Journal: International journal of cancer

Article Title: Extracellular vesicles secreted by triple-negative breast cancer stem cells trigger premetastatic niche remodeling and metastatic growth in the lungs.

doi: 10.1002/ijc.34447

Figure Lengend Snippet: FIGURE 1 EVsCSC and EVsDCC isolation and characterization. (A) Representative images of CSCs and DCCs analyzed for tdTomato fluorescence, scale bar = 25 μm. (B) Gene expression of stemness reporters ALDH1A1, Nanog and Oct-4 detected by qPCR for MDA-MB-231 subpopulations. CSC refers to tdTomato + cells cultured in stem cell maintenance media while DCC indicates cells cultured with a minimal presence of CSC (1%-2%) to avoid de-differentiation. CNRQ stands for Calibrated Normalized Relative Quantity with respect to GAPDH and Actin housekeeping genes. (C) 2D laminin invasion assay comparing CSC and DCC cells. (D) CryoTEM imaging of isolated EVsCSC and EVsDCC. (E) Stochastic Optical Resolution Microscopy (STORM) imaging of isolated EVs previously labeled with DiD. (F) Size distribution by nanoparticle tracking analysis (NTA) of EVsCSC and EVsDCC. (G) EVs typical markers, CD81, TSG101 and ALIX and cell lysate control, β-tubulin identified by Western blot. Twenty micrograms of total protein was loaded per lane and cell extracts (CE) were included as controls. (H) ALDH1A1 and CD44 protein cargo in EVCSC and EVsDCC examined by Western blot. (I) Protein cargo of EVCSC and EVsDCC assayed with a Human Pluripotent Stem Cell Array, showing higher content of OCT4, SOX2 and E- Cadherin in EVCSC. In H and I, quantification of blots are shown as the fold change in the band/dot intensity of EVsCSC/EVsDCC. Differences are significant for ALDH1A1, CD44, SOX2 and E-Cadherin (*P < .05)

Article Snippet: Human pluripotent stem cell antibody array (R&D Systems, ARY010) was performed in accordance with the manufacturer's instructions.

Techniques: Isolation, Fluorescence, Gene Expression, Cell Culture, Invasion Assay, Imaging, Microscopy, Labeling, Control, Western Blot