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Telomerase-Immortalized Human Mammary Stem/ Progenitor Cells with Ability to Self-Renew and Differentiate

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Telomerase-Immortalized Human Mammary Stem/ Progenitor Cells with Ability to Self-Renew and Differentiate Telomerase-immortalized human mammary stem/ progenitor cells with ability to self-renew and differentiate Xiangshan Zhaoa, Gautam K. Malhotraa, Subodh M. Leleb, Manjiri S. Leleb, William W. Westb, James D. Eudya, Hamid Banda,b,c,d, and Vimla Banda,d,1 Departments of aGenetics, Cell Biology, and Anatomy, bMicrobiology and Pathology, and cBiochemistry and Molecular Biology, College of Medicine, and dEppley Institute for Cancer and Allied Diseases and University of Nebraska Medical Center–Eppley Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198 Communicated by Arthur B. Pardee, Dana–Farber Cancer Institute, Boston, MA, June 29, 2010 (received for review May 21, 2010) There is increasing evidence that breast and other cancers originate Recent progress in cancer biology has been substantially facili- from and are maintained by a small fraction of stem/progenitor cells tated by in vitro models. Strategies to culture and efficiently im- with self-renewal properties. Whether such cancer stem/progenitor mortalize human mammary epithelial cells (hMECs) led to cells originate from normal stem cells based on initiation of a de novo landmark findings that expression of a limited set of oncogenes stem cell program, by reprogramming of a more differentiated cell and/or loss of tumor suppressor genes together with imparting type by oncogenic insults, orboth remains unresolved. A majorhurdle immortal behavior was sufficient to induce malignant trans- in addressing these issues is lack of immortal human stem/progenitor formation of normal hMECs (4). Use of culture models has also cells that can be deliberately manipulated in vitro. We present suggested the possibility for reprogramming hMECs as demon- evidence that normal and human telomerase reverse transcriptase strated by the attainment of stem cell-like epithelial mesenchymal (hTERT)-immortalized human mammary epithelial cells (hMECs) iso- transition (EMT) upon manipulation of gene expression (5). Un- lated and maintained in Dana-Farber Cancer Institute 1 (DFCI-1) me- fortunately, the fundamentally important questions related to or- dium retain a fraction with progenitor cell properties. These cells igin of cancers have been difficult to address due to lack of coexpress basal (K5, K14, and vimentin), luminal (E-cadherin, K8, tractable cellular models of mammary stem/progenitor cells that K18, or K19), and stem/progenitor (CD49f, CD29, CD44, and p63) cell can be continuously cultured in vitro and would be suitable for markers. Clonal derivatives of progenitors coexpressing these detailed cellular and biochemical analyses and to interrogate their − markers fall into two distinct types—aK5+/K19 type and a K5+/ potential for oncogenic transformation. K19+ type. We show that both types of progenitor cells have self- Here, we have isolated and characterized human telomerase renewal and differentiation ability. Microarray analyses confirmed reverse transcriptase (hTERT)-immortalized clonal cell pop- the differential expression of components of stem/progenitor- ulations that are enriched for markers of normal mammary stem/ associated pathways, such as Notch, Wnt, Hedgehog, and LIF, in pro- progenitors cells. We show that the immortalized stem/progenitor genitor cells compared with differentiated cells. Given the emerging cells maintain their self-renewal ability but can also be induced to evidence that stem/progenitor cells serve as precursors for cancers, differentiate. The presence of related cell populations in the these cellular reagents represent a timely and invaluable resource to normal mammary gland together with gene expression analyses explore unresolved questions related to stem/progenitor origin of further support the idea that immortalized cell lines characterized breast cancer. here represent genuine human mammary stem/progenitor cells. These unique cellular tools should prove invaluable in future immortalization | in vitro stem cell model | mammary epithelial cells | self- studies to answer important questions related to breast cancer, renewal | stem cell such as the origin of breast cancer stem/progenitor cells and mechanisms of heterogeneity in breast cancers. reast cancer is a genetically and clinically heterogeneous dis- Bease (1). It is unclear whether different target cells contribute Results to this heterogeneity and which cell types are most susceptible to Primary and hTERT-Immortalized hMECs Isolated and Cultured in oncogenesis. Recent molecular profiling has identified five major DFCI-1 Medium Express Stem/Progenitor Cell Markers. We have subtypes of breast cancers: a basal epithelium-like group, an previously developed a medium designated as Dana-Farber Can- ErbB2-overexpressing group, a normal breast epithelium-like cer Institute 1 (DFCI-1) to isolate and propagate hMECs from group, and two luminal epithelial cell types with significantly dif- mammoplasty and mastectomy tissues (6, 7). We have shown that ferent outcomes for patients belonging to various groups (2). The hMECs cultured in DFCI-1 medium proliferate for 10–20 pop- correspondence of some breast cancer subtypes with cell types ulation doublings, followed by a selection (crisis) and emergence of present in the normal mammary gland (such as basal and luminal) a postselection cell population with a typical life span of about 60 strongly supports the idea that breast tumor subtypes may repre- sent malignancies of biologically distinct cell subtypes. Alterna- tively, different types of breast cancers may arise from a common Author contributions: X.Z., G.K.M., S.M.L., M.S.L., W.W.W., J.D.E., H.B., and V.B. designed precursor, based on distinct pathways of oncogene-driven repro- research; X.Z. and G.K.M. performed research; X.Z. and G.K.M. contributed new reagents/ analytic tools; X.Z., G.K.M., S.M.L., M.S.L., W.W.W., J.D.E., H.B., and V.B. analyzed data; gramming. Heterogeneity in cancers is ascribed to clonal evolution and X.Z., G.K.M., H.B., and V.B. wrote the paper. as a result of inherent genomic instability of tumor cells and tu- The authors declare no conflict of interest. mor–host interactions (3). The stem cell hypothesis suggests an Freely available online through the PNAS open access option. alternate explanation with tumor heterogeneity reflecting the Data deposition: The microarray data reported in this paper have been deposited in the relative fraction of cancer stem/progenitor cells and differences in Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. their abilities to produce progeny at various stages of differentia- GSE22580). tion. Although current literature supports each of these ideas, 1To whom correspondence should be addressed. E-mail: [email protected]. fi de nitive studies to favor one or the other model, or both hy- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. potheses, are lacking. 1073/pnas.1009030107/-/DCSupplemental. 14146–14151 | PNAS | August 10, 2010 | vol. 107 | no. 32 www.pnas.org/cgi/doi/10.1073/pnas.1009030107 Downloaded by guest on September 25, 2021 population doublings before senescence (6, 8–10). Our earlier 2-D on non-overlapping cells. Notably, every cell in primary and im- gel analyses showed that the hMECs coexpress both basal (K5 and mortal culture expressed K5, K14, vimentin, and E-cadherin, as K14) and luminal (K8 and K18) types of cytokeratins, suggestive of well as a known stem cell marker CD49f (21) (Fig. 1B), indic- their being progenitor/stem cells (11, 12). Given their limited life ating that stem/progenitor characteristics were maintained through span, we used retroviral infection to introduce a cellular gene immortalization. hTERT into preselection hMECs at passage 2 to isolate immor- talized hMECs and assessed the expression of hMEC lineage- Isolation of Distinct hMEC Stem/Progenitors Through Cloning. related and stem/progenitor cell markers using Western blotting. Whereas most luminal breast cancers are K19 positive (14), we These analyses demonstrated that two distinct primary hMECs found K19 expression on primary as well as TERT-immortalized and their TERT-immortalized derivatives propagated in DFCI-1 hMECs to be variable. K19 was essentially undetectable in 76N medium express both basal (K5, K14, p63, and vimentin) and lu- and 76N.TERT lines, whereas 70N cells showed easily detectable minal (K8, K18, and E-cadherin) cell markers; one cell strain K19 (Fig. 1A). Further immunostaining analysis showed hetero- (70N) also expressed K19, a known luminal cell marker (Fig.1A). geneous K19 expression (Fig. S1). To isolate K19-positive pro- In addition both hMECs and their TERT-immortalized deriva- genitor/stem cells, we seeded 70N.TERT cells at low density in tives expressed CD29, a known stem cell marker (13), but did not 3-D Matrigel and characterized the individual clonal isolates for − express detectable levels of differentiated luminal cell marker K19 expression. We obtained 86 K5+/K19 and 7 K5+/K19+ − MUC1 (14, 15) or differentiated myoepithelial cell marker clones (Fig. 2 A and B). Three independent clones of K5+/K19 α-smooth muscle actin (α-SMA) (14, 16–18) (Fig.1A). Consistent and K5+/K19+ cell types showed expression pattern of indicated with other findings, despite expression of several luminal cell proteins was identical to their parental 70N.TERT cells, except markers, the primary and TERT-immortalized hMECs lacked the K19 expression (Fig. 2 B and C). There was no apparent mor- − expression of estrogen receptor α (ER-α) (19, 20). phological difference between K5+/K19+ and K5+/K19 cells. Next, we carried out immumofluorescence staining to assess Stem/progenitor cells isolated from the same specimen differ- whether different lineage markers were expressed on same cells or ing in K19 expression have not been previously described. Im- 32 yrammaM T A T ts R R alborbif ET.N67 ET.N07 M- 1 D74T AD N07 N67 M-B K5 K8 K14 K18 K19 CD29 p63 MUC1 ER-α E-cadherin Vimentin α-SMA β-actin B 70N 76N 70N.TERT 76N.TERT MDA-MB-231 T47D K5 K14 Fig. 1. Analyses of stem/ progenitor cell markers in CD49f parental and hTERT immor- talized human mammary epithelial cells isolated and cultured in DFCI-1 medium.
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