CSCs and NSCCs Generated During the Transformation Process Have Distinct MicroRNA Profiles.

Although CSCs and NSCCs are formed from nontransformed cells concomitantly upon the induction of transformation, profiling of 365 microRNAs indicates that two cell populations have distinct expression patterns (Fig. 2A). Nineteen microRNAs are expressed at lower levels in CSCs than NSCCs, and three microRNAs are expressed at higher levels. As expected, the same set of microRNAs is also differentially expressed in mammospheres compared with the mixed transformed population (Fig. 2A). The miR-200 and let-7 families together with miR-145 and miR-146 are also differentially expressed in CSCs and NSCCs isolated from genetically distinct breast cancer cell lines and human breast tumors described here (Fig. 2B and Figs. S1 and S2) and elsewhere (20).

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Fig. 2.

MicroRNAs differentially regulated in CSCs vs. NSCCs. (A) Relative expression levels (fold-effect) of microRNAs down-regulated (- numbers) or up-regulated (+ numbers) in isolated CSCs vs. NSCCs (black bars) or mammospheres vs. nonsorted transformed cells (gray bars). (B) Relative expression levels of the indicated microRNAs in NSCCs (defined as 1.0) and CSCs from MCF7, MDA-MB-231, and five human beast tumors. (C) Heat map representations of up-regulated (red) and down-regulated (green) microRNAs in CSCs (compared with NSCCs) or at different time points during ER-Src transformation (24), with the three different classes of microRNAs indicated.

Importantly, the set of 22 microRNAs differentially expressed in CSCs vs. NSCCs is quite different from the set of 29 microRNAs independently shown (24) to be differentially affected in the nontransformed vs. transformed population (Fig. 2C). Indeed, most microRNAs differentially expressed in CSCs vs. NSCCs have similar expression levels during the process of cellular transformation (i.e., at various time points after TAM addition). Conversely, most microRNAs differentially expressed during the process of cellular transformation are expressed at comparable levels in sorted CSCs and NSCCs. However, the let-7 family and miR-335 are down-regulated during transformation and further down-regulated in CSCs, whereas miR-210 is up-regulated in both situations. Thus, in some respects (let-7, miR-335, and miR-210), CSCs represent a more extreme version of transformed cells and, indeed, CSCs have a more robust inflammatory feedback loop (high NF-κB, high Lin28, low let-7, and high IL6) than NSCCs (21). More generally, however, CSCs and NSCCs represent two distinct cell types within a transformed population generated through a common induction step.

CSC Formation Depends on, but Is Not Required for, Transformation.

Cellular transformation and CSC formation are both induced upon addition of TAM to ER-Src cells, but it is unclear if one cell type is a precursor of the other or whether they arise independently. To address this issue, we took advantage of previous observations that miR-200, which is strongly down-regulated in CSCs, inhibits CSC growth and the epithelial-mesenchymal transition (27–31). Addition of miR-200b before TAM treatment essentially abolishes CSC formation, but it has no detectable effect on cellular transformation [assayed morphologically (22) or by colony formation in soft agar; Fig. 3A], indicating that CSC formation is not required for transformation. Conversely, addition of miR-200b antisense RNA to parental ER-Src cells does not result in transformation or formation of CSCs. The observation that CSC formation depends on, but is not required for, transformation, suggests that NSCCs are precursors of CSCs.

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Fig. 3.

Relationship of CSC and NSCC formation and stability of the isolated cell types. (A) Transformation assays (morphology or colony formation in soft agar) of ER-Src cells that were or were not treated with TAM in the presence or absence of miR-200b or anti-sense miR-200b. (B) Number of CSCs (black) or NSCCs (gray) after growth of isolated CSCs (Left) or isolated NSCCs (Right) from ER-Src transformed cells for the indicated number of days. (C) Same as B except that sorted CSCs and NSCCs were derived from a breast tumor.

CSCs Rapidly Differentiate into NSCCs, but NSCCs Are Not Easily Converted to CSCs.

There are two basic models by which the proportion of CSCs within a transformed cell population remains constant over multiple generations. In one model, CSCs and NSCCs represent distinct epigenetically stable cell types that copropagate independently. Alternatively, the two cell types can switch from one type to the other in a dynamic equilibrium that maintains the proportion of CSCs. To distinguish between these possibilities, we sorted a transformed population of ER-Src cells (36 h after TAM addition) into CSCs and NSCCs and examined the distribution of cell types after multiple generations of growth. Sorted CSCs rapidly differentiate into NSCCs, such that after 9 d, the population was ~15% CSCs and 85% NSCCs (Fig. 3B). Over more extended times, the CSC population is maintained at the 10% level typical of the transformed cell line. In contrast, the sorted NSCCs generate very few CSCs over the same period (Fig. 3B). Similar results were obtained with CSCs and NSCCs derived directly from a breast tumor (Fig. 3C). Thus, although NSCCs and CSCs can form concomitantly during the process of cellular transformation, the conversion of NSCCs to CSCs is very inefficient. The failure of the sorted CSCs to be stably propagated under these conditions indicates that CSCs do not represent a stable epigenetic state.

Conversion of NSCCs to CSCs Within Tumors in Mouse Xenografts.

The nonreciprocal conversion between sorted NSCCs and CSCs should result in ever-decreasing numbers of CSCs and, hence, is paradoxical in light of the constant proportion of CSCs within the transformed population. This apparent paradox suggests that CSCs and NSCCs interact with each other in a mixed population in a manner that does not occur when the cell types are propagated separately. As a first test of this hypothesis, we performed a mixed xenograft experiment (Fig. 4A) involving coinjection of NSCCs from an ER-negative, PKCα-positive cell line (MDA-MB-231) and CSCs from ER-Src cells (ER-positive, PKCα-negative). In comparison with tumors generated by ER-Src CSCs alone, the CSC population in the tumor derived by coinjection had 5- to 10-fold fewer ER-positive cells (assayed by ER DNA and RNA levels) and 10-fold higher PKCα-positive cells (assayed by RNA levels) (Fig. 4B), indicating that most of the CSCs in the tumor were derived from the ER-negative NSCCs. Because injection of the same number of ER-negative NSCCs is insufficient to generate tumors (Fig. S1), this experiment suggests that ER-negative NSCCs can be converted to CSCs in the presence of the ER-positive CSCs during tumor formation.

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Fig. 4.

Conversion of NSCCs to CSCs in tumors in mouse xenografts. (A) Scheme of mixed xenograft experiment. The 10⁴ ER-Src CSCs were combined (or not) with 10⁴ NSCCs from MDA-MB-231, injected into nude mice. CSCs were obtained by sorting cells from excised tumors 15 d after injection, and the resulting material examined for ER and PKCα RNA levels or ER copy number (B).

Sorting of CSCs and NSCCs Subpopulations from Cancer Cells.

For ER-Src–transformed cells, MCF7 and MDA-MB-231 cancer cells, to separate CSCss from NSCCs, flow cytometric cell sorting was performed on single-cell suspensions that were stained with CD44 antibody (FITC-conjugated) (555478; BD Biosciences) and with CD24 antibody (PE-conjugated) (555428; BD Biosciences) for 30 min (21, 22, 26). As used throughout this work, CSCs are defined by the minority CD44^high/CD24^low population, whereas NSCCs are defined by the majority CD44^low/CD24^high.

To separate CSCs from NSCCs for PC3 prostate cells, flow cytometric cell sorting was performed on single-cell suspensions that were stained with CD44 antibody (FITC-conjugated) (555478; BD Biosciences) and CD133 antibody (PE-conjugated) (239C3; Miltenyi Biotech Ltd.) for 20 min. Prostate CSCs were defined by the minority CD133^high/CD44^high population, whereas NSCCs are defined by the majority CD133^low/CD44^low population.

Purification and Experiments Using CSCs and NSCCs from Human Breast Tissues.

Five human invasive ductal carcinoma tissues (stage III) were purchased from AMS Biotechnology and Biochain Inc. All these tissues were negative for ER, PR, and HER2 expression (triple negative). Immunomagnetic purification of CSCs and NSCCs was performed according to Shipitsin et al. (36). Briefly, the breast tissues were minced into small pieces (1 mm) by using a sterile razor blade. The tissues were digested with 2 mg/mL collagenase I (C0130; Sigma) and 2 mg/mL hyalurinidase (H3506; Sigma) in 37 °C for 3 h. Cells were filtered, washed with PBS, and followed by Percoll gradient centrifugation. The first purification step was to remove the immune cells by immunomagnetic purification by using an equal mix of CD45 (leukocytes), CD15 (granulocytes), CD14 (monocytes), and CD19 (B cells) Dynabeads (Invitrogen). The second purification step was to isolate fibroblasts from the cell population by using CD10 beads for magnetic purification. The third step was to isolate the endothelial cells by using an “endothelial cocktail” beads (CD31, BD Pharmingen cat no. 555444; CD146 P1H12 MCAM, BD Pharmingen cat no. 550314; CD105, Abcam cat no. Ab2529; Cadherin 5, Immunotech cat no. 1597; and CD34, BD Pharmingen cat no. 555820). In the final step, from remaining cell population, only the CD44^high cells were purified by using CD44 beads. These cells were sorted for CD44^high/CD24^low (CSC) cells. On the other hand, CD24^high cells were purified by using CD24 beads. These cells were sorted for CD44^low/CD24^high (NSCCs) cells. These CSC and NSCC populations were sorted again to increase their purity (>99.2% in all cases).

Mammosphere Formation Assay.

Mammospheres were generated by placing transformed cell lines in suspension (1,000 cells per mL) in serum-free DMEM/F12 media, supplemented with B27 (1:50, Invitrogen), 0.4% BSA, 20 ng/mL EGF, and 4 μg/mL insulin. After 6 d of incubation, mammospheres were typically >75 mM in size with ~97% being CD44^high/CD24^low. For serial passaging, 6-d-old mammospheres were harvested by using a 70-μm cell strainer, whereupon they were dissociated to single cells with trypsin (37), and then regrown in suspension for 6 d.

Chemotherapy Treatment of CSCs and NSCCs.

CSCs and NSCCs were sorted from ER-Src transformed (36 h tam-treated) cells, seeded in monolayer culture, and treated with different doses of paclitaxel, doxorubicin, and 5-fluorouracil for 24 h and cell viability was assessed by the CCK8 assay (Dojindo).

MicroRNA Transfection Experiments.

ER-Src MCF10A cells were transfected with 100 nM microRNA negative control (miR NC) or miR-200b by using siPORT NeoFX transfection agent. In these cells, 24 h later, tamoxifen was added for 36 h. After that, the cells were sorted for CD44 and CD24 antigens. In addition, untransformed or transformed (36 tam-treated) ER-Src cells were treated with 100 nM miR-200b or as-miR-200b for 48 h, and then the cells were plated in soft agar. The number of colonies was counted 15 d later.

Conditions for Differentiation of CSCs.

For differentiation experiments, CSCs sorted from ER-Src MCF10A transformed (+TAM for 36 h) cells were plated at 1 × 10⁵ cells per mL on six-well plates precoated with Collagen IV (BD BioSciences) in DMEM/F12 supplemented with 5% serum without growth factors and passaged when they reached >95% confluence. CSC differentiation was monitored every 6 d and tested by flow cytometry analysis.

MicroRNA Analysis.

RNA extracted from untreated (0 h) or tamoxifen-treated (1, 2, 4, 8, 12, 16, 24, 36 h) ER-Src cells together with RNA extracted from CSCs derived from tamoxifen-treated (36 h) ER-Src cells were used for testing the expression levels of 365 microRNAs (microRNA TLDA v1.0 card; Applied Biosystems) in the Dana–Farber Molecular Diagnostics Facility. In addition, microRNA expression levels were tested by using the mirVana qRT–PCR miRNA Detection Kit and qRT–PCR Primer Sets, according to the manufacturer's instructions (Ambion). RNU48 expression was used as an internal control. Specifically, microRNA expression levels by quanitative RT-PCR (qRT-PCR) were tested in: (i) 6-d mammospheres derived from ER-Src transformed (36 h tam-treated) cells; (ii) sorted CSCs and NSCCs from MCF7 and MDA-MB-231 breast cancer cells; and (iii) CSCs and NSCCs isolated by immunomagnetic purification followed by cell sorting.

We thank Marianne Lindahl-Allen for help with the kinetic analysis of CSC formation and for useful comments on the work, and Philip N. Tsichlis for providing facilities for performing the xenograft experiments. This work was supported by a postdoctoral fellowship from the American Cancer Society (to H.A.H.) and National Institutes of Health research Grant CA 107486 (to K.S.).