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 represent 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 immortalized 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

K14

Fig. 1. Analyses of stem/ progenitor cell markers in CD49f parental and hTERT immortalized human mammary epithelial cells isolated and cultured in DFCI-1 medium. μ (A)Atotalof50 gofcell CELL BIOLOGY ER-α lysates were Western blot- ted using indicated antibodies. Breast cancer cell lines MDA-MB-231 and T47D E-cadherin are used as controls. β-Actin was used as loading control. (B) Immunoﬂuorenscence staining of normal and im- Vimentin mortal hMECs using indicated markers (red).

Zhao et al. PNAS | August 10, 2010 | vol. 107 | no. 32 | 14147 Downloaded by guest on September 25, 2021 munohistochemical staining of human breast tissue clearly dem- (Figs. 1B and 3B), the spindle-shaped cells forming the peripheral onstrated the existence of discrete K5+ cells that are either K19+ halo were K5 negative (Fig. 3C) and acquired several well-known − or K19 in human breast (Fig. 2D), consistent with the idea that myoepithelial cell markers (α-SMA, CD10, and Thy-1) that were both cell types of hMEC clones that we have isolated likely rep- absent on the parental cells as well as in the central compact part of resent physiologically relevant hMEC populations. colonies grown in MEGM (Figs. 1A,2C,and3C). Similar staining results were obtained with K5+/K19+-type hMECs plated in − K5+/K19 and K5+/K19+ Cell Types Indefinitely Maintain Self-Renewal MEGM medium (Figs. S2 and S3). The antibodies used to es- and Differentiation Abilities. Defining properties of stem/pro- tablish the identity of peripheral cells as myoepithelial were indeed genitor cells deduced from studies of embryonic as well as adult specific as demonstrated by their specific staining of myoepithelial stem/progenitor cells include the ability to self-renew as well as to components in healthy breast tissue (Fig. S4). Notably, differential produce cells at further stages of differentiation. Using hMECs trypsinization allowed us to isolate the central compact colonies derived from tissues without initial in vitro culture, Dontu et al. separately from the cells forming the peripheral halo followed by showed multicellular spheres starting from single cells when cul- replating to assess their ability to continue to proliferate: the tured in low-attachment plates and that a fraction of these cells central compact part of colonies could be repeatedly plated to retained the ability to form secondary and tertiary mammo- regenerate similar colonies as well as to generate differentiated spheres thereby providing an assay of self-renewal (16). We have myoepithelial halos in MEGM medium. In contrast, the differ- made repeated attempts to test the self-renewal and differentia- entiated myoepithelial cells in the peripheral halo quickly lost the tion of our putative stem/progenitor cells in this assay but we were ability to attach and failed to proliferate. We have continuously unable to generate mammospheres from a single cell. Although maintained these cell cultures for more than 1 y and observed that the precise reasons for the inability of our cells to grow as mam- the tight cells continued to proliferate and exhibit self-renewal and mospheres remain unknown, it is possible that mammosphere differentiation abilities. The retention of these abilities strongly − formation reflects a property of stem/progenitor cells from freshly support the conclusion that the immortalized K5+/K19 and K5+/ isolated mammary tissue that is lost when stem/progenitor cells K19+ cell types have stem/progenitor properties. are cultured in vitro. Furthermore, a subset of cells within the center of the colonies Although our cells were unable to form mammospheres from formed in MEGM medium became mucin-1 (MUC1) positive a single cell, we are able to observe their ability to self-renew and consistent with their differentiation along the luminal cell lineage − − differentiate. When K5+/K19 and K5+/K19+ cell types were (Fig. 4A). Additionally, when we cultured the K5+/K19 and K5+/ cultured in mammary epithelial cell growth medium (MEGM) on K19+ cell types in our defined medium DFCI-2 (6, 7) we did not 2-D plastic substratum, these cells proliferate as tightly packed observe myoepithelial differentiation but consistently observed epithelial colonies (Fig.3A) and subsequently a proportion of cells appearance of MUC1-positive and vimentin-negative cells (Fig. near the periphery adopted a spindle-shaped morphology sur- 4B); these results suggest that DFCI-2 medium may favor luminal rounding the tight epithelial cell colony in the center (Fig. 3A). differentiation or selectively inhibit myoepithelial differentiation. Notably, while the cells in the center expressed the basal, luminal, Notably, FACS-sorted MUC1-positive cells from both cell types and stem cell markers like the cells cultured in DFCI-1 medium failed to attach or proliferate and therefore cannot be used for

DAPI K19 Merge A K5+/K19- K5+/K19+ B

K5+/K19-

K5+/K19+ tsalborbif C yrammaM

K5+/K19- K5+/K19+ DAPI K5 K19 Merge K5 D

K14

K18

K19 CD29 p63

α -SMA

Ecadherin Vimentin

Pan-cytokeratin

β -actin

− − Fig. 2. Isolation of K5+/K19 and K5+/K19+ cell types from 70N.TERT cell line. (A) Morphology of K5+/K19 and K5+/K19+ cell type cultured in 2-D culture in − − − DFCI-1 medium is shown. (B) Immunoﬂuorenscence staining of K5+/K19 andK5+/ K19 is shown. (C) Western blotting of K5+/K19 and K5+/K19+ cell types using indicated antibodies (D) Immunoﬂuorescence staining of human mammary tissue specimen with anti-K5 (green), and anti-K19 (red) double staining. K5+/K19− (shown by arrow) and K5+/K19+ (shown by arrowhead).

14148 | www.pnas.org/cgi/doi/10.1073/pnas.1009030107 Zhao et al. Downloaded by guest on September 25, 2021 A K5+/K19- K5+/K19+ A DAPI K5 MUC1 Merge

K5+/K19- Before differentiation

K5+/K19+

After DAPI K5 MUC1 Merge differentiation B K5+/K19-

B K5+/K19+ CK8 (red) K5(green)

DAPI VIM MUC1 Merge E-cadherin (red) K5 (green) K5+/K19-

K19(red) K5(green) K5+/K19+

CD49f (red) K5(green) − Fig. 4. Analyses of luminal cell differentiation. K5+/K19 and K5+/K19+ cell types cultured in MEGM and DFCI-2 media were stained with luminal cell marker C MUC1. Both cell types cultured in MEGM (A)orDFCI-1(B) were stained with anti- α-SMA (red) MUC1 antibody followed by costaining with rabbit-anti human K5 (green) and K5 (green) mouse anti-human MUC1 (red) and anti-vimentin (green). VIM, vimentin. DAPI (blue)

with stem/progenitor cell populations whose expression was also Thy-1 (red) K5 (green) modulated upon myoepithelial cell differentiation including DAPI (blue) components of Notch, Wnt, hedgehog pathways, and other genes such as LIF and KLF5 (Table 1). Notably, aldehyde dehydrogenase 1A3 enzyme (ALDH1A3), a marker used to isolate nor- CD10 (red) ≈ K5 (green) mal and tumor mammary stem cells (23, 24), was 400- to 500- + − + + DAPI (blue) fold higher in K5 /K19 and K5 /K19 cell types, relative to differentiated myoepithelial cells (Table 1). Indeed, we confirmed − Fig. 3. In vitro self-renewal and myoepithelial cell differentiation of K5+/ changes in ALDH mRNA levels between K5+/K19 cell type and − K19 and K5+/K19+ cell types in MEGM medium. (A) Morphology of cells their differentiated myoepithelial progeny by real-time PCR and fi before and after start of differentiation; loose cells with broblastic mor- ALDEFLUOR FACS analyses (Fig. S5). These results further phology represent myoepithelial cells and center tight colony represent fi + − + + undifferentiated cells. (B) Immunostaining of cells with rabbit-anti human substantiate our ndings that K5 /K19 and K5 /K19 cell types K5 (green) and mouse anti-human CD49f, K8, E-cadherin (red). (C) Immu- represent stem/progenitor cells. + − + + nofluorescence staining of myoepithelial cells. The cells were costained with Although K5 /K19 and K5 /K19 cell types were isolated on rabbit-anti human K5 (green) and three myoepithelial cell markers: mouse the basis of differences in only K19 expression, microarray analyses anti-human α-SMA, Thy-1, and CD10 (red). demonstrated a substantial number of gene expression differences between these two cell types (Table S1) consistent with the possibility that these cells may reflect distinct progenitor stages. further analyses. Taken together these results support the conclu- + − + + sion that immortalized K5 /K19 and K5 /K19 cell types retain Discussion mammary stem or progenitor properties with capability of self-re- It is now generally accepted that breast cancers harbor cells with newal and differentiation along luminal and myoepithelial lineages. stem cell markers suggesting their role in the initiation and/or maintenance of tumors (25–28). It is however unclear whether such Changes in Well-Established Stem Cell Pathways Observed upon CELL BIOLOGY Differentiation of K5+/K19− and K5+/K19+Cells. Maintenance of cancer initiating or cancer stem cells originate from normal stem stem/progenitor properties depends on a well-regulated tran- cells or represent reprogramming of tumor cells to attain stem cell- scriptional program that undergoes defined changes upon dif- like phenotypes. In fact, deliberate oncogenic transformation of ferentiation along specific pathways (15, 22). Next, we performed a human mammary stem cell has not been possible. A critical Affymetrix-based genomewide mRNA expression profiling of barrier to address these basic issues is a lack of well-defined hMEC − both K5+/K19 and K5+/K19+ cell types, and differentiated stem/progenitor cells that can be subjected to oncogenesis studies. myoepithelial cells (NCBI Gene Expression Omnibus accession Here, we report the characterization of hTERT-immortalized − no. GSE22580). These analyses demonstrated that K5+/K19 and hMECs with stem/progenitor properties that provide tools for K5+/K19+ cells express a number of genes previously associated such studies.

Zhao et al. PNAS | August 10, 2010 | vol. 107 | no. 32 | 14149 Downloaded by guest on September 25, 2021 Table 1. Data from microarray analyses reproducibly form a sheath of elongated spindle-shaped cells − K5+K19 / K5+K19+/ around the central epithelial colony attain myoepithelial markers. Gene symbol Gene title myoepi myoepithelial Furthermore, a small number of cells within the central mass of colonies in differentiation cultures become MUC1 positive/ Notch signaling pathway vimentin negative, consistent with differentiation along the lu- − JAG1 Jagged 1 (Alagille syndrome) 13 14 minal pathway. Both K5+/K19 and K5+/K19+ cell types do not JAG2 Jagged 2 34 28 represent a peculiarity of in vitro cell culture but such cells exist in HES2 Hairy and enhancer of split 2 80 90 vivo in normal human mammary gland. The immortalized clonal (Drosophila) hMECs we have obtained exhibit markers of stem cells similar to HES4 Hairy and enhancer of split 4 154 83 those defined by others (13, 19, 21, 23, 29). It is remarkable, (Drosophila) however, that immortal hMECs maintain their stem/progenitor Wnt signaling pathway characteristics in 2-D culture apparently without need for any WNT5A Wingless-type MMTV integration −11 −20 additional cell type. site family, member 5A It is notable that we have been able to identify two distinct types WNT5B Wingless-type MMTV integration −5 −5 of hMEC stem/progenitors, initially based on K19 expression but site family, member 5B with additional differences as suggested by gene expression WNT7A Wingless-type MMTV integration 41 37 analyses (Table S1), and these cells are present in human mam- site family, member 7A mary tissues (Fig. 2D). In a previous study, cell sorting based on WNT10A Wingless-type MMTV integration 10 12 surface antigen markers (MUC1 and ESA) was shown to identify − site family, member 10A MUC /ESA+/K19+ cells that were able to self-renew and dif- − − Sonic hedgehog signaling pathway ferentiate into MUC+/ESA+/K19 cells, suggesting that MUC / GLI3 GLI-Kruppel family member GLI3 −2 −2 ESA+/K19+ cells represent mammary stem cells (14). Although − DISP1 Dispatched homolog 1 −4 −2 we were able to identify both K19+ and K19 stem/progenitor − (Drosophila) cells, we were unable to show that K5+/K19 cells were able to GLIS1 GLIS family zinc finger 1 −23 −15 differentiate into K5+/K19+ cells or vice versa. These results GLIS3 GLIS family zinc finger 3 −5 −2 suggest that both might represent relatively early progenitors. Genes involved in embryonic stem cell self-renewal Whether these are related in a hierarchical commitment scheme LIFR Leukemia inhibitory factor −17 −13 will require further investigations. − receptor alpha It is notable that progeny of immortalized K5+/K19 and K5+/ LIF Leukemia inhibitory factor 105 288 K19+ cell types differentiated along a luminal pathway could not (cholinergic differentiation factor) be propagated further, suggesting near-terminal differentiation. KLF5 Kruppel-like factor 5 (intestinal) 7 7 In contrast, some myoepithelial cells were able to continuously ALDH gene proliferate when transferred from MEGM to DFCI-1 medium; ALDH1A3 Aldehyde dehydrogenase 1 462 539 however, others were unable to proliferate, suggesting that both family, member A3 cell types are able to differentiate into myoepithelial progenitor as well as terminally differentiated myoepithelial cells. These results Fold changes in the expression levels of selected genes from well-established stem cell pathways upon myoepithelial differentiation of K5+/K19− and K5+/ suggest that in addition to differentiation programs assessed using K19+ cells. available markers, these cells are able to undergo irreversible cell cycle arrest associated with differentiation despite ectopic hTERT expression. Thus, the cells isolated here may be useful to Definitive demonstration of mammary stem cells in the mouse study events associated with terminal differentiation of hMECs predicts that similar cells exist in human mammary gland. This into myoepithelial and luminal cells. idea is supported by immunohistochemical studies of human A number of key gene regulatory pathways including compo- mammary gland as well as demonstration of stem cell-like traits, nents of the Wnt, Notch, and hedgehog pathways, and other stem including stem cell marker expression, limited in vitro self- cell control genes are involved in the maintenance of embryonic renewal in mammospheres, induced lineage differentiation, and stem cell self-renewal and are also modulated upon adult stem cell – an ability to form mammary tissue when xeno-transplanted in self-renewal and differentiation (15, 30 37). Our gene expression fi mouse mammary fat pad with freshly isolated hMECs. However, pro ling demonstrates that these pathways are expressed in stem/ normal hMECs with such stem cell characteristics have not been progenitor hMECs and notably are prominently modulated upon propagated in long-term culture. Thus, our indefinite propaga- differentiation along the myoepithelial lineage (Table 1). Due to a relatively low frequency of cells differentiating along the luminal tion of two different types of immortal hMECs with stem/pro- pathway and an inability of these differentiated cells to be prop- genitor characteristics represents a significant step forward in agated has thus far prevented us from comparing specific alter- breast cancer cell biology. ations in gene expression associated with luminal pathway of The concurrent presence of markers associated with basal, lu- differentiation. Further studies to define conditions that yield minal, and stem/progenitor cell compartments in both primary high proportion of luminal lineage differentiated cells would hMEC cultures and in hTERT-immortalized hMECs suggested therefore be instructive. The propagation of distinct immortal that our culture conditions promoted the maintenance of stem/ clonal hMEC lines that appear to represent relatively early stem/ progenitor cell properties potentially through their self-renewal. progenitor cells, together with our ability to generate myoepi- Analyses of clonally derived cell lines not only established that this thelial and luminal progeny upon differentiation of both cell types fi was the case but also led to the identi cation of distinct stem/ suggest that our culture conditions allow stable maintenance of progenitor cell types. These cells demonstrate an ability to con- hMECs at multiple stem/progenitor stages, something that will be tinuously proliferate and maintain composite marker profiles of of significance in deliberate efforts to establish immortal mam- basal, luminal, and stem cells. When these cells are cultured in mary progenitors “frozen” at different stages along the differen- media known to promote lineage differentiation of freshly iso- tiation hierarchy. lated hMECs, a subset of epithelium-like cells perpetuate the The present findings provide proof of principle that it is pos- marker profiles of parental clones and can be propagated, sug- sible to isolate hMECs that represent stem/progenitor stages as gesting their capability to self-renewal. Notably, the cells that immortal lines that can be perpetuated and induced into differ-

14150 | www.pnas.org/cgi/doi/10.1073/pnas.1009030107 Zhao et al. Downloaded by guest on September 25, 2021 entiation. Use of hTERT a cellular gene rather than viral onco- (Sigma). DFCI-2 and mammary epithelial growth medium (MEGM) have been genes provides a more physiological model for understanding described previously (6, 7). stem cell biology. Future efforts using this approach may help + − + + identify additional stem/progenitor stages in hMECs. More im- Isolation of K5 /K19 and K5 /K19 Cell Type Clones. 70N.TERT cells were portantly, these immortal stem/progenitors will be suitable for seeded in at low density (300 cells/100-mm dish) in a 3-D Matrigel cultures (BD Bioscience) (44, 45) or in regular 2-D culture in 96-well plates (1 cell/well) in DFCI- deliberate oncogenesis studies to test whether indeed their − 1 medium. A total of 93 clones were isolated and characterized as K5+/K19 (86 transformation can lead to tumors and if such transformed cells clones) and K5+/K19+ (7 clones); those used in further experiments were will resemble tumor stem cells. Although stem/progenitor analy- subcloned three times to ensure that each clone was derived from a single cell. ses were not carried out, earlier studies by Weinberg’s group have shown that primary hMECs can be rendered fully tumorigenic Self-Renewal and Differentiation. Cells were grown in serum-free MEGM upon introduction of a deﬁned number of oncogenes and loss of medium (Lonza) supplemented with B27 (10 mL/500 mL medium, Invitrogen), tumor suppressors (4). We and others have previously shown that 20 ng/mL EGF (Invitrogen), 20 ng/mL bFGF (BD Biosciences), and 4 μg/mL hMECs are highly susceptible to immortalization upon ectopic heparin (Sigma) without bovine pituitary extract (16). Both types of cells were expression of cellular genes (10, 38–41), viral genes (7, 38), car- also cultured in DFCI-2 medium (6, 7) to obtain luminal differentiation. cinogens (42), and radiation (43). We also noted that primary The details of the materials and methods for antibodies used in the study, hMECs at early or late passages exhibited differential suscepti- retroviral infection and immortalization of hMECs, Western blotting, im- bility to immortalization by human papilloma virus (HPV) E6 and munoﬂuorescence of cells and tissues, Affymetrix microarray analyses, real- E7 or their combinations (7, 8, 12). It is reasonable to suggest that time PCR, and FACS analysis of ALDH expression can be found in SI Materials the propensity of hMECs to be transformed by ectopic oncogene and Methods. expression and/or down-regulation of tumor suppressors may in- ACKNOWLEDGMENTS. We thank Dr. Ruth Sager (deceased) for stimulating deed be related to the susceptibility of stem/progenitor cells to discussions that helped formulate the ideas investigated in this paper. This oncogenesis. The cell lines established here should allow direct work was supported by the National Institutes of Health Grants CA96844 examination of these critical issues. and CA94143 and Department of Defense Grant W81XWH-07-1-0351 (to V.B.) and NIH grants CA87986, CA105489, CA99163, and CA116552 (to H.B.). The Materials and Methods University of Nebraska Medical Center Microarray Core Facility receives partial support from National Institutes of Health Grant P20 RR016469 from the IDeA Cell Strains and Cell Culture. hMEC strains 76N and 70N were grown in DFCI-1 Networks of Biomedical Research Excellence Program of the National Center medium, as described earlier (6–8). Human telomerase (hTERT)-immortalized for Research Resources and a University of Nebraska Medical Center-Eppley cells were grown in DFCI-1 medium supplemented with 100 μg/mL G418 Cancer Center core grant from the National Cancer Institute.

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