The conventional versus a new integrative CSC model for breast cancer.

<p>(A) In luminal epithelial cell-lines (e.g. in HMLE, HMLER, MCF7) the conventional model describes breast CSCs as M cells and ‘non cancer stem cells’ as E cells. Paradoxically, in more basal mesenchymal cancer cell-lines (MDA-MB231 cells, MCF10ACA1, 4T1) the more E gene-expressing cells are associated with CSC-properties. Thus, the identity of CSCs appears to be context-dependent. (B) Our model for CSCs integrates and explains these paradoxes by the existence of stem-like intermediate hybrid E/M cells independent of the tumor cell line. We propose that compared to more ‘polarized’ differentiated E (capable of plasticity) and M cell-types (capable of self-renewal), undifferentiated hybrid E/M cells can generate more mammospheres and heterogeneous progeny due to their capacity of both, self-renewal and plasticity. Arrows indicate possible state transitions (incomplete EMT and MET) between the instable hybrid E/M state and the extreme stable E and M states. Stemness of the intermediate E/M state is reflected both by presence of stem-like hybrid E/M cells and by co-presence of E and M cell-types due to cooperation. Context-independency of the stemness of the intermediate E/M state explains the paradoxical context-dependent meaning of E genes in basal tumors and M cell lines and stemness of M genes in luminal B tumors and E cell-lines (A).</p

Co-culture of E and M cell-types synergistically increases mammosphere formation.

<p>(A) Representative light microscope images (10x objective) from mammospheres of 500 E5, 500 M5, and 500 E5 + 500 M5-derived mammospheres after two weeks suspension culture. (B) Total number of primary mammospheres derived from 500 (1x) or 1000 (2x) cells per cell-type seeded (from A) after two weeks suspension culture. (C) Total number of primary mammospheres grown from 500 (1x) freshly sorted CD24+/CD44-(E) cells, 500 CD24-/CD44+(M) cells, the co-culture, or 500 un-gated HP cells (‘all HP’) after two weeks of mammosphere formation. (D) Relative number of primary (seeded 2,000 cells per cell line per well) and secondary mammospheres from M4 grown with or without E5 cells or HP cells. After two weeks 10% of total number of dissociated cells from the indicated primary mammosphere samples were reseeded for secondary mammospheres and counted after one week. Mammospheres per well relative to that of M4-derived spheres are shown.</p

CD24+/CD44+ cells are hybrid E/M cells and have increased stem-like properties.

<p>(A) CD24/CD44 flow cytometry profile of HP_late cells. (B) Single cell qPCR analysis of 20 individual cells sorted from each of the six indicated CD24/CD44 subpopulations (from two independently passaged HP_late cell-lines). Mean expression of 10 E and 7 M genes (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126522#pone.0126522.g003" target="_blank">Fig 3</a>) is visualized in the E-M state space. The crosses mark the average expression within the three different CD24/CD44 subpopulations (40 cells). Indicated p-values were determined using a cross-match test by comparing mean of E gene expression and/or M gene expression between indicated groups of single cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126522#pone.0126522.s012" target="_blank">S5 Table</a>). (C) Principal component (PC) projections of the 120 individual cells as tested in (B) for the three different subpopulations colored according to their CD24/CD44 origin are shown. (D) PC loading projections of the 48 genes as tested in (B), showing the contribution of each gene to the first two PCs. E genes are colored in orange, M genes in blue, pluripotency genes in green, and housekeeping genes in black. (E) CD24/CD44 flow cytometry analysis of cells cultured under standard adhesion conditions for 10 days that had been freshly sorted from 100 cells of the three subpopulations from (A). (F) Assessment of ALDH1 activity in cultured cells derived from the three subpopulations (E) using the ALDEFLUOR assay. Gate shows percentage of ALDH1+ cells. Small insets show the negative control cells incubated with DEAB. (G) Mammosphere-forming ability per 1,000 sorted cells of the three HP_late derived cell populations (A) after two weeks in suspension.</p

Co-expression of E and M genes predicts poor breast cancer patient survival.

<p>Kaplan-Meier plots showing time of overall survival (OS) and relapse-free survival (RFS) of breast cancer patients with different intrinsic breast cancer subtypes using the indicated gene expression signatures consisting of 60 genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126522#pone.0126522.s009" target="_blank">S2 Table</a>) and the Kaplan-Meier-Plotter online tool (database 2014). Numbers of patients within tumor subtype for OS or RFS are indicated on top. Hazard ratio (HR) and respective logrank p-values (P) that discriminate high (red line) and low (black line) expressing patient groups are shown.</p

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

<p>(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 (<i>CDH1</i>, <i>CD24</i>, <i>EPCAM</i>, <i>IL1B</i>, <i>KRT5</i>, <i>LCN2</i>, <i>TP63</i>, <i>TRAIL</i>, <i>SLPI</i>, <i>S100A8</i>) and 7 M-specific genes (<i>ABCA6</i>, <i>DCN</i>, <i>IL1R1</i>, <i>PCOLCE</i>, <i>WNT5A</i>, <i>VIM</i>, <i>ZEB2</i>) 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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126522#pone.0126522.s012" target="_blank">S5 Table</a>). 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.</p