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: Scientific Reports

Article Title: Characterization of transcriptome diversity and in vitro behavior of primary human high-risk breast cells

doi: 10.1038/s41598-022-10246-4

Figure Lengend Snippet: Relative expression levels of cell cycle related target genes of E2F transcription factors. Relative expression levels of cell cycle related target genes of E2F transcription factors (HALLMARK_E2F_TARGETS) are shown with samples arrayed left to right according to increasing viability. Italicized samples ( IPSI6, IPSI7, IPSI8 ) were mycoplasma negative on sequence evaluation and grew in MEGM but had no material for biochemical mycoplasma testing and were therefore excluded from presentation of viability and mammosphere numbers. IPSI ipsilateral non-cancer, T tumor cancer. Color coding: Dark blue to yellow with increasing expression.

Article Snippet: Cell viability and mammosphere formation after seven days growth were determined in three different mammary specific media; MEGM, EpiCultTM-C Human Media (STEMCELL Technologies) supplemented with 10 ng/mL EGF (cat no: PHG0311) and bFGF (cat no: PHG0261) (Thermofischer Scientific) and 0.48 μg/mL Hydrocortisone (STEMCELL Technologies, MammoCultTM Human Media (MammoC) (STEMCELL Technologies) supplemented with 4 μg/mL Heparin (STEMCELL Technologies) and 0.48 μg/mL Hydrocortisone, and Conditioned Media (CM) .

Techniques: Expressing, Sequencing

Journal: Scientific Reports

Article Title: Characterization of transcriptome diversity and in vitro behavior of primary human high-risk breast cells

doi: 10.1038/s41598-022-10246-4

Figure Lengend Snippet: Viability and mammosphere formation in different media. ( a ) Scatter plot illustrating age distribution of samples isolated and not isolated in CRC with and without prior neoadjuvant therapy (Rx). Sample numbers, mean and standard error of the mean (SEM), and p values < 0.05 shown. Ordinary one-way ANOVA, p = 0.0035 F = 5.218, Brown-Forsythe test F = 0.6113, DFn = 3 DFd = 45, Sidak’s multiple comparisons test padj = 0.0296 [Isolation in CRC No prior Rx n = 20, No isolation in CRC No prior Rx n = 21], padj = 0.0168 [Isolation in CRC Prior Rx n = 7, No isolation in CRC Prior Rx n = 4]. ( b) Scatter plot illustrating age distribution of samples passaged and not passaged in MEGM with and without prior neoadjuvant therapy (Rx). Sample numbers and mean and SEM shown. ( c ) Regression scatter plot: patient age (years) and viability measured in MEGM. n = 6 replicates. Regression equation, p value and R squared shown. ( d) Bar graphs presenting cell viability of primary cells measured seven days following plating in different media (n = 6 replicates/condition with exceptions IPSI9 n = 3 replicates/EpiCult, IPSI24 n = 5 replicates/CRC Conditioned Media). ( e) Bar graphs presenting numbers of mammospheres formed counted seven days following plating in different media (n = 6 replicates/condition with exceptions IPSI9 n = 3 replicates/EpiCult, IPSI24 n = 5 replicates/CRC Conditioned Media). * p < 0.05. 2way ANOVAs demonstrated an interactive effect between medium and samples accounting for 17.03% of the variance ( p < 0.0001 F = 193.21 DFn = 21 DFd156) in viability for samples tested in MEGM, EpiC, MammoC and CRC Conditioned Media with sample accounting for 66.54% (p < 0.0001 F = 2264.44 DFn = 7 DFd = 156) and medium 12.33% ( p < 0.0001 F = 979.30 DFn = 3 DFd = 156) with 7.535% of the variance for samples tested in MEGM, EpiC and MammoC due to an interactive effect ( p < 0.0001 F = 6.43 DFn = 26 DFd = 204) with sample accounting for 82.71% of the variance ( p < 0.0001 F = 141.05 DFn = 13 DFd = 204) and medium 0.5213% ( p < 0.0036 F = 5.78 DFn = 2 DFd = 204). 2way ANOVAs showed an interactive effect accounting for 32.94% of variance ( p < 0.0001 F = 21.85 DFn = 21 DFd = 157) in mammosphere numbers for samples tested in MEGM, EpiC, MammoC and CRC Conditioned Media with sample accounting for 35.11% ( p < 0.0001 F = 69.89 DFn = 7 DFd = 157) and medium 20.51% ( p < 0.001 F = 95.28 DFn = 3 DFd = 157) with 34.01% of the samples tested in MEGM, EpiC and MammoC due to an interactive effect ( p < 0.001 F = 14.67 DFn = 24 DFd = 195) with sample variance accounting for 44.58% of the variance (p < 0.0001 F = 38.46 Dfn = 12 Dfd = 195) and medium 2.581% (p < 0.0001 F = 13.36 DFn = 2 DFd = 195). Cell viability measured utilizing CellTiter-Glo® 3D with relative viability expressed as CellTiter-Glo luminescent cell viability score. Color coding: Black: Phenol red-free MEGM™. Red: EpiCult™. Blue: MammoCult™. Green: CRC Conditioned Media. IPSI ipsilateral non-cancer, T tumor cancer.

Article Snippet: Cell viability and mammosphere formation after seven days growth were determined in three different mammary specific media; MEGM, EpiCultTM-C Human Media (STEMCELL Technologies) supplemented with 10 ng/mL EGF (cat no: PHG0311) and bFGF (cat no: PHG0261) (Thermofischer Scientific) and 0.48 μg/mL Hydrocortisone (STEMCELL Technologies, MammoCultTM Human Media (MammoC) (STEMCELL Technologies) supplemented with 4 μg/mL Heparin (STEMCELL Technologies) and 0.48 μg/mL Hydrocortisone, and Conditioned Media (CM) .

Techniques: Isolation