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: Cancer immunology research

Article Title: Inflammation-induced, abnormal expression of self-molecules on epithelial cells: targets for tumor immunoprevention

doi: 10.1158/2326-6066.CIR-19-0870

Figure Lengend Snippet: (A) MUC1, Her-2/neu, hypoglycolysated MUC1 (hyp-MUC1), and CEA expression on untreated MEPiC (black) and following treatment for 72 hours with different cytokines or their combinations (indicated by different colors). MFI, mean fluorescence intensity. (B) Fold-increase in the expression of MUC1, hyp-MUC1, Her-2/neu, and CEA on MEPiC treated with different pro-inflammatory cytokines (indicated by different colors) for different durations of time, over untreated cells (dashed line). Results are presented as mean values±SEM of three experiments. Dunnett’s multiple comparisons test: ***p<0.001, **p<0.01, *p<0.05. The following cytokine concentrations were used: IL6 (50 ng/mL), IL1β (12.5 ng/mL), and TNFα (12.5 ng/mL).

Article Snippet: Human primary mammary epithelial cells (MEPic) were purchased from ScienCell Research Laboratories, Inc. (Carlsbad, CA, USA) and cultured according to supplier’s instructions.

Techniques: Expressing, Fluorescence

Journal: Cancer immunology research

Article Title: Inflammation-induced, abnormal expression of self-molecules on epithelial cells: targets for tumor immunoprevention

doi: 10.1158/2326-6066.CIR-19-0870

Figure Lengend Snippet: (A) Volcano plot displaying differentially expressed genes in treated mammary epithelial cells compared to untreated cells. Names of mRNA probes indicate genes that reached significance in difference of expression. The horizontal lines show the threshold of p<0.01 (continuous) or p<0.001(dashed). Vertical dashed lines represent the threshold for upregulation (Log2 FC>1) and downregulation (Log2 FC<−1). (B) Differential expression of the top 10 gene sets in treated cells compared to untreated cells. (C) Heatmap representation of expression of 12 genes involved in NF-κB signaling in MEPiC (orange) and MCF10A (purple) treated (yellow) or untreated (blue) cells. Names of genes significantly overexpressed in treated cells are framed in red. The color key was provided by the software and shows a gradient from low (log2 FC <–1) to high (log2 FC>1) expression compared to untreated cells. (D) Venn diagram of the highest differentially expressed genes in treated cells over untreated cells shared between MCF10A and MEPiC cells. Treated cells were exposed to IL6 (50 ng/mL), IL1β (12.5 ng/mL), and TNFα (12.5 ng/mL) for 72 hours.

Article Snippet: Human primary mammary epithelial cells (MEPic) were purchased from ScienCell Research Laboratories, Inc. (Carlsbad, CA, USA) and cultured according to supplier’s instructions.

Techniques: Expressing, Quantitative Proteomics, Software

Journal: Nucleic Acids Research

Article Title: Phototoxic aptamers selectively enter and kill epithelial cancer cells

doi: 10.1093/nar/gkn967

Figure Lengend Snippet: DNA aptamers labelled with the photodynamic agent chlorin e 6 selectively kill epithelial cancer cells. Cell viability curves were constructed for eight MUC1 + human cancer cells (human breast cancer cell lines T47D and MCF-7, human pancreatic cancer cell lines PANC-1 and BxPC3, human lung cancer cell lines A549 and MGH13, human ovarian cancer cell line OVCAR-3, colon cancer cell line HT-29), two MUC1 − cell lines (Chinese Hamster Ovary cells [CHO], human malignant glioma cell line U87MG) as well as fully glycosylated MUC1 + normal human mammary epithelial cells (HMEC). Cells were incubated in the presence of increasing concentrations of chlorin e 6 alone (x), or either chlorin e 6 -labelled Ce6-5TR1 aptamer (open square), Ce6-5TRG2 aptamer (open triangle), Ce6-GalNAc3 aptamer (open circle), unmodified 5TR1 aptamer (filled square), 5TRG2 aptamer (filled triangle), GalNAc3 aptamer (filled circle), or a Ce6-irrelevant aptamer (open diamond). Cell viability was determined for cells exposed (full graph) or not (captioned graph within each graph) to photoirradiation and reported as the percentage surviving cells measured in a MTT assay. The term concentration refers to the molar concentration of chlorin e 6 either as a free drug or conjugated to aptamers. The irrelevant DNA aptamer used was a 25 base-long oligonucleotide composed of GATC repeats and labelled with chlorin e 6 and was shown not to be toxic to MUC1 − and MUC1 + cells.

Article Snippet: Aliquots of 5 × 10 3 cells of either human cancer cells (T47D, MCF-7, PANC-1, BxPC3, A549, MGH13, OVCAR-3, HT-29, U87MG), CHO cells or normal human primary mammary epithelial cells (HMEC) (Clonetics; Lonza, Basel, Switzerland) suspended in 200 μl growth medium were seeded into wells of blackened 96-well plates with clear bottoms (Corning Costar Corp., Cambridge, MA) and cultured for 6 h at 37°C in the presence of 5% CO 2 .

Techniques: Construct, Incubation, MTT Assay, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Theranostics Using MCM-41-Based Mesoporous Silica Nanoparticles: Integrating Magnetic Resonance Imaging and Novel Chemotherapy for Breast Cancer Treatment

doi: 10.3390/ijms25158097

Figure Lengend Snippet: MTT assay results showing the cell viability of ( A ) MDA-MB-231 breast cancer cells and ( B ) normal human mammary epithelial cells (HMECs) after 96 h of treatment with increasing concentrations of MCM-41-NH 2 -DTPA-Gd 3+ ( ● ) and MCM-41-NH 2 -DTPA-Gd 3+ -MIH ( ● ) nanoparticles. The dashed lines represent 50% normalized cell viability. Each data point represents the mean ± standard deviation of three replicates.

Article Snippet: Human Primary Mammary Epithelial Cells (H-6035) were obtained from Cell Biologics, Inc. (Chicago, IL, USA) and cultured in collagen-coated tissue culture flasks using Gelatin-Based Coating Solution (H6950); Cell Biologics) with complete Human Epithelial Cell Medium (H6621; Cell Biologics), All cells were maintained at 37 °C in a 5% CO 2 humidified incubator and passaged before reaching 90% cell confluency.

Techniques: MTT Assay, Standard Deviation