The quest for the melanoma stem cell: still more questions than answers

Cutaneous malignant melanoma is generally accepted as being clonal despite presenting molecular and cellular heterogeneity. These skin melanomas can be very aggressive and often display multi-drug-resistance characteristics. Molecular and cellular mechanisms occurring during embryonic development, homeostasis and oncological transformation were found to share similarities. Before addressing the question of the melanoma stem cell, let us define the melanoblast during embryonic development and the melanocyte stem cell during homeostasis. During embryonic development, a very small number of melanoblasts are determined from a subpopulation of pluripotent neural crest cell derivatives. Substantial proliferation and migration of melanoblasts are required for the colonization of the entire skin by birth. At the end of embryogenesis, melanoblasts are found in the dermis and epidermis. In the epidermis, they are located between hair follicles, in the bulge and bulb of the hair. After reaching their final destinations, the proliferation of melanoblasts slows down. Around birth, skin melanoblasts fully differentiate into melanocytes; the terminal differentiation of melanoblasts can be defined by the production of active tyrosinase, the production of melanin in melanosomes and the transfer of melanin to keratinocytes. The establishment of the melanocyte lineage is reproducibly dependent on a spatial and temporal harmony of various cellular and molecular events. Cell renewal involves the activation of a limited number of quiescent stem cells, the melanocyte stem cells, residing in their niche in the bulge of the hair. These bipotent cells divide asymmetrically, giving rise to a quiescent stem cell and to the first amplifying cell; this amplifying cell can proliferate actively for a limited number of cell cycles before terminal differentiation. The cellular origin of melanoma remains unclear and there are diverse possibilities. A straightforward and possibly simplistic view is that they may be derived from non-fully determined melanoblasts, melanoblasts, dermal/epidermal melanocytes and/or melanocyte stem cells. The cause of the initial transformation of such cells can be intrinsic (e.g., genetic predisposition), extrinsic (e.g., exposure to a physical or chemical agent), or both. After melanoma initiation (including early proliferation and immortalization), which is mainly cell-autonomous, progression requires an important non cell-autonomous component. Melanoma progression may be dependent on the initial site of transformation, and also on heterotypic cell–cell interactions, angiogenesis, immune responses and possibly other factors. The microenvironments, especially as concerns these factors, of individual melanoma cells in the tumor differ partly due to their location within the tumor; in particular, they differ in their distances from the periphery and the center of the tumor. As a result, various melanoma cells exhibit diverse repertoires of proteins, lipids and sugars in the same tumor. Such heterogeneity is associated with various cellular behaviors including cell death, cell proliferation, senescence and quiescence. Most cells are proliferating cells. There are nevertheless senescent and quiescent cells that we can call non-dividing cells. Are such non-dividing cells numerous? Are they able to change their behavior? Are they able to regenerate a mixed population? Are they located in a specific place/environment in the tumor? Melanoma cells show various degrees of chemo/radio/immuno/hormono resistance. In addition to the obvious issue of differential accessibility of melanoma cells within the tumor to treatment, there may also be intrinsic cellular diversity. The mechanism of action of a treatment and its efficiency clearly depend on either specific cell behavior or on the molecular content of the cells. It also appears that various therapeutic treatments seem to be more effective against proliferative cells than resting cells; this indicates that the resistant cells are the non-dividing cells. After therapeutic treatment, most melanoma cells are killed, but some survive, leading to a resurgent melanoma. The surviving cells are sometimes called ‘residual cells’, suggesting that they are limited in number. However, it is not clear whether all residual cells are non-dividing cells, or that they ‘hide’ to escape from the treatment. After the selection period, are they able to produce a diversity of behavior including the capacity to proliferate and to rest? During normal development and homeostasis, spatial and temporal controls are specifically required. These controls do not operate during melanoma progression/resurgence. However, molecular and cellular parallels can be drawn for development/homeostasis and melanoma progression/resurgence. This led to the start of the quest for melanoma stem cells. The definition of cancer stem cells is known: a cell capable of self-renewal, with indefinite proliferation and differentiation potential. Without raising the issue of the meaning of differentiation in developmental terms, this definition is based on a hypothesis, which should be experimentally demonstrated to avoid the genesis of a ‘myth’. Indeed, many other terms for melanoma stem cell have been used to avoid philosophical debates, terms such as melanoma-initiating cells or individual melanoma-propagating cells. Such terms must be carefully defined and vetted to prevent confusion. Melanoma stem cells may exist in progressing and resurgent melanomas for a certain type of melanoma (nodular, acral, etc.). Progressing and resurgent melanoma stem cells should possess the characteristics of the given definition (self-renewal, with indefinite proliferation and differentiation potential) plus others, which are potentially different for progressing and resurgent melanoma stem cells. Are these differences of importance for subsequent therapeutic treatment? It is certainly an exciting period and all these questions cannot be answered without addressing them experimentally. Melanoma stem cells are currently defined according to their ability to grow in immunodeficient mice or by their molecular behavior. At the molecular level, not one but four subpopulations of melanoma stem cells have been described: CD20-positive cells, CD133-positive cells, side population cells excluding dye using ABC transporters, and slow cycling cells (Zabierowski and Herlyn, 2008). By xenografting human tumors into immunodeficient mice, Frank’s laboratory was able to isolate a subpopulation of cells growing in vivo that produced the surface protein ABCB5, which is involved in multi-drug resistance and is produced by quiescent cells (Schatton et al., 2008). They found that only one of every 106 cells was growing in vivo, indicating that the emergence of such cells was a rare event. It would be valuable to determine the melanoma stem cell function of ABCB5 by, for example, specific inactivation in a spontaneous immunocompetent mouse melanoma model known to produce ABCB5. The same year, Morrison’s laboratory (Quintana et al., 2008) reported that 27 of 100 human melanoma cells (and not one of 106 cells) were able to grow in immunodeficient mice. It appears that the growth of these melanomas is independent of a series of proteins found at the surface of the cells. These antigens were A2B5, KIT, CD44, CD49B, CD49D, CD49f, CD54, CD133, CD166, CDH1, HNK-1, L1CAM, MCAM, CDH2 and p75. It also appears that the growth of these cells in immunodeficient mice was also independent of the presence of ABCB5 (6th SMR meeting in Boston; Fisher et al., 2010). Thus, there is a discrepancy between the findings of these two groups. There are at least two significant differences in the experimental design. (i) The immunodeficient mice used were different: NOD/SCID mice were used by Schatton et al. (2008) and NOD/SCID IL2rγ−/− by Quintana et al. (2008). (ii) The cells were injected subcutaneously in both experiments, but were ‘protected’ by the presence of Matrigel in the experiments performed by Quintana and colleagues. One explanation for the high number of melanoma stem cells may be associated with the ability of cells to switch reciprocally from a proliferative to a non-proliferative status. Such a possibility is highly probable, as supporting evidence can be found in the literature. For instance, the Brn2 (Pou3f2)–Mitf equilibrium may be important in controlling the division of non-dividing, early dividing, transit amplifying and differentiated cells (Goodall et al., 2008). Recently, Bosenberg’s laboratory identified an alternative melanoma ‘stem cell’-like subpopulation which they called melanoma-propagating cells, or MPCs (Held et al., 2010). According to the parallel with normal embryonic development and homeostasis, these cells may correspond to the transit-amplifying cells during homeostasis. The experiments did not include human melanoma cells but did include three spontaneous mouse melanoma models: (i) Tyr::CreERt2/°; Cdkn2aloxP/loxP; PtenloxP/loxP; β-cateninex3−loxP/+, (ii) Tyr::CreERT2/°; Cdkn2aloxP/loxP; PtenloxP/loxP and (iii) Tyr::CreERt2/°; BrafCA−loxP/+; PtenloxP/loxP. Melanoma cells were separated from the tumors and FACS-sorted for CD34 and p75, giving rise to four subpopulations: CD34+ p75+, CD34+ p75−, CD34− p75+, and CD34− p75−. These four classes of cells were subjected to five tests, two in vivo and three in vitro: growth when seeded at one cell per well, assessment of the expression profile for CD34 and p75 after growth as single cells in vitro, evaluation of resistance to drugs (temozolomide and cisplatin) in vitro, tumor formation when injected as single cell into nude mice in the presence of Matrigel, and determination of the expression of CD34 and p75 by these tumors. Held et al. (2010) observed that cells producing p75 were not able to grow in culture or in mice and were sensitive to drugs. Therefore, inducing p75 expression in melanomas or maintaining p75 in melanocytes may be beneficial! These findings raise the issue of the status of p75 (and also CD34) in cells of the melanocyte lineage during development and homeostasis. A p75− cell can grow in vitro and in vivo irrespective of the presence or absence of CD34, although the probability of growth was higher when CD34 was present at the surface (100%) compared with absent (70%). The maintenance (or lack) of CD34 and p75 production after in vivo or in vitro proliferation was evaluated. CD34+ p75− cells produce only CD34+ p75− cells. The proliferation of CD34− p75− cells resulted in a heterogeneous population of proliferative CD34+ p75− and CD34− p75−cells, and non-proliferative CD34+ p75+ and CD34− p75+ cells. Moreover, CD34+ p75− cells appeared to be the most resistant to drugs. These results led the authors to put the spotlight on p75− cells and CD34− cells; it might be risky to base a classification on the absence of a surface marker expression. It might have been more appropriate to classify these based on relative expression levels such as ‘high’ and ‘low’. The good news is that the antibodies directed against p75 and CD34 are robust. From this battery of five tests, no major difference was observed between the three spontaneous mouse melanoma models, which is surprising; the function of these proteins is clearly different and it may hide a lack of sensitivity in some of the assays. However, in the presence of a mutated form of β-catenin, melanoma cells appear to be slightly more resistant to drugs. The immunological response was not addressed in that study, although it is undoubtedly of crucial importance. We can imagine two key experimental approaches. The first approach would be to inject melanoma cells into immunocompetent mice. The second would be to produce primary melanoma on a NOD/SCID IL2rγ−/− background. We are convinced that this type of issue will have to be addressed in collaboration with our immunologist colleagues. Held et al. (2010) have enriched the list of melanoma-associated stem cell-like cells by using their own markers, in vitro tests and mouse melanoma models. Once their stem cell-like criteria in human melanomas have been evaluated, we will be one step closer to elucidating the ‘melanoma stem cell potential’– essential if we wish to improve melanoma therapy.

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