Colon cancer cell-derived 12(S)-HETE induces the retraction of cancer-associated fibroblast via MLC2, RHO/ROCK and Ca2+ signalling

Retraction of mesenchymal stromal cells supports the invasion of colorectal cancer cells (CRC) into the adjacent compartment. CRC-secreted 12(S)-HETE enhances the retraction of cancer-associated fibroblasts (CAFs) and therefore, 12(S)-HETE may enforce invasivity of CRC. Understanding the mechanisms of metastatic CRC is crucial for successful intervention. Therefore, we studied pro-invasive contributions of stromal cells in physiologically relevant three-dimensional in vitro assays consisting of CRC spheroids, CAFs, extracellular matrix and endothelial cells, as well as in reductionist models. In order to elucidate how CAFs support CRC invasion, tumour spheroid-induced CAF retraction and free intracellular Ca2+ levels were measured and pharmacological-or siRNA-based inhibition of selected signalling cascades was performed. CRC spheroids caused the retraction of CAFs, generating entry gates in the adjacent surrogate stroma. The responsible trigger factor 12(S)-HETE provoked a signal, which was transduced by PLC, IP3, free intracellular Ca2+, Ca(2+)calmodulin-kinase-II, RHO/ROCK and MYLK which led to the activation of myosin light chain 2, and subsequent CAF mobility. RHO activity was observed downstream as well as upstream of Ca2+ release. Thus, Ca2+ signalling served as central signal amplifier. Treatment with the FDA-approved drugs carbamazepine, cinnarizine, nifedipine and bepridil HCl, which reportedly interfere with cellular calcium availability, inhibited CAF-retraction. The elucidation of signalling pathways and identification of approved inhibitory drugs warrant development of intervention strategies targeting tumour-stroma interaction

An adapted passive model of anti-MPO dependent crescentic glomerulonephritis reveals matrix dysregulation and is amenable to modulation by CXCR4 inhibition

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) are severe inflammatory disorders that often involve focal necrotizing glomerulonephritis (FNGN) and consequent glomerular scarring, interstitial fibrosis, and chronic kidney disease. Robust murine models of scarring in FNGN that may help to further our understanding of deleterious processes are still lacking. Here, we present a murine model of severe FNGN based on combined administration of antibodies against the glomerular basement membrane (GBM) and myeloperoxidase (MPO), and bacterial lipopolysaccharides (LPS), that recapitulates acute injury and was adapted to investigate subsequent glomerular and interstitial scarring. Hematuria without involvement of other organs occurs consistently and rapidly, glomerular necrosis and crescent formation are evident at 12 days, and consequent glomerular and interstitial scarring at 29 days after initial treatment. Using mass-spectrometric proteome analysis, we provide a detailed overview of matrisomal and cellular changes in our model. We observed increased expression of the matrisome including collagens, fibronectin, tenascin-C, in accordance with human AAV as deduced from analysis of gene expression microarrays and tissue staining. Moreover, we observed tissue infiltration by neutrophils, macrophages, T cells and myofibroblasts upon injury. Experimental inhibition of CXCR4 using AMD3100 led to a sustained histological presence of fibrin extravasate, reduced chemokine expression and leukocyte activation, but did not markedly affect ECM composition. Altogether, we demonstrate an adapted FNGN model that enables the study of matrisomal changes both in disease and upon intervention, as exemplified via CXCR4 inhibition

Lymphatic exosomes promote dendritic cell migration along guidance cues

Lymphatic endothelial cells (LECs) release extracellular chemokines to guide the migration of dendritic cells. In this study, we report that LECs also release basolateral exosome-rich endothelial vesicles (EEVs) that are secreted in greater numbers in the presence of inflammatory cytokines and accumulate in the perivascular stroma of small lymphatic vessels in human chronic inflammatory diseases. Proteomic analyses of EEV fractions identified >1,700 cargo proteins and revealed a dominant motility-promoting protein signature. In vitro and ex vivo EEV fractions augmented cellular protrusion formation in a CX3CL1/fractalkine-dependent fashion and enhanced the directional migratory response of human dendritic cells along guidance cues. We conclude that perilymphatic LEC exosomes enhance exploratory behavior and thus promote directional migration of CX3CR1-expressing cells in complex tissue environments.Peer reviewe

Glomerular Collagen Deposition and Lipocalin-2 Expression Are Early Signs of Renal Injury in Prediabetic Obese Rats

Feeding rats with high-fat diet (HFD) with a single streptozotocin (STZ) injection induced obesity, slightly elevated fasting blood glucose and impaired glucose and insulin tolerance, and caused cardiac hypertrophy and mild diastolic dysfunction as published before by Koncsos et al. in 2016. Here we aimed to explore the renal consequences in the same groups of rats. Male Long-Evans rats were fed normal chow (CON; n = 9) or HFD containing 40% lard and were administered STZ at 20 mg/kg (i.p.) at week four (prediabetic rats, PRED, n = 9). At week 21 blood and urine samples were taken and kidney and liver samples were collected for histology, immunohistochemistry and for analysis of gene expression. HFD and STZ increased body weight and visceral adiposity and plasma leptin concentration. Despite hyperleptinemia, plasma C-reactive protein concentration decreased in PRED rats. Immunohistochemistry revealed elevated collagen IV protein expression in the glomeruli, and Lcn2 mRNA expression increased, while Il-1β mRNA expression decreased in both the renal cortex and medulla in PRED vs. CON rats. Kidney histology, urinary protein excretion, plasma creatinine, glomerular Feret diameter, desmin protein expression, and cortical and medullary mRNA expression of TGF-β1, Nrf2, and PPARγ were similar in CON and PRED rats. Reduced AMPKα phosphorylation of the autophagy regulator Akt was the first sign of liver damage, while plasma lipid and liver enzyme concentrations were similar. In conclusion, glomerular collagen deposition and increased lipocalin-2 expression were the early signs of kidney injury, while most biomarkers of inflammation, oxidative stress and fibrosis were negative in the kidneys of obese, prediabetic rats with mild heart and liver injury

Podocyte RNA sequencing reveals Wnt- and ECM-associated genes as central in FSGS.

Loss of podocyte differentiation can cause nephrotic-range proteinuria and Focal and Segmental Glomerulosclerosis (FSGS). As specific therapy is still lacking, FSGS frequently progresses to end-stage renal disease. The exact molecular mechanisms of FSGS and gene expression changes in podocytes are complex and widely unknown as marker changes have mostly been assessed on the glomerular level. To gain a better insight, we isolated podocytes of miR-193a overexpressing mice, which suffer from FSGS due to suppression of the podocyte master regulator Wt1. We characterised the podocytic gene expression changes by RNAseq and identified many novel candidate genes not linked to FSGS so far. This included strong upregulation of the receptor tyrosine kinase EphA6 and a massive dysregulation of circadian genes including the loss of the transcriptional activator Arntl. By comparison with podocyte-specific changes in other FSGS models we found a shared dysregulation of genes associated with the Wnt signaling cascade, while classical podocyte-specific genes appeared widely unaltered. An overlap with gene expression screens from human FSGS patients revealed a strong enrichment in genes associated with extra-cellular matrix (ECM) and metabolism. Our data suggest that FSGS progression might frequently depend on pathways that are often overlooked when considering podocyte homeostasis

Selection of scFv Antibody Fragments Binding to Human Blood versus Lymphatic Endothelial Surface Antigens by Direct Cell Phage Display

<div><p>The identification of marker molecules specific for blood and lymphatic endothelium may provide new diagnostic tools and identify new targets for therapy of immune, microvascular and cancerous diseases. Here, we used a phage display library expressing human randomized single-chain Fv (scFv) antibodies for direct panning against live cultures of blood (BECs) and lymphatic (LECs) endothelial cells in solution. After six panning rounds, out of 944 sequenced antibody clones, we retrieved 166 unique/diverse scFv fragments, as indicated by the V-region sequences. Specificities of these phage clone antibodies for respective compartments were individually tested by direct cell ELISA, indicating that mainly pan-endothelial cell (EC) binders had been selected, but also revealing a subset of BEC-specific scFv antibodies. The specific staining pattern was recapitulated by twelve phage-independently expressed scFv antibodies. Binding capacity to BECs and LECs and differential staining of BEC versus LEC by a subset of eight scFv antibodies was confirmed by immunofluorescence staining. As one antigen, CD146 was identified by immunoprecipitation with phage-independent scFv fragment. This antibody, B6-11, specifically bound to recombinant CD146, and to native CD146 expressed by BECs, melanoma cells and blood vessels. Further, binding capacity of B6-11 to CD146 was fully retained after fusion to a mouse Fc portion, which enabled eukaryotic cell expression. Beyond visualization and diagnosis, this antibody might be used as a functional tool. Overall, our approach provided a method to select antibodies specific for endothelial surface determinants in their native configuration. We successfully selected antibodies that bind to antigens expressed on the human endothelial cell surfaces <i>in situ</i>, showing that BECs and LECs share a majority of surface antigens, which is complemented by cell-type specific, unique markers.</p></div

Expression of Merkelcell polyomavirus (MCPyV) large T-antigen in Merkel cell carcinoma lymph node metastases predicts poor outcome.

The aim of this study was to determine the prevalence of MCPyV in Merkel cell carcinoma (MCC) primaries versus lymph node metastasis and to evaluate possible prognostic factors.Samples of MCC primaries and lymph node metastases were stained immunohistochemically for the MCPyV large T-antigen and expression was compared to patients´ clinical outcome.41 MCC patients were included. 33 (61%) out of 54 specimens were MCPyV-positive in the immunohistochemistry. 15 (47%) out of 32 primary tumors were positive compared to 18 (82%) out of 22 lymph node metastases. Eleven patients with positive polyomavirus expression died from the carcinoma compared to 4 patients without virus expression. Cox regression analysis showed worse disease-free survival in patients with MCPyV compared to virus-negative lymph nodes (p = 0.002).To our knowledge this is the first study to describe a negative prognostic effect of the MCPyV expression in lymph node metastasis in MCC patients

Enrichment of phage antibodies that bind to BECs and LECs with successive biopanning rounds.

<p>A: Numbers of recovered phage particles after each panning round on BECs and LECs. For each selection round, the phage output/input titer ratios are given on the y-axis, showing an increase on BECs (red line) and LECs (green line) when adding polyclonal ETH-2 phage. In contrast, control wild-type VCS-M13 (grey and black lines) phage did not shift significantly on either cell population. B: Enrichment of BEC- and LEC-binding phage antibodies as seen by FACS-analysis of phage antibody pools. Shown are representative histograms of BECs and LECs stained with phage antibodies after respective panning rounds (black line) and WT phage as negative control (grey-filled graphs). The number of cells (counts: Y-axis) is given as function of the fluorescence intensity of phage antibody staining of the cells (FL1-H: X-axis). Percentage of BECs and LECs shifted by phage binding is depicted in the graphics. In all experiments, cells were incubated with phage antibody pools, and cell-binding was detected by anti-M13 antibody and FITC-conjugated anti-rabbit antibody. Grey-filled graphs show WT phage binding as negative control. C: <i>BstN</i>I DNA fingerprint analysis of single scFv clones. Representative gel images of respective panning rounds (#1 - #6) performed on BECs and LECs are shown. Over the course of panning rounds, the number of intact clones is increasing. Phagemid DNA isolated from single clones was digested with <i>BstN</i>I and analyzed in agarose gel electrophoresis. M: 1 kbp molecular weight marker.</p

LC-MS/MS identification of CD146 binding to scFv B6-11, and antigen confirmation by immunoprecipitation and ELISA.

<p>A: Alignment of LC-MS/MS identified peptides (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127169#pone.0127169.s004" target="_blank">S4 Fig</a>) with the sequence of MCAM/CD146MUC18. The eluates from scFv B6-11 immunoprecipitation were subjected to trypsin digestion (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127169#sec002" target="_blank">Materials and Methods</a> section) and subsequently analyzed by LC-MS/MS. B: Immunoprecipitation of CD146 by soluble scFv B6-11 from BEC lysates. Immune complexes were tested by Western blot in reducing conditions using commercial anti-CD146 antibody. Lane 1: input BEC lysate, lanes 2–5: immunoprecipitates with scFv B6-11, lanes 2 and 4: under addition of PNGase F. Treatment of BEC lysates with PNGase prior or after addition of scFv B6-11 had no influence on co-immunoprecipitation capacity of scFv B6-11, showing that scFv B6-11 binding to CD146 is glycosylation-independent. C: scFv B6-11 binds to immobilized extracellular domain of recombinant human CD146 in ELISA. An irrelevant antigen (BSA), a non-binding scFv and uncoated wells served as controls. scFv binding was detected with peroxidase-conjugated anti-His tag antibody (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127169#pone.0127169.s011" target="_blank">S1 Table</a>), and absorbance was measured at 450nm. Mean and standard deviation of triplicate experiments are given. D: CD146 expression of different cell lines as shown by immunoprobing with anti-CD146 antibody. CD146 is expressed in BECs, in A375, CRL1676, HTB71 melanoma cells, but not in primary LECs and HEK293 cells. The same blot was probed with anti-tubulin antibody for control of equal protein loads. E: scFv B6-11 stains cell lines expressing CD146 with similar intensity as commercial anti-CD146 antibody in ELISA. Negative controls were a non-binding scFv and 2^nd antibody only. F: Similar to commercial anti-CD146 antibody, scFv B6-11 stains BECs (upper lane, red) but not LECs (lower lane, green) in immunofluorescence. Size bars: 50μm. G: scFv B6-11 stains blood, but not lymphatic vessels in human skin. Double immunofluorescence staining of skin sample with Cy3-labeled scFv-B6-11 or anti-CD146 (red) and anti-PDPN (green) antibodies. Blood (BV) and lymphatic (LV) vessels are indicated by lines. Nuclei were counterstained with DAPI. Size bars: 50μm.</p

Fusion antibodies scFv-Fc B6-11, B6-112 and B6-117 stain blood vessels in the dermis.

<p>A: Representative images of double immunofluorescence stainings of frozen human skin sections with scFv-Fc fusion antibodies (red) in combination with anti-CD31 (green) show overlapping staining, but B: not with anti-PDPN antibody (green) as control. Nuclei were counter-stained with DAPI. Size bars: 50μm.</p