Figure 4. Met analysis in tumor and BM cells.

(a) Western blot analysis of Met and phospho-Met in B16-F1 and B16-F10 exosomes and cells (b) QRT-PCR analysis of Met and Cd44 in lineage-negative BM cells after B16-F10 or B16-F1 exosome education; error bars represent s.e.m.; NS = not significant by ANOVA. (c) Flow cytometric analysis of c-Kit and Met expression on BM cells after overnight incubation with fluorescently labeled exosomes (20 μg ml⁻¹, PKH26⁺). FL2 fluorescence indicates exosome uptake (right panel). (d) Flow cytometric analysis of Met expression in BM c-Kit⁺Tie2⁺ cells (upper panel) and Lin^–c-Kit⁺Tie2⁺ circulating blood cells (lower panel) of mice educated with B16-F10 and B16-F1 exosomes (red area=percentage of Met⁺ cells). Error bars represent s.e.m.; NS = not significant by ANOVA. (e) Analysis of metastasis in mice educated with 5μg of B16-F10, B16-F10shMet and control. Scale bar, 200 μm. Quantification of total photon flux 21d post B16-F10-luciferase cell injection (lower panel). Error bars represent s.e.m.; P value by ANOVA. (f) Multiplex protein analysis of MET and phospho-MET (Tyr1349) in the circulating exosomes from a retrospective series of frozen plasma derived from melanoma subjects and controls. Controls (n = 7); Stage III (n = 24); Stage IV (n = 15). P values by ANOVA. (g) Flow cytometric analysis depicting the percentage of MET⁺ BM progenitor cells in the blood of individuals with melanoma. Controls (n = 7); stage I–III (n = 10); stage IV (n = 9). Error bars represent s.e.m; P values by ANOVA.