Journal: Oncogene

Article Title: Induction of cells with cancer stem cell properties from nontumorigenic human mammary epithelial cells by defined reprogramming factors

doi: 10.1038/onc.2012.614

Figure Lengend Snippet: Reprogramming of human MCF-10A mammary epithelial cells. (a) Experimental scheme for the reprogramming of MCF-10A cells. (b) Phase-contrast images and immunofluorescence images of iPS-like colonies from MCF-10A cells (iPSL-10A) and normal human iPSCs stained with antibodies against OCT4, SOX2, TRA-1-60 and Nanog. Scale bar, 500 µm. (c) Semiquantitative reverse transcriptase–PCR (RT–PCR) analysis of iPSC markers in iPSL-10A cell clones 1–4, normal human iPSCs and MCF-10A cells. SOX2 and OCT4 are endogenously derived. (d) Immunoblotting of the stem cell marker proteins in iPSL-10A cell clones 1–4, normal human iPSCs and MCF-10A cells. (e) DNA methylation ‘heat map’ of iPSL-10A cells. DNA methylation analysis was performed using an Illumina Human Methylation 27 Beads Chip (MBL) with genomic DNA extracted from iPSL-10A clones 1 and 2, normal human iPSCs and MCF-10A cells. The β-value was calculated by a quantitative measure of the DNA methylation levels at specific CpG islands. Average β-values were subjected to unsupervised hierarchical clustering based on the Manhattan distance and average linkage. (f) High-resolution insertion-site analysis by linear amplification-mediated PCR (LAM PCR). Genomic DNA was prepared using phenol/chloroform extraction and subjected to LAM PCR. Amplicons were validated by sequencing. (g) Standard G-band chromosome analysis of MCF-10A and iPSL-10A cells. Arrows indicate identifiable aberrations common to both cell types.

Article Snippet: Human primary mammary epithelial cells were purchased from Kurabo Industrial (Osaka, Japan).

Techniques: Immunofluorescence, Staining, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Clone Assay, Derivative Assay, Western Blot, Marker, DNA Methylation Assay, Methylation, Amplification, Extraction, Sequencing

Journal: Oncogene

Article Title: Induction of cells with cancer stem cell properties from nontumorigenic human mammary epithelial cells by defined reprogramming factors

doi: 10.1038/onc.2012.614

Figure Lengend Snippet: Characterization of the CSC properties of iCSCL-10A clones. (a) Flow cytometric analysis of CD44 and CD24 expression in the MCF-10A, iCSCL-10A and MCF7 cell lines. The numbers indicate the percentage of each sub-population according to the CD44/CD24 expression profile. (b, c) Tumor sphere formation assays of MCF-10A-Ras, iCSCL-10A and MCF7 cell lines. Phase-contrast images of tumor spheres are shown (b). Values represent the mean ± s.e.m. (n=3, c). (d) Semiquantitative reverse transcriptase–PCR (RT–PCR) analysis of the expression of CSC- or epithelial-to-mesenchymal transition (EMT)-related genes. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was analyzed as a control. (e) Viability of MCF-10A-Ras, iCSCL-10A and MCF7 cell lines treated with various chemotherapeutic agents for 72 h by MTT assay. Values represent the mean ± s.e.m. (n=3). (f, g) iCSCL-10A and parental MCF-10A cells were treated with Juglone (5 µm) for 24 h and subjected to TUNEL (terminal deoxyribonucleotidyl transferase-mediated dUTP nick end-labeling) assay (f, brown color). TUNEL-positive cells were scored from triplicate independent experiments (g). Values represent the mean ± s.e.m. (n=3).

Article Snippet: Human primary mammary epithelial cells were purchased from Kurabo Industrial (Osaka, Japan).

Techniques: Clone Assay, Expressing, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Control, MTT Assay, TUNEL Assay, End Labeling

Journal: Oncogene

Article Title: Induction of cells with cancer stem cell properties from nontumorigenic human mammary epithelial cells by defined reprogramming factors

doi: 10.1038/onc.2012.614

Figure Lengend Snippet: iCSCL-10A cells form hierarchically organized tumors in vivo. (a) Tumor-seeding ability of iCSCL-10A, MCF-10A-Ras parental MCF-10A cells and iPSC-EBD. The indicated numbers of each cell type were injected into immunocompromised mice. The tumor-initiation ability per injection was then monitored. (b) Hematoxylin and eosin (H&E) staining of primary tumor tissues. Scale bar, 500 µm. (c) Immunohistochemical analysis of primary tumor tissues derived from iCSCL-10A cells using antibodies targeting hCD34 (endothelial), smooth muscle actin (SMA; myoblastic), β3-tubulin (neural), cytokeratin (CAM5.2, epithelial), vimentin (mesenchymal) and osteopontin (osteoblastic). Scale bar, 500 µm. (d) Immunofluorescent analysis with antibodies targeting SOX2 and cytokeratin (AE1/AE3). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar, 500 µm.

Article Snippet: Human primary mammary epithelial cells were purchased from Kurabo Industrial (Osaka, Japan).

Techniques: In Vivo, Injection, Staining, Immunohistochemical staining, Derivative Assay

Journal: Oncogene

Article Title: Elevated leptin disrupts epithelial polarity and promotes premalignant alterations in the mammary gland

doi: 10.1038/s41388-019-0687-8

Figure Lengend Snippet: Leptin primes mammary epithelial cells for proliferation. a Bright field micrographs of differentiated S1 acini treated for 72h with vehicle or leptin. Scale bar, 100 µm. b Representative confocal images and quantification of Ki67 staining in acini treated for 72h with vehicle, with leptin (100 ng/ml), with IGF-1 (0.1 µg/ml) and elevated insulin (500 ng/ml), or with the combination of the three factors. Scale bar, 20 µm. *, P < 0.05 and **, P < 0.01 (one-way ANOVA and Tukey's; n = 5). c Sizes (cross sections) of acini treated as in B. *, P < 0.05 (compared to all other treatments, one-way ANOVA and Fisher’s LSD, n = 3). d Apical localization of ZO-1 in acini treated as in B. **, P < 0.01, n.s., P > 0.05 (one-way ANOVA and Tukey's; n = 3). e Analysis of Ki67 in S1 acini after treatments with leptin and growth factors. Treatments were dissociated in time, as indicated in the schematic. Values were normalized to control (vehicle/vehicle). *, P < 0.05 (Kruskal-Wallis and Dunn’s, n = 4).

Article Snippet: Human mammary epithelial cells (post-stasis; psHMEC) were obtained from Lonza (Walkersville MD, USA) and cultured on Matrigel-coated surfaces with the supplier’s Epithelial Cell Medium for up to 5 passages.

Techniques: Staining, Control

Journal: Oncogene

Article Title: Elevated leptin disrupts epithelial polarity and promotes premalignant alterations in the mammary gland

doi: 10.1038/s41388-019-0687-8

Figure Lengend Snippet: Leptin leads to mitotic spindle misalignment. a Detection of ZO-1 and NuMA by immunofluorescence in polarized monolayers of S1 cells. Scale bar, 10 µm. b Mitotic spindle orientation in S1 cell monolayers treated with vehicle or leptin, as measured by confocal microscopy. Images on top represent maximal intensity projections whereas bottom images (a-d) are representative orthogonal views of the dual NuMA-DAPI staining. Angles between the two spindle poles (dashed lines) and the horizontal are quantified in the graph. **, P < 0.01 (unpaired t-test, n = 30 cells from two independent experiments). c Histology of mammary glands from mice with diet-induced obesity (DIO) and mice fed a control diet. Arrows indicate multilayered acini, defined as having ≥ 3 cell layers. d Correlation between apical polarization (Par3 score) and multilayering in the mammary epithelium from control and DIO mice. The exact P value (two-tailed Spearman test) was computed. The graphs represent mean ± SEM (B-E) or mean ± SD (G). e Schematic summarizing the effect of leptin on epithelial polarity and the functional consequences of polarity loss in the premalignant context.

Article Snippet: Human mammary epithelial cells (post-stasis; psHMEC) were obtained from Lonza (Walkersville MD, USA) and cultured on Matrigel-coated surfaces with the supplier’s Epithelial Cell Medium for up to 5 passages.

Techniques: Immunofluorescence, Confocal Microscopy, Staining, Control, Two Tailed Test, Functional Assay

Journal: Oncogene

Article Title: Elevated leptin disrupts epithelial polarity and promotes premalignant alterations in the mammary gland

doi: 10.1038/s41388-019-0687-8

Figure Lengend Snippet: Apical polarity correlates with epithelial multilayering and adipokine imbalance in human breast tissue. a Confocal microscopy images of ZO-1 and Par3 immunostaining in frozen sections from normal breast tissue from two different patients (003 and 005). Cell nuclei were conterstained with DAPI. Scale bars, 50 µm. b Association between apical polarity scores derived from ZO-1 and Par3 staining. The Pearson correlation coefficient ( r s ) and P value are shown. Each dot on the graph represents one patient (n = 13). Error bars are SEM. c Association between apical polarity scores (ZO-1 or Par3) and the number of epithelial cell layers, determined based on DAPI images. Mean ± SEM are shown for each patient (n = 13) with r s and P values. d-e Apical polarity in normal breast tissue classified based on their relative leptin/adiponectin transcript abundance (high, above median; low, below median). Apical polarity (AP) loss was defined as patient score averages below the 25 th percentile (ZO-1 and Par3) or below the 50 th percentile (actin). AP results for ZO-1, Par3, and actin are shown individually ( d ) or in combination, by adding scores values of each marker ( e ; mean ± SD). #, P < 0.05 (two tailed Chi-square test); *, P < 0.05 (two tailed Mann-Whitney test).

Article Snippet: Human mammary epithelial cells (post-stasis; psHMEC) were obtained from Lonza (Walkersville MD, USA) and cultured on Matrigel-coated surfaces with the supplier’s Epithelial Cell Medium for up to 5 passages.

Techniques: Confocal Microscopy, Immunostaining, Derivative Assay, Staining, Marker, Two Tailed Test, MANN-WHITNEY