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Keywords:

  • breast cancer;
  • stem cells

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Mammary epithelial stem cells
  5. Candidate populations
  6. Cancer stem cells
  7. References

Proliferation in continuously renewing tissues, including the mammary gland, is hierarchically organized with a small number of slowly dividing stem cells and a greater number of more rapidly proliferating ‘transit amplifying’ cells. Mammary stem cells have been recently identified and purified based on their surface antigen expression. The recognition of mammary epithelial stem cells had led to the hypothesis that these may be at the root of breast cancer. In support of this, a highly tumorigenic subpopulation of cancer cells – cancer stem cells – has recently been identified in primary and metastatic breast cancer samples and in a number of established breast cancer cell lines. The existence of cancer stem cells would explain why only a small minority of cancer cells is capable of extensive proliferation and transferral of the tumour. In this article we aim to review the evidence in support of the existence of both normal mammary stem cells and breast cancer stem cells, and provide further insight into how taking this subpopulation of cells into account may affect the way we treat epithelial cancers in the future.


Abbreviations:
ABC

ATP-binding cassette

AML

acute myeloid leukaemia

BrdU

bromodeoxyuridine

CK

cytokeratin

ECM

extracellular matrix

ESA

epithelial specific antigen

HSCs

haematopoietic stem cells

LRC

label-retaining cell

Ma-CFC

mammary colony forming cell

MMTV

mouse mammary tumour virus

MRU

mammary repopulating unit

NOD/SCID

non-obese diabetic/severe combined immunodeficiency

PCR

polymerase chain reaction

SLC

small light cell

SP

side population

TDLU

terminal duct lobular unit

TGF

transforming growth factor

ULLC

undifferentiated large light cells

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Mammary epithelial stem cells
  5. Candidate populations
  6. Cancer stem cells
  7. References

Stem cells, as classically defined, are cells with a capacity to self-renew and to generate daughter cells that can differentiate down different cell lineages to form all the cell types that are found in the mature tissue.1 In order to maintain this ability indefinitely, they must be able to perform asymmetric cell divisions, each cell therefore generating one that is identical to it as well as another which is different, in that it is more committed towards a certain differentiation pattern. The identical cell maintains the stem cell compartment through time; the distinct cell undergoes a series of divisions and differentiation steps that result in the generation of terminally differentiated cell populations. The cells in intermediate states between the stem cell and the terminally differentiated cell are usually referred to as progenitors, transit cells or transit amplifying cells.2 Although both stem cells and transit amplifying cells divide and produce similar end-products (a range of differentiated progeny), they differ in their ability to proliferate and maintain an undifferentiated state for an extended period of time (Figure 1).3 This hierarchical organization is best established for the murine haematopoietic system, where a single cell with the Lin−, c-Kit+, Sca-1+ (LSK) phenotype is capable of long-term reconstitution of a lethally irradiated recipient.4 A similar organization seems to exist in other renewing tissues such as the intestinal epithelium and the epidermis.5,6 In order to maintain tissue homeostasis, the number of daughter cells that maintain stem cell identity must be strictly controlled. Regulation of haematopoietic stem cells (HSCs), for example, is under the control of multiple genes.7 Emerging evidence suggests that a specialized microenvironment is one of the factors regulating normal stem cell maintenance and self-renewal, by controlling the crucial choice between self-renewal and initiation of differentiation.8 Furthermore, stem cells that replenish and repair adult tissues must be able to withstand the stress of events associated with tissue damage. Experimental evidence suggests that stem cells residing in adult tissues are extremely resilient to fluctuations in temperature, pH and exposure to toxicants.9,10

image

Figure 1.  Stem cell compartment hierarchy. A stem cell is capable of going through asymmetric cell division to generate one cell which is identical to itself and one that it is more committed towards a certain differentiation pattern. The formation of the identical cell ensures that the stem cell compartment is maintained through time; the distinct cell undergoes a series of divisions and differentiation steps that result in the generation of a terminally differentiated cell population. The cells that form the intermediate states are usually referred to as progenitors, transit cells or transit amplifying cells.

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The concept that cancers contain a subpopulation of cells similar to epithelial stem cells was proposed several years ago, and the expansive growth of malignant lesions indicates the presence of cells with at least the stem cell property of indefinite proliferation.11 There is emerging evidence that some blood cell malignancies and solid tumours may contain a cancer cell hierarchy similar to that observed in the normal tissue from which the tumour arose, with a cancer stem cell producing a progeny with limited replication potential.12–14 Cancer stem cells may therefore drive the growth and spread of the tumour. It has been suggested that cancer stem cells may arise in either one of two ways (Figure 2). In the first, oncogenic mutations in normal stem cells may cause alterations in the mechanisms that put constraints on normal stem cell expansion, such as stem cell dependence on the niche (either by expansion of the niche itself (Figure 2B) or by acquisition of independence from niche signalling (Figure 2A)). In the second situation, oncogenic mutations allow transit-amplifying cells to continue proliferating without entering a post-mitotic differentiated state (Figure 2C), therefore allowing aberrant activation of stem cell self-renewal machinery in these cells.3 Regardless of its mechanism of origin, if a cancer stem cell existed within a tumour, it would have significant therapeutic implications, as failure to target this cell would set the stage for local and systemic recurrence (Figure 3).

image

Figure 2.  Origins of cancer stem cells. Cancer stem cells could arise in one of two ways. In the first, oncogenic mutations could arise in normal stem cells, causing alterations in the mechanisms that put constraints on normal stem cell expansion, such as stem cell dependence on the niche. This could be due to the fact that cancer stem cells may become independent of niche signalling for activation of self-renewal pathways (A). Alternatively, recruitment of niche-forming cells within the tumour could result in expansion of the niche itself, and therefore of the stem cell compartment (B). In the second situation, oncogenic mutations may allow transit amplifying cells to continue proliferating without entering a post-mitotic differentiated state, therefore allowing aberrant activation of stem cell self-renewal machinery in these cells (C).

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image

Figure 3.  Therapeutic implications of the cancer stem cell hypothesis. The efficacy of cancer treatment is often measured by the extent of pathological response. As cancer stem cells form a very small proportion of a tumour, this may not necessarily select for drugs that act specifically on the stem cells. The cancer stem cell hypothesis suggests that conventional chemotherapeutic approaches act on differentiated or differentiating cells, which form the bulk of the tumour, but are unable to propagate the cancer. A population of cancer stem cells, which gave rise to it, remains untouched and may cause a relapse of the disease (A). With the emergence of the cancer stem cell paradigm has come the realization that, because only a small proportion of tumour cells have the capacity to self-renew and propagate the tumour, it is these cells rather than transit amplifying or differentiated cells that must be targeted to achieve long-term cure (B).

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Mammary epithelial stem cells

  1. Top of page
  2. Abstract
  3. Introduction
  4. Mammary epithelial stem cells
  5. Candidate populations
  6. Cancer stem cells
  7. References

The mammary gland is a structurally dynamic organ, varying with age, menstrual cycle and reproductive status.15 In the adult, it consists of a tree-like structure of branching ducts and lobulo-alveolar units, the latter arising during pregnancy.16 The inner layer of luminal epithelial cells is surrounded by an outer layer of myoepithelial cells that secretes the basal lamina separating the mammary parenchyma from the stroma.17,18 The intralobular stroma consists of cellular loose connective tissue with a zone of hormone-sensitive fibroblasts surrounding the epithelial components. These are thought to take part in epithelial/basement membrane/stromal inductive interactions during morphogenesis and differentiation.15

Several features of the developing and adult mammary gland appear to require the existence of a stem cell compartment.10 The existence of mammary stem cells is indicated by (i) the expansion and regenerative ability of the gland during puberty and successive reproductive cycles;19 (ii) the existence of two different cell lineages arising from a common progenitor;19 and (iii) the replacement of cells that are shed from the epithelium into the lumen during routine cell turnover.10

The existence of mammary gland stem cells was first suggested by transplantation studies conducted by DeOme et al. It was shown in the mouse that epithelium isolated from different regions of a mammary gland at various stages of postnatal development was capable of generating fully functional mammary epithelial outgrowths containing ducts, lobules and myoepithelial cells. Thus, the mammary gland resembles the haematopoietic system, in that they can both be generated by transplantation.20 Furthermore, it was later shown that a fragment could be taken from this regenerated gland and serially transplanted to another cleared mammary fat pad.21 Normal mammary epithelial stem cells showed ductal growth senescence on serial transplantation after six generations.22,23

Kordon and Smith have subsequently shown, by retroviral tagging, that progeny of a single multipotent stem cell can produce an entire mammary gland. Neonatal mice were infected with mouse mammary tumour virus (MMTV), and randomly chosen fragments of the gland were transplanted from multiparous adult animals to the cleared mammary fat pad of non-infected recipients. Because MMTV integrates at random into the genome of the infected cell, clones derived from individual infected cells can be distinguished from one another. Southern analysis of these unique virus–host junction restriction fragments has demonstrated clonality of the regenerated glands. Three distinct progenitor populations have been identified, one capable of producing the entire epithelial compartment, and two downstream progenitors with limited potency that can produce either secretory lobules or branching ducts.24,25 It must be pointed out that although clonality of a whole gland is possible under experimental conditions, it may not occur in the normal gland.26

Further support for the existence of pluripotent epithelial stem cells in the mammary gland comes from limiting dilution transplantation studies.25,26 When small numbers of dissociated mammary epithelial cells are transplanted, a minority of fat pads develop epithelial outgrowths, most derived from a single mammary epithelial cell. However, three types of outgrowths can be distinguished. The first comprises a limited network of ducts without lobules and another a limited outgrowth of lobules without branching ducts. The final outgrowth has both ductal and lobular development and completely fills the mammary fat pad.25,26

Intuitively, it seems likely that human mammary epithelium is organized along similar lines to the mouse; results support this premise. Clonal patches of X-inactivation27 and loss of heterozygosity28 affect large, contiguous regions of breast epithelium. This has been interpreted as reflecting origin from a common pluripotent stem cell, although the alternative possibility that these alterations arise during development29,30 cannot be formally excluded. Nevertheless, X-linked inactivation analysis of normal breast samples supports the hypothesis that the mammary gland is organized into distinct stem cell-derived monoclonal patches and that terminal duct lobular units (TDLUs) are monoclonal in origin.31 Therefore, the biology of the mammary gland indicates a role for stem cells, and data support their existence.

Candidate populations

  1. Top of page
  2. Abstract
  3. Introduction
  4. Mammary epithelial stem cells
  5. Candidate populations
  6. Cancer stem cells
  7. References

Several approaches have been employed to identify, isolate and enrich mammary epithelial stem cells. It must be noted that, according to recent reports, the population of murine mammary epithelial cells initiating colony formation in vitro is distinct from the population that repopulates the cleared mammary fat pad.32 This strongly suggests that these in vitro studies are characterizing committed progenitors rather than pluripotent stem cells. Moreover, until recently there have been no certain stem/transit amplifying cell markers for the mammary gland. Therefore, researchers have applied knowledge obtained in the field of haematopoietic, neural and epidermal stem cells, and applied markers borrowed from these areas to identify prospectively stem/progenitor cells in the mammary gland.

Ultrastructural studies performed in mouse and rat mammary epithelium have examined the properties and locations of morphologically distinct ‘putative’ stem cells among the more differentiated cells in the mammary gland. Four types of cells, small light cells (SLCs), undifferentiated large light cells (ULLCs), differentiated large light cells and large dark cells, have been defined by electron microscopy.25 SLCs were hypothesized to be mammary epithelial stem cells, based on the presence of mitotic figures and their basal location within the gland. Furthermore, these cells occurred in side by side homogeneous, as well as heterogeneous pairs, suggesting that they might undergo both symmetric as well as asymmetric cell division. ULLCs were division competent, contained secretory granules and appeared capable of both symmetric and asymmetric division; these were suggested to be the secondary progenitors to the SLCs. Increased numbers of SLCs and ULLCs also appeared to be present in some human and mouse mammary tumours, suggesting that they might play a role in the aetiology of breast cancer.25 Of note, similar cells have been identified in the human mammary gland.33

Several investigators have used label-retention studies to identify mammary stem cells. During the labelling period, bromodeoxyuridine (BrdU) is incorporated into the DNA as cells replicate and is then diluted with each cell cycle after the labelling period. Long-term maintenance of BrdU indicates that cells have a very slow rate of proliferation, a putative characteristic of stem cells that enables them to maintain indefinite proliferative potential.34,35 Some label-retaining cells (LRCs) were negative for differentiation markers such as progesterone receptor or cytokeratin (CK) markers, indicating that they represent a less differentiated state. Others were positive for the markers, indicating a transition from an undifferentiated LRC stem cell, through a differentiated transit-cell population.35 More recently, by labelling mice with two different molecules, first with 3H-thymidine and subsequently with a pulse of BrdU, Smith has been able to confirm that LRCs selectively retain their original template DNA strand, while passing any newly synthesized daughter strand to their progeny during asymmetric divisions.34

The ability to exclude fluorescent dyes such as Hoechst 33342 from the cytoplasm has been employed to isolate a ‘side population’ (SP) of haematopoietic cells that is highly enriched in HSCs.36 Dye exclusion is thought to result from the activity of membrane protein pumps belonging to the ABC transporter family (also known as breast cancer resistance protein), which appear to be more active (as a defence mechanism) in stem/progenitor cells than in terminally differentiated cells.37,38 An analogous population has been identified in primary cultured murine mammary epithelial cells and uncultured human and murine mammary epithelial cells.37,39,40 It has been proposed that this SP is enriched in mammary epithelial stem cells. In support of this hypothesis, murine SP cells give rise to all three mammary epithelial cell lineages when transplanted to cleared mammary fat pads.35,40 Moreover, human SP cells form much larger branching colonies in matrigel than non-SP cells.39 SP cells are also highly enriched for the ability to grow in non-adherent conditions as spheroids, a feature considered to belong to stem/progenitor cells.41 On the other hand, only a small proportion (<10%) of cells with the capacity to repopulate the cleared mammary fat pad are contained within the SP, and LRCs constitute only a small proportion of SP cells.19,32,35

Analysis of cell surface antigens, in order to identify prospectively cell subpopulations, as well as the existence of a robust and well-characterized repopulation assay in the form of bone marrow transplantation, has brought great progress in the field of HSCs.42 Similar approaches have been used in attempts to characterize further mammary epithelial stem cells. Several studies have described bi-potent human mammary epithelial progenitor cells in normal adult breast tissue based on immuno-sorting using epithelial specific antigen (ESA), mucin-1 (MUC-1) and α6-integrin.43,44 ESA+/MUC-1+ cells cultured in vitro gave rise to clones that expressed only luminal-specific keratin markers. Cells expressing CD10, a myoepithelial surface marker, generated clones that expressed only myoepithelial markers. However, cells that were ESA+/MUC-1−/weak/CD10+/weak were able to generate clones containing cells of both lineages.43 These were also positive for α6-integrin, implying a basal or suprabasal location within the luminal cell layer.44 Successive studies have isolated two subpopulations of epithelial cells from human breast samples, a larger ESA+/MUC-1+ group and a smaller ESA+/MUC-1−/CK19+ population which occupied a suprabasal position. Following immortalization and in vitro culture, the ESA+/MUC-1+ line was luminal epithelial restricted, whereas the ESA+/MUC-1− line was also able to give rise to myoepithelial cells. In three-dimensional cultures and transplantation studies these cells formed elaborate branching structures resembling uncultured TDLUs, indicating that these cells are TDLU precursors in the human breast.45

More recently, using multiparametric cell sorting and limiting dilution mammary fat pad transplant analysis, two groups have purified a rare subset of adult mouse mammary cells, named ‘mammary repopulating units’ (MRUs). MRUs are able, individually, to regenerate an entire mammary gland, while at the same time undergoing up to 10 symmetrical cell divisions, therefore resulting in self-enrichment within the newly generated gland.19,32 Following removal of cells of non-epithelial lineages, MRUs could be prospectively purified from mammary epithelial cells on the basis of expression of either CD24+/CD29high19 or CD24+/CD49fhigh/Sca-1low.32 These MRUs are phenotypically distinct from, and give rise to, progenitor cells that are able to produce adherent colonies in vitro, mammary colony forming cells (Ma-CFCs), which are detectable at a 20-fold higher frequency within the gland (approximately 1400 MRUs/gland; 30 000Ma-CFCs/gland). For both kinds of MRUs, a single cell could give rise to epithelial outgrowths in cleared mammary fat pads that contained all mammary epithelial lineages, thereby demonstrating pluripotency. Serial transplantation has proved the ability of these cells to self-renew.19,32 This represents not only the first report of prospective identification of mammary epithelial stem cells, but indeed of any mammalian epithelial stem cells.

Characterization of prospectively purified MRUs offers insights into their phenotype. Flow cytometry has revealed that a large proportion of stem cell candidates with the CD24+CD29hi phenotype express the myoepithelial-specific marker CK14.19 Similarly, oligonucleotide array studies and quantitative polymerase chain reaction (PCR) have found higher levels of CK14, and other myoepithelial markers, in the CD24medCD49fhi population. However, further examination of the CD24medCD49fhi population at the single-cell level has revealed that, although a proportion of cells expressed luminal-specific markers and others expressed myoepithelial-specific markers, these were never co-expressed at the single-cell level. Of interest, half of these cells did not express a marker of either lineage.32

Sleeman and colleagues independently found MRUs to be enriched in the CD24lo population, as opposed to the CD24hi/−.46 Cytokeratin expression and PCR have revealed that CD24−, CD24low and CD24hi populations correspond to non-epithelial, basal/myoepithelial and luminal epithelial cells, respectively. The highest concentration of MRUs has been found in the CD24lo population, suggesting that mammary epithelial stem cells might have a basal/myoepithelial phenotype.46 It is essential to remember that the frequency of MRUs in the CD24+CD29hi and CD24medCD49fhi populations is estimated at 1/64 and 1/60, respectively. Therefore, a large majority of the cells within these populations are transit amplifying cells rather than stem cells. Furthermore, although the contents of the two candidate stem cell populations overlap, in that they both contain MRUs, it is unlikely that the identity of the non-stem cells in these two populations corresponds exactly. Thus, until more highly purified and characterized populations are available, caution must be employed when inferring the properties of mammary epithelial stem cells from these prospectively isolated populations.

Comparison of DNA and RNA content in CD24medCD49fhi stem cell candidate populations has revealed that the vast majority of these cells are actively cycling. Despite the caveat noted above, that only a small proportion of these cells are actually stem cells, this would not be an unexpected finding, because double-labelling experiments have shown that a large proportion of intestinal and mammary epithelial LRCs, which are believed to correspond to stem cells, are actively cycling.2,34

The isolation and propagation of normal human mammary progenitor cells from primary tissue has been historically limited by the lack of suitable systems that would allow the propagation of these cells in an undifferentiated state. When cultured on solid substrata, primary cultures undergo limited replication and differentiate in a process regulated by hormonal factors, extracellular matrix (ECM) and cell–cell interactions.47–50 A significant advancement was achieved in neural stem cell research when it was found that an undifferentiated multipotent population of neural cells could be grown in suspension as neurospheres. Neurospheres have been shown to consist of 4–20% stem cells, the rest of the population representing progenitor cells in various stages of differentiation.50 A similar strategy has been developed for the cultivation of undifferentiated human mammary epithelial cells, which form spherical colonies named ‘non-adherent mammospheres’. Either the sphere-initiating cells, or their undifferentiated progeny, secrete and deposit ECM proteins that contribute to the composition of mammospheres. Most of these ECM components are associated with embryonic development.41 Mammospheres have proven to be highly enriched in undifferentiated cells, to be able to form at clonal densities and do not depend on cell aggregation, as shown by limiting dilution experiments.41

Cancer stem cells

  1. Top of page
  2. Abstract
  3. Introduction
  4. Mammary epithelial stem cells
  5. Candidate populations
  6. Cancer stem cells
  7. References

Cancers are believed to arise from a series of sequential mutations that occur as a result of genetic instability and/or environmental factors. There is substantial evidence that certain types of leukaemia arise from mutations that accumulate in HSCs. Cells capable of initiating acute myeloid leukaemia (AML) in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice have a CD34+/CD38− phenotype in most AML subtypes, a phenotype similar to normal HSCs.51 The most frequent chromosomal abnormalities in AML involve the (8;21) translocation, and the same transcripts have been identified in a fraction of normal HSCs in the bone marrow. Nevertheless, these displayed a normal ability to differentiate to normal myeloerythroid cells in vitro, indicating that the translocation occurred originally in normal HSCs and that additional mutations in a subset of these cells, or their progeny, subsequently led to leukaemia.52

In solid tumours, cells are phenotypically heterogeneous and only a small proportion are clonogenic in vitro and in vivo.11,53–57 Only one in 1000 to one in 5000 lung cancer, ovarian cancer or neuroblastoma cancer cells have been found to form colonies in soft agar.11 Furthermore, large numbers of cells must typically be transplanted to form tumours in xenograft models.58 There are two possible explanations for these observations. One is that every cell is capable of proliferation and has the ability to form new tumours, but the probability of an individual cell to complete all the necessary steps is small. Conversely, it could be that only a rare, phenotypically distinct subset of cells has the capacity to proliferate significantly and form new tumours, but cells within this subset do so very efficiently. Recent evidence seems to support this second hypothesis.14,59

Work by Al-Hajj and colleagues has provided some evidence for the existence of a putative stem cell-like population within human breast cancer-derived malignant pleural effusions, defined by the presence or absence of two cell surface markers, CD24 and CD44. The CD44+/CD24− cell population lacks differentiated breast epithelial cell lineage markers and has a 10–50-fold increase in ability to form tumours in NOD/SCID mice when compared with the bulk of tumour cells. CD44+CD24−/loLin− cells isolated from tumours initiated by these cells were able to transfer the tumour to secondary and subsequent hosts, demonstrating the capacity for self-renewal.14

Using an anchorage-independent in vitro growth assay, Ponti et al. have been able to grow mammospheres from single-cell suspensions obtained from the dissociation of primary breast tumours. The vast majority of cells in culture were CD44+/CD24−, and approximately 10–20% of these showed the ability to self-renew. Mammospheres were able to initiate tumours in the cleared mammary fat pad of immunodeficient mice at 1000-fold greater dilutions than established breast cancer-derived cell lines.59 Furthermore, subpopulations of cells displaying putative stem cell features, such as containing a side-population or bearing the ability to grow in anchorage independence, have been identified in a number of established breast cancer cell lines60,61. These subpopulations have been reported to show greater resistance to both radiation at clinically relevant doses60 and to tamoxifen at high doses in vitro61. Mammosphere-forming cells within the MCF-7 cell line displayed greater tumorigenic ability61.

Translational studies have shown how a higher fraction of CD24−/CD44+ cells is associated with shorter disease-free interval and overall survival and with greater incidence of distant metastasis.62,63 Furthermore, a recent study looking into the overall gene signature of CD44+ and CD24+ cells (normal and malignant) has shown how CD44+ cells are very similar, in terms of gene expression, to stem cells, and that normal and malignant CD44+ cells are more similar to each other than to CD24+ cells from the same tissue. Moreover, tumours rich in CD44+ cells conferred a significantly worse clinical outcome and were characterized by activation of transforming growth factor (TGF)-β.64

Further evidence in support of a role for stem cells in solid tumours has also emerged from the study of brain tumours, where clonogenic cells could be identified and purified on the basis of cell surface markers and in vitro cultivation systems (neurosphere formation).13,65,66

Recent work suggests that many signalling pathways thought to be involved in the maintenance of normal stem cells are found to be mutated in human cancers, including those regulated by Wnt, β-catenin, phosphatase and tensin homologue, TGF-β, sonic Hedgehog, Notch, Bmi-1 and Oct-4.10,67–70 It has been observed that tumours induced in mice by expression of the Wnt pathway express markers of both luminal and basal/myoepithelial lineages.71 This is not seen with other oncogenes such as Ras or neu and suggests origin from a pluripotent precursor. In support of this, the number of stem cell candidates in Wnt-overexpressing mice was increased.19

The existence of cancer stem cells has significant implications in breast cancer treatment. The efficacy of chemotherapy regimens, particularly in the neoadjuvant setting, can be gauged by the proportion of tumour cells killed, as reflected clinically by a reduction in tumour size. Since cancer stem cells represent a small proportion of the cells within a breast tumour, a particular treatment modality could, theoretically, kill a large proportion of differentiated cells while leaving the tumour stem cells intact. In fact, CD34+CD38− cells isolated from patients with AML are more resistant to daunorubicin than more differentiated progenitors,72 suggesting that cancer stem cells may have relative resistance to chemotherapy regimens. As noted above, cancer stem cells within established breast cancer cell lines display resistance to both radiotherapeutic and tamoxifen treatment at clinically relevant doses60,61. The ability prospectively to identify and characterize cancer stem cells within a tissue sample will be invaluable in permitting identification of treatment regimens that efficiently eliminate cancer stem cells. It will also be instructive to determine whether the proportion of stem versus transit amplifying cells in a given tumour has implications for prognosis; initial results raise the possibility that the proportion of cancer stem cells within a breast tumour may relate to risk of local and distant recurrence.62–64

In essence, since only a small proportion of cells have the capacity to self-renew and propagate the tumour it is these, rather than transit amplifying or differentiated cells, that must be targeted to achieve long-term cure for malignant tumours, including breast cancer. One approach for optimizing such treatment might be to sensitize breast cancer stem cells to existing chemotherapy regimens. For many years multidrug resistance has been recognized as a hindrance to effective chemotherapy. This is the phenomenon whereby exposure of cancer cells to a cytotoxic compound leads to resistance not only to that compound, but also to a range of structurally unrelated drugs.73 Several mechanisms, in particular resistance to DNA damage and the efflux of toxic compounds, are also advantageous to tissue stem cells. Their activation in malignancy may reflect not only somatic mutation but also intrinsic activation in the tissue stem cells from which cancer arises. Moreover, since cancer stem cells are at the top of the differentiation hierarchy and cycle less frequently than other subpopulations of tumour cells, they therefore have a greater resistance to anti-mitotic therapies. Attention has focused on up-regulation of cell surface transporters of the ATP-binding cassette family (ABC), in particular ABCG2. This was initially identified as a transcript overexpressed in multiresistant breast cancer, hence the alternative name of breast cancer resistance protein (BCRP).74 As noted above, in order to achieve long-term remission it is cancer stem cells rather than transit amplifying cells that must be destroyed; however, there is evidence that cancer stem cells have greater resistance to chemotherapeutic agents.72 Co-administration of inhibitors of the ABC family of transporters and other inhibitors of multidrug resistance may selectively sensitize breast cancer stem cells to chemotherapy. Furthermore, signalling pathways implicated in the self-renewal of breast cancer stem cells may provide novel therapeutic targets, and this has stimulated attempts to characterize these pathways.

In conclusion, there is substantial evidence that proliferation within the normal mammary epithelium is organized in a hierarchical manner. Repopulation experiments and lineage analysis support the existence of pluripotent mammary epithelial stem cells. Cell surface markers have been identified to facilitate their identification and purification. Similar techniques have been employed to isolate prospectively breast cancer stem cells. Although a substantial amount of work remains in the further characterization of these cells, there is little doubt that these new discoveries have profound implications for the tailoring of current treatment strategies and for the development of the next generation of targeted therapies.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Mammary epithelial stem cells
  5. Candidate populations
  6. Cancer stem cells
  7. References
  • 1
    Seaberg RM, Van Der Kooy D. Stem and progenitor cells: the premature desertion of rigorous definitions. Trends Neurosci. 2003; 26; 125131.
  • 2
    Potten CS, Loeffler M. Stem cells: attributes, cycles, spirals, pitfalls and uncertainties. Lessons for and from the crypt. Development 1990; 110; 10011020.
  • 3
    Clarke MF, Fuller M. Stem cells and cancer: two faces of eve. Cell 2006; 124; 11111115.
  • 4
    Osawa M, Hanada K, Hamada H, Nakauchi H. Long-term lymphohematopoietic reconstitution by a single CD34 low/negative stem cell. Science 1996; 273; 242245.
  • 5
    Janes SM, Lowell S, Hutter C. Epidermal stem cells. J. Pathol. 2002; 197; 479491.
  • 6
    Marshman E, Booth C, Potten CS. The intestinal epithelial stem cell. Bioessays 2002; 24; 9198.
  • 7
    Morrison SJ, Qian D, Jerabek L et al. A genetic determinant that specifically regulates the frequency of hematopoietic stem cells. J. Immunol. 2002; 168; 635642.
  • 8
    Spradling A, Drummond-Barbosa D, Kai T. Stem cells find their niche. Nature 2001; 414; 98104.
  • 9
    Miyagi K, Yamazaki T, Tsujino I et al. Application of hypothermia to autologous stem cell purging. Cryobiology 2001; 42; 190195.
  • 10
    Woodward WA, Chen MS, Behbod F, Rosen JM. On mammary stem cells. J. Cell Sci. 2005; 118; 35853594.
  • 11
    Hamburger AW, Salmon SE. Primary bioassay of human tumor stem cells. Science 1977; 197; 461463.
  • 12
    Lapidot T, Sirard C, Vormoor J et al. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 1994; 367; 645648.
  • 13
    Singh SK, Clarke ID, Terasaki M et al. Identification of a cancer stem cell in human brain tumours. Cancer Res. 2003; 63; 58215828.
  • 14
    Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc. Natl Acad. Sci. U. S. A. 2003; 100; 39833988.
  • 15
    Osborne MP. Breast anatomy and development. In HarrisJR ed. Diseases of the breas, 2nd edn, Vol. 1. Philadelphia: Lippincott Williams & Wilkins, 2000; 113.
  • 16
    Henninghausen L, Robinson GW. Think globally, act locally: the making of a mouse mammary gland. Genes Dev. 1998; 12; 449455.
  • 17
    Richert MM, Schwertfeger KL, Ryder JW, Anderson SM. An atlas of mouse mammary gland development. J. Mammary Gland Biol. Neoplasia 2000; 5; 227241.
  • 18
    Williams JM, Daniel CW. Mammary ductal elongation: differentiation of myoepithelium and basal lamina during branching morphogenesis. Dev. Biol. 1983; 97; 274290.
  • 19
    Shackleton M, Vaillant F, Simpson KJ et al. Generation of a functional mammary gland from a single stem cell. Nature 2006; 439; 8488.
  • 20
    DeOme KB, Faulkin LJJ, Bern HA, Blair PB. Development of mammary tumours from hyperplastic alveolar nodules transplanted into gland-free mammary fat pads of female CH3 mice. Cancer Res. 1959; 19; 515520.
  • 21
    Hoshino K, Gardner WU. Transplantability and life span of mammary gland during serial transplantation in mice. Nature 1967; 213; 193194.
  • 22
    Smith GH, Strickland P, Daniel CW. Putative epithelial stem cell loss corresponds with mammary growth senescence. Cell Tissue Res. 2002; 10; 313320.
  • 23
    Young LJ, Medina D, DeOme KB, Daniel CW. The influence of host and tissue age on life span and growth rate of serially transplanted mouse mammary gland. Exp. Gerontol. 1971; 6; 4956.
  • 24
    Kordon EC, Smith GH. An entire functional mammary gland may comprise the progeny from a single cell. Development 1998; 125; 19211930.
  • 25
    Chepko G, Smith GH. Three division-competent, structurally distinct cell populations contribute to mammary epithelial renewal. Tissue Cell 1997; 29; 239253.
  • 26
    Smith GH. Experimental mammary epithelial morphogenesis in an in vivo model: evidence for distinct cellular progenitors of the ductal and lobular phenotype. Breast Cancer Res. 1996; 39; 2131.
  • 27
    Tsai YC, Lu Y, Nichols PW, Zlotnikov G, Jones PA, Smith HS. Contiguous patches of normal human mammary epithelium derived from a single stem cell: implications for breast carcinogenesis. Cancer Res. 1996; 56; 402404.
  • 28
    Deng G, Lu Y, Zlotnikov G, Thor AD, Smith HS. Loss of heterozygosity in normal tissue adjacent to breast carcinomas. Science 1996; 274; 20572059.
  • 29
    Frank SA, Nowak MA. Cell biology: developmental predisposition to cancer. Nature 2003; 422; 494.
  • 30
    Novelli M, Cossu A, Oukrif D et al. X-inactivation patch size in human female tissue confounds the assessment of tumor clonality. Proc. Natl Acad. Sci. U. S. A. 2003; 100; 33113314.
  • 31
    Diallo R, Schaefer KL, Poremba C et al. Monoclonality in normal epithelium and in hyperplastic and neoplastic lesions of the breast. J. Pathol. 2001; 193; 2732.
  • 32
    Stingl J, Eirew P, Ricketson I et al. Purification and unique properties of mammary epithelial stem cells. Nature 2006; 439; 993997.
  • 33
    Smith CA, Monaghan P, Neville AM. Basal clear cells of the normal human breast. Virchows Arch. A Pathol. Anat. Histopathol. 1984; 402; 319329.
  • 34
    Smith GH. Label-retaining epithelial cells in the mammary gland divide asymmetrically and retain their template DNA strands. Development 2005; 132; 681687.
  • 35
    Welm BE, Tepera SB, Venezia T, Graubert TA, Rosen JM, Goodell MA. Sca-1(pos) cells in the mouse mammary gland represent an enriched progenitor cell population. Dev. Biol. 2002; 245; 4256.
  • 36
    Goodell MA, Brose K, Paradis G, Conner AS, Mulligan RC. Isolation and functional properties of murine haematopoietic stem cells that are replicating in vivo. J. Exp. Med. 1996; 183; 17971806.
  • 37
    Clayton H, Titley I, Vivanco M. Growth and differentiation of progenitor/stem cells derived from the human mammary gland. Exp. Cell Res. 2004; 297; 444460.
  • 38
    Petersen TW, Ibrahim SF, Diercks AH, Van Den Engh G. Chromatic shifts in the fluorescence emitted by murine thymocytes stained with Hoechst 33342. Cytometry A 2004; 60; 173181.
  • 39
    Clarke RB, Spence K, Anderson E, Howell A, Okano H, Potten CS. A putative human breast stem cell population is enriched for steroid receptor-positive cells. Dev. Biol. 2005; 277; 443456.
  • 40
    Alvi AJ, Clayton H, Joshi C et al. Functional and molecular characterization of mammary side population cells. Breast Cancer Res. 2003; 5; R1R8.
  • 41
    Dontu G, Abdallah WM, Foley JM et al. In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev. 2003; 17; 12531270.
  • 42
    Spangrude GJ, Heimfeld S, Weissman IL. Purification and characterization of mouse haematopoietic stem cells. Science 1988; 241; 5862.
  • 43
    Stingl J, Eaves CJ, Kuusk U, Emerman JT. Phenotypic and functional characterization in vitro of a multipotent epithelial cell present in the normal adult human breast. Differentiation 1998; 63; 201213.
  • 44
    Stingl J, Eaves CJ, Zandieh I, Emerman JT. Characterization of bipotent mammary epithelial progenitor cells in normal adult human breast tissue. Breast Cancer Res. Treat. 2001; 67; 93109.
  • 45
    Gudjonsson T, Villadsen R, Nielsen HL, Ronnov-Jessen L, Bissel MJ, Petersen OW. Isolation, immortalization, and characterization of a human breast epithelial cell line with stem cell properties. Genes Dev. 2002; 16; 693706.
  • 46
    Sleeman KE, Kendrick H, Ashworth A, Isacke CM, Smalley MJ. CD24 staining of mouse mammary gland cells defines luminal epithelial, myoepithelial/basal and non-epithelial cells. Breast Cancer Res. 2006; 8; R7.
  • 47
    Simian M, Hiray Y, Navre M, Werb Z, Lochter A, Bissel MJ. The interplay of matrix metalloproteinases, morphogens and growth factors is necessary for branching of mammary epithelial cells. Development 2001; 128; 31173131.
  • 48
    Romanov SR, Kozakiewicz BK, Holst CR, Stampfer MR, Haupt LM, Tlsty TD. Normal human mammary epithelial cells spontaneously escape senescence and acquire genomic changes. Nature 2001; 409; 633637.
  • 49
    Muschler J, Lochter A, Roskelley CD, Yurchenco P, Bissel MJ. Division of labor among the alpha6beta4 integrin, beta1 integrins, and E3 laminin receptor to signal morphogenesis and beta-casein expression in mammary epithelial cells. Mol. Biol. Cell 1999; 10; 28172828.
  • 50
    Reynolds BA, Weiss S. Clonal and population analyses demonstrate that EGF-responsive mammalian embryonic CNS precursor is a stem cell. Dev. Biol. 1996; 175; 113.
  • 51
    Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat. Med. 1997; 3; 730737.
  • 52
    Miyamoto T, Weissman IL, Akashi K. AML1/ETO-expressing nonleukemic stem cells in acute myelogenous leukemia with 8;21 chromosomal translocation. Proc. Natl Acad. Sci. U. S. A. 2000; 97; 75217526.
  • 53
    Fidler IJ, Kripke ML. Metastasis results from pre-existing variant cells within a malignant tumour. Science 1977; 197; 893895.
  • 54
    Fidler IJ, Hart IR. Biological diversity in metastatic neoplasms: origins and implications. Science 1982; 217; 9981003.
  • 55
    Nowell PC. Mechanisms of tumour progression. Cancer Res. 1986; 46; 22032207.
  • 56
    Heppner GH. Tumor heterogeneity. Cancer Res. 1984; 44; 22592265.
  • 57
    Weisenthal LM, Lippman ME. Clonogenic and nonclonogenic in vitro chemosensitivity assays. Cancer Treat. Rep. 1985; 69; 615632.
  • 58
    Masters JR. Human cancer cell lines: fact and fantasy. Nat. Rev. Mol. Cell Biol. 2000; 1; 233236.
  • 59
    Ponti D, Costa A, Zaffaroni N et al. Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/cell progenitors. Cancer Res. 2005; 65; 55065511.
  • 60
    Phillips TM, McBride WH, Pajonk F. The response of CD24(-/low)/CD44 +  breast cancer-initiating cells to radiation. J. Natl Cancer Inst. 2006; 98; 17771785.
  • 61
    Cariati M, Naderi A, Brown JP et al. Alpha-6 integrin is necessary for the tumourigenicity of a stem cell-like subpopulation within the MCF-7 breast cancer cell line. Int. J. Cancer 2007; 122; 298304.
  • 62
    Abraham BK, Fritz P, McClellan M, Hauptvogel P, Athelogou M, Brauch H. Prevalence of CD44+/CD24-/low cells in breast cancer may not be associated with clinical outcome but may favor distant metastasis. Clin. Cancer Res. 2005; 11; 11541159.
  • 63
    Glinsky GV, Berezovska O, Glinskii AB. Microarray analysis identifies a death-from-cancer signature predicting therapy failure in patients with multiple types of cancer. J. Clin. Invest. 2005; 115; 15031521.
  • 64
    Shipitsin M, Campbell LL, Argani P et al. Molecular definition of breast tumor heterogeneity. Cancer Cell 2007; 11; 259273.
  • 65
    Ignatova TN, Kukekov VG, Laywell ED, Suslov ON, Vrionis FD, Steindler DA. Human cortical glial tumours contain neural-stem-like cells expressing astroglial and neuronal markers in vitro. Glia 2002; 39; 193206.
  • 66
    Hemmati HD, Nakano I, Lazareff JA et al. Cancerous stem cells can arise from pediatric brain tumours. Proc. Natl Acad. Sci. U. S. A. 2003; 100; 1517815183.
  • 67
    Beachy PA, Karhadkar SS, Berman DM. Tissue repair and stem cell renewal in carcinogenesis. Nature 2004; 432; 324331.
  • 68
    Valk-Lingbeek ME, Bruggeman SW, Van Lohuizen M. Stem cells and cancer; the polycomb connection. Cell 2004; 118; 409418.
  • 69
    Hochedlinger K, Yamada Y, Beard C, Jaenisch R. Ectopic expression of Oct-4 blocks progenitor-cell differentiation and causes dysplasia in epithelial tissues. Cell 2005; 121; 465477.
  • 70
    Liu S, Dontu G, Mantle ID et al. Hedgehog signaling and Bmi-1 regulate self-renewal of normal and malignant human mammary stem cells. Cancer Res. 2006; 66; 60636071.
  • 71
    Li Y, Welm B, Podsypanina K et al. Evidence that transgenes encoding components of the Wnt signaling pathway preferentially induce mammary cancers from progenitor cells. Proc. Natl Acad. Sci. U. S. A. 2003; 100; 1585315858.
  • 72
    Costello RT, Mallet F, Gaugler B et al. Human acute myeloid leukemia CD34+/CD38- progenitor cells have decreased sensitivity to chemotherapy and Fas-induced apoptosis, reduced immunogenicity, and impaired dendritic cell transformation capacities. Cancer Res. 2000; 60; 44034411.
  • 73
    Norgaard JM, Olesen LH, Hokland P. Changing picture of cellular drug resistance in human leukemia. Crit. Rev. Oncol. Hematol. 2004; 50; 3949.
  • 74
    Doyle LA, Yang W, Abruzzo LV et al. A multidrug resistance transporter from human MCF-7 breast cancer cells. Proc. Natl Acad. Sci. U. S. A. 1998; 95; 1566515670.