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

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

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

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

Production of scFv-Fc B6-11 fusion antibody and reconfirmation of binding to CD146.

<p>A: Western Blot analysis showing molecular size of scFv-Fc fusion antibodies B6-11, B6-112 and B6-117. The scFv-region of the phagemids was cloned into the pFUSE-mIgG2B-vector (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127169#pone.0127169.s005" target="_blank">S5 Fig</a>). Constructs were transfected into HEK293-cells. Cell culture supernatants were loaded on reducing (R) and non-reducing (NR) 10% SDS-PAGE, and nitrocellulose blots were probed with peroxidase-labeled anti-mouseFc antibodies. scFv-Fc fusion antibodies are secreted as approximate 140 kDa dimers, as seen under non-reducing condition (-DTT). Control: Fc only protein, produced from pFUSE vector without scFv insert. B: Immunoprecipitation with scFv-Fc B6-11 reconfirms CD146-binding. G1S1 lysates were incubated with scFv-Fc B6-11 and Fc only as control. Blots of immunoprecipitates were probed with anti-CD146 and anti-mouseFc antibodies. C: scFv-Fc B6-11 binds to recombinant CD146 in ELISA. scFv-Fc B6-11, commercial anti-CD146 antibody and Fc only were used on respective dilutions of recombinant human CD146 or BSA as control antigen coated on 96-well ELISA plates. Absorbance was measured at 450nm. D: scFv-Fc B6-11 fusion antibody binds to cells expressing CD146 in ELISA. Purified scFv-Fc B6-11 (light grey bars), commercial anti-CD146 antibody (dark grey bars) and Fc only (black bars) were added to monolayers of respective cell lines. Bound antibodies were detected using anti-Fc and HRP-conjugated anti-rabbit antibodies, and absorbance was measured at 450nm. Mean and standard deviation of triplicate experiments are given. E: On HDMECs, scFv-Fc B6-11 fusion antibody shows the same reactivity as a commercial anti-CD146 antibody. F: scFv-Fc fusion antibodies (red) reveal diverse membraneous labling patterns in co-immunofluorescent stainings with anti-CD31 antibody (green). Nuclei were counterstained with DAPI. Size bars: 50μm.</p

Frequency of diverse scFv antibody sequences among 557 intact clones.

<p>Out of 994 sequenced phage clones, 557 intact scFv sequences were derived, among which 166 diverse scFv sequences were identified (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127169#pone.0127169.s012" target="_blank">S2 Table</a>).</p><p>^a Frequency of unique scFv antibody sequences</p><p>^b Number of different scFv sequences</p><p>^c Number of scFv sequences with respective sequence diversity</p><p>^d Sequence occurrence was calculated as percentage of sequence count.</p><p>Frequency of diverse scFv antibody sequences among 557 intact clones.</p

Purity control of HDMEC, BEC and LEC cultures used for <i>in vitro</i> cell panning experiments.

<p>Light microscopy and double-immunofluorescence stainings of HDMECs that were separated into BECs and LECs by sorting with magnetic beads coated with rabbit anti-podoplanin (PDPN) antibody. A: HDMECs contain subcultures of BECs and LECs. B: BECs are CD31+ / PDPN-. C: LECs are CD31+ / PDPN+. Nuclei were counterstained with DAPI. Size bars: 50μm.</p