Journal: International Journal of Molecular Sciences

Article Title: Disseminated Tumor Cells (DTCs) in Patients with Cervical Cancer Reveal Mesenchymal Properties and Potential Therapeutic Targets—A New Perspective?

doi: 10.3390/ijms27114875

Figure Lengend Snippet: Positive and negative control cells for both marker panels. ( A ) HeLa (white arrows): in the first panel positive for cytokeratin (CK), Vimentin (Vim), VEGF and in the second panel positive for p16INK4A, negative for PD-L1 and CD45. ( B ) T98G (red arrows): positive for Vim. ( C ) CaSki (yellow arrows): positive for CK, Vim, VEGF, PD-L1 and p16INK4A. ( D ) MCF-7 (green arrows): positive for CK. All cell lines were negative for CD45. Only hematopoietic cells showed CD45 positive staining. Scale bar = 10 μm. Images were processed using Zeiss ZEN 3.7 software and the figure was assembled using Microsoft PowerPoint.

Article Snippet: In the first round of staining, anti-human Pan-Cytokeratin APC-conjugated REAdye_lease antibody (Cat. No. 130-123-091, Miltenyi Biotech, Bergisch Gladbach, Germany), anti-human vimentin FITC-conjugated REAdye_lease antibody (Cat. No. 130-127-022, Miltenyi Biotech, Bergisch Gladbach, Germany) and anti-human VEGF PE-conjugated REAdye_lease antibody (Cat. No. 130-118-061, Miltenyi Biotech, Bergisch Gladbach, Germany) were applied.

Techniques: Negative Control, Marker, Staining, Software

Journal: International Journal of Molecular Sciences

Article Title: Disseminated Tumor Cells (DTCs) in Patients with Cervical Cancer Reveal Mesenchymal Properties and Potential Therapeutic Targets—A New Perspective?

doi: 10.3390/ijms27114875

Figure Lengend Snippet: Representative images of patient-derived DTCs with three different profiles. ( A ) DTC positive for Vim, VEGF and p16, negative for CK, PD-L1 and CD45. ( B ) CK and Vim positive while negative for VEGF, PD-L1, p16 and CD45. ( C ) DTC positive for Vim and PD-L1, negative for all other markers. Scale bar = 10 μm. Images were processed using Zeiss ZEN 3.7 software and figure was assembled using Microsoft PowerPoint.

Article Snippet: In the first round of staining, anti-human Pan-Cytokeratin APC-conjugated REAdye_lease antibody (Cat. No. 130-123-091, Miltenyi Biotech, Bergisch Gladbach, Germany), anti-human vimentin FITC-conjugated REAdye_lease antibody (Cat. No. 130-127-022, Miltenyi Biotech, Bergisch Gladbach, Germany) and anti-human VEGF PE-conjugated REAdye_lease antibody (Cat. No. 130-118-061, Miltenyi Biotech, Bergisch Gladbach, Germany) were applied.

Techniques: Derivative Assay, Software

Journal: International Journal of Molecular Sciences

Article Title: Disseminated Tumor Cells (DTCs) in Patients with Cervical Cancer Reveal Mesenchymal Properties and Potential Therapeutic Targets—A New Perspective?

doi: 10.3390/ijms27114875

Figure Lengend Snippet: Proportion of DTC Phenotypes. The majority of detected DTCs (70%, n = 174) was positive for Vimentin without CK expression, hence revealing a mesenchymal phenotype. An epithelial phenotype was found in 13% ( n = 31) of the DTCs and 15% ( n = 37) presented epithelial–mesenchymal transition (EMT)-like properties. Chart generated using Microsoft Excel.

Article Snippet: In the first round of staining, anti-human Pan-Cytokeratin APC-conjugated REAdye_lease antibody (Cat. No. 130-123-091, Miltenyi Biotech, Bergisch Gladbach, Germany), anti-human vimentin FITC-conjugated REAdye_lease antibody (Cat. No. 130-127-022, Miltenyi Biotech, Bergisch Gladbach, Germany) and anti-human VEGF PE-conjugated REAdye_lease antibody (Cat. No. 130-118-061, Miltenyi Biotech, Bergisch Gladbach, Germany) were applied.

Techniques: Expressing, Generated

Journal: International Journal of Molecular Sciences

Article Title: Disseminated Tumor Cells (DTCs) in Patients with Cervical Cancer Reveal Mesenchymal Properties and Potential Therapeutic Targets—A New Perspective?

doi: 10.3390/ijms27114875

Figure Lengend Snippet: Sequential multi-parameter immunofluorescent staining. After applying the first antibody panel (CK, Vim and VEGF), the slides were scanned and DTC profiles were assessed. Subsequently, releasable fluorochrome-conjugates were digested and the slides were subjected to the second antibody panel (PD-L1, p16, CD45) followed by scanning and DTC detection. Created with BioRender.com.

Article Snippet: In the first round of staining, anti-human Pan-Cytokeratin APC-conjugated REAdye_lease antibody (Cat. No. 130-123-091, Miltenyi Biotech, Bergisch Gladbach, Germany), anti-human vimentin FITC-conjugated REAdye_lease antibody (Cat. No. 130-127-022, Miltenyi Biotech, Bergisch Gladbach, Germany) and anti-human VEGF PE-conjugated REAdye_lease antibody (Cat. No. 130-118-061, Miltenyi Biotech, Bergisch Gladbach, Germany) were applied.

Techniques: Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting CXCR4 by a selective peptide antagonist modulates tumor microenvironment and microglia reactivity in a human glioblastoma model

doi: 10.1186/s13046-016-0326-y

Figure Lengend Snippet: In vivo effects of peptide R treatment of U87MG orthotopic mouse model. a Tumor volume measures obtained by MRI analyses performed on mice at day 10, 15 and 23 after U87MG cells implantation and drug administration. Curves represent mean value of tumor volumes measures of three independent experiments obtained from mice treated with vehicle (PBS) ● CTRL, ■ Plerixafor and ▲ peptide R ( n = 10 animals per group, error bars ± SD). b Representative brain sections of vehicle-treated (CTRL) or peptide R- or Plerixafor-treated mice stained by immunohistochemistry with the anti-vimentin antibody. Scale bars, 75 μM. c Representative CLSM images of Vimentin expression ( green ) of tumor-free contralateral hemispheres of vehicle-treated mice (CTRL), peptide R- and Plerixafor-treated mice. Arrows represent Vimentin + cells. Nuclei were stained with DAPI ( blue ). Scale bars 75 μM

Article Snippet: The following primary antibodies were used in overnight incubations: rat anti-mouse CD11b (1:100, Serotec), rabbit anti-human CD68 (1:200, able to detect mouse, rat and human CD68, Santa Cruz), mouse anti-mouse Arg-1 (1:200, BD Biosciences), rabbit anti-mouse anti-iNOS (1:2000, Millipore), goat anti-human vimentin (1:40, R&D Systems), rabbit anti-mouse/human glial fibrillary acidic protein, GFAP (1:200, Dako), rat anti-mouse CD31 (1:200, Serotec), mouse anti-human/mouse vascular endothelial growth factor, VEGF (1:50, R&D Systems).

Techniques: In Vivo, Staining, Immunohistochemistry, Expressing

Journal: International Journal of Medical Sciences

Article Title: Establishment and Characterization of an Immortalized Human Hepatic Stellate Cell Line for Applications in Co-Culturing with Immortalized Human Hepatocytes

doi: 10.7150/ijms.11002

Figure Lengend Snippet: Immunofluorescence staining of HSC-Li cells and DiI-LDL uptake . A. The expression of SV40LT proteins was positive in the HSC-Li cells. It was localized in the nucleus of HSC-Li cells. Cells were costained with DAPI to identify nuclei. Original magnification: ×400. B-D. The expression of α-SMA, vimentin, and PDGFR-β proteins was also positive in the HSC-Li cells. Cells were costained with DAPI to identify nuclei. Original magnification: ×400. E.The HSC-Li cells strongly endocytosed LDL after 24 h of culture with DiI-LDL. Original magnification: ×200.

Article Snippet: The primary antibodies included anti-human vimentin antibody (R&D Systems, USA), anti-human α-smooth muscle actin (α-SMA) antibody (R&D Systems, USA), platelet-derived growth factor receptor-beta (PDGFR-β) (Santa Cruz, USA) and mouse monoclonal anti-SV40LT antibody (Santa Cruz, USA).

Techniques: Immunofluorescence, Staining, Expressing

Journal: Scientific Reports

Article Title: Metformin exerts anti-cancerogenic effects and reverses epithelial-to-mesenchymal transition trait in primary human intrahepatic cholangiocarcinoma cells

doi: 10.1038/s41598-021-81172-0

Figure Lengend Snippet: Phenotypic characterization of iCCA primary cell cultures and effect of Metformin on cell proliferation of iCCA primary cultures. ( A ) Immunofluorescence for Vimentin, Cytokeratin-19 and E-Cadherin in Large duct-type iCCA (up) and Small duct-type iCCA (down) iCCA primary cell cultures; nuclei were stained with DAPI. The bar graph indicates the percentage of positive cells. More than 70% of cells stained positive for Vimentin while express very low level of Cytokeratin-19 (< 6% of positive cells) and were negative for E-Cadherin. (See also Supplementary Figure A). Figures are representative of N = 5 different cell cultures. Data in the bar graph represent mean ± SD of N = 5 independent experiments. ( B ) Large duct-type iCCA (up) and Small duct-type iCCA (down) primary iCCA cell cultures were exposed for 24, 48, 72, 96 h to increasing concentration of Metformin (0–1000 µM); cell proliferation was evaluated by MTS assay and expressed as ratio with respect to controls. Metformin significantly reduced cell proliferation in a dose-dependent manner. In most experiments the inhibitory effect of Metformin on both Large and Small duct-type iCCA started at 5–10 µM. * p < 0.05 versus controls. Data represent mean ± SD of N = 5 independent experiments.

Article Snippet: Primary cell cultures were fixed in 1:1 Acetone/Methanol (10 min at room temperature), incubated in 20% fetal bovine serum (FBS) in Dulbecco’s Phosphate Buffered Saline (DPBS) for 30 min, and then incubated for 1 h at room temperature with primary antibodies anti Human Vimentin RV202 (sc-32322, Santa Cruz Biotechnology Dallas, Texas, USA), Human Cytokeratin-19 (sc-6278, Santa Cruz Biotechnology), Human Vimentin (AlexaFluor 488 conjugated, goat IgG, IC8104G, R&D System, Minneapolis, MN, USA), Human Cytokeratin-19 (AlexaFluor 594 clone A53-B/A2, 628,504, Biolegend San Diego, CA, USA).

Techniques: Immunofluorescence, Staining, Concentration Assay, MTS Assay

Journal: Scientific Reports

Article Title: Metformin exerts anti-cancerogenic effects and reverses epithelial-to-mesenchymal transition trait in primary human intrahepatic cholangiocarcinoma cells

doi: 10.1038/s41598-021-81172-0

Figure Lengend Snippet: Effect of Metformin on gene expression of mesenchymal, EMT, epithelial markers, AMPK and FOXO3 in iCCA primary cells and nuclear translocation of FOXO3 in iCCA primary cell cultures. ( A ) The gene expression of mesenchymal and EMT markers (Vimentin, SNAIL1, SNAIL2, TWIST1) and epithelial markers (E-Cadherin, Cytokeratin-19) was analysed by RT-qPCR in Large and Small duct-type iCCA primary cultures exposed to Metformin 10 µM for 48 and 96 h and normalized to the expression of GAPDH (housekeeping gene). In both iCCA subtypes, the gene expression of Vimentin was significantly decreased by Metformin only after 96 h while the expression of SNAIL1, SNAIL2 and TWIST1 decreased after 48 and 96 h. In contrast, the gene expression of the epithelial marker E-Cadherin significantly increased after 48 and 96 h in both Large and Small duct-type iCCA while Cytokeratin-19 increased only in Large duct-type iCCA after 96 h of exposure to Metformin. Data represent mean ± SD of N = 5 independent experiments. * p < 0.05. ( B ) The gene expression of AMPK and FOXO3, two signalling molecules involved in the mechanism of action of Metformin, was analysed by RT-qPCR and normalized to the expression of GAPDH (housekeeping gene). After 48 and 96 h, Metformin 10 µM significantly induced the gene expression of AMPK and FOXO3 in both Large and Small duct-type iCCA primary cultures. Data represent mean ± SD of N = 5 independent experiments. * p < 0.05. ( C ) IF analysis of FOXO3 in Large and Small duct-type iCCA primary cultures exposed for 96 h to Metformin 10 µM. Migration of FOXO3 from the cytoplasm to the nucleus is evident and occurred in 64 ± 10% of the cells. Nuclei were stained with DAPI. Magnification 20 × and 10 × ; representative images of N = 3 independent experiments.

Article Snippet: Primary cell cultures were fixed in 1:1 Acetone/Methanol (10 min at room temperature), incubated in 20% fetal bovine serum (FBS) in Dulbecco’s Phosphate Buffered Saline (DPBS) for 30 min, and then incubated for 1 h at room temperature with primary antibodies anti Human Vimentin RV202 (sc-32322, Santa Cruz Biotechnology Dallas, Texas, USA), Human Cytokeratin-19 (sc-6278, Santa Cruz Biotechnology), Human Vimentin (AlexaFluor 488 conjugated, goat IgG, IC8104G, R&D System, Minneapolis, MN, USA), Human Cytokeratin-19 (AlexaFluor 594 clone A53-B/A2, 628,504, Biolegend San Diego, CA, USA).

Techniques: Gene Expression, Translocation Assay, Quantitative RT-PCR, Expressing, Marker, Migration, Staining

Journal: Scientific Reports

Article Title: Metformin exerts anti-cancerogenic effects and reverses epithelial-to-mesenchymal transition trait in primary human intrahepatic cholangiocarcinoma cells

doi: 10.1038/s41598-021-81172-0

Figure Lengend Snippet: Effect of prolonged treatment (12 and 57 days) of iCCA primary cells with Metformin on the gene expression of mesenchymal, EMT and epithelial markers. The gene expression of mesenchymal and EMT markers (Vimentin, SNAIL1, SNAIL2, TWIST1) and epithelial markers (E-Cadherin, Cytokeratin-19) was analysed by RT-qPCR in Large duct-type iCCA ( A ) and Small duct-type iCCA ( B ) primary cultures exposed to Metformin 10 µM for 12 or 57 days and normalized to the expression of GAPDH (housekeeping gene). The mesenchymal and EMT markers, Vimentin, SNAIL1, SNAIL2 and TWIST1, were markedly down-regulated with respect to controls; in some case almost completely suppressed (i.e. Vimentin and SNAIL2 in Large duct-type iCCA and SNAIL2 in Small duct-type iCCA). The epithelial markers, E-Cadherin and Cytokeratin-19 were markedly upregulated in both Large and Small duct-type iCCA. Data represent mean ± SD of N = 5 independent experiments; * p < 0.05 versus controls.

Article Snippet: Primary cell cultures were fixed in 1:1 Acetone/Methanol (10 min at room temperature), incubated in 20% fetal bovine serum (FBS) in Dulbecco’s Phosphate Buffered Saline (DPBS) for 30 min, and then incubated for 1 h at room temperature with primary antibodies anti Human Vimentin RV202 (sc-32322, Santa Cruz Biotechnology Dallas, Texas, USA), Human Cytokeratin-19 (sc-6278, Santa Cruz Biotechnology), Human Vimentin (AlexaFluor 488 conjugated, goat IgG, IC8104G, R&D System, Minneapolis, MN, USA), Human Cytokeratin-19 (AlexaFluor 594 clone A53-B/A2, 628,504, Biolegend San Diego, CA, USA).

Techniques: Gene Expression, Quantitative RT-PCR, Expressing

Journal: Scientific Reports

Article Title: Metformin exerts anti-cancerogenic effects and reverses epithelial-to-mesenchymal transition trait in primary human intrahepatic cholangiocarcinoma cells

doi: 10.1038/s41598-021-81172-0

Figure Lengend Snippet: IF and western blot analysis of Vimentin and Cytokeratin-19 in primary cultures of Large duct-type iCCA treated for 12 or 57 days with Metformin. ( A ) Vimentin and Cytokeratin-19 were analysed by IF (left panels), in Large duct-type iCCA primary cultures treated with Metformin 10 µM for 12 and 57 days. Metformin induced a marked IF positivity for Cytokeratin-19 and a marked decrease of Vimentin expression that were not observed after 48–96 h of treatment. By western blot (right panels, cropped gels/blots are displayed), the protein expression of Vimentin decreased while that of Cytokeratin-19 increased after prolonged (12 and 57 days) treatment with Metformin 10 µM. * p < 0.05 versus controls. Data represent mean ± SD of N = 3 independent experiments. ( B ) In contrast with Large duct-type iCCA, in the Small duct-type iCCA cells the Cytokeratin-19 expression was not observed neither by IF not by western blot (not detectable, ND). However, by western blot analysis a decreased level of Vimentin was observed. * p < 0.05 versus controls. Mean ± SD of N = 3 independent experiments.

Article Snippet: Primary cell cultures were fixed in 1:1 Acetone/Methanol (10 min at room temperature), incubated in 20% fetal bovine serum (FBS) in Dulbecco’s Phosphate Buffered Saline (DPBS) for 30 min, and then incubated for 1 h at room temperature with primary antibodies anti Human Vimentin RV202 (sc-32322, Santa Cruz Biotechnology Dallas, Texas, USA), Human Cytokeratin-19 (sc-6278, Santa Cruz Biotechnology), Human Vimentin (AlexaFluor 488 conjugated, goat IgG, IC8104G, R&D System, Minneapolis, MN, USA), Human Cytokeratin-19 (AlexaFluor 594 clone A53-B/A2, 628,504, Biolegend San Diego, CA, USA).

Techniques: Western Blot, Expressing