Journal: Cell reports

Article Title: Disseminated Melanoma Cells Transdifferentiate into Endothelial Cells in Intravascular Niches at Metastatic Sites.

doi: 10.1016/j.celrep.2020.107765

Figure Lengend Snippet: Figure 4. EndT and EndMT of Metastatic Melanoma Cells in Mouse (A) Whole-mount staining of pulmonary artery with GFP+ cells located at the endothelium (Videos S1, S2, and S3). Bars: 10 mm. (B and C) EndT occurred in lymph nodes. EndT occurred in LYVE-1+ lymphatic vessles (B) and CD31+ blood vessels (C) in lymph nodes. Bars: 20 mm. (D) LYVE-1 immunostaining on lung cryosections. Bar: 20 mm. (E–J) Immunostaining indicated EndT is transient during tumor progression. At primary tumor sites, GFP+ cells were CD31-, VE-cadherin+ (E and H), while in the lung blood vessels, GFP+ cells were both CD31+ and VE-cadherin+ (F and I). GFP+ cells lost both endothelial markers when populating metastases in the lung (G and J). Bars: 20 mm. (K) CD31+/a-SMA+/GFP+ cell (arrowhead) inside the alveolar capillary. CD31+/aSMA+/GFP cell (arrow) near the GFP+ cell showed the same phenotype. It is possible that this is an authentic vascular endothelial cell undergoing EndMT or a metastatic cell that was not labeled with GFP. Bars: 10 mm. (A–K) Mice, n R 3. (L) Correlation between the number of GFP+ cells per vessel and the number of metastasis foci or the size of metastasis foci per mouse. The number of GFP+ cells/vessel vs. number of metastasis foci, r = 0.7950, p = 0.03*; number of GFP+ cells/vessel vs. metastasis size, r = 0.8012, *p = 0.03. Mice, n = 7.

Article Snippet: Human pulmonary artery endothelial cells (PAECs; ATCC PCS-100-022) were cultured according to the supplier’s instructions.

Techniques: Staining, Immunostaining, Labeling

Journal: Cell reports

Article Title: Disseminated Melanoma Cells Transdifferentiate into Endothelial Cells in Intravascular Niches at Metastatic Sites.

doi: 10.1016/j.celrep.2020.107765

Figure Lengend Snippet: Figure 5. In Vitro System of Melanoma Cell EndT (A–C) Melanoma/endothelial marker immunostaining of Q-YUWERA cells (cells with green dots) cultured alone (A, VE-cadherin showed background staining in nuclei) or 5 days after co-culturing with PAECs (B and C). CD31 and HMB45 double staining in (B), VE-cadherin and HMB45 double staining in (C). Bars: 20 mm. Independent experiments, n R 3. (D) Imaging flow cytometry confirmed the expression of CD31 by individual YUWERA (APC+) cells after the co-culture. BF, bright field; SSC, side scatter; APC, CellTrace far red-labeled YUWERA cells. (E) YUWERA cells were labeled with a CFSE proliferation kit and co-cultured with PAECs for 5 days with 300 nM sunitinib treatment or control solution. Cells were subjected to CD31 staining followed by flow cytometry analysis. Independent experiments, n = 3; replicates, n = 3 in each experiment. 300 nM sunitinib treatment significantly increased the percentage of CD31+ YUWERA cells of the overall YUWERA cells in the co-culture (control group 2.878% ± 0.1309%, sunitinib group 4.677% ± 0.3125%,*p = 0.0357, shown as mean ± SEM by a Mann-Whitney test).

Article Snippet: Human pulmonary artery endothelial cells (PAECs; ATCC PCS-100-022) were cultured according to the supplier’s instructions.

Techniques: In Vitro, Marker, Immunostaining, Cell Culture, Staining, Double Staining, Imaging, Cytometry, Expressing, Co-Culture Assay, Labeling, Control, MANN-WHITNEY

Journal: Cell reports

Article Title: Disseminated Melanoma Cells Transdifferentiate into Endothelial Cells in Intravascular Niches at Metastatic Sites.

doi: 10.1016/j.celrep.2020.107765

Figure Lengend Snippet: Figure 6. EndT Inferred from Single-Cell RNA-Seq Data and Immunofluorescence in Metastatic Human Melanoma Biopsies (A) tSNE plot from single-cell RNA-seq showing metastatic melanoma cells of different patients (23). Malignant melanoma cells were selected based on aberrant copy number profiles, which are mutually exclusive from authentic endothelial cells, and plotted based on their pigmentation activity score, based on AUCell (37). The zoom shows cells of a melanoma patient that does not show pigmentation activity but had rare cells with high expressional activity for an endothelial gene signature. (B–I) Immunofluorescence on metastatic melanoma biopsies from BRAFV600E-harboring patients. (B and C) Representative double-immunofluorescence mi- crographs for BRAFV600E and CD31 of metastatic melanoma biopsies in the lung (B) and in the brain (C). (D and E) BRAFV600E+/CD31+ cells (arrows) localized inside the vasculature of metastatic melanoma biopsies in the lung (D) and in the brain (E). Bars: 50 mm. (F and G) Representative triple-immunofluorescence micrographs for BRAFV600E, MITF and CD31 of metastatic melanoma biopsies in the lung F) and in the brain (G). (H and I) BRAFV600E+/CD31+ cells inside the vasculature of metastatic melanoma biopsies in the lung (H) and in the brain (I) were negative for melanocytic marker MITF (arrows). Bars: 50 mm.

Article Snippet: Human pulmonary artery endothelial cells (PAECs; ATCC PCS-100-022) were cultured according to the supplier’s instructions.

Techniques: RNA Sequencing, Activity Assay, Marker

Journal: The Journal of Cell Biology

Article Title: Monocyte Adhesion to Activated Aortic Endothelium: Role of L-Selectin and Heparan Sulfate Proteoglycans

doi:

Figure Lengend Snippet: Monocyte attachment to unstimulated or TNF-α–activated aortic endothelium under rotation: inhibition by mAbs. Endothelial monolayers were activated for 8 h with TNF-α (100 U/ml). BAEC were preincubated with medium or anti–VCAM-1 mAb (HAE-2). Monocytes were preincubated with medium, blocking anti–L-selectin mAb anti–LAM1-3 or control mAb anti–LAM1-11. Adhesion assays were carried out under rotation for 30 min at 4°C. Data are expressed as means ± SD. Results are representative of those obtained in six experiments. * P < 0.01. **Statistically significant ( P < 0.005) difference in adhesion relative to control.

Article Snippet: Norgard-Sumnicht et al. ( , ) have previously reported the presence of heparan sulfate in a calf pulmonary artery endothelial cell line (American Type Culture Collection CCL 209).

Techniques: Inhibition, Blocking Assay, Control

Journal: The Journal of Cell Biology

Article Title: Monocyte Adhesion to Activated Aortic Endothelium: Role of L-Selectin and Heparan Sulfate Proteoglycans

doi:

Figure Lengend Snippet: Monocyte attachment to unstimulated or TNF-α–activated aortic endothelium under rotation: inhibition by chimeric proteins. Endothelial monolayers were activated for 8 h with TNF-α (100 U/ml). Unstimulated and TNF-activated BAEC were then preincubated with medium, L-selectin/μ, or CD4/μ. Adhesion assays were carried out under rotation for 30 min at 4°C. Data are expressed as means ± SD. Results are representative of three experiments. ** P < 0.005.

Article Snippet: Norgard-Sumnicht et al. ( , ) have previously reported the presence of heparan sulfate in a calf pulmonary artery endothelial cell line (American Type Culture Collection CCL 209).

Techniques: Inhibition

Journal: The Journal of Cell Biology

Article Title: Monocyte Adhesion to Activated Aortic Endothelium: Role of L-Selectin and Heparan Sulfate Proteoglycans

doi:

Figure Lengend Snippet: Kinetics of monocyte attachment to TNF-α–activated aortic endothelium under rotation. Endothelial monolayers were stimulated with TNF-α (100 U/ml) for 0–8 h at 37°C before the addition of monocytes. The L-selectin–dependent component of monocyte adhesion was determined using function-blocking mAb anti–LAM1-3. Adhesion assays were performed under rotation for 30 min at 4°C. Solid circles represent adhesion of untreated monocytes. Open circles represent adhesion of monocytes pretreated with anti–LAM1-3 mAb. Data are expressed as means ± SD. Results are representative of two experiments.

Article Snippet: Norgard-Sumnicht et al. ( , ) have previously reported the presence of heparan sulfate in a calf pulmonary artery endothelial cell line (American Type Culture Collection CCL 209).

Techniques: Blocking Assay

Journal: The Journal of Cell Biology

Article Title: Monocyte Adhesion to Activated Aortic Endothelium: Role of L-Selectin and Heparan Sulfate Proteoglycans

doi:

Figure Lengend Snippet: Interaction of L-selectin with suspended aortic endothelial cells. Unactivated or TNF-α–activated BAEC (8 h, 100 U/ ml) were examined by indirect immunofluorescence analysis for the expression of L-selectin ligands. L-selectin/μ was used as the probe ( solid lines ), and CD4/μ, an isotype-matched chimeric protein, was used as the control ( dotted lines ). The data are representative of three experiments.

Article Snippet: Norgard-Sumnicht et al. ( , ) have previously reported the presence of heparan sulfate in a calf pulmonary artery endothelial cell line (American Type Culture Collection CCL 209).

Techniques: Immunofluorescence, Expressing, Control

Journal: The Journal of Cell Biology

Article Title: Monocyte Adhesion to Activated Aortic Endothelium: Role of L-Selectin and Heparan Sulfate Proteoglycans

doi:

Figure Lengend Snippet: Time course of aortic endothelial cell activation. Endothelial cells were activated with TNF-α (100 U/ml). At the indicated times, indirect immunofluorescence analysis was performed with L-selectin/μ ( solid lines ) and CD4/μ ( dotted lines ). The data are representative of three experiments.

Article Snippet: Norgard-Sumnicht et al. ( , ) have previously reported the presence of heparan sulfate in a calf pulmonary artery endothelial cell line (American Type Culture Collection CCL 209).

Techniques: Activation Assay, Immunofluorescence

Journal: The Journal of Cell Biology

Article Title: Monocyte Adhesion to Activated Aortic Endothelium: Role of L-Selectin and Heparan Sulfate Proteoglycans

doi:

Figure Lengend Snippet: Interaction of L-selectin with suspended aortic endothelial cells: effect of treating unstimulated BAEC with heparinase I, heparitinase II, chondroitinase ABC, hyaluronidase, or trypsin. Unactivated BAEC were examined by indirect immunofluorescence analysis with L-selectin/μ ( solid lines ) and CD4/μ ( dotted lines ). Identical results were obtained by treating BAEC with heparinase I, II, or III. The data are representative of six experiments. Percentages of BAEC that bound to L-selectin/μ were as follows: control, 86%; heparinase I, 54%; heparitinase II, 56%; chondroitinase, 89%; hyaluronidase, 90%; trypsin, 7%. The background staining with CD4/μ chimera was <1%.

Article Snippet: Norgard-Sumnicht et al. ( , ) have previously reported the presence of heparan sulfate in a calf pulmonary artery endothelial cell line (American Type Culture Collection CCL 209).

Techniques: Immunofluorescence, Control, Staining

Journal: The Journal of Cell Biology

Article Title: Monocyte Adhesion to Activated Aortic Endothelium: Role of L-Selectin and Heparan Sulfate Proteoglycans

doi:

Figure Lengend Snippet: Interaction of L-selectin with suspended aortic endothelial cells: effect of treating TNF-α–activated BAEC (8 h, 100 U/ml) with heparinase I, heparitinase II, chondroitinase ABC, hyaluronidase, or trypsin. Unactivated BAEC were examined by indirect immunofluorescence analysis with L-selectin/μ ( solid lines ) and CD4/μ ( dotted lines ). Identical results were obtained by treating BAEC with heparinase I, II, or III. The data are representative of six experiments. Percentages of BAEC that bound to L-selectin/ μ are as follows: control, 87%; heparinase I, 39%; heparitinase II, 47%; chondroitinase, 89%; hyaluronidase, 82%; trypsin, 4%.

Article Snippet: Norgard-Sumnicht et al. ( , ) have previously reported the presence of heparan sulfate in a calf pulmonary artery endothelial cell line (American Type Culture Collection CCL 209).

Techniques: Immunofluorescence, Control

Journal: The Journal of Cell Biology

Article Title: Monocyte Adhesion to Activated Aortic Endothelium: Role of L-Selectin and Heparan Sulfate Proteoglycans

doi:

Figure Lengend Snippet: Interaction of L-selectin with suspended aortic endothelial cells: effect of treating unstimulated or TNF-α–activated BAEC (6 h, 100 U/ml) with cycloheximide (10 μg/ml), V. cholerae neuraminidase (750 U/ml), or sodium chlorate (10 mM, 24 h). BAEC were examined by indirect immunofluorescence analysis with L-selectin/μ ( solid lines ) and CD4/μ ( dotted lines ). The data are representative of six experiments. Percentages of unactivated BAEC that bound to L-selectin/μ are as follows: control, 86%; cycloheximide, 22%; V. cholerae , 91%; chlorate, 13%. Percentages of TNF-α–activated BAEC that bound to L-selectin/μ are as follows: control, 77%; cycloheximide, 11%; V. cholerae , 89%; chlorate, 17%.

Article Snippet: Norgard-Sumnicht et al. ( , ) have previously reported the presence of heparan sulfate in a calf pulmonary artery endothelial cell line (American Type Culture Collection CCL 209).

Techniques: Immunofluorescence, Control

Journal: The Journal of Cell Biology

Article Title: Monocyte Adhesion to Activated Aortic Endothelium: Role of L-Selectin and Heparan Sulfate Proteoglycans

doi:

Figure Lengend Snippet: Inhibition of monocyte adhesion to 8-h TNF-α–activated endothelium under flow (wall shear stress estimated at 1.8 dynes/cm 2 ). Endothelial monolayers were activated for 8 h with TNF-α (100 U/ml), washed, and then preincubated for 45 min at 37°C with heparinase I (1,600 mU/ml) or hyaluronidase (200 mU/ml). The adhesion assay was performed in a flow chamber, and adherent monocytes were counted as described in the legend to Fig. . Data are expressed as means ± SD. Results are representative of three experiments. ** P < 0.001.

Article Snippet: Norgard-Sumnicht et al. ( , ) have previously reported the presence of heparan sulfate in a calf pulmonary artery endothelial cell line (American Type Culture Collection CCL 209).

Techniques: Inhibition, Shear, Cell Adhesion Assay