The more things change ... the more things change: developmental plasticity of tumor-initiating mammary epithelial cells

In our haste to find and eliminate breast cancer stem cells, it appears as though we may have missed something. Contrary to current thought, a recent paper by Meyer and colleagues demonstrates developmental plasticity of breast cancer cells with respect to the CD24 cell surface marker, such that CD44pos; CD24pos and CD44pos; CD24low/- cells are able to give rise to one another in an activin/nodal-dependent manner, and that cells derived from single cells of either phenotype are capable of forming tumors as xenografts. If confirmed clinically, these data imply that simply targeting the CD44pos; CD24low/- breast cancer stem cell for breast cancer treatment may be destined to fail unless this plasticity is taken into account and prevented.

A recent paper by Meyer and colleagues reminds us of this statement, and suggests we may have forgotten something in our rush to exploit the fi rst set of breast cancer stem cell markers identifi ed [1]. Namely, a full analysis of the developmental and tumor-initiating potential of the CD44 pos ;CD24 pos breast cancer cell.
In 2003, two cell surface markers -CD44 high and CD24 low/--were associated with breast cancer stem cells [2]. In xenograft transplantation studies, this cell population regenerated tumors at high frequency whereas other cell populations were depleted for this function.
Subsequent work in breast cancer cell lines showed that CD44 high ;CD24 low/cells were present in culture, and were also tumorigenic upon transplantation, with CD24 positivity being associated with decreased invasiveness (for example [3,4]). Naturally, this work set off a fl urry of activity to characterize the CD44 high ;CD24 low/population molecularly relative to other populations present in tumors [5][6][7][8], and to evaluate their response to treatment (for example [9,10]).
In the wake of this fl urry of activity, it appears we may have forgotten something -to determine the full develop mental and tumor-initiating potential of the CD44 pos ;CD24 pos cell. Th e recent paper by Meyer and colleagues confi rms that CD44 pos ;CD24 low/cells in a number of cell lines can give rise to CD44 pos ;CD24 pos cells, and can yield total populations characteristic of the parental line [1]. Th is fi nding is not surprising, and in fact is as expected for a cancer stem cell. Using fl ow cytometry and single cell culture, however, these authors went on to show that the converse can also occur -CD44 pos ;CD24 pos cells can give rise to their CD44 pos ;CD24 low/counterparts, and are subse quently also capable of initiating tumors as xenografts with high effi ciency. Further, their paper shows that the developmental potential for either CD44 pos cell population to regenerate the other was dependent on activin/nodal signaling.
While the analysis was limited to established cell lines, what these data imply is that the status of CD24 is dynamically regulated in a developmental context, and suggests that the CD24 status may ultimately be immaterial as to whether or not the CD44 pos population is capable of initiating tumors. In addition, these data also imply that current eff orts by many groups to develop agents that specifi cally target the CD44 high ;CD24 low/population may be destined to fail unless activin/nodal signaling is also prevented.
Aside from the potential implications on translational research, if confi rmed clinically, these results beg the

Abstract
In our haste to fi nd and eliminate breast cancer stem cells, it appears as though we may have missed something. Contrary to current thought, a recent paper by Meyer and colleagues demonstrates developmental plasticity of breast cancer cells with respect to the CD24 cell surface marker, such that CD44 pos ;CD24 pos and CD44 pos ;CD24 low/cells are able to give rise to one another in an activin/nodal-dependent manner, and that cells derived from single cells of either phenotype are capable of forming tumors as xenografts. If confi rmed clinically, these data imply that simply targeting the CD44 pos ;CD24 low/breast cancer stem cell for breast cancer treatment may be destined to fail unless this plasticity is taken into account and prevented. question of why we tend to consider mammary tumorinitiating cells as static entities given the fact that the plasticity of normal mammary epithelial cells is, in some circles, the stuff of legends [11].
In the normal gland, plasticity comes in a couple of diff erent forms. First, cellular plasticity -the character of a given cell can change dramatically over the course of gland development (for example, virgin ducts versus preg nancy, or lactation versus involution), and in response to treatment with a bioactive agent (for example, hormones, growth factors). Similarly, there is developmental plasticity -the observation that subsets of mammary epithelial cells retain the ability to give rise to multiple cell types at defi ned phases of development, although they do not express this ability until needed.
As an example of cellular plasticity, cells already present within the duct of a virgin mouse (and probably of human) in early pregnancy are induced to proliferate and ultimately give rise to alveolar structures capable of producing copious amounts of milk. At least some of these cells can be parity identifi ed after the fact using elegant genetic tagging methods sensitive to at least some degree of alveolar diff erentiation. Transplantation of these tagged populations suggests that a subset of diff erentiated alveolar epithelial cells survive the involution process after weaning and retain a high degree of regenerative and multilineage diff erentiation capacity upon transplantation [12,13].
With respect to developmental plasticity, we know that regenerative stem cells are present throughout the mature mammary gland in the virgin animal. Small fragments of duct derived from any portion of the gland are capable of regenerating a functional mammary gland when transplanted into a mammary fat pad lacking its endogenous epithelium [14,15]. If a fragment of duct is transplanted into an intact mammary fat pad already containing epithelium, however, it does not regenerate. Further, actively growing terminal end buds in the mammary gland, which by defi nition contain regenerative stem cells, do not run into each other and, in fact, are regularly spaced from one another throughout the mammary gland [14].
Th e inference is that regenerative stem cells resident in the mature duct are not generally actively engaged in stem cell behaviors, and are strongly growth-inhibited by the presence of neighboring normal mammary epithelium. Th e behavior of regenerative stem cells is thus entirely dependent on the environment in which it fi nds itself. Further, there is high probability that gene expression in an actively regenerating stem cell is probably quite diff erent from gene expression in a quiescent stem cell.
If normal mammary epithelial cells are plastic, why should we not expect malignant epithelium to share this characteristic?
We are clearly in desperate need of new, rigorously validated, markers of normal and malignant stem cells. Perhaps most importantly, however, we need to ensure that newly emerging therapeutics intended to target tumor-initiating cancer stem cells are evaluated carefully for their ability to eliminate all sources of such cells completely, lest they fi nd a way to express the developmental plasticity with which they appear to be endowed.
Change -count on it.