Single cell analysis of EMT and MET state transitions during mammosphere formation.

(A) Flow cytometry profiles of the expression of CD24 and CD44 cell surface antigens in adherent grown parental HMLER (HP) cells. Cells were sorted from the indicated gates and (B) cultured in adhesion until confluence or (C) as mammospheres for two weeks in suspension. Cells were individualized, stained for CD24 and CD44 and again analyzed by flow cytometry. Data shown are from biological duplicates and are representative of at least three independent experiments. (D) Heat map of single cell qPCR analysis (BioMark Fluidigm) from sorted cells of E (HP, E5) and M (M4, M5) populations under adherent and mammosphere conditions for two days (2d) and three weeks (3w), using E and M-specific genes. Columns represent individual cells. Gene expression measured below background is shown in gray. (E) Mean expression per cell of 10 E-specific (CDH1, CD24, EPCAM, IL1B, KRT5, LCN2, TP63, TRAIL, SLPI, S100A8) and 7 M-specific genes (ABCA6, DCN, IL1R1, PCOLCE, WNT5A, VIM, ZEB2) in the E/M state space of individual cells grown in adhesion and three weeks as mammospheres (F). Crosses indicate mean expression of all 12 measured cells. P-values between the indicated groups of cells were determined using a cross-match test to discriminate by mean of E and M gene expression (S5 Table). Results with respect to statistically significant difference between gene expression in adherent E and M cells were observed in three independent experiments for single cells as well as well as for 100 cell pooled samples.