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: International Journal of Molecular Sciences

Article Title: Endothelial Cells Tissue-Specific Origins Affects Their Responsiveness to TGF-β2 during Endothelial-to-Mesenchymal Transition

doi: 10.3390/ijms20030458

Figure Lengend Snippet: Endothelial cells characterization regarding morphological, immunophenotyping, and vessel-like structures assay. ( A ) Phase contrast micrography demonstrating the polygonal morphology of aortic artery endothelial cells (PAEC), coronary artery endothelial cells (CAEC), human umbilical vein endothelial cells (HUVEC), and pulmonary artery endothelial cells (HPAEC) cells (100× magnification). ( B ) Immunophenotyping of ECs by flow cytometry. ( C ) All endothelial cells (PAEC, CAEC, HUVEC, and HPAEC) were able to form vessel-like structures when grown in matrigel, evidencing characteristics typical of CEs (40× and 100× magnification).

Article Snippet: We used distinct types of endothelial cells (ECs): CAEC (coronary artery endothelial cells, ATCC ® -Catalog No. PCS-100-020), PAEC (aortic artery endothelial cells, ATCC ® -Catalog No. PCS-100-011), HPAEC (pulmonary artery endothelial cells, ATCC ® -Catalog No. PCS-100-022) and HUVEC (human umbilical vein endothelial cells).

Techniques: Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Endothelial Cells Tissue-Specific Origins Affects Their Responsiveness to TGF-β2 during Endothelial-to-Mesenchymal Transition

doi: 10.3390/ijms20030458

Figure Lengend Snippet: Characterization of EndMT induction by TGF-β2 (10 ng/mL) in cell lines ( A ) PAEC, ( B ) CAEC, ( C ) HPAEC, and ( D ) HUVECs (non-treated or treated with TGF-β2). Immunofluorescence microscopy of cell lines induced to EndMT shows a decrease in the fluorescent intensity of CD31 (green) in PAECs, CAECs, and HUVECs cells. The nuclei were stained with DAPI (blue) and F-actin were stained with Phalloidin (red) (scale bar 50 µM; representative image of one replicate of each sample).

Article Snippet: We used distinct types of endothelial cells (ECs): CAEC (coronary artery endothelial cells, ATCC ® -Catalog No. PCS-100-020), PAEC (aortic artery endothelial cells, ATCC ® -Catalog No. PCS-100-011), HPAEC (pulmonary artery endothelial cells, ATCC ® -Catalog No. PCS-100-022) and HUVEC (human umbilical vein endothelial cells).

Techniques: Immunofluorescence, Microscopy, Staining

Journal: International Journal of Molecular Sciences

Article Title: Endothelial Cells Tissue-Specific Origins Affects Their Responsiveness to TGF-β2 during Endothelial-to-Mesenchymal Transition

doi: 10.3390/ijms20030458

Figure Lengend Snippet: TGF-β2 decrease formation of vessel-like structures in the cell lines (CAEC, PAEC, HPAEC, and HUVEC). The cells were treated with TGF-β2 and evaluated the capacity formation of vessel-like structures. This inhibitory effect was observed mainly in PAECs (representative image of one replicate; n = 3).

Article Snippet: We used distinct types of endothelial cells (ECs): CAEC (coronary artery endothelial cells, ATCC ® -Catalog No. PCS-100-020), PAEC (aortic artery endothelial cells, ATCC ® -Catalog No. PCS-100-011), HPAEC (pulmonary artery endothelial cells, ATCC ® -Catalog No. PCS-100-022) and HUVEC (human umbilical vein endothelial cells).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Endothelial Cells Tissue-Specific Origins Affects Their Responsiveness to TGF-β2 during Endothelial-to-Mesenchymal Transition

doi: 10.3390/ijms20030458

Figure Lengend Snippet: Effect of EndMT on the activation of the Erk pathway. The cells (CAEC, PAEC, HUVEC and HPAEC) were cultured for five days in presence TGF-β2 (10 ng/mL). Aliquots were withdrawn after the treatment and evaluated by ( A ) Multiplex technique analysis Array Kit ( n = 3, * p ≤ 0.05) and ( B ) western blotting using phospho-Erk1/2 (Thr202/Tyr204) and ERK1/2. β-actin were used as endogenous controls (representative image of one replicate of each sample). ( C ) Chemical inhibitor against MEK1/2 (U0126; 1 μM) inhibits the increase of ERK1/2 phosphorylation in the PAECs treated with TGF-β2. 1) U0126; 2) U0126-15′ TGF-β2; 3) U0126-30′ TGF-β2; 4) TGF-β2-15′; 5) TGF-β2-30′. GAPDH were used as endogenous controls (representative image of one replicate of each sample).

Article Snippet: We used distinct types of endothelial cells (ECs): CAEC (coronary artery endothelial cells, ATCC ® -Catalog No. PCS-100-020), PAEC (aortic artery endothelial cells, ATCC ® -Catalog No. PCS-100-011), HPAEC (pulmonary artery endothelial cells, ATCC ® -Catalog No. PCS-100-022) and HUVEC (human umbilical vein endothelial cells).

Techniques: Activation Assay, Cell Culture, Multiplex Assay, Western Blot, Phospho-proteomics