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: Pharmaceuticals

Article Title: Tunicamycin Protects against LPS-Induced Lung Injury

doi: 10.3390/ph15020134

Figure Lengend Snippet: Effects of tunicamycin and LPS on lung endothelial permeability. ( A ) BPAECs were grown on gold-plated ECIS arrays to form a confluent monolayer. Those cells were exposed to either vehicle (VEH) (0.1% DMSO) or tunicamycin (TM) (0.5 μM). A gradual decrease in BPAEC permeability (increased TEER) was observed in the TM-treated cells. n = 3 per group; means ± SEM. ( B ) BPAECs were treated with either vehicle (0.1% DMSO) or TM (0.5 μM) for 24 h prior to treatment with either vehicle (PBS) or LPS (10 μg/mL). LPS exposure decreased TEER values (increased permeability), while TM pretreatment prevented LPS-triggered barrier dysfunction as reflected in the higher TEER values. * p < 0.05 vs. vehicle (VEH) and $ p < 0.05 vs. LPS. n = 3 per group; means ± SEM. ( C ) BPAECs were seeded onto transwell inserts of a 24-well culture plate. After 24 h, the cells were treated with either vehicle (VEH) (0.1% DMSO) or TM (1 µM) for 48 h. LPS (1 µg/mL) was added in the media for 1 h; followed by the addition of 70 kDa FITC-dextran (1 mg/mL). 20 min after FITC-dextran addition, 100 mL of basal media was removed and the fluorescence intensity was measured. * p < 0.05 vs. vehicle (VEH) and $ p < 0.05 vs. LPS. Means ± SEM, n = 3. ( D ) Western blot analysis of phosphorylated VE-cadherin (pVE-cad) and VE-cadherin (VE-cad) in BPAEC treated with either vehicle (0.1% DMSO) or TM (1 μΜ) prior to a 1 h exposure to either vehicle (PBS) or LPS (1 μg/mL). The blots shown are representative of three independent experiments. The signal intensity of the protein bands was analyzed by densitometry. Protein levels of pVE-cad were normalized to total VE-cad. * p < 0.05 vs. vehicle (VEH) and $ p < 0.05 vs. LPS. Means ± SEM.

Article Snippet: Bovine pulmonary artery endothelial cells (BPAEC) were purchased from Genlantis (San Diego, CA, USA).

Techniques: Permeability, Fluorescence, Western Blot

Journal: Pharmaceuticals

Article Title: Tunicamycin Protects against LPS-Induced Lung Injury

doi: 10.3390/ph15020134

Figure Lengend Snippet: Effects of tunicamycin in LPS-induced lung endothelial inflammation. ( A ) Western Blot analysis of BiP and β-actin in BPAEC treated with either vehicle (0.1% DMSO) or tunicamycin (TM) (1 μΜ) prior to treatment with either vehicle (PBS) or LPS (1 μg/mL) for 1 h. The blots shown are representative of four independent experiments. The signal intensity of the protein bands was analyzed by densitometry. Protein levels of BiP were normalized to β-actin. * p < 0.05 vs. vehicle (VEH) and $ p < 0.05 vs. LPS. Means ± SEM. Western Blot analysis of ( B ) phosphorylated cofilin (pCofilin) and cofilin, ( C ) phosphorylated MLC2 (pMLC2) and MLC2, ( D ) phosphorylated STAT3 and STAT3. BPAEC were pre-treated with either vehicle (0.1% DMSO) or TM (1 μΜ) for 24 h and post-treated with either vehicle (PBS) or LPS (1 μg/mL) for 1 h. The blots shown are representative of three independent experiments. The signal intensity of the protein bands was analyzed by densitometry. Protein levels of p-Cofilin, pMLC2, and pSTAT3 were normalized to cofilin, MLC2 and STAT3, respectively. * p < 0.05 vs. vehicle (VEH) and $ p < 0.05 vs. LPS. Means ± SEM.

Article Snippet: Bovine pulmonary artery endothelial cells (BPAEC) were purchased from Genlantis (San Diego, CA, USA).

Techniques: Western Blot