Normal Breast-Derived Epithelial Cells with Luminal and Intrinsic Subtype-Enriched Gene Expression Document Interindividual Differences in Their Differentiation Cascade

Abstract

Cell-type origin is one of the factors that determine molecular features of tumors, but resources to validate this concept are scarce because of technical difficulties in propagating major cell types of adult organs. Previous attempts to generate such resources to study breast cancer have yielded predominantly basal-type cell lines. We have created a panel of immortalized cell lines from core breast biopsies of ancestry-mapped healthy women that form ductal structures similar to normal breast in 3D cultures and expressed markers of major cell types, including the luminal-differentiated cell-enriched ERα-FOXA1-GATA3 transcription factor network. We have also created cell lines from PROCR (CD201)/EpCAM cells that are likely the "normal" counterpart of the claudin-low subtype of breast cancers. RNA-seq and PAM50-intrinsic subtype clustering identified these cell lines as the "normal" counterparts of luminal A, basal, and normal-like subtypes and validated via immunostaining with basal-enriched KRT14 and luminal-enriched KRT19. We further characterized these cell lines by flow cytometry for distribution patterns of stem/basal, luminal-progenitor, mature/differentiated, multipotent PROCR cells, and organogenesis-enriched epithelial/mesenchymal hybrid cells using CD44/CD24, CD49f/EpCAM, CD271/EpCAM, CD201/EpCAM, and ALDEFLUOR assays and E-cadherin/vimentin double staining. These cell lines showed interindividual heterogeneity in stemness/differentiation capabilities and baseline activity of signaling molecules such as NF-κB, AKT2, pERK, and BRD4. These resources can be used to test the emerging concept that genetic variations in regulatory regions contribute to widespread differences in gene expression in "normal" conditions among the general population and can delineate the impact of cell-type origin on tumor progression. In addition to providing a valuable resource for the breast cancer research community to investigate cell-type origin of different subtypes of breast cancer, this study highlights interindividual differences in normal breast, emphasizing the need to use "normal" cells from multiple sources as controls to decipher the effects of cancer-specific genomic aberrations. .

Genetic Ancestry–dependent Differences in Breast Cancer–induced Field Defects in the Tumor-adjacent Normal Breast

“…These observations along with our unique observations of genetic ancestry-dependent differences in ZEB1 þ cells in the normal breast, elevated number of ZEB1 þ cells in NATs compared with healthy breast tissues of women of EA ancestry, and its localization outside the ductal structures raise several questions about the function of ZEB1 þ cells in the normal and tumor adjacent normal breast. We have shown previously that cytokeratin-positive, PROCR þ /EpCAM À cells of the normal breast, which are enriched in the normal breast of AA women compared with EA women, express 50-fold higher ZEB1 compared with cytokeratinpositive, PROCR À /EpCAM þ cells of the breast (12,43). Thus, we suspect that ZEB1 þ cells in the normal breast correspond to PROCR þ /EpCAM À cells and that cancer-induced field effect leads to expansion/proliferation of such cells in the breast of EA women.…”

Dual TGFβ/BMP Pathway Inhibition Enables Expansion and Characterization of Multiple Epithelial Cell Types of the Normal and Cancerous Breast

“…Floating collagen scaffold was prepared on the basis of a protocol from Linnermann and colleagues, with some modifications as detailed in Kumar and colleagues (2,11). Matrigel basement membrane (354234, Corning) was slowly thawed on ice.…”

“…Several protocols have been developed that support the propagation of breast epithelial cells, which, in large part, are biased toward outgrowth of basal cells (summarized in ref. 2). Several of these culture protocols utilize irradiated mouse embryonic fibroblast or human foreskin fibroblasts as feeder cells to maintain the pluripotent state of stem cells (3,4).…”

High‐Throughput Automated Single‐Cell Imaging Analysis Reveals Dynamics of Glioblastoma Stem Cell Population During State Transition

“…Our results revealed a minor sub‐population of GSCs that is stable in a GSC propagating medium. Previous studies have suggested that multiple CSC subtypes may co‐exist in the same tumor . Observed increase in this minor SOX2‐low sub‐population followed BMP4‐induced state transition suggests that these cells may be further on a differentiation spectrum.…”