E4-ECs promote mammosphere enrichment.

<p><b>A</b>) Schematic representation of sphere forming assay for enriching CSCs. PKH26-BCCs were cultured with and without GFP⁺E4-ECs (1∶10 ratio) under low adherent condition for 5 days and the rate of mammo-angiosphere or mammosphere enrichment was evaluated to determine the role of endothelial cells in CSC propagation. <b>B</b>) Phase contrast microscopy images of sphere forming assay illustrating the effect of E4-ECs on mammary stem cell development. <b>C</b>) Quantitative analysis of mammosphere formation grown with and without E4-ECs. When E4-ECs mingled with BCCs, the rate of mammosphere formation increased by 3 to 4-fold (***<i>p</i><0.001, mean ± SEM). <b>D</b>) Quantitative analysis of secondary mammosphere formation. Initially, primary mammospheres or mammo-angiospheres were cultivated for 5 days, then dissociated and flow cytometry-sorted mammospheroids were plated under low attachment condition to obtain secondary mammosphere (***<i>p</i><0.001, mean ± SEM). <b>E</b>) Phase contrast microscopy showing significant increase in secondary mammosphere growth in spheroids pre-exposed to E4-ECs. <b>F</b>) Immunofluorescent live confocal imaging of the daily process of mammo-angiosphere formation. GFP⁺E4-ECs serve as a core for the accumulation and enrichment of mammary stem cells (red). PKH26^High CSCs remain in close vicinity of E4-ECs (white arrowheads).