In mammospheres E and M cell lines converge to an intermediate E/M gene expression state.
(A) Phase-contrast (20x) images showing a typical HMLER CD24+/CD44-(E) clone and a typical HMLER CD24-/CD44+(M) clone. Orange color refers to epithelial cells, and blue color to mesenchymal cells throughout manuscript. (B) Heat map of the E and M-specific gene expression defined as the 150 most extreme differentially expressed genes between morphological E clones and M clones (E_HMLER (150), and M_HMLER (150), S1 Table) in different HMLER cell-lines. RNA for gene expression arrays shown was isolated from different cell line passages. Gene expression data were relative-normalized towards the intermediate expression between E and M clones. Overlap with other E and M gene sets from different cell-types (HMLE cells and lung cancer) is shown on the right. (C) Phase-contrast (20x) images showing mammospheres grown in suspension of epithelial HP cells and the mesenchymal M4 clone. (D) Heat map of E and M-specific genes expressed in HP(E) and M4(M) cells when grown under adhesion and suspension mammospheres conditions. Normalization of gene expression was as in B, biological duplicate arrays are shown. (E) GSEA of gene expression dynamics between cells grown in adhesion versus suspension from (D), assessed for the E- and M-specific genes. (Normalized) Enrichment Scores (NES and ES) were based on duplicate arrays, had a false discovery rate q-value of < 0.0001 and a family wise p-value < 0.01 (S3 Table).(F) Pearson correlation coefficient between whole transcriptomes from (D) for global profiles. Numbers in heatmap represent the correlation average.