Figure 4

ECs ^Mes provide a pro-tumoral niche for tumor growth and development. A) Schematic representation of the experimental procedures carried out for evaluating the role of ECs^Mes in tumor development. ECs^Mes were acquired following the steps described in Figure 3A. Next, ECs^Mes and ECs^Norm were co-cultured with BCCs to compare their effects on cancer cell survival, cancer stem cell enrichment and cancer cell invasiveness. B) Cell proliferation assay and phase contrast imaging showing survival or proliferation of MDA-231 cancer cells when cultured alone or co-cultured with ECs^Mes or ECs^Norm. Once MDA-231 cells were cultured alone under complete starvation, not only didn't they proliferate, they minimally survived the culture condition. However, once grown with ECs, they showed significantly higher proliferation rate. ECs^Mes were capable of increasing MDA-231 cell proliferation around 1.5-fold higher than ECs^Norm. C) Mammosphere assay used for enriching cancer stem cells. MDA-231 cells were co-cultivated with either ECs^Norm or ECs^Mes under anchorage-independent condition for five days and their mammosphere-enriching capacity was evaluated by considering the number and size of the mammospheres grown in combination with ECs^Norm or ECs^Mes. Angiospheres were excluded from quantification by their GFP signal. Around 1.6-fold increase was observed in the rate of mammosphere (white arrowhead) formation when they mingled with ECs^Mes (black arrowhead). D) Wound healing assay performed to assess the migratory capacity of breast tumor cells when grown and sorted from ECs^Mes or ECs^Norm. BCCs exposed and sorted from ECs^Norm (ECs^Norm-exposed) or ECs^Mes (ECs^Mes-exposed) were used in a 48-hour wound healing assay under complete starvation to exclude the possibility of cell proliferation. BCCs that were grown and sorted from ECs^Mes were capable of closing the wound around 1.5-fold more efficiently than those sorted from EC^Norm. (*** p < 0.001, mean ± SEM, n = 3).