Fig. 1
Melanoma endothelium in heterogeneous. a Schematic diagram demonstrating experimental set up using vascular lineage tracing Cdh5-CreER RosaYFP mice. Flow cytometry plots showing cells dissociated from B16-F0 tumors harbor distinct CD34 positive, lineage (LIN) negative populations (red gate) as determined using strict fluorescence-minus-one (FMO) analysis. >98% of CD34+LIN- cells are YFP+. Three distinct populations were observed based on CD31 and VEGFR2 expression in tumors (from left to right: EVP, TA, and D) amongst CD34+LIN-YFP+cells (n = 4). b Schematic diagram demonstrating the isolation of GFP+ EVP, TA, and D from tumors, which were subsequently re-transplanted in a 1:1 ratio with B16-F0 cells into a wild-type host. c, d Flow cytometry plots showing only GFP+ EVP cells re-transplanted were able to persist and engraft in secondary tumors. TA and D cells inoculated in secondary tumors could not be recovered 14 days later (**p < 0.01 vs TA and D; Mann–Whitney T-Test) (n = 6). e Immunofluorescence images of only GFP+ EVP cells (white arrows) engrafting and surviving transplantation with B16-F0 (scale bar represents 100 µm). f Delivery of B16-F0 cells with GFP+ EVP resulted in larger tumors (increase in weight by 34% compared to B16-F0 alone), whereas tumor cells delivered with D cells alone resulted in smaller tumors (decrease in weight by 33% compared to B16-F0 alone) (*p < 0.05; Mann–Whitney T-Test). Results presented as mean ± SEM. EVP endovascular progenitor, TA transit amplifying, D definitive differentiated, CON control (no GFP cells)
