Journal: bioRxiv

Article Title: Single-cell analysis identifies EpCAM + /CDH6 + /TROP-2 − cells as human liver progenitors

doi: 10.1101/294272

Figure Lengend Snippet: (A) Overview of FACS strategy in adult human liver to isolate cells for scRNA-seq. (B) 2D t-SNE visualisation of single cells isolated from adult human liver included in the study coloured by FACS parent population. (C) 2D t-SNE visualisation of single cells isolated from adult human liver t-SNE plot points coloured by K-mean cluster and labelled A1-A2. (D) Violin plots detailing expression levels of selected marker genes in each cluster identified in adult liver scRNA-seq analysis detailing distinct cell types. Scale is log10 transcripts per million (TPM). (E) Expression of selected enriched ‘Adult progenitor’ (A2) marker transcripts overlaid on the 2D t-SNE space of adult liver scRNA-seq analysis. (F) Schematic of known and novel markers expressed in adult clusters A1 and A2 labelled as ‘Adult hepatocyte and ‘Adult progenitor’ cell types respectively. (G) Duplex RNA-ISH for CDH6(red)/STAT1(blue) in adult human intra-hepatic bile ducts (BD) and limiting plate (LP), detail of the red/blue dots expanded in the square. Scale bars represent 50 μm, 25 μm in blown up squares. (I) Immunofluorescence (IF) staining on Endoscopic retrograde cholangiopancreatography procedure (ERCP) intra-hepatic biliary brushing primary human samples for EpCAM (purple) with CLDN3 (red) (i) and KRT7 (magenta) with CDH6 (green) (ii), after 2 days in Matrigel with liver expansion (LE) media. Scale bars represent 25 μm.

Article Snippet: Cells were resuspended in 3% B.S.A with 0.1mM EDTA (15575020, Gibco, Life technologies) and stained for the following conjugated primary antibodies, CD235a (349104, FITC, mouse; Biolegend), CD45 (304050, BV711, mouse; Biolegend), EpCAM (324221, PE/Cy7, mouse, 1:100, Biolegend), Claudin-3 (130-110-834, PE, REA, 1:50, Miltenyl Biotech), CDH6 (130-113-099, APC, REA, 1:50, Miltenyl Biotech) and CD133 (372808, BV 421, mouse, 1:100, Biolegend) for 30 minutes at 4 ̊C.

Techniques: Isolation, Expressing, Marker, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Single-cell analysis identifies EpCAM + /CDH6 + /TROP-2 − cells as human liver progenitors

doi: 10.1101/294272

Figure Lengend Snippet: (A) RNA-ISH for CDH6 and STAT1 on human foetal liver ductal plate (DP) and bile ducts (BD) regions (detail of the red dots expanded in the squares). Scale bars represent 50 μm, 25 μm in blown up squares. (B) Immunohistochemistry (IHC) of EpCAM, CK19, CDH6 and STAT1 in BD and DP regions of human foetal liver. Scale bars represent 50 μm. (C) Immunofluorescence (IF) staining of CDH6 (yellow) and CLDN3 (magenta) (i) and CDH6 (yellow), CLDN3 (magenta) and STAT1 (grey) (ii) co-expression in human 2 nd trimester (15-21 pcw) foetal liver slides. Slides counterstained in DAPI (cyan). (D) Representative FACS plot of MAC-sorted total (left), EpCAM enriched(middle) and EpCAM depleted (right) cells from dissociated human foetal liver. All gates were set on fluorescence minus one (FMO) controls Gate percentages are representative of n = 3 isolations. (E) Phase-contrast and IF staining of foetal intra-hepatic organoids (f-IHOs) derived from EpCAM enriched foetal liver cells in liver expansion (LE) media. All structures are counterstained with DAPI (blue). P0 refers to cells isolated directly from primary tissue. All IF staining performed between passage 3-5. (F) IF staining of f-IHOs in LE media. All structures are counterstained with DAPI (blue). All IF staining performed between passage 3-5. (G) IF staining of f-IHOs for albumin (magenta) and KRT19 (red) cultured for 7 days after passage in LE media or Hepatic differentiation (HD) media. (H) IF staining of f-IHOs and foetal ex-hepatic organoids (f-EHOs) derived from foetal gallbladder for CDH6 (yellow) and biliary marker KRT7 (magenta). Counterstained with DAPI (blue). (I) Representative scatter plot of FAC-sorting strategy for isolation of EpCAM + /CLDN3 + /CDH6 + cells. Top plot displays EPCAM (X-axis) vs CLDN3 (y-axis) of Live/CD235a − /CD45 − negatively selected cells. Middle plot displays CDH6 (x-axis) and CD133 (y-axis) of EPCAM + /CLDN3 + cells. Bottom plot displays CDH6 (x-axis) and CD133 (y-axis) of EPCAM + /CLDN3 − cells. All plots gated on fluorescence minus one (FMO) staining controls. (J) Phase contrast and IF imaging of FAC-isolated EpCAM + /CLDN3 − /CDH6 − /CD133-(i) (scale bars represent 300 μm) and EpCAM + /CLDN3 + /CDH6 + /CD133 − (ii) (scale bars represent 200 μm) cells cultured for 10 days on RFP-HUVECs in LE media, with corresponding IF images. RFP negative regions circled in white. All scale bars represent 25 μm unless stated otherwise. FACS plots are representative of at least 3 independent experiments.

Article Snippet: Cells were resuspended in 3% B.S.A with 0.1mM EDTA (15575020, Gibco, Life technologies) and stained for the following conjugated primary antibodies, CD235a (349104, FITC, mouse; Biolegend), CD45 (304050, BV711, mouse; Biolegend), EpCAM (324221, PE/Cy7, mouse, 1:100, Biolegend), Claudin-3 (130-110-834, PE, REA, 1:50, Miltenyl Biotech), CDH6 (130-113-099, APC, REA, 1:50, Miltenyl Biotech) and CD133 (372808, BV 421, mouse, 1:100, Biolegend) for 30 minutes at 4 ̊C.

Techniques: Immunohistochemistry, Immunofluorescence, Staining, Expressing, Fluorescence, Derivative Assay, Isolation, Cell Culture, Marker, Imaging

Journal: bioRxiv

Article Title: Single-cell analysis identifies EpCAM + /CDH6 + /TROP-2 − cells as human liver progenitors

doi: 10.1101/294272

Figure Lengend Snippet: (A) 2D t-SNE representation of all single cells included in this scRNA-seq study. Cells differentially coloured by K-means cluster (adult or foetal). Adult and foetal hLPCs shading (blue) represents single K-means cluster. (B) Heat map showing the spearman correlation coefficient of pairwise comparison between all single cells in data set. The spearman correlation coefficient between samples was calculated based on Log10(TPM) of each gene. Columns are labelled by foetal and adult K-means clusters. (C) PCA plot of Monocle pseudo-time analysis on all cells of foetal and adult hLPC cell clusters, colored by Pseudo state. (D) Heat maps of significantly expressed genes between pseudo states 1,2,3 (i) and 1,2,4 (ii). Gene expression in Log10(TPM). (E) Schematic of hepatic and cholangiocyte liver progenitor lineage based on monocle-derived ‘pseudo state’. (F) Scatter and bar plots of Gene expression Vs Pseudo state for selected genes enriched in pseudo states 1 (i), 2 (ii), 3 (iii) and 4 (iv). (G) PCA plot of Monocle pseudo-time analysis on hLPC cells with overlay of TROP-2 and CDH6 transcript expression. Gene expression in Log10(TPM). (H) Duplex RNA-ISH for TROP-2(red)/STAT1(blue) and CDH6(red)/STAT1(blue) in foetal human bile duct (BD) and ductal plate (DP) structures, detail of the red/blue dots expanded in the square. Scale bars represent 50 μm, 25 μm in blown up squares. (I) Duplex RNA-ISH for TROP-2(red)/STAT1(blue) and CDH6(red)/STAT1(blue) in adult human intra-portal BD and limiting plate (LP) regions. Scale bars represent 25 μm.

Article Snippet: Cells were resuspended in 3% B.S.A with 0.1mM EDTA (15575020, Gibco, Life technologies) and stained for the following conjugated primary antibodies, CD235a (349104, FITC, mouse; Biolegend), CD45 (304050, BV711, mouse; Biolegend), EpCAM (324221, PE/Cy7, mouse, 1:100, Biolegend), Claudin-3 (130-110-834, PE, REA, 1:50, Miltenyl Biotech), CDH6 (130-113-099, APC, REA, 1:50, Miltenyl Biotech) and CD133 (372808, BV 421, mouse, 1:100, Biolegend) for 30 minutes at 4 ̊C.

Techniques: Comparison, Gene Expression, Derivative Assay, Expressing

Journal: bioRxiv

Article Title: Single-cell analysis identifies EpCAM + /CDH6 + /TROP-2 − cells as human liver progenitors

doi: 10.1101/294272

Figure Lengend Snippet: (A) Hematoxylin and eosin (H&E) staining with RNA-ISH for CDH6 and TROP-2 transcript expression in tissue overlay of normal human adult liver tissue. Arrows indicate bile duct and limiting plate structures. (B) H&E staining with RNA-ISH for CDH6 and TROP-2 transcript expression in adult liver tumour samples from hepatocellular carcinoma (HCC) with dedifferentiated morphology and intra-hepatic cholangiocarcinoma (ICC). (C) H&E staining with RNA-ISH for CDH6 and TROP-2 transcript expression in tissue overlay of human adult subacute liver failure bile duct and ductular reactions. (D) H&E staining with RNA-ISH for CDH6 and TROP-2 transcript expression in tissue overlay of human cirrhotic liver bile duct and ductular reactions. All scale bars represent 50 μm.

Article Snippet: Cells were resuspended in 3% B.S.A with 0.1mM EDTA (15575020, Gibco, Life technologies) and stained for the following conjugated primary antibodies, CD235a (349104, FITC, mouse; Biolegend), CD45 (304050, BV711, mouse; Biolegend), EpCAM (324221, PE/Cy7, mouse, 1:100, Biolegend), Claudin-3 (130-110-834, PE, REA, 1:50, Miltenyl Biotech), CDH6 (130-113-099, APC, REA, 1:50, Miltenyl Biotech) and CD133 (372808, BV 421, mouse, 1:100, Biolegend) for 30 minutes at 4 ̊C.

Techniques: Staining, Expressing

Journal: bioRxiv

Article Title: Single-cell analysis identifies EpCAM + /CDH6 + /TROP-2 − cells as human liver progenitors

doi: 10.1101/294272

Figure Lengend Snippet: Schematic of human CDH6 and TROP-2 expression in normal, tumour and liver injury models.

Article Snippet: Cells were resuspended in 3% B.S.A with 0.1mM EDTA (15575020, Gibco, Life technologies) and stained for the following conjugated primary antibodies, CD235a (349104, FITC, mouse; Biolegend), CD45 (304050, BV711, mouse; Biolegend), EpCAM (324221, PE/Cy7, mouse, 1:100, Biolegend), Claudin-3 (130-110-834, PE, REA, 1:50, Miltenyl Biotech), CDH6 (130-113-099, APC, REA, 1:50, Miltenyl Biotech) and CD133 (372808, BV 421, mouse, 1:100, Biolegend) for 30 minutes at 4 ̊C.

Techniques: Expressing

Journal: Molecular Oncology

Article Title: CDH6‐activated αIIbβ3 crosstalks with α2β1 to trigger cellular adhesion and invasion in metastatic ovarian and renal cancers

doi: 10.1002/1878-0261.12947

Figure Lengend Snippet: Expression of RGD Cadherins and integrins in renal and ovarian cancer cells. (A) Western blot analysis of the expression of CDH6, CDH17, and the indicated integrin subunits in renal cancer, ovarian cancer, and colorectal cancer cells. β‐Tubulin expression was used as loading control. (B) Flow cytometry analysis of cadherin and integrin expression in the indicated cell lines. (C) Changes in the expression of the indicated cadherins and integrin subunits as a function of SKOV‐3 cell confluency by flow cytometry (left) and Western blot analysis (right). All results are representative of at least three independent experiments; error bars indicate standard deviation.

Article Snippet: CDH6 antibody (M06353) for immunofluorescence was from Boster (Pleasanton, CA, USA), whereas antibodies for Src (AF3389) and CDH6 (AF2715) for Western blot and flow cytometry were from R&D Systems (Minneapolis, MN, USA). β‐Tubulin antibody (ab21057) and phospho‐focal adhesion kinase (611722) were from Abcam (Cambridge, UK) and BD Transduction Laboratories (Franklin Lakes, NJ, USA), respectively. β1 and β3 integrin‐specific antibodies for high‐affinity conformation were from BD Biosciences, San Diego, CA, USA (HUTS21) and Merck, Kenilworth, NJ, USA (MABT27), respectively.

Techniques: Expressing, Western Blot, Control, Flow Cytometry, Standard Deviation

Journal: Molecular Oncology

Article Title: CDH6‐activated αIIbβ3 crosstalks with α2β1 to trigger cellular adhesion and invasion in metastatic ovarian and renal cancers

doi: 10.1002/1878-0261.12947

Figure Lengend Snippet: CDH6 binds αIIbβ3 or α2β1 integrins in different cell lines. (A, B, C) The indicated cells were immunoprecipitated using anti‐CDH6, anti‐CDH17, anti‐αIIb, or control antibodies. Immunoprecipitates were analyzed by Western blot using the indicated antibodies. (D) SKOV‐3 and OVCAR3 cells were used to determine CDH6, α2, and αIIb integrin subunits colocalization by confocal microscopy. Pearson’s correlation coefficient was calculated for each colocalization experiment (right). Bar size: 20 µm.

Article Snippet: CDH6 antibody (M06353) for immunofluorescence was from Boster (Pleasanton, CA, USA), whereas antibodies for Src (AF3389) and CDH6 (AF2715) for Western blot and flow cytometry were from R&D Systems (Minneapolis, MN, USA). β‐Tubulin antibody (ab21057) and phospho‐focal adhesion kinase (611722) were from Abcam (Cambridge, UK) and BD Transduction Laboratories (Franklin Lakes, NJ, USA), respectively. β1 and β3 integrin‐specific antibodies for high‐affinity conformation were from BD Biosciences, San Diego, CA, USA (HUTS21) and Merck, Kenilworth, NJ, USA (MABT27), respectively.

Techniques: Immunoprecipitation, Control, Western Blot, Confocal Microscopy

Journal: Molecular Oncology

Article Title: CDH6‐activated αIIbβ3 crosstalks with α2β1 to trigger cellular adhesion and invasion in metastatic ovarian and renal cancers

doi: 10.1002/1878-0261.12947

Figure Lengend Snippet: CDH6 and αIIbβ3 integrin regulate cell adhesion, migration, invasion, and proliferation in ovarian and renal cancer cells. (A) SKOV‐3 and 786‐O cells were transfected with siRNAs for the indicated genes and subjected to cell adhesion, wound healing, cell invasion, or MTT assays. Transient silencing of CDH6, αIIb, or β3 integrin subunits caused a significant decrease in cell adhesion/migratory speed/cell invasion/proliferation (* P < 0.05; ** P < 0.01; *** P < 0.001), according to ANOVA tests. (B) The same transfectants were analyzed by Western blot to examine the phosphorylation status of the indicated signaling proteins. Blots were re‐probed with antibodies against the total signaling proteins as loading controls. All results are representative of at least three independent experiments; error bars indicate standard deviation.

Article Snippet: CDH6 antibody (M06353) for immunofluorescence was from Boster (Pleasanton, CA, USA), whereas antibodies for Src (AF3389) and CDH6 (AF2715) for Western blot and flow cytometry were from R&D Systems (Minneapolis, MN, USA). β‐Tubulin antibody (ab21057) and phospho‐focal adhesion kinase (611722) were from Abcam (Cambridge, UK) and BD Transduction Laboratories (Franklin Lakes, NJ, USA), respectively. β1 and β3 integrin‐specific antibodies for high‐affinity conformation were from BD Biosciences, San Diego, CA, USA (HUTS21) and Merck, Kenilworth, NJ, USA (MABT27), respectively.

Techniques: Migration, Transfection, Western Blot, Phospho-proteomics, Standard Deviation

Journal: Molecular Oncology

Article Title: CDH6‐activated αIIbβ3 crosstalks with α2β1 to trigger cellular adhesion and invasion in metastatic ovarian and renal cancers

doi: 10.1002/1878-0261.12947

Figure Lengend Snippet: CDH6 and CDH17 promote the activation of β1 and β3 integrins. (A) Ovarian and renal cancer cell lines were exposed to RGD peptides of CDH6 or CDH17 and subjected to flow cytometry analyses to assess the activation status of β1 and β3 integrins. (B) SKOV‐3 cells were silenced for CDH6, α2, or αIIb integrin subunits, exposed to the RGD peptides and subjected to flow cytometry assays to detect β1 or β3 in high‐affinity conformation. (C) The same transfectants exposed to the indicated peptides were subjected to cell adhesion assays to Matrigel. (D) SKOV‐3 and 786‐O cells were silenced for α2 or αIIb integrin subunits and subjected to adhesion to collagen type I. Integrin activation or cell adhesion was significantly increased by the addition of RGD peptides (** P < 0.01; *** P < 0.001) or the silencing of α2 integrin subunit ($ P < 0.05; $$ P < 0.01) and significantly decreased by the silencing of CDH6, α2, or αIIb (◊ P < 0.05; ◊◊◊ P < 0.001), according to ANOVA tests. All results are representative of at least three independent experiments; error bars indicate standard deviation.

Article Snippet: CDH6 antibody (M06353) for immunofluorescence was from Boster (Pleasanton, CA, USA), whereas antibodies for Src (AF3389) and CDH6 (AF2715) for Western blot and flow cytometry were from R&D Systems (Minneapolis, MN, USA). β‐Tubulin antibody (ab21057) and phospho‐focal adhesion kinase (611722) were from Abcam (Cambridge, UK) and BD Transduction Laboratories (Franklin Lakes, NJ, USA), respectively. β1 and β3 integrin‐specific antibodies for high‐affinity conformation were from BD Biosciences, San Diego, CA, USA (HUTS21) and Merck, Kenilworth, NJ, USA (MABT27), respectively.

Techniques: Activation Assay, Flow Cytometry, Standard Deviation

Journal: Molecular Oncology

Article Title: CDH6‐activated αIIbβ3 crosstalks with α2β1 to trigger cellular adhesion and invasion in metastatic ovarian and renal cancers

doi: 10.1002/1878-0261.12947

Figure Lengend Snippet: CDH6 and αIIb/α2 integrins are required for lung homing in ovarian and renal cancer. (A) RNA was isolated from liver or lungs of mice inoculated with SKOV‐3 cells previously silenced for the indicated genes and subjected to RT‐PCR assays to detect human GAPDH as surrogate of cell colonization. Murine β‐actin was used as loading control. (B) Model of integrin crosstalk. According to our results, the presence of CDH6 activates αIIbβ3 integrin, which induces the activation of α2β1 integrin. The activation of integrins promotes cell adhesion, invasion, and proliferation, leading to the metastatic dissemination of cancer cells.

Article Snippet: CDH6 antibody (M06353) for immunofluorescence was from Boster (Pleasanton, CA, USA), whereas antibodies for Src (AF3389) and CDH6 (AF2715) for Western blot and flow cytometry were from R&D Systems (Minneapolis, MN, USA). β‐Tubulin antibody (ab21057) and phospho‐focal adhesion kinase (611722) were from Abcam (Cambridge, UK) and BD Transduction Laboratories (Franklin Lakes, NJ, USA), respectively. β1 and β3 integrin‐specific antibodies for high‐affinity conformation were from BD Biosciences, San Diego, CA, USA (HUTS21) and Merck, Kenilworth, NJ, USA (MABT27), respectively.

Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction, Control, Activation Assay