Appraisal of progenitor markers in the context of molecular classification of breast cancers

Clinical management of breast cancer relies on case stratification, which increasingly employs molecular markers. The motivation behind delineating breast epithelial differentiation is to better target cancer cases through innate sensitivities bequeathed to the cancer from its normal progenitor state. A combination of histopathological and molecular classification of breast cancer cases suggests a role for progenitors in particular breast cancer cases. Although a remarkable fraction of the real tissue repertoire is maintained within a population of independent cell line cultures, some steps that are closer to the terminal differentiation state and that form a majority of primary human breast tissues are missing in the cell line cultures. This raises concerns about current breast cancer models.

Th e Heisenberg uncertainty principle has a great impact on medical research by drawing our attention to the bias introduced by our experimental tools. In a recent issue of Breast Cancer Research, Keller and colleagues [1] report an example of this principle: sustained propagation of large numbers of cells, through the establishment of cell lines, disrupts the normal balance between diff erentiated cells and their progenitors, as observed in fresh biological specimens. Th e work of these authors contributes another piece in a contentious fi eld that combines tissue morphology and immuno histo chemical phenotypes [2,3], molecular classifi cation of breast cancer tissues [4], and cell biological assays aimed at the tumor-initiating cell (TIC) phenotype [5]. Sorting cells according to their respective cell surface markers, CD44 + /CD24 −/low , results in the enrichment of TIC activities, including mammospheres [6] and trans plan ta tion effi ciency in mouse xenografts [7]. Establishing xeno graft growth could be the product of several system-specifi c selections other than breast progenitor phenotypes. However, further molecular profi ling of these cell populations -in which CD44 + /CD24 −/low -sorted cells expressed low levels of luminal diff erentiation markers (such as MUC1, CD24, or CDH1) and elevated levels of epithelial-mesenchymal transition markers (such as VIM, collagens, TWIST1, SNAI1/2, and Zeb1/2) -indicated a link between epithelial-mesen chymal transi tion, TIC, and basal-like [6,8] or claudin-low [9,10]-specifi c breast cancer molecular subtypes. More recently, however, a more comprehen sive interrogation of pluripotent self-renewal identifi ed a population high for CD24, or luminal progenitors [9,[11][12][13], capable of giving rise to mesenchymal or basallike tumors, at least in the context of BrCa1 mutations. Given the variability of single markers within single individuals, the diff erent sensitivities each cell biological assay presents with, and the consistency across other genes (which are more likely to be drivers of the phenotypes rather than eff ective surrogate markers), the more recent work presents compel ling evidence that, admittedly, neither CD24 nor CD44 populations are homogenous or contain pure diff erentiated of progenitor populations, respectively. A hypothetical linear sequential diff erentiation track that would fl ip back and forth with respect to CD24 expression and appear as a hidden subpopulation in a majority of cells from another diff erentiation stage could explain this discrepancy. A more comprehensive whole-genome mRNA profi ling analysis of the relatedness between luminal progenitors (CD49f hi / EpCAM + ), stem cells (CD49f hi /EpCAM − ), and CD44 + / CD24 −/low popula tions is necessary to assess this hypothesis. Th is point is emphasized by the fact that stem cell marker ALDH1 [14] is expressed by only some of the cells in either fractions described above yet ALDH1 + cells exhibit the greatest TIC capacity.
Th e underlying hypothesis assumes that, within the dynamic steady state of breast epithelial maintenance, self-renewal, and diff erentiation (as it responds to

Abstract
Clinical management of breast cancer relies on case stratifi cation, which increasingly employs molecular markers. The motivation behind delineating breast epithelial diff erentiation is to better target cancer cases through innate sensitivities bequeathed to the cancer from its normal progenitor state. A combination of histopathological and molecular classifi cation of breast cancer cases suggests a role for progenitors in particular breast cancer cases. Although a remarkable fraction of the real tissue repertoire is maintained within a population of independent cell line cultures, some steps that are closer to the terminal diff erentiation state and that form a majority of primary human breast tissues are missing in the cell line cultures. This raises concerns about current breast cancer models. lactation-related breast augmentation) and the successive involution, cancer arises from specifi c intermediary states and somehow maintains the molecular profi le of its cell of origin [15]. It is indeed remarkable that breast cancer cell lines can be subdivided into the same molecular subtypes as primary cancer [16]. However, these observations are good in only fi rst-degree approximation since molecular profi ling of tissue whole mass cannot capture the incredible heterogeneity of cancer populations [17]. Although cellular heterogeneity severely hinders our ability to assign stem cell phenotype and markers on the single-cell level, recent advances in expression profi ling of single cells [18] may shed more light on this mystery. Nevertheless, population analysis still informs our under standing of TIC markers. By employing fl uorescence-activated cell sorting (FACS) and carefully appraising each marker, Keller and colleagues [1] fi nd that CD44 is a relatively promiscuous marker whereas EpCAM, CD24, and CD49f demonstrate extensive heterogeneity within cultured populations of the investigated cell lines. Interpreting these results in terms of self-renewal and morphological phenotypes (such as mammosphere and xenograft growth effi ciency) or mesenchymal appearance further demonstrated the complexity of diff erentiation states, as judged by a handful of markers. Th e authors benchmark the progenitor cell population by assuming that the overall self-renewal phenotype of a given cell line's mixed population should correlate with the abundance of the particular population allegedly capable of the self-renewal. Further support to their cell fraction-self-renewal assignment is still needed from direct cell population subfractionation by using FACS similar to that performed by Al-Hajj and colleagues [7].
Stem cell 'purifi cation' may gain insight from a biochemical purifi cation scheme, in which successive fractionation results in diminishing yields and increasing specifi c activity. For example, consider the purifi cation of mitosis-promoting factor (MPF). For a long time, confl icting reports claimed that MPF depends on or is attenuated by phosphatase activity. Ultimately, it was recog nized that the activity was dependent, in a sequential fashion, on both the kinase activity of MPF and the phosphatase activity of CDC25. It is agreed that, in normal tissue, progenitors are regulated by signals from their respective niche. However, assays for the activity of self-renewal, which not only mimic the niche more eff ectively but potentially involve mixing back the isolated cells with other cell populations at the onset of the assay (much to the same eff ect as mixing homogenous basal transcription factors in an in vitro reconstituted transcription reaction), have not yet been employed. Consequently, as was the case for MPF, it is possible that all current reports rely on mixed populations. In support of this notion, the claudin-low candidate TIC-like cancers are also elevated with leukocyte infi ltrate signature [10], which could refl ect the role of heterotypic interactions that regulate progenitor phenotype in vivo, but not in current model systems. Such rigorous reconstitution experiments, with trackable cell progeny, may off er new handles with which to control, rather than monitor, breast stem cells.

Competing interests
The author declares that he has no competing interests.