Figure 1.
Identification and characterization of isolated exosomes.
Exosomes were isolated by the ExoQuick-TC Precipitation Solution. (A) Representative morphological characterization of exosomes derived from brain metastatic 70 W melanoma cells by transmission electron microscopy. Round particles with characteristic exosomal size (30–100 nm) and shape were observed (arrows) immersed in the Exoquick solution. Scale bar is 50 nm. (B) Molecular confirmation of exosomes markers by Western blotting analysis. Exosome preparations were found to be positive for the exosomal markers CD9, CD63, and CD81 while negative for proteins from the endoplasmic reticulum (calnexin) and the Golgi apparatus (GM130) which were found to be present in cells lysates.
Figure 2.
Differential miRNA profiles of exosomes from brain metastatic versus non-brain metastatic cells.
Pathway-focused miScript miRNA PCR array was used to analyze the miRNAs contained within the exosomes. MiRNAs with statistically significant fold changes between brain metastatic (BM) and non-BM cell-derived exosomes were represented. Asterisks (*) denote statistically significant differences (p<0.05). MiR-210 was significantly enriched in all three BM cell-derived exosomes compared to non-BM, while two miRNAs were significantly down-regulated: miR-19a and miR-29c.
Figure 3.
Differential protein profiles of exosomes compared to respective cells.
Proteomic analyses were conducted using the Reverse Phase Protein Array by the RPPA Core Facility at MD Anderson Cancer Center (Houston, TX). Fold change of protein content in cells versus exosomes is represented by a histogram. The brown bars show the group of proteins that are present at high levels in exosomes compared to cells (0 to 3-fold change), the blue bars represent the bulk of the proteins (3 to 26-fold change), and the green bar shows the group of proteins detected at low quantities in exosomes (fold change higher than 26).
Figure 4.
Differential protein profiles of brain metastatic versus non-brain metastatic cell-derived exosomes.
The normalized expression of proteins detected in exosomes is represented according to the RPPA data. Five proteins up-regulated and four proteins down-regulated in the three BM exosomes compared with non-BM exosomes were identified. Of note, the highest expression among the up-regulated and the lowest expression among the down-regulated proteins occurred in the CTC1BMSM cell line in all cases.
Figure 5.
Non-brain metastatic cells uptake exosomes from their derivatives brain metastatic (BM) cell lines.
Exosomes from the BM cell lines were labeled with green lipophilic fluorescent dye PKH67 and incubated for 5-BM cells transduced with Tubulin-RFP and visualized by fluorescence microscopy. (A) MDA-MB-231P (231P) cells with MDA-MB-231BR-derived exosomes. (B) CTC1P cells with CTC1BMSM-derived exosomes. (C) MeWo cells with 70 W-derived exosomes. Numerous green fluorescent labeled exosomes were observed inside the cells, mostly located at the perinuclear region.
Figure 6.
Cells acquire a higher adhesive and invasive potential through uptaking exosomes.
(A) Tubulin-RFP transduced non-BM cell lines with and without exosomes from BM cells were plated over a human brain microvascular endothelial cells (HBMEC) monolayer. All non-BM cell lines increased their adhesive potential when exosomes are added compared to cells without exosomes. (B) Cells without exosomes, cells incubated with their own exosomes (parental cell line-derived exosomes), and cells incubated with the exosomes from their cell variants (BM cell line-derived exosomes) were plated onto invasion chambers coated with Matrigel™ artificial basement membrane. Cells incubated with exosomes showed a higher invasive capability compared to the cells without exosomes. Asterisks (*) denote statistically significant differences (p<0.05).