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Clinical Updates

Christelle Charbel

Melanoma-initiating Cells: Do They Exist?

Christelle Charbel

Wednesday, February 16, 2011

The cancer stem cell (CSC) theory derives from data obtained from malignant hemopathies, and postulates that solid tumors can be viewed as the result of a central abnormality in organ stem cells.1 In the CSC model, cancers are composed of functionally distinct cells: (i) those with the potential for tumor formation and (ii) those that derive from tumorigenic cells and may even retain some proliferative activity, but have lost the potential to form tumors. Cancers (myeloid melanoma, teratocarcinoma, breast cancer, etc) that follow a CSC model contain tumorigenic cells, as well as non-tumorigenic cells that derive from them. Central to the CSC model is the idea that non-tumorigenic cells in a cancer derive from parent tumorigenic cells in a hierarchical and stable manner that parallels, in concept, the development of differentiated cells from stem cells in normal tissue development and homeostasis. Normal stem cells and tumorigenic cancer cells arising from the same tissues need not express the same markers.2

Based on this hypothesis, researchers have conducted studies of several malignancies, including melanoma. Using various tools and methods based on in vivo xenotransplantation (injection of human melanoma cells into immunosuppressed mice) and in vitro assays (melanospheres culture and investigation of their stemness), they found a subset of cells believed to represent melanoma stem cells.

Thus, it is presently unclear what role a sufficiently characterized population of melanoma stem cells plays in cancer promotion and progression. Here, we review the emerging melanoma stem cell model, and discuss the phenotypic characterization of melanoma-initiating cells (MICs).

Definition and Concept of Melanoma-initiating Cells

If we view CSCs as being similar to physiological stem cells, then they should be capable of self-renewal and differentiation, having gained the potential for unlimited proliferation - a process through which they could cause tumor growth (Figure 1).

 

Figure 1. CSCs are characterized by two defining properties: (i) the ability for long-term self-renewal and tumorigenic growth; and (ii) the potential to give rise to differentiated tumor bulk populations devoid of CSC characteristics.

Developmental biology allowed the discovery of cell-surface markers in cancer cells that phenotypically distinguish stem cells from their differentiated progeny, and tumorigenic cells from non-tumorigenic cells. CD133 (prominin1) was originally identified as a marker of tumorigenic cells in brain cancers that follow a CSC model.3 More recently, the same marker was used to enrich tumorigenic cells in colon cancer4 and in melanoma.5 ABCB5 (subfamily B [MDR/TAP]) is a human ABC transporter principally expressed in the skin and a chemoresistance mediator that has been recently been identified as a marker of MICs.6 CD271 (neural crest nerve growth factor receptor) has been found in a number of human neural-crest-derived tissues and in some human cancers, including melanomas, which, like normal melanocytes, derive from the neural crest lineage.7

Are There Stem Cells in Human Melanoma?

Accumulating evidence supports the presence and involvement of "stem cells" in the tumor initiation and progression, chemoresistance and therapeutic failure seen in human melanoma.8 Similar to physiological tissues, melanomas consist of phenotypically heterogeneous cell populations.9 Highly aggressive melanoma cell subsets have been associated with molecular signatures that resemble those of pluripotent stem cells.10 In addition, stem cell- and progenitor cell-associated proteins, such as cancer testis antigens,11 bone morphogenetic proteins,12 Notch receptors,13 Wnt proteins,14 or the ABCB5, CD133, CD166, CD34, nestin or c-kit stem-cell antigens, have all been detected in melanoma.15 Recent findings of melanoma subsets with embryonic-like differentiation plasticity and increased tumorigenic potential further suggest the existence of "melanoma stem cells", which may contribute to the natural progression and therapeutic failure seen in this disease.8

Are Melanoma-initiating Cells Rare or Not?

Although the CSC theory does not necessarily imply that CSCs are rare,16 early experiments appeared to confirm that melanoma stem cells, in a similar manner to normal stem cells, represent a rare population within the tumour.17 To analyze the presence of such rare cells, researchers recently developed powerful transplantation tools. One such method involves the injection of human melanoma cells into the flank of immunosuppressed mice. If the xenografted population contains tumor-initiating cells, a melanoma develops at the injection site. In a study using this new method, it was calculated that the number of MICs was less than one per million based on the tumour development frequency per number of injected cells.6 However, the notion that these initiating cells represented a rare population within the tumor was later challenged. Indeed, by taking unselected melanoma cells directly from patients, but embedded in MatrigelTM, and then injecting them into the most severely immunocompromised NOD/SCID IL-2rγc mice, researchers found that up to 25% of cells showed tumorigenesis.18 However, it appears that the outcome can be affected by technical issues, such as the severity of the immune defect of the mice, the processing of the human tumor cells and the precise site of injection. Furthermore, it is important to question whether mechanical forces of transplantation, apoptosis, or senescence owing to a lack of survival/proliferation signals prevented some initiating cells from forming tumors.


Do Melanoma-initiating Cells Have a Specific Phenotype?

In Vivo Assays

These new tools allow us to better analyze the characteristics of cells that are more able to induce tumors. Researchers have been able to sort cells, depending on the membrane antigens expressed, and then test their ability to initiate melanoma in immunosuppressed mice. Using this method, Monzani et al. compared the abilities of CD133+ versus CD133- melanoma cells to initiate tumor formation in vivo.5 Importantly, primary-tumor-initiating properties were exclusively associated with melanoma cell subsets characterized by the expression of CD133, as CD133- melanoma cells were found to lack tumorigenicity.5 The ATP binding cassette (ABC) B5 transporter is heterogeneously expressed in primary and metastatic melanomas, but never in nevi - it was therefore tested to see if it was a marker for tumor-initiating properties. Accordingly, ABCB5+ cells derived from human melanomas were able to form tumors comprising both ABCB5+ and ABCB5- populations following transplantation into immunosuppressed mice.6 In contrast, ABCB5- cells formed tumors much less efficiently. By re-analyzing this population, Boiko et al.  found that CD271+ (Ngfr/p75) cells also increased tumor formation in severely immunocompromised mice when compared with their CD271- counterparts (70% versus 7%).7 The CD271+ population generated heterogeneous tumors comprising both CD271+ and CD271- cells. Recently, Roesch et al. showed that the expression of the histone demethylase family JARID1B on melanoma cells corresponded with slow proliferation and was associated with tumor maintenance.19 Likewise, ABCG2 served as another candidate marker for tumorigenic cancer subpopulations,6 but the molecule was not found to serve any such function.20

Although these data indicate that a specific phenotype is able to distinguish cells capable of tumor initiation, the study by Quintana et al.  found no correlation between tumor-forming potential and any of the identified melanoma-initiating stem cell markers, such as CD271/p75/Ngfr, ABCB5 and CD133, suggesting that these markers are reversibly expressed by tumorigenic melanoma cells rather than distinguishing cells at different levels of hierarchy. These results support the phenotypic plasticity of stem cell markers in melanoma.21 Hence, the debate surrounding the precise phenotype of MICs continues. 

In Vitro Assays

In addition to in vivo xenografting, other methods have also been used to investigate whether stem cells exist in human melanomas. Fang et al. described melanoma cell subsets capable of forming non-adherent spheres when cultured in growth medium for human embryonic stem cells.8 These sphere-forming subpopulations of human melanoma cells, which differentiated under appropriate conditions into multiple cell lineages and preferentially expressed the CD20 marker, were more tumorigenic and had an increased differentiation plasticity compared with their adherent counterparts when grafted into mice.8 Sphere-forming melanoma cells persisted after dissociation and re-plating of xenografts formed in vivo and in long-term cultures in vitro, highlighting their competence at self-renewal. Based on these in vitro findings, the authors proposed that melanomas also contain a subpopulation of stem-like cells that contribute to heterogeneity and tumorigenesis.11 Under standard culture conditions in vitro, a melanoma cell line was found to include a majority of CD133+ cells expressing markers of melanoma cell plasticity10 and low levels of the side-population determinant ABCG2.22 In contrast to the findings by Fang et al.,8 a greater number of tumorigenic CD133+ melanoma subsets were confined to adherent cell populations, whereas melanoma sphere-associated cells were devoid of CD133.5

Conclusions and Perspectives

The identification of molecularly defined MICs should shed light on the cellular events driving melanomagenesis and may provide improved experimental systems for the discovery of MIC-targeted novel therapeutic strategies. Indeed, these cells seem to frequently express transporters implicated in resistance to chemotherapy, and may ultimately be responsible for recurrence and progression. Specific targeting of MICs via a molecular marker could provide more potent and selective forms of melanoma therapy.

References

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