Journal: Breast Cancer Research : BCR

Article Title: The hypoxic tumor microenvironment in vivo selects the cancer stem cell fate of breast cancer cells

doi: 10.1186/s13058-018-0944-8

Figure Lengend Snippet: The hypoxia-sensing human breast cancer xenograft model. a , b MDA-MB-231 cells stably expressing the HRE-EGPF reporter gene are implanted either orthotopically in mammary fat pads ( a ) or ectopically in the hind back ( b ) of female athymic mice. Hypoxic regions are visualized by immunostaining of the Hypoxyprobe (red). Expression of the hypoxia reporter gene is shown by fluorescence of enhanced green fluorescent protein (EGFP). Nuclei are counterstained with Hoechst 33342. c The hypoxic populations from the MDA-MB-231/HRE-EGFP and MCF7/HRE-EGFP xenografts, respectively, are analyzed by fluorescence-activated cell sorting. d Microarray analysis shows that expression of a panel of commonly observed hypoxia-induced genes is significantly upregulated in the EGFP + cells freshly isolated from the MDA-MB-231/HRE-EGFP xenografts, compared to the EGFP − cells from the same xenografts ( n = 3; analysis of variance, p < 0.05). SSC, side scatter

Article Snippet: Emergence of CD44 + /CD24 + MCF7 cells, also positive for another stem cell marker CD49f, could result from increased stemcell-like populations in the hypoxic tumor microenvironment in vivo.

Techniques: Stable Transfection, Expressing, Immunostaining, Fluorescence, FACS, Microarray, Isolation

Journal: Breast Cancer Research : BCR

Article Title: The hypoxic tumor microenvironment in vivo selects the cancer stem cell fate of breast cancer cells

doi: 10.1186/s13058-018-0944-8

Figure Lengend Snippet: Cancer stem cell (CSC)-like cells are enriched in the hypoxic populations freshly isolated from xenografts. Tumor cells are enzymatically dissociated and isolated from either the MDA-MB-231/HRE-EGFP ( a - c ) or MCF7/HRE-EGFP ( d - f ) xenografts. Stem cell characteristics are evaluated by fluorescence-activated cell sorting (FACS) for the expression of CSC-associated surface markers CD24, CD44 and CD49f. Representative FACS plots are shown in a , c , d and f . Quantitative population analyses are shown in b ( n = 4–5; * p < 0.05, *** p < 0.001, Student’s t test) and e (n = 4; *** p < 0.001, Student’s t test). These results are confirmed by three or more independent experiments. EGFP, enhanced green fluorescent protein; SSC, side scatter

Article Snippet: Emergence of CD44 + /CD24 + MCF7 cells, also positive for another stem cell marker CD49f, could result from increased stemcell-like populations in the hypoxic tumor microenvironment in vivo.

Techniques: Isolation, Fluorescence, FACS, Expressing

Journal: Breast Cancer Research : BCR

Article Title: The hypoxic tumor microenvironment in vivo selects the cancer stem cell fate of breast cancer cells

doi: 10.1186/s13058-018-0944-8

Figure Lengend Snippet: The cancer stem cell (CSC)-like population is further enriched in secondary xenografts derived from the enhanced green fluorescent protein (EGFP) + MDA-MB-231 cells. a Generation of the secondary xenografts. b , c Surface levels of CD24 and CD44 are analyzed by fluorescence-activated cell sorting. b Average CD24 + populations from six individual tumors (*** p < 0.001, Student’s t test). c Average CD44 ++ (right-pointing arrow) populations from three individual tumors (* p < 0.05, Student’s t test). d Quantitative RT-PCR analysis of expression of CD24 and CD44 genes in the EGFP + and EGFP − cells freshly isolated from either the 2 nd or 1 st xenografts (n = 3; * p < 0.05, ** p < 0.01, Student’s t test). e Clonogenic growth of sorted EGFP + and EGFP − cells freshly isolated from the 2 nd xenografts in comparison to the unsorted tumor cells from the 1 st xenografts (n = 3; * p < 0.05, *** p < 0.0001, Student’s t test)

Article Snippet: Emergence of CD44 + /CD24 + MCF7 cells, also positive for another stem cell marker CD49f, could result from increased stemcell-like populations in the hypoxic tumor microenvironment in vivo.

Techniques: Derivative Assay, Fluorescence, FACS, Quantitative RT-PCR, Expressing, Isolation, Comparison

Journal: Breast Cancer Research : BCR

Article Title: The hypoxic tumor microenvironment in vivo selects the cancer stem cell fate of breast cancer cells

doi: 10.1186/s13058-018-0944-8

Figure Lengend Snippet: The cancer stem cell (CSC)-like characteristics of tumor cells isolated from the secondary enhanced green fluorescent protein (EGFP) + MCF7/HRE-EGFP xenografts. a Generation of secondary MCF7/HRE-EGFP xenografts by re-implantation of sorted EGFP + and EGFP − cells isolated from the primary MCF7/HRE-EGFP xenografts. Tumor cells freshly isolated from the secondary xenografts were sorted into EGFP + and EGFP − populations for ( b ) fluorescence-activated cell sorting (FACS) analysis of the CD44 + /CD24 + and CD44 + populations ( n = 5 for EGFP + cells, n = 4 for EGFP − cells; *** p < 0.001, Student’s t test). c Side population (SP) of the secondary xenograft-derived tumor cells. MCF7 tumor cells were isolated from the secondary xenografts derived from EGFP + and EGFP - tumor cells, respectively, and expanded in vitro for three passages. Cells were stained with Hoechst 33342 for side population analysis by FACS. Verapamil (50 μM) was used to block nuclear export of Hoechst 33342. These results were validated in two independent experiments. d Clonogenic potential of the freshly sorted EGFP + and EGFP − populations from the secondary xenografts ( n = 6; **** p < 0.0001, *** p < 0.001, Student’s t test)

Article Snippet: Emergence of CD44 + /CD24 + MCF7 cells, also positive for another stem cell marker CD49f, could result from increased stemcell-like populations in the hypoxic tumor microenvironment in vivo.

Techniques: Isolation, Fluorescence, FACS, Derivative Assay, In Vitro, Staining, Blocking Assay

Journal: Breast Cancer Research : BCR

Article Title: The hypoxic tumor microenvironment in vivo selects the cancer stem cell fate of breast cancer cells

doi: 10.1186/s13058-018-0944-8

Figure Lengend Snippet: The PI3K/AKT pathway is required for maintenance of the CD44 + /CD24 − cancer stem cell (CSC) phenotype. The enhanced green fluorescent protein (EGFP) + and EGFP − cells sorted from the 1 st MDA-MB-231 xenografts underwent serum starvation overnight. After serum stimulation, phosphorylation of AKT ( a ), PI3Kp85 ( b ), mTOR ( b ), and ERK1/2 ( c ) was analyzed using Western blots. d Serum-stimulated AKT phosphorylation in the parental MDA-MB-231/HRE-EGFP cells under normoxia and hypoxia (1% O 2 ). e Increase in the CD24 + population induced by the PI3K inhibitor LY294002 (20 μM). f Quantitative RT-PCR analysis of expression of CD24 and CD44 genes in response to LY294002. These observations are confirmed by independent experiments

Article Snippet: Emergence of CD44 + /CD24 + MCF7 cells, also positive for another stem cell marker CD49f, could result from increased stemcell-like populations in the hypoxic tumor microenvironment in vivo.

Techniques: Phospho-proteomics, Western Blot, Quantitative RT-PCR, Expressing