Our experiments suggest that melanoma does not adopt a hierarchy consisting of a minor subpopulation of tumorigenic cells and a majority population of non-tumorigenic cells. Melanomas consistently contained high frequencies of tumorigenic cells, irrespective of whether they were primary cutaneous or metastatic melanomas, whether they were from stage II, III, or IV disease, and whether they were obtained directly from patients or after xenografting (, , , ). All tumorigenic cells appeared to have the capacity to proliferate indefinitely upon serial transplantation assay ().
We have not been able to identify any marker that robustly distinguishes tumorigenic from non-tumorigenic melanoma cells despite examining 85 markers and carefully studying the tumorigenic potential of cells that differ in their expression of 22 heterogeneously expressed markers, including ABCB5 (), CD271 (; Table S1
) and CD133 (Shackleton et al., 2009
). For all of the markers we studied, tumors with similar growth rates readily arose from all fractions of cells (; Figure S4
). Despite subdividing melanoma cells using many markers we have been unable to identify any large subpopulation of melanoma cells that lacks tumorigenic potential. Thus, tumorigenic capacity is not restricted to a small subpopulation of melanoma cells but is widely shared among phenotypically diverse cells.
Many phenotypically distinct melanoma cells had the capacity to form tumors that recapitulated the phenotypic diversity of the tumors from which they derived (; Figure S5-10
). This suggests that tumorigenic cells appeared to undergo reversible changes in the expression of many markers in vivo. This contrasts with models that attribute phenotypic heterogeneity to the hierarchical differentiation of cancer stem cells into non-tumorigenic progeny or to irreversible genetic changes that arise through clonal evolution.
Our results are compatible with the idea that tumorigenic competence might reflect a reversible state in melanoma. Studies of breast cancer cell lines have suggested that tumorigenic activity correlates with the capacity to undergo an epithelial to mesenchymal transition and that cells might reversibly undergo such transitions (Mani et al., 2008
). Studies of other cell lines have suggested that therapy resistance can also reflect a reversible state (Sharma et al., 2010
). Recent studies of melanoma cell lines have indicated that the JARID1B histone demethylase, Brn2, and pigment are reversibly turned on and off within lineages of melanoma cells in a manner related to cell function (Pinner et al., 2009
; Roesch et al., 2010
). Transient exposure of glioblastoma cells to perivascular nitric oxide confers tumorigenic competence and stem cell properties, raising the possibility that these attributes reflect a reversible state in brain tumor cells (Charles et al., 2010
). These studies make the prediction that in some cancers many cells will be capable of forming phenotypically diverse tumors, without robust hierarchical organization. Our study comprehensively tests this prediction in tumors from patients in vivo, finding that many phenotypic differences among melanoma cells reversibly change within lineages of tumorigenic cells rather than being hierarchically organized.
Although no marker robustly distinguished tumorigenic from non-tumorigenic melanoma cells, we observed little tumorigenic activity among CD271+
cells from two primary cutaneous melanomas. In this regard, our data are similar to results from primary mouse melanomas (Held et al., 2010
) in that both studies found tumorigenic activity in a high percentage of single cells, but CD271-
cells were more likely to form tumors. However, neither our results nor the results from Held et al. were consistent with the cancer stem cell model because Held et al. found that the tumorigenic cells they studied often did not recapitulate the heterogeneity of parental tumors, and the CD271+ cells with limited tumorigenic activity in two tumors in our study represented only 2 to 12% of tumor cells, a minor subpopulation of cancer cells.
Our results with CD271 are different from the results reported by Boiko et al. (2010)
even though both studies used the same anti-CD271 antibody, both studied a similar spectrum of melanoma stages (mainly stage III), and both studied a combination of xenografted tumors and tumors obtained directly from patients. The most obvious potential explanation for the difference in results lies in the different assays used in the two studies: different enzymatic dissociation conditions (22 minutes in our study versus up to 3 hours in their study), different injection sites (subcutaneous versus intradermal) and different recipient mice (NSG versus Rag-/-
). An average of 1 in 50,000 unfractionated melanoma cells formed tumors in the study performed by Boiko et al. (using 4 stage III, 1 stage IV, and 1 stage II melanoma, see Suppl. Table 3 in Boiko et al., 2010
). In contrast, when we transplanted unfractionated melanoma cells directly from 6 stage III patients in our prior study, an average of 1 in 4 cells formed tumors (Quintana et al., 2008
). In our current study, an average of 28% of single, unfractionated melanoma cells obtained directly from 5 stage III melanoma patients () and 17% (1 in 6) of melanoma cells obtained directly from a stage II melanoma patient () formed tumors. Thus, the assay we used appears to be approximately 10,000-fold more sensitive than the assay used by Boiko et al. Using this more sensitive assay, we find that CD271-
cells have at least as much ability to form tumors as CD271+
cells. It will now be critical for other labs to independently assess whether they also observe tumor formation by CD271-
While our results argue against the cancer stem cell model in melanoma, they do not mean that other cancers do not follow a stem cell model. We and others have found that most chronic myeloid leukemias (Eisterer et al., 2005
; Jamieson et al., 2004
; Neering et al., 2007
; Oravecz-Wilson et al., 2009
) and acute myeloid leukemias (Bonnet and Dick, 1997
; Lapidot et al., 1994
; Yilmaz et al., 2006
) do follow a cancer stem cell model in which leukemogenic cells are rare, phenotypically distinct from the vast majority of other leukemia cells, and robustly hierarchically organized. It will be critical to determine which cancers follow a stem cell model and which do not, so therapies designed to target rare subpopulations of cells are not inappropriately tested in patients whose disease is driven by many diverse cancer cells.
If a marker is identified in future that robustly distinguishes tumorigenic from non-tumorigenic melanoma cells, melanoma would still be quite different from cancers, such as myeloid leukemia, that follow a stem cell model. Our observation that many markers are reversibly expressed by tumorigenic melanoma cells contrasts with the obvious morphologic and phenotypic differences between leukemogenic and non-leukemogenic cells. Thus, even if some melanomas do contain a hierarchy, it would be a shallow hierarchy that includes abundant and diverse tumorigenic cells rather than a steep hierarchy driven by rare tumorigenic cells, as described so far in cancers found to follow a stem cell model.
Some have suggested that cancer stem cells might be distinguished from non-tumorigenic cancer cells by reduced immunogenicity, allowing them to proliferate more extensively by escaping immune detection (Schatton and Frank, 2009
). However, this hypothesis is not testable in human cancers because they cannot be transplanted autologously into patients or into immunocompetent mice. Immunocompetent mice mount a powerful xenogeneic immune response against human cells, making it impossible to assess whether a failure to engraft reflects immune rejection or an intrinsic lack of tumorigenic capacity. Furthermore, no xenotransplantation model recapitulates the autologous anti-cancer immune response that occurs in some patients against their own cancers because the xenogeneic immune response is far more powerful and driven by very different mechanisms than autologous immune responses. For these reasons, thirty years of research has found that the preferred system for studying the potential of normal human hematopoietic stem cells (Ito et al., 2002
; Shultz et al., 2005
) and human leukemic stem cells (Agliano et al., 2008
; Sanchez et al., 2009
) is NSG mice that are not only highly immunocompromised, but also irradiated to further promote the engraftment of human cells. The failure of human hematopoietic stem cells and leukemic stem cells to engraft in immunocompetent mice does not mean theses cells are normally regulated by autologous immune responses in patients because the xenogeneic immune response that rejects these cells from mice uses very different mechanisms.
To identify the spectrum of human cancer cells that have the potential to contribute to disease, these cells must be studied in highly immunocompromised mice. Once the spectrum of cells capable of contributing to disease is identified, a separate and context-dependent question concerns which of these cells are actually fated to contribute to disease in a patient. This question can only be addressed in mouse cancers because no xenograft model reflects the anti-cancer immune response, or certain other aspects of the environment, in patients.
Our results demonstrate that phenotypic heterogeneity in melanomas obtained from patients is largely driven by reversible changes in a broad range of markers that turn on and off within lineages of tumorigenic cells. This phenotypic plasticity contrasts with both the cancer stem cell and clonal evolution models, which largely attribute heterogeneity to irreversible epigenetic and genetic changes. So while clonal evolution occurs in many cancers, including melanoma, and some cancers follow a stem cell model, our results raise the possibility that pervasive phenotypic plasticity is an independent source of heterogeneity in many cancers.