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Keywords:

  • Tissue-specific stem cells;
  • Clonal assays;
  • Progenitor cells;
  • Breast cancer

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Elevated aldehyde dehydrogenase (ALDH) expression/activity has been identified as an important biomarker of primitive cells in various normal and malignant human tissues. Here we examined the level and type of ALDH expression and activity in different subsets of phenotypically and functionally defined normal human mammary cells. We find that the most primitive human mammary stem and progenitor cell types with bilineage differentiation potential show low ALDH activity but undergo a marked, selective, and transient upregulation of ALDH activity at the point of commitment to the luminal lineage. This mirrors a corresponding change in transcripts and protein levels of ALDH1A3, an enzyme involved in retinoic acid synthesis and the most highly expressed ALDH gene in normal human mammary tissue. In contrast, ALDH1A1 is expressed at low levels in all mammary epithelial cells. These findings raise interesting questions about the reported association of ALDH activity with breast cancer stem cells and breast cancer prognosis. STEM CELLS 2012; 30:344–348.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Much attention is now being focused on elucidating the molecular mechanisms that regulate the different early stages of normal mammary cell differentiation, with the goal of identifying those relevant to the cells that maintain human breast cancers or that may serve as useful markers of these cells. The aldehyde dehydrogenases (ALDHs), which are encoded by a large gene family (http://www.aldh.org), are of interest in this regard [1], although the results of analyses of their expression in normal and neoplastic human breast cells remain controversial (reviewed in [2]). Here we analyzed the ALDH activity and isoform expression in different phenotypically and functionally defined subsets of normal human mammary cells. We show that ALDH activity in the normal human mammary gland is largely due to ALDH1A3 and is low or absent in normal human mammary stem cells but is then elevated when these transition into progenitor cells committed to the luminal lineage.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

We first analyzed the ALDH activity present in each of the five biologically and phenotypically distinct populations that are found in dissociated normal human mammary tissue and can be separately isolated based on their differential expression of CD49f, epithelial cell adhesion molecule (EpCAM, also known as CD326), CD31, and CD45 (Fig. 1A) [3–5]. Three of these populations are subsets of mammary epithelial cells: (a) a basal (CD49f+EpCAM−/lowCD31CD45) fraction that contains all of the mammary stem cells, all of the bilineage- and myoepithelial-restricted progenitors, and all of the mature myoepithelial cells; (b) a primitive luminal (or “luminal precursor-enriched”) (CD49f+EpCAM+CD31CD45) fraction that contains all of the luminal-restricted progenitors and some of the more differentiated luminal cells, and (c) a mature luminal (CD49fEpCAM+CD31CD45) fraction that consists of fully differentiated luminal cells. Because mammary stem cells cannot be identified directly, they are referred to as “mammary repopulating units” (MRUs) based on their ability to individually regenerate a full mammary gland structure within 4–6 weeks when transplanted into an appropriate environment in immunodeficient mice [3, 4]. Mammary progenitors are likewise referred to operationally as mammary “colony-forming cells” (CFCs), based on their ability to produce colonies within 8–10 days in vitro that contain differentiated adherent cells of one or both mammary lineages. CD49f/EpCAM/CD31/CD45 staining also yields another two populations of cells that are present in high numbers in normal breast tissue but are not epithelial, that is, a stromal (CD49fEpCAMCD31CD45) fraction and a fraction that contains a mixture of hematopoietic (CD45+) and endothelial (CD31+) cells.

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Figure 1. Elevated aldehyde dehydrogenase activity is specific to the primitive luminal epithelial and stromal cell fractions. (A): Fluorescence-activated cell sorting gating approach used to analyze fractions of the different cell types present in dissociated normal human breast tissue, based on differential expression of EpCAM, CD49f, CD31, and CD45, as previously described [4, 5]. (B): Representative BAA fluorescence profiles of the fractions identified in Panel (A) after incubation with Aldefluor in the presence (solid gray) or absence (open profiles) of DEAB and then with antibodies. (C): MFI values for the BAA fluorescence of cells in each fraction in Panel (B) expressed relative to the MFI of the corresponding basal fraction. Bars show the mean ± SEM for mammoplasty samples that were freshly processed (green bars, n = 4) or previously cryopreserved (yellow bars, n = 7). Abbreviations: BAA, bodipy aminoacetate; DEAB, diethylaminobenzaldehyde; EpCAM, epithelial cell adhesion molecule; FSC, forward scatter; MFI, median fluorescence intensity.

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We incubated normal human mammary cells with Aldefluor (a fluorescent reagent that is activated by ALDH to produce an intracellularly retained fluorescent derivative, bodipy aminoacetate [BAA]) and then with fluorescently conjugated antibodies. Comparison of the resulting fluorescence profiles obtained in the presence and absence of diethylaminobenzaldehyde (DEAB), a specific inhibitor of ALDH enzyme activity, showed that the cells in all fractions had DEAB-sensitive ALDH activity, as indicated by their decreased fluorescence when DEAB was present (Fig. 1B, Supporting Information Fig. 1). However, in all tissue samples examined (four fresh and seven previously cryopreserved), we found the median fluorescence intensity (MFI) to be much higher (on average, 18-fold, p < .001) in the primitive luminal fraction when compared with the basal fraction (Fig. 1C) and this difference was sustained when CD10 was used as an additional marker to discriminate the luminal progenitor-enriched and basal fractions [6, 7] (with a 17-fold higher MFI in CD49f+EpCAM+CD10 population on average when compared with the CD49f+EpCAM−/lowCD10+ cells; Supporting Information Fig. 2). The mature luminal cells exhibited intermediate activity (on average, fourfold higher MFI than the basal cells, p < .001, Fig. 1C). In the same cell suspensions, the MFI of the stromal cell fraction was also much higher (on average, 13-fold, p < .001) than the MFI of the basal fraction. Thus, the elevated ALDH activity within bulk populations of human mammary cells is determined predominantly by the prevalence of primitive luminal cells and stromal cells, both typically present in high numbers. As a result, even after fractionation, minor contamination of the more primitive basal fraction with either of these subpopulations would give false-positive readings.

Examination of two published global gene expression datasets [5, 8] for these subpopulations of cells that used microarray or LongSAGE technologies suggested that ALDH1A3 is the most prominently expressed ALDH of all the members of this gene family in the normal human breast and, in both datasets, ALDH1A3 expression is highest in the CD49f+EpCAM+ (or CD24+) primitive luminal populations (Supporting Information Fig. 3A). We also compared these datasets with another gene expression microarray dataset generated on similarly fractionated cells obtained from 3-day cultures of dissociated primary mammoplasty tissue cells [6]. The results were largely confirmatory, ALDH1A3 remaining as one of the most highly expressed ALDH family members in these cultured cells, with highest ALDH1A3 expression again in the CD49f+EpCAM+(CD133/Muc1)+ luminal progenitor-enriched fraction (Supporting Information Fig. 3B). However, some notable differences were seen (e.g., marked upregulation of ALDH1B1, 3B1, 3B2, 5A1, and 16A1 expression in the cultured vs. freshly isolated mammary cells), highlighting the likelihood that mammary cells maintained in culture may not retain all of the phenotypic properties they possess in vivo.

To obtain a more accurate assessment of the changes in expression of ALDH1A3 during the differentiation of human mammary stem cells in vivo, we performed quantitative reverse trancriptase polymerase chain reaction and Western blotting analyses for ALDH1A3 transcripts and protein on cells isolated from three normal breast tissue samples. The results (Fig. 2A, 2B) consistently paralleled the ALDH activity profiles determined by Aldefluor staining (Fig. 1B, 1C). In contrast, the ALDH1A1 transcript and protein levels were consistently low in all epithelial subsets (Fig. 2A, 2B). Interestingly, we found that the stromal cells present in mammary tissue express high levels of ALDH1A1 transcripts and protein compared with any of the subsets of mammary epithelial cells and low levels of ALDH1A3 compared with the luminal subsets (Fig. 2A, 2B).

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Figure 2. ALDH1A3 transcript and protein levels are elevated in the primitive luminal cell fraction. (A):ALDH1A3 and ALDH1A1 transcript levels in the different fluorescence-activated cell sorted fractions of cells are determined by quantitative reverse transcription polymerase chain reaction and normalized relative to GAPDH. Mean ± SEM values for three independently studied mammoplasty samples are shown. (B): Representative Western blots showing different ALDH1A3 and ALDH1A1 protein levels in the different fractions of cells. Histone H3 is shown as the loading control. Abbreviation: GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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To determine directly the ALDH activity of CFCs and MRUs, we subjected cells dissociated from another eight normal breast tissue samples to the same surface marker- and Aldefluor-based fractionation strategy (Fig. 3A, 3B, left and middle panels) and then further subdivided the cells as follows. In the first set of experiments, all of the three mammary epithelial subsets plus the stromal cells were separated as a single group into an ALDH+ and ALDH fraction using a single stringent gate to discriminate between DEAB-sensitive and DEAB-insensitive BAA fluorescence. In the second set of experiments, we subdivided the individually defined primitive luminal and basal subsets (only) into low, medium, and highly BAA-fluorescent fractions. The cells from each of the eight fractions from both designs (2 + 6) were then isolated by fluorescence-activated cell sorting (FACS) and assayed for CFC activity in vitro and MRU activity in vivo (the latter using a subrenal capsule xenotransplantation assay that facilitates sensitive quantitative measurements of human MRU frequencies [4, 9]). The results we obtained from the first experiments showed that more than 90% of the MRUs as well as the bilineage and myoepithelial CFCs are in the ALDH (BAAlow-med) fraction, whereas most (76 ± 10%) of the luminal CFCs are in the ALDH+ (BAAhigh) fraction (right panel of Fig. 3A; Supporting Information Table 1). The results of the second experiments confirmed the segregation of luminal versus basal CFCs and MRUs in the EpCAM+ and EpCAM subsets of CD49f+ cells, respectively (right panel of Fig. 3B; Supporting Information Table 2). In addition, they showed the luminal CFCs to be split between the three ALDH activity fractions in a ratio of 5:62:33 (BAAlow:BAAmed:BAAhigh), with a reverse distribution (75:24:1 and 81:18:1) for the myoepithelial plus bilineage CFCs and the MRUs, respectively (right panel of Fig. 3B; Supporting Information Table 2).

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Figure 3. Human MRUs and bipotent CFCs have low aldehyde dehydrogenase (ALDH) activity whereas luminal CFCs exhibit high ALDH activity. Both left and middle panels of (A) and (B) show the two gating approaches used to isolate the fractions to be subsequently assessed functionally for their CFC and MRU content as described in Supporting Information Methods. The proportions of total cells in each of these fractions (mean ± SEM of non-diethylaminobenzaldehyde [DEAB]-treated cells) are indicated in the middle panels. The right panels show the distributions of luminal CFCs (red diamonds), combined myoepithelial plus bipotent CFCs (blue diamonds), and MRUs (blue circles) in each set of fractions assayed. Each symbol shows the result from an individual experiment, and the bars show the mean of the values measured (n = 5 samples in (A), n = 3 samples in (B)). Abbreviations: BAA, bodipy aminoacetate; CFC, colony-forming cell; EpCAM, epithelial cell adhesion molecule; MRU, mammary repopulating unit.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

These results establish that the most primitive normal human mammary epithelial cell types defined by their bilineage developmental potential are characterized by low ALDH activity, and that this activity is first and selectively increased by more than an order of magnitude at the time of commitment to the luminal lineage before further proliferative ability is lost. A similar pattern occurs in bovine mammary tissue where the stem cells are detected using the same in vivo transplant assay as used here for human MRUs [10]. The present findings are also in agreement with those found in the mouse mammary gland where ALDH1A3 expression has been found to be upregulated at the point of luminal progenitor generation [11]. ALDH1A3 transcripts are also detected at high levels in luminal progenitor-enriched fractions obtained from short-term adherent cultures of freshly isolated normal human mammary cells [6]; however, a more detailed study of different in vitro systems is required to investigate the stability of this phenotype.

ALDH1A3 plays a catalytic role in the biosynthesis of retinoic acid in mammary cells [12] and approximately two-thirds of human genes with binding sites for Gata-3 (a transcription factor that regulates the luminal differentiation program [13]) contain retinoic acid regulatory elements [14]. Activation of retinoic acid signaling (using all-trans retinoic acid) in breast cancer cell lines was reported to result in a reduced frequency of cells with mammosphere-forming activity, and blocking of ALDH activity (using DEAB) had the reverse effect [15]. These findings encourage future investigation of the possibility that ALDH activity plays a role in activating the luminal cell differentiation program in the mammary gland through retinoid signaling pathways.

The high ALDH activity reported for cells with breast cancer-initiating properties in xenograft assays [16–18] stands in direct contrast with the results for normal mammary stem cells and correlates better with normal luminal progenitors. This adds further weight to the possibility that cells with breast cancer stem cell activity in clinical samples may be more closely related to luminal progenitor cells. A case for this model has recently been made in BRCA1-associated breast cancer, in which aberrant luminal progenitor activity has been described in human breast tissue that is still phenotypically normal [5]. It is noteworthy that more ALDH+ cells have been reported in breast tissue from BRCA1 mutation carriers [19], where they appear to have a luminal rather than a basal location. Alternatively, it may be that in certain breast cancers, the oncogenic process itself perturbs the control of ALDH expression and/or activity. Examples of the latter have been demonstrated in leukemia [20–23], suggesting that the picture may be equally heterogeneous in breast cancer.

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Our study demonstrates that elevated ALDH activity characterizes luminal progenitor cells in the normal human mammary epithelium.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

We acknowledge the excellent technical contributions of D. Wilkinson, G. Edin, and the staff of the Flow Cytometry Facilities of the Terry Fox Laboratory and the Walter and Eliza Hall Institute. Mammoplasty tissue, generously donated by patients, was obtained with the assistance of Drs. J. Sproul, P. Lennox, N. Van Laeken, and R. Warren (Canada) and the Victorian Cancer Biobank (Australia). This work was supported by the Canadian Breast Cancer Research Alliance (Grant CBCRA 019343), the U.S. Department of Defense Breast Cancer Research Program (Predoctoral Fellowship number W81XWH-06-1-0702), the Canadian Breast Cancer Foundation BC/Yukon (Fellowship to N. Kannan), the National Health and Medical Research Council (Australia, Grants #461224 and #461221), the Victorian Government through the Victorian Cancer Agency/Victorian Breast Cancer Research Consortium and an Operational Infrastructure Support grant, and the Australian Cancer Research Foundation.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
STEM_1001_sm_supplFig1.pdf155KSupplementary Figure 1. Fluorescence with BAA is sensitive to DEAB in all mammary fractions Plot shows MFI with BAA in the presence of DEAB divided by the MFI with BAA in the absence of DEAB, showing mean±SEM of this ratio for each mammary fractions (7 previously cryopreserved samples analyzed). Fractions are defined by the gating approach depicted in Fig. 1A.
STEM_1001_sm_supplFig2.pdf330KSupplementary Figure 2. Comparison of fluorescence with BAA in basal and primitive luminal fractions using CD10 as an additional phenotypic marker (A) Representative FACS profiles. Previously cryopreserved mammoplasty tissue was dissociated to form suspensions of single cells, incubated with Aldefluor in the presence or absence of DEAB, then stained with antibodies against CD49f, EpCAM, CD10, CD31 and CD45. Two fractions were identified based on relative expression of EpCAM and CD10, after gating on CD49f+CD31−CD45− cells; an EpCAM−/lowCD10+CD49f+CD31−CD45− and an EpCAM+CD10−CD49f+CD31−CD45− fraction, previously shown to be enriched for basal cells and primitive luminal cells respectively9. Lower panel displays the fluorescence with BAA in the 2 fractions in the presence (solid grey) or absence (open profiles) of DEAB. (B) Quantitation of the relative fluorescence intensity with BAA in the two fractions. Plot shows the MFI with BAA in the EpCAM+CD10−CD49f+CD31−CD45− fraction divided by the MFI with BAA in the EpCAM−/lowCD10+CD49f+CD31−CD45− fraction, in the absence of DEAB (mean±SEM of this ratio for 4 previously cryopreserved samples). The former (luminal progenitor enriched) exhibited a MFI that was 17±6 fold higher than the latter (basal) fraction. (C) Plot shows the MFI with BAA in the presence of DEAB divided by the MFI with BAA in the absence of DEAB (mean±SEM of this ratio for the two fractions for 4 previously cryopreserved samples).
STEM_1001_sm_supplFig3.pdf314KSupplementary Figure 3. Relative transcripts levels of all ALDH gene family members in different subsets of normal human mammary cells (A) Comparison of ALDH gene expression levels in 2 previously published whole transcriptome analyses of non-cultured primary human mammoplasty tissue samples. Upper panel: expression levels from array hybridization dataset3 for different fractions of cells isolated by FACS based on the expression of CD49f and EpCAM. Values plotted are the normalized (limma) signal intensity for each gene in each fraction divided by the average normalized (limma) signal intensity of a group of 7 housekeeping genes, averaged across all of the biological samples in the dataset. Lower panel: expression levels derived from LongSAGE dataset8 for different fractions of cells isolated immunomagnetically based on the expression of CD24, Muc1, CD44 and CD10. Values plotted are the tag counts for each gene in each fraction divided by the average tag count of a group of 7 housekeeping genes, averaged across all of the biological samples in the dataset. (B) Comparison of ALDH gene expression in previously published whole transcriptome array hybridization analysis of primary human mammoplasty tissue samples cells following 3 days adherent culture7. Different fractions of cells were subsequently isolated by FACS based on the expression of CD49f and EpCAM. Values plotted are the normalized (GC-RMA) signal intensity for each gene in each fraction divided by the average normalized signal intensity of a group of 7 housekeeping genes, averaged across all of the biological samples in the dataset.
STEM_1001_sm_supplMethods.pdf35KSupplementary Data
STEM_1001_sm_supplTables1-2.pdf54KSupplementary Tables 1 and 2. Results of 2° CFC assays used as regenerative endpoints for MRU assays

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