Defining the cellular precursors to human breast cancer - PubMed (original) (raw)

. 2012 Feb 21;109(8):2772-7.

doi: 10.1073/pnas.1017626108. Epub 2011 Sep 21.

Lisa M Arendt, Adam Skibinski, Tanya Logvinenko, Ina Klebba, Shumin Dong, Avi E Smith, Aleix Prat, Charles M Perou, Hannah Gilmore, Stuart Schnitt, Stephen P Naber, Jonathan A Garlick, Charlotte Kuperwasser

Affiliations

Defining the cellular precursors to human breast cancer

Patricia J Keller et al. Proc Natl Acad Sci U S A. 2012.

Abstract

Human breast cancers are broadly classified based on their gene-expression profiles into luminal- and basal-type tumors. These two major tumor subtypes express markers corresponding to the major differentiation states of epithelial cells in the breast: luminal (EpCAM(+)) and basal/myoepithelial (CD10(+)). However, there are also rare types of breast cancers, such as metaplastic carcinomas, where tumor cells exhibit features of alternate cell types that no longer resemble breast epithelium. Until now, it has been difficult to identify the cell type(s) in the human breast that gives rise to these various forms of breast cancer. Here we report that transformation of EpCAM(+) epithelial cells results in the formation of common forms of human breast cancer, including estrogen receptor-positive and estrogen receptor-negative tumors with luminal and basal-like characteristics, respectively, whereas transformation of CD10(+) cells results in the development of rare metaplastic tumors reminiscent of the claudin-low subtype. We also demonstrate the existence of CD10(+) breast cells with metaplastic traits that can give rise to skin and epidermal tissues. Furthermore, we show that the development of metaplastic breast cancer is attributable, in part, to the transformation of these metaplastic breast epithelial cells. These findings identify normal cellular precursors to human breast cancers and reveal the existence of a population of cells with epidermal progenitor activity within adult human breast tissues.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Enrichment of basal/ME and luminal populations from primary human breast tissue. (A) Single-cell suspensions of human breast epithelial cells analyzed by flow cytometry for expression of EpCAM, CD49f, and CD10 (n = 5 patient samples). (Left) Representative dot plot of EpCAM and CD49f stained cells. Five fractions of cells were gated and analyzed for CD10 content (%) as shown in the histograms on the Right. (B) Schematic of immunomagnetic sorting strategy. (C) Unsorted cells and fractions recovered after immunomagnetic sorting were analyzed by flow cytometry for expression of EpCAM and CD49f (n = 3). Representative dot plots from unsorted (patient 641), CD10+, and depleted cells are shown. (Lower Right) Overlay of unsorted EpCAMhi, sorted CD10+, and depleted fractions. Sorted EpCAM+CD10− cells did not stain with the fluorescently conjugated EpCAM antibody because of occupation of antigen sites from bead sorting; however, the luminal EpCAMhi clouds from unsorted cells (red) are clearly missing from the depleted fraction (blue). Enrichment of the EpCAMloCD49f+ population within the sorted CD10+ fraction is shown in green.

Fig. 2.

Fig. 2.

EpCAM+CD10− luminal and CD10+ basal/ME sorted populations contain cells with distinct functional characteristics. (A and B) Unsorted or sorted fractions of cells were plated in adherent conditions (A) or nonadherent conditions (B) and allowed to grow for 7–10 d. Graphed data represent average fold ± SE from independently sorted patient samples (n = 4–5). P values were calculated by Student's t test. (A Left) Representative image of suspension colonies (arrows) and adherent colonies. (Bar: 200 μm.) (Right) Representative images of crystal violet-stained colonies. (B) Immunofluorescence staining for CK8/18 (red) and CK14 (green) in spheres formed in nonadherent conditions. (Bar: 100 μm.) (C) Outgrowth from unsorted cells or sorted fractions plated on collagen I gels. (Upper) Representative images of the three structures formed. (Bar: 100 μm.) Graphed data represent average number ± SE from independently sorted patient samples (n = 3). P values were calculated by Student's t test. (D) Immunohistochemistry for luminal (CK19 and ERα) and basal/ME (CK14 and αSMA) differentiation markers in bilayered structures formed in vivo in the HIM model by sorted fractions. (Bar: 25 μm.)

Fig. 3.

Fig. 3.

Tumors formed from sorted fractions have distinct phenotypes. (A) Representative images from tumors derived from unsorted and sorted fractions transformed with the SV40/Ras oncogene combination stained for ERα, CK14 and CK8/18, and CK19. (Bars: 200 μm.) (B) Quantification of CK14 and CK19 expression (n = 8–9 tumors per group; immunofluorescence) and ERα expression (n = 4–7 tumors per group; immunohistochemistry) across tumors from 4onc and SV40/Ras models. Graphs represent average ± SE. P values were calculated by one-way ANOVA (CK19 and CK14) or Student's t test (ERα). (C) Heat map of hierarchical clustering of global gene-expression data collected from SV40/Ras tumors (n = 20) arising from unsorted (blue) or sorted CD10+ (green), EpCAM+ (red), EpCAM+/CD49f+ (yellow), or EpCAM+/CD49f− (magenta) cells. (D) Differentiation status of the various SV40/Ras tumors along a normal breast development axis using the Genomic Differentiation Predictor described in ref. . The P value was calculated by comparing gene-expression means across unsorted and sorted tumor groups. (E) Enrichment of the CD10 Signature derived from CD10+ tumors across a panel of breast tumor intrinsic subtypes from the UNC337 data set (Gene Expression Omnibus accession no. GSE18229). P value was calculated by comparing gene-expression means across all subtypes.

Fig. 4.

Fig. 4.

vCD10+ cells spontaneously lose mammary fate specification and gain ability to form skin tissues. (A) Representative graph of long-term culture of sorted CD10+ and EpCAM+ cells grown in MEGM showing the formation of vHMEC cells (vCD10+) preferentially from sorted CD10+ cells (n = 5 independently sorted patient samples). (B) Summary of mammary fate gene expression as analyzed by custom qRT-PCR array in P1 CD10+ compared with vCD10+ cells (n = 3 from independently sorted patient samples). (C) Outgrowth from P1 CD10+, vCD10+, MCF10A, and HaCAT cells plated on collagen I gels. (D) Representative images of sections from HSE assays stained for H&E, CK1/10, involucrin, E-cadherin, and laminin V (n = 3 from independently sorted or immortalized patient samples for each condition). (Bars: 100 μm.) (Inset) An example of ductal-like growths into the collagen gels from P1 CD10+ cells is shown. (E) Heat map of hierarchical clustering of global gene-expression data collected from a panel of HMECs: primary vHMECs (vHMEC-1, vHMEC-2, and vHMEC-3) isolated from three patients, immortalized HMECs (HME-CC and ME16C), and MCF10A, MCF12F, and MCF12A cells. Comparison with the CD10 Signature is shown on the right.

Fig. 5.

Fig. 5.

Transformed vHMEC cells give rise to metaplastic tumors. (A) Immortalized vHMECs from unsorted cells give rise to disorganized sebaceous-like growths (Upper Left) as well as tumors with medullary (Upper Right), spindle/giant-cell (Lower Left; arrows indicate giant cells), and squamous (Lower Right) histologies when transformed with SV40/Ras. (Bars: 100 μm.) (B) Enrichment of the CD10 Signature, derived from CD10+ tumors, across a panel of 11 special histological types of breast cancer from ref. 23.

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