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Fig. 1 | Breast Cancer Research

Fig. 1

From: Transformation of enriched mammary cell populations with polyomavirus middle T antigen influences tumor subtype and metastatic potential

Fig. 1

A model to assess the influence of the cell of origin on tumor phenotype. a Freshly isolated mammary epithelial cells (MECs) were transduced with the EF1α-PyMT-ZsGreen lentivirus, sorted into distinct populations by fluorescence-activated cell sorting (FACS), and transplanted into the cleared mammary fat pads of syngeneic mice. b Transduced MECs were sorted into basal, luminal, and stem cell populations based on the expression of the cell surface markers CD49f and CD24 (right). Luminal cells were further sorted according to CD133 expression into hormone receptor–positive (CD133+) and hormone receptor–negative (CD133−) populations (left). The collected populations are indicated by red gates. APC allophycocyanin, PE phycoerythrin. c FACS-enriched populations were evaluated for expression of basal keratin 14 (K14; red), luminal keratin 8 (K8; green), and 4′,6-diamidino-2-phenylindole dihydrochloride (blue) by immunofluorescence (scale bar = 20 μm). Inset shows representative K14/K8 double-positive cells from the stem cell enriched population (scale bar = 10 μm). d Quantification of the cytokeratin profile for each MEC subgroup (n = total number of cells imaged). e Real-time quantitative PCR (RT-qPCR) quantification of relative differences in cytokeratin (K8, K14), hormone receptor (p rogesterone [Pgr], estrogen receptor [Esr1]), transcription factor (p63, Slug), and tyrosine-protein kinase (c-Kit) RNA expression levels between sorted MEC populations normalized to the GAPDH housekeeping gene. Expression levels were compared (by t test) with RNA from unsorted controls (dashed line). *p < 0.05, **p < 0.01, ***p < 0.0005, ****p < 0.0001; ^expression values for p63 and Slug in the luminal CD133+ population could not be determined, as amplification only rarely occurred below 40 cycles. Each data point is a mean ± SEM for triplicate qPCR reactions from three independent cDNA synthesis/preamplification reactions (n = 9). f Kaplan-Meier curves of mice receiving orthotopic transplants of distinct MEC subgroups. Mice were killed when tumors reached 2 cm in diameter (n = number of mice). g RT-qPCR quantification of relative PyMT mRNA expression levels in averaged luminal CD133+ cell, luminal CD133− cell, basal cell, and stem cell tumors normalized to the Rplp0 housekeeping gene. No significant differences between tumor groups were detected (by t test, n = number of tumors). h Average ratio of phosphorylated (pAKT) to AKT, phosphorylated extracellular signal-regulated kinase (pERK) to ERK, and phosphorylated SRC (pSRC) to Src protein expression in luminal CD133+, luminal CD133−, basal, and stem cell tumors normalized to the β-actin. No significant differences were detected between tumor groups (by analysis of variance for multiple comparisons)

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