Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Notch3 Signaling-Mediated Melanoma-Endothelial Crosstalk Regulates Melanoma Stem-Like Cell Homeostasis and Niche Morphogenesis

doi: 10.1038/labinvest.2017.1

Figure Lengend Snippet: Two dimensional (2D) melanoma-EC co-culture model recapitulates MSLC niche in vitro . A. Schematic representation of the 2D MSLC niche model in vitro . GFP-labeled CD133 − non-MSLCs were co-cultured with RFP-labeled human umbilical vein endothelial cells (HUVECs) at 1:1 or 1:4 ratios for 5 days. B. In this model, ECs aligned to form branching tubular networks, reminiscent of the vascular niche in vivo (Magnification, ×100; scale bar, 200 μm). Co-cultured melanoma cells were then segregated from ECs by flow cytometry. C. MSLC (e.g., CD133 and CD271) and VM (e.g., CD144) markers were up-regulated in co-cultured melanoma cells compared to their mono-culture counter parts using qRT-PCR, simulating “dynamic stemness” and VM morphogenesis in vitro . D. Such a niche-inducing phenomenon was most pronounced when melanoma-EC contact is permitted, while EC extracellular matrix (ECM) or soluble factors in transwell cultures alone exhibited limited/partial effects. *, P < 0.05.

Article Snippet: Blots were probed with anti-Notch3 (Cell Signaling Technology, D1118 rabbit mAb, Danvers, MA), anti-CD133 (Miltenyi Biotech Inc., clone W6B3C1), anti-CD271 (Alomone Labs, Jerusalem, Israel), anti-CD144 (Cell Signaling Technology) Abs.

Techniques: Co-Culture Assay, In Vitro, Labeling, Cell Culture, In Vivo, Flow Cytometry, Quantitative RT-PCR

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Notch3 Signaling-Mediated Melanoma-Endothelial Crosstalk Regulates Melanoma Stem-Like Cell Homeostasis and Niche Morphogenesis

doi: 10.1038/labinvest.2017.1

Figure Lengend Snippet: Effective and functional Notch3 KD depletes MSLCs and VM-engaging melanoma cells in 1205 Lu xenografts. A. Western blot analyses demonstrated that stem cell markers (e.g., CD133 and CD271) as well as VM maker, CD144, were down-regulated in Notch3 KD 1205 Lu tumors, compared to non-target control (A). Consistent with the observed VM inhibition by Notch3 KD above, the expression of Tie-1, a VM-associated gene, was also down-regulated in Nocth3 KD 1205Lu xenografts by qRT-PCR (B). Of note, the efficacy of Notch3 KD was maintained in the tumor xenografts as shown by Western blotting (A). On the other hand, in A375 melanoma cells where no change in tumorigenicity was appreciated following Notch3 KD, the expression of stem-like cell marker, CD271, was not affected (C). Attempts to further validate CD133 and CD144 in Notch3 KD A375 xenografts using Western blot analysis (C8161 lysate included as a positive control) were unsuccessful due to sensitivity issue ( ; note that CD133 and CD144 signals were undetectable both in the control and KD xenografts despite maximal loading and prolonged overnight exposure). *, P < 0.05.

Article Snippet: Blots were probed with anti-Notch3 (Cell Signaling Technology, D1118 rabbit mAb, Danvers, MA), anti-CD133 (Miltenyi Biotech Inc., clone W6B3C1), anti-CD271 (Alomone Labs, Jerusalem, Israel), anti-CD144 (Cell Signaling Technology) Abs.

Techniques: Functional Assay, Western Blot, Inhibition, Expressing, Quantitative RT-PCR, Marker, Positive Control