Fig. 7.

Characterization of VM formation and EMT pathways in study groups. (A) Representative micrographs of CD31/PAS dual staining of VM during melanomagenesis in non-neoplastic nevus patient No. NN06, primary melanoma patient No. PM08, and metastatic melanoma patient No. MM05 tissues (Supplemental Table S5). The CD31 staining is shown in brown color (yellow arrow) and PAS staining is shown deep blue color (black arrow). Melanoma cross-section shows the presence of a PAS+/CD31+ endothelial-dependent vessels (black cycle) and the presence of PAS+ “patterned structures” that have been speculated to represent VM (yellow cycle). This in vivo experiment results show that VM tends to increase in patients with a high stage or grade of melanoma. Original magnification, × 200. Black arrow: structure of VM; Yellow arrow: structure of endothelial-dependent vessels. (B) The quantification of vasculogenic mimicry density (VMD) in different groups by comparing the microvessel density (MVD) and VMD in tumors. The MVD and VMD levels were significantly higher in patients with late-stage melanoma than in those with non-neoplastic nevus. (C) EMT-TFs, NOTCH1, SLUG, and ZEB1 expression levels were detected in melanoma tissues and matched with the corresponding paired adjacent normal tissues through qRT-PCR. The average EMT-TFs NOTCH1, SLUG, and ZEB1 expression levels were significantly higher in tissues of metastatic melanoma than in tissues of Nevi and primary melanoma. EMT-TF expression levels were compared between primary cancer and paired adjacent tissues in each case after normalization to β-actin housekeeping gene via the 2^−ΔΔCT method. Nevi refers to patients diagnosed with any abnormal, congenital formation or mark on the skin or neighboring mucosa that does not show neoplastic growth. ZEB1, zinc-finger E-box-binding-1; SLUG, zinc finger protein SNAI2; NOTCH1, Notch homolog 1, translocation-associated (Drosophila). *p < 0.05 and **p < 0.001 vs the Nevi group; #p < 0.05 and ##p < 0.001 vs the primary group (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).