|Application:||Gene expression microarray analysis|
|Number of samples:||32|
|Release date:||Mar 17 2015|
|Last update date:||May 15 2017|
|Diseases:||Breast Neoplasms, Neoplasms, Brain Stem Neoplasms|
|Dataset link||Identification of signatures specific to the epithelial or mesenchymal phenotypes from the heterogeneous mammary epithelial HMLER cells|
We chose to identify genes characteristic of the E and M phenotypes isolated from the same individual, which are therefore independent of genetic differences that are the usual problem when comparing cancer cell lines derived from different individuals. HMLER cells are derived from normal mammary epithelial cells from a healthy individual (HMEC) and immortalized and transformed by transduction with hTERT, SV40LT and RAS oncogenes (Elenbaas et al., 2001). The parental HMLER cell line (HP) is heterogeneous as it usually contains both epithelial and mesenchymal subpopulations that can be identified and discriminated by inverse expression of CD24 and CD44 cell surface markers. Single HMLER cells were sorted using flow cytometry and expanded in adhesion cultures. Morphology with respect to cobble stone-like epithelial phenotype or fibroblast-like solitary growing mesenchymal phenotypes was assessed. Epithelial clones were designated as E clones and mesenchymal clones were designated as M clones. Expression of the cell surface markers CD24 and CD44 strongly correlated with the E (CD24+/CD44-) and M (CD24-/CD44+) phenotypes, and only clones with 95% homogeneity for CD24+/CD44- or CD24-/CD44+ were used for determination of E versus M genes. In order to determine genes specific for the respective epithelial or mesenchymal phenotypes, we generated gene expression arrays (Agilent) by comparing stable (with respect to morphology and cell surface CD24/CD44 profile) E clones (E1 to E6) with M clones (M2-M5), which were collected as biological replicates by at least two passages and usually a freezing cycle to test biological reproducibility of the gene expression profiles. Clones E1 to E3 and M2 and M3 were generated in a first batch of single cell-cloning, and after another freezing cycle of the parental HMLER cell line, clones E4 to E6 and M1, M4 and M5 were generated. Data normalization was performed in Genedata Analyst using central tendency followed by relative normalization. Transcripts showing differential expression (p value < 0.01) between two different morphological phenotypes (E clones E1, E2, E3, E4, E5, E6 and M clones M2, M3, M4, M5) were identified by Limma and ranked according to their effect size. As control for genes that are not specific for homogeneous single cell-derived E clones or M clones, we generated gene expression arrays from the adherent heterogeneous parental HMLER cells (=HP) that has also an epithelial phenotype and about 10-30% CD24+/CD44- cells. M1 was a CD44+ clone that also contained a stable subpopulation of CD24+/CD44+ cells, could change phenotype and had either E or M morphology, which was thus excluded from the E versus M comparison. As a control for genes that are not specific for adhesion cultures (supposedly differentiated cultures, _adh) but rather for stem cell-enriched populations, we analyzed gene expression profiles from mammospheres (_sus) from the epithelial HP cell line and the mesenchymal clone M4. Biological replicate adherent E cell lines - E1 cell line: E1_adh_0629, E1_adh_0826; E2 cell line: E2_adh_0616, E2_adh_0629, E2_adh_0826; E3 cell line: E3_adh, E3_adh_0629, E3_adh_0826; E4 cell line: E4_adh_0210, E4_adh_0607; E5 cell line: E5_adh_0210, E5_adh_0607; HP cell line: HP_adh_2210, HP_adh_0607c, HP_adh_0607sc, HP_adh_0629. Biological replicates adherent M cell lines - M1: M1_adh_0210, M1_adh_0607; M2 cell line: M2_adh, M2_adh_0629, M2_adh_2210; M3 cell line: M3_adh, M3_adh_2210, M3_adh_2810; M4 cell line: M4_adh_0607, M4_adh_0210. Biological replicates mammospheres - HP cells: HP_sus_0603_1, HP_sus_0603_2; M4 cells: M4_sus_0603_1, M4_sus_0603_2.
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