Ethnicity-Dependent and -Independent Heterogeneity in Healthy Normal Breast Hierarchy Impacts Tumor Characterization

Abstract

Recent reports of widespread genetic variation affecting regulation of gene expression raise the possibility of significant inter-individual differences in stem-progenitor-mature cell hierarchy in adult organs. This has not been explored because of paucity of methods to quantitatively assess subpopulation of normal epithelial cells on individual basis. We report the remarkable inter-individual differences in differentiation capabilities as documented by phenotypic heterogeneity in stem-progenitor-mature cell hierarchy of the normal breast. Ethnicity and genetic predisposition are partly responsible for this heterogeneity, evidenced by the finding that CD44+/CD24- and PROCR+/EpCAM- multi-potent stem cells were elevated significantly in African American women compared with Caucasians. ALDEFLUOR+ luminal stem/progenitor cells were lower in BRCA1-mutation carriers compared with cells from healthy donors (p = 0.0014). Moreover, tumor and adjoining-normal breast cells of the same patients showed distinct CD49f+/EpCAM+ progenitor, CD271+/EpCAM- basal, and ALDEFLUOR+ cell profiles. These inter-individual differences in the rate of differentiation in the normal breast may contribute to a substantial proportion of transcriptome, epigenome, and signaling pathway alterations and consequently has the potential to spuriously magnify the extent of documented tumor-specific gene expression. Therefore, comparative analysis of phenotypically defined subpopulations of normal and tumor cells on an individual basis may be required to identify cancer-specific aberrations.

A system for detecting high impact-low frequency mutations in primary tumors and metastases

“…1A and Supplementary Table S1, respectively. Briefly, freshly obtained or cryopreserved (described previously 45 ) tumors were minced and divided into two parts. One part was used for DNA isolation and the other was placed in culture.…”

“…One part was used for DNA isolation and the other was placed in culture. Once epithelial cells were sufficiently expanded, typically ~one million cells within five passages, epithelial cells were sorted by flow cytometry using Jam-A/EpCAM antibodies 45 , an antibody combination demonstrated to separate epithelial cells from stromal and feeder layer fibroblasts 45 . Sorted cells were used to prepare DNA and RNA.…”

“…Primary tumor cells were propagated using epithelial reprogramming conditions as described previously 40 . Characterization of these cells by flow cytometry and antibodies used for flow cytometry has been described previously 45 . Antibodies used are CD44-APC (Cat#559942, BD Pharmingen), CD24-PE (555428, BD Pharmingen), CD49f-APC (FAB13501A, R&D Systems), EpCAM-PE (130-091-253, Miltenyi Biotech), EpCAM-APC (130-091-253, Miltenyi Biotech), Jam-A-PE (552556, BD Pharmingen) and CD271-APC (345108, Biolegand).…”

Individualized Breast Cancer Characterization through Single-Cell Analysis of Tumor and Adjacent Normal Cells

“…Unlike previously reported mammary epithelial growth conditions, which favors outgrowth of basal epithelial cells, reprogramming assay allows growth of stem, luminal progenitor and mature cells (12–14). Assays that allow growth of breast epithelial cells of different differentiation state are essential because most breast cancers including basal-like breast cancers are suggested to originate from luminal progenitors and then differentiate/dedifferentiate into specific subtypes (15–18).…”

Distinct Effects of Adipose-Derived Stem Cells and Adipocytes on Normal and Cancer Cell Hierarchy

“…Concentrations of cytokines/chemokines/growth factors used in mammosphere assays are indicated in figure legends. Antibodies against CD49f, CD44, CD24, and EpCAM used for flow cytometry have been described in our recent publication (23). Mammospheres were collected, washed, and exposed briefly to trypsin to obtain single-cell suspensions.…”