrospective case-control study including myocardial autopsy samples from 24 patients with end-stage CKD and in vitro studies in cardiac fibroblasts and myocytes, we investigated the pro-fibrotic properties of FGF23. Results: The accumulation of fibrillar collagens I and III was increased in myocardial tissue of CKD patients and correlated with dialysis vintage, klotho deficiency and enhanced cardiac angiotensinogen (AGT) expression. Using human fibrosis RT2 Profiler PCR array analysis, transforming growth factor (TGF)-ß and its related TGF-ß receptor/Smad complexes, extracellular matrix remodeling enzymes and pro-fibrotic growth factors were upregulated in myocardial tissue of CKD patients. FGF23 stimulated cell proliferation, migration, pro-fibrotic TGF-ß receptor/Smad complexes and collagen synthesis in cultured cardiac fibroblasts. In isolated cardiac myocytes, FGF23 enhanced collagen remodeling, expression of pro-inflammatory genes and pro-survival pathways and induced pro-hypertrophic genes. FGF23 stimulated AGT expression in cardiac myocytes and angiotensin II and aldosterone, as components of the renin-angiotensin-aldosterone system (RAAS), induced FGF23 in cardiac myocytes. Conclusions: Our data demonstrate that activated RAAS induces FGF23 expression in cardiac myocytes and thereby stimulates a pro-fibrotic crosstalk between cardiac myocytes and fibroblasts, which may contribute to myocardial fibrosis in CKD.

Focal adhesion kinase activity is required for actomyosin contractility-based invasion of cells into dense 3D matrices.

The focal adhesion kinase (FAK) regulates the dynamics of integrin-based cell adhesions important for motility. FAK's activity regulation is involved in stress-sensing and focal-adhesion turnover. The effect of FAK on 3D migration and cellular mechanics is unclear. We analyzed FAK knock-out mouse embryonic fibroblasts and cells expressing a kinase-dead FAK mutant, R454-FAK, in comparison to FAK wild-type cells. FAK knock-out and FAKR454/R454 cells invade dense 3D matrices less efficiently. These results are supported by FAK knock-down in wild-type fibroblasts and MDA-MB-231 human breast cancer cells showing reduced invasiveness. Pharmacological interventions indicate that in 3D matrices, cells deficient in FAK or kinase-activity behave similarly to wild-type cells treated with inhibitors of Src-activity or actomyosin-contractility. Using magnetic tweezers experiments, FAKR454/R454 cells are shown to be softer and exhibit impaired adhesion to fibronectin and collagen, which is consistent with their reduced 3D invasiveness. In line with this, FAKR454/R454 cells cannot contract the matrix in contrast to FAK wild-type cells. Finally, our findings demonstrate that active FAK facilitates 3D matrix invasion through increased cellular stiffness and transmission of actomyosin-dependent contractile force in dense 3D extracellular matrices.