Mapping the cellular and molecular heterogeneity of normal and malignant breast tissues and cultured cell lines

Abstract

Introduction: Normal and neoplastic breast tissues are comprised of heterogeneous populations of epithelial cells involving various degrees of maturation and differentiation. While cultured cell lines have been derived from both normal and malignant tissues, it remains unclear whether they retain a similar cellular heterogeneity as to that found within breast tissues.

Methods: We used 12 reduction mammoplasty tissues, 15 primary breast cancer tissues, and 20 human breast epithelial cell lines (16 cancer lines, 4 normal lines) to perform flow cytometry for CD44, CD24, epithelial cell adhesion molecule (EpCAM), and CD49f expression as well as immunohistochemistry, and in vivo tumor xenograft formation studies to extensively analyze the molecular and cellular characteristics of breast epithelial cell lineages.

Results: Human breast tissues contain four distinguishable epithelial differentiation states (two luminal phenotypes and two basal phenotypes) that differ on the basis of CD24, EpCAM and CD49f expression. Primary human breast cancer tissues also contain these four cellular states, but in altered proportions compared to normal tissues. In contrast, cultured cancer cell lines are enriched for rare basal and mesenchymal phenotypes, which are normally present in small numbers within human tissues. Similarly, cultured normal human mammary epithelial cell lines were enriched for rare basal and mesenchymal phenotypes that represent a minor fraction of cells within reduction mammoplasty tissues. Although normal human mammary epithelial cell lines exhibited features of bi-potent progenitor cells they were unable to differentiate into mature luminal breast epithelial cells.

Conclusions: As a group breast cancer cell lines represent the heterogeneity of human breast tumors, but individually they exhibit increased lineage-restricted profiles that fall short of truly representing the intratumoral heterogeneity of individual breast tumors. Additionally, normal human mammary epithelial cell lines fail to retain much of the cellular diversity found in human breast tissues and are enriched for differentiation states that are a minority in breast tissues, although they do exhibit features of bi-potent basal progenitor cells. These findings suggest that collections of cell lines representing multiple cell types can be used to model the cellular heterogeneity of tissues.

Figure 1

CD44 or CD24 expression alone does not classify breast cancer cell lines into tumor subytpes. Breast cancer cell lines are grouped based on tumor subtype classification defined by [7] as Luminal, Basal A (Bas A), Basal B (Bas B), or those that have not been previously classified (NC). The percentage of cells in breast cancer cell lines expressing either CD44 (a) or CD24 (b) is variable and does not correlate with tumor classification. (c) The percentage of cells in cancer cell lines expressing the CD44⁺/CD24^- phenotype correlates with spindle/mesenchymal features, not tumor subtype. Surface marker expression was quantified by flow cytometry (mean ± S.E.) as described in Materials and methods. Phase contrast, bright-field photomicrographs of representative cell lines exhibiting epithelial versus spindle morphology.

Figure 2

Normal human breast tissue contains four distinct epithelial subtypes. (a) Freshly dissociated breast epithelial cells from reduction mammoplasty can be divided into four epithelial differentiation states. Primary breast epithelial cells (n = 12) were isolated, lineage depleted, stained with EpCAM, CD24, and CD49f, and quantified as described in Materials and methods. Representative dot plots of EpCAM vs. CD49f staining (left) and CD24 vs. CD49f staining in EpCAM/CD49f populations (middle) are shown. Quantification of CD24 staining in Luminal 1/2, Basal and Mesenchymal populations from 12 patient samples (right, mean ± S.E.). Quantification of immunofluorescence from a representative patient sample is shown (% of total DAPI stained cells, minimum 50 cells analyzed). Luminal 1 and Luminal 2 cells from reduction mammoplasty tissue are predominantly CK 8/18 positive. Basal and Mesenchymal cells expressed CK14, VIM, and αSMA. (b) Freshly dissociated epithelial cells from reduction mammoplasty tissue were stained for EpCAM and CD49f expression, sorted by FACS and cytospun on onto slides for characterization of lineage markers by immunofluorescence. Cytokeratin (CK) 18, 14, smooth muscle actin (αSMA) and vimentin (VIM) immunofluorescence staining and quantification of sorted populations indicates luminal and basal/myoepithelial cell enrichment.

Figure 3

Human breast cancers are heterogeneous tissues comprised of all four cell phenotypes. Primary breast tumors exhibited a similar cellular heterogeneity to normal reduction mammoplasty tissues when examined by flow cytometry using the markers EpCAM, CD24, and CD49f as described in Materials and methods. (a) Representative flow cytometry dot plots of EpCAM and CD49f expression in reduction mammoplasty tissues (RM) and primary human breast tumors of different subtypes. Quantification of flow cytometry tumors (n = 8) classified clinically by expression of ER, PR and Her2 expression. (b) Primary breast cancers demonstrated heterogeneous expression of markers used to characterize cancer cell line xenografts by immunohistochemistry as in Figure 2. Bar = 100 μm.

Figure 4

Surface markers EpCAM, CD24, and CD49f classify breast cancer cell lines into distinct differentiation states. (a) Cell surface markers EpCAM, CD24, and CD49f distinguish four classes of cell lines that map to differentiation states found in normal precursor cells. Surface marker expression was quantified by flow cytometry (mean ± S.D., n = 3 to 4 biological replicates per cell line) as described in Materials and Methods. (b) Luminal 1, Luminal 2, Basal, and Mesenchymal cell lines identified by EpCAM, CD24, and CD49f expression were classified on the basis of CK14, CK8/18, ERα, EpCAM, and vimentin expression. Representative immunofluorescence images are shown from MCF7 (Luminal 1), BT20 (Luminal 2), HCC1806 (Basal), and MDA.MB.231 (Mesenchymal). Nuclei were counterstained with DAPI (blue). ER expression of Luminal II cells is heterogeneous across cell lines. Cell lines T47 D, HCC1500, and MDA.MB.361 all express ER (MDA.MB.361 is shown in inset), while BT20, SUM225 and HCC1419 are ER-negative. Original magnification: 200×. Bar = 100 μm.

Figure 5

Cell line marker profiles correlate with established biomarkers in tumor xenografts. (a) Tumor growth curves of cell line-derived xenografts over time. Tumorigenicity of breast cancer cell lines was not correlated with their individual cellular profiles when injected into mammary glands of NOD/SCID mice. (b) Cell line marker profile correlated with resulting tumor histology and expression of biomarkers. Representative H&E stained sections of tumor xenografts from Luminal 1 and 2 cell lines (MCF7, SUM225) showing intraductal and comedo-like DCIS patterns of growth respectively, as well as, Basal cell line (HCC1806) showing solid carcinoma growth with squamous differentiation, and Mesenchymal cell lines (SUM159) showing spindle-cell metaplastic-like growth. Original magnification: 100×. Immunohistochemistry was used to stain tumor xenografts for expression of ERα, Her2, p53, CK14, CK18 and vimentin. Representative images shown from tumors of Luminal 1 (MCF7), Luminal 2 (MDA.MB.361), Basal (SUM 149), and Mesenchymal (MDA.MB.231) cell lines. Bar = 100 μm.

Figure 6

Human breast cell lines are enriched for basal and mesenchymal phenotypes. (a) Normal breast cell lines demonstrate loss of EpCAM⁺/CD49f- and EpCAM⁺/CD24⁺/CD49f⁺ populations compared to primary breast epithelial cells isolated from reduction mammoplasty. Reduction mammoplasty tissues (RM) and normal breast cells lines as well as matched RM with HME cell lines were stained with EpCAM, CD24 and CD49f and quantified by flow cytometry as described in Materials and methods. (b) Quantification of mammospheres formed in non-adherent culture by HME II, MCF10A and MCF10F cell lines (left) and representative images (right). Bar = 100 μm. (c, d) Addition of 5% serum to the culture conditions of HME cells increases differentiation to a more luminal state as assessed by flow cytometry for EpCAM, CD49f and CD24. Representative dot plots are shown in C and quantification in D. (e) Changes in Basal/Luminal differentiation were assessed by immunofluorescence staining for CK8/18, EpCAM, CK14 and vimentin following treatment with serum. Cells were counterstained with DAPI (blue). HME I cells are shown, bar = 100 μm.