Loss of inter-cellular cooperation by complete epithelial-mesenchymal transition supports favorable outcomes in basal breast cancer patients.

According to the sequential metastasis model, aggressive mesenchymal (M) metastasis-initiating cells (MICs) are generated by an epithelial-mesenchymal transition (EMT) which eventually is reversed by a mesenchymal-epithelial transition (MET) and outgrowth of life-threatening epithelial (E) macrometastases. Paradoxically, in breast cancer M signatures are linked with more favorable outcomes than E signatures, and M cells are often dispensable for metastasis in mouse models. Here we present evidence at the cellular and patient level for the cooperation metastasis model, according to which E cells are MICs, while M cells merely support E cell persistence through cooperation. We tracked the fates of co-cultured E and M clones and of fluorescent CDH1-promoter-driven cell lines reporting the E state derived from basal breast cancer HMLER cells. Cells were placed in suspension state and allowed to reattach and select an EMT cell fate. Flow cytometry, single cell and bulk gene expression analyses revealed that only pre-existing E cells generated E cells, mixed E/M populations, or stem-like hybrid E/M cells after suspension and that complete EMT manifest in M clones and CDH1-negative reporter cells resulted in loss of cell plasticity, suggesting full transdifferentiation. Mechanistically, E-M coculture experiments supported the persistence of pre-existing E cells where M cells inhibited EMT of E cells in a mutual cooperation via direct cell-cell contact. Consistently, M signatures were associated with more favorable patient outcomes compared to E signatures in breast cancer, specifically in basal breast cancer patients. These findings suggest a potential benefit of complete EMT for basal breast cancer patients

Stemness of the hybrid Epithelial/Mesenchymal State in Breast Cancer and Its Association with Poor Survival.

Breast cancer stem cells (CSCs) are thought to drive recurrence and metastasis. Their identity has been linked to the epithelial to mesenchymal transition (EMT) but remains highly controversial since-depending on the cell-line studied-either epithelial (E) or mesenchymal (M) markers, alone or together have been associated with stemness. Using distinct transcript expression signatures characterizing the three different E, M and hybrid E/M cell-types, our data support a novel model that links a mixed EM signature with stemness in 1) individual cells, 2) luminal and basal cell lines, 3) in vivo xenograft mouse models, and 4) in all breast cancer subtypes. In particular, we found that co-expression of E and M signatures was associated with poorest outcome in luminal and basal breast cancer patients as well as with enrichment for stem-like cells in both E and M breast cell-lines. This link between a mixed EM expression signature and stemness was explained by two findings: first, mixed cultures of E and M cells showed increased cooperation in mammosphere formation (indicative of stemness) compared to the more differentiated E and M cell-types. Second, single-cell qPCR analysis revealed that E and M genes could be co-expressed in the same cell. These hybrid E/M cells were generated by both E or M cells and had a combination of several stem-like traits since they displayed increased plasticity, self-renewal, mammosphere formation, and produced ALDH1+ progenies, while more differentiated M cells showed less plasticity and E cells showed less self-renewal. Thus, the hybrid E/M state reflecting stemness and its promotion by E-M cooperation offers a dual biological rationale for the robust association of the mixed EM signature with poor prognosis, independent of cellular origin. Together, our model explains previous paradoxical findings that breast CSCs appear to be M in luminal cell-lines but E in basal breast cancer cell-lines. Our results suggest that targeting E/M heterogeneity by eliminating hybrid E/M cells and cooperation between E and M cell-types could improve breast cancer patient survival independent of breast cancer-subtype

<i>Francisella tularensis</i> subsp. <i>tularensis</i> Induces a Unique Pulmonary Inflammatory Response: Role of Bacterial Gene Expression in Temporal Regulation of Host Defense Responses

<div><p>Pulmonary exposure to <i>Francisella tularensis</i> is associated with severe lung pathology and a high mortality rate. The lack of induction of classical inflammatory mediators, including IL1-β and TNF-α, during early infection has led to the suggestion that <i>F. tularensis</i> evades detection by host innate immune surveillance and/or actively suppresses inflammation. To gain more insight into the host response to <i>Francisella</i> infection during the acute stage, transcriptomic analysis was performed on lung tissue from mice exposed to virulent (<i>Francisella tularensis</i> ssp <i>tularensis</i> SchuS4). Despite an extensive transcriptional response in the lungs of animals as early as 4 hrs post-exposure, <i>Francisella tularensis</i> was associated with an almost complete lack of induction of immune-related genes during the initial 24 hrs post-exposure. This broad subversion of innate immune responses was particularly evident when compared to the pulmonary inflammatory response induced by other lethal (<i>Yersinia pestis</i>) and non-lethal (<i>Legionella pneumophila, Pseudomonas aeruginosa</i>) pulmonary infections. However, the unique induction of a subset of inflammation-related genes suggests a role for dysregulation of lymphocyte function and anti-inflammatory pathways in the extreme virulence of <i>Francisella</i>. Subsequent activation of a classical inflammatory response 48 hrs post-exposure was associated with altered abundance of <i>Francisella</i>-specific transcripts, including those associated with bacterial surface components. In summary, virulent <i>Francisella</i> induces a unique pulmonary inflammatory response characterized by temporal regulation of innate immune pathways correlating with altered bacterial gene expression patterns. This study represents the first simultaneous measurement of both host and <i>Francisella</i> transcriptome changes that occur during <i>in vivo</i> infection and identifies potential bacterial virulence factors responsible for regulation of host inflammatory pathways.</p></div

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

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-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

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

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