A fundamental question in cancer biology is whether cells with tumorigenic potential are common or rare within human cancers. Studies on diverse cancers, including melanoma, have indicated that only rare human cancer cells (0.1% to 0.0001%) have tumorigenic potential when transplanted into NOD/SCID mice. However, the extent to which NOD/SCID mice underestimate the frequency of tumorigenic human cancer cells has been uncertain. Here we show that modified xenotransplantation assay conditions, including the use of more highly immunocompromised NOD/SCID IL2Rγnull mice, can increase the detection of tumorigenic melanoma cells by several orders-of-magnitude. In limiting dilution assays, approximately 25% of unselected melanoma cells from 12 different patients, including cells from primary and metastatic melanomas obtained directly from patients, formed tumors under these more permissive conditions. In single cell transplants, an average of 27% of unselected melanoma cells from four different patients formed tumors. Xenotransplantation assay modifications can therefore dramatically increase the detectable frequency of tumorigenic cells, demonstrating that they are common in some human cancers.

The identification and characterization of cancer stem cells might lead to more effective treatments for some cancers by focusing therapy on the most malignant cells. To achieve this goal it will be necessary to determine which cancers follow a cancer stem cell model and which do not, to address technical issues related to tumorigenesis assays, and to test the extent to which cancer cell heterogeneity arises from genetic versus epigenetic differences.

Summary We investigated whether melanoma is hierarchically organized into phenotypically distinct subpopulations of tumorigenic and non-tumorigenic cells, or whether most melanoma cells retain tumorigenic capacity, irrespective of their phenotype. We found 28% of single melanoma cells obtained directly from patients formed tumors in NOD/SCID IL2Rγnull mice. All stage II, III, and IV melanomas obtained directly from patients had common tumorigenic cells. All tumorigenic cells appeared to have unlimited tumorigenic capacity upon serial transplantation. We were unable to find any large subpopulation of melanoma cells that lacked tumorigenic potential. None of 22 heterogeneously-expressed markers, including CD271 and ABCB5, enriched tumorigenic cells. Some melanomas metastasized in mice, irrespective of whether they arose from CD271- or CD271+ cells. Many markers appeared to be reversibly expressed by tumorigenic melanoma cells. Significance In cancers that follow a stem cell model, phenotypically distinct tumorigenic cells form abundant and phenotypically diverse non-tumorigenic progeny in a hierarchical manner that resembles normal stem cell differentiation. In contrast to this model, our results indicate that primary cutaneous or metastatic melanomas from patients have common and phenotypically diverse tumorigenic cells that undergo reversible phenotypic changes in vivo. Most of the phenotypic heterogeneity in melanoma is therefore not associated with a loss of tumorigenic potential or organized in stable hierarchies. These data suggest a phenotypic plasticity model in which phenotypic heterogeneity is driven largely by reversible changes within lineages of tumorigenic cells rather than by irreversible epigenetic or genetic changes.

It is unclear whether neurogenesis occurs in the adult mammalian enteric nervous system (ENS). Neural crestderived cells capable of forming multilineage colonies in culture, and neurons and glia upon transplantation into chick embryos, persist throughout adult life in the mammalian ENS. In this study we sought to determine the physiological function of these cells. We discovered that these cells could be identified based on CD49b expression and that they had characteristics of enteric glia, including p75, GFAP, S100B, and SOX10 expression. To test whether new neurons or glia arise in the adult gut under physiological conditions, we marked dividing progenitors with a thymidine analog in rodents under steady-state conditions, or during aging, pregnancy, dietary changes, hyperglycemia, or exercise. We also tested gut injuries including inflammation, irradiation, benzalkonium chloride treatment, partial gut stenosis, and glial ablation. We readily observed neurogenesis in a neurogenic region of the central nervous system, but not reproducibly in the adult ENS. Lineage tracing of glial cells with GFAP-Cre and GFAP-CreERT2 also detected little or no adult ENS neurogenesis. Neurogenesis in the adult gut is therefore very limited under the conditions we studied. In contrast, ENS gliogenesis was readily observed under steady-state conditions and after injury. Adult enteric glia thus have the potential to form neurons and glia in culture but are fated to form mainly glia under physiological conditions and after the injuries we studied.