Iterative sorting reveals CD133+ and CD133- melanoma cells as phenotypically distinct populations

Abstract

BackgroundThe heterogeneity and tumourigenicity of metastatic melanoma is attributed to a cancer stem cell model, with CD133 considered to be a cancer stem cell marker in melanoma as well as other tumours, but its role has remained controversial.MethodsWe iteratively sorted CD133+ and CD133- cells from 3 metastatic melanoma cell lines, and observed tumourigenicity and phenotypic characteristics over 7 generations of serial xeno-transplantation in NOD/SCID mice.ResultsWe demonstrate that iterative sorting is required to make highly pure populations of CD133+ and CD133- cells from metastatic melanoma, and that these two populations have distinct characteristics not related to the cancer stem cell phenotype. In vitro, gene set enrichment analysis indicated CD133+ cells were related to a proliferative phenotype, whereas CD133- cells were of an invasive phenotype. However, in vivo, serial transplantation of CD133+ and CD133- tumours over 7 generations showed that both populations were equally able to initiate and propagate tumours. Despite this, both populations remained phenotypically distinct, with CD133- cells only able to express CD133 in vivo and not in vitro. Loss of CD133 from the surface of a CD133+ cell was observed in vitro and in vivo, however CD133- cells derived from CD133+ retained the CD133+ phenotype, even in the presence of signals from the tumour microenvironment.ConclusionWe show for the first time the necessity of iterative sorting to isolate pure marker-positive and marker-negative populations for comparative studies, and present evidence that despite CD133+ and CD133- cells being equally tumourigenic, they display distinct phenotypic differences, suggesting CD133 may define a distinct lineage in melanoma.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2759-2) contains supplementary material, which is available to authorized users.

Suitability of CD133 as a Marker for Cancer Stem Cells in Melanoma

“…Most studies have reported a rare subset of CD133-positive cells in melanoma comprising less than 1% of total tumor cells (Monzani et al, 2007). The D10 cell line expresses CD133 more frequently than typical melanoma (approximately 10.7%); this high rate appears to be associated with the metastatic state of this cell line (Grasso et al, 2016). However, there are contradictory statements regarding the ability of CD133-negative cells to induce tumors.…”

Cervical cancer stem cell‑associated genes: Prognostic implications in cervical cancer (Review)

“…The surface markers CD133, CD34, CD44, CD26 and CD90 are used to identify and isolate CSCs from tumor cell populations. Additionally, these cells possess metastatic, invasive and chemoresistance abilities (10,34–37). Several potential cervical epithelial stem cell markers, including MSI1, ALDH1, SOX2 and CD49f, have been used to identify CCSCs (10,15).…”

The DLK1-MEG3 locus in malignant cells of proposed primordial germ cell origins.

“…Recent reports offer a new perspective on this phenomenon by providing evidence that, like previous studies in hematopoietic stem cells and Chinese hamster ovary cells, the expression of putative CSC markers fluctuate over time [70][71][72][73][74]. Surprisingly, the isolation of potential CSC subpopulations from the ovarian carcinoma cell line A2780 based on CD24 and CD44 surface expression In summary, the quiescence-associated hypomethylation of the IGF2-H19 locus is surprisingly found in NTera2 cells as well as EC-containing mixed TGCT tissues.…”

“…These CSC subpopulations are often identified based on their expression of certain surface antigens which also exist on normal stem cell populations, including SSEA4, CD133, and DLK1 [11,[67][68][69]. However, several studies have challenged the nature of true CSCs within tumors by providing evidence that these CSC-like subpopulations of cells dynamically express CSC-associated antigens [70,71]. Notably, low-passage cell lines from human metastatic melanoma patients were recently shown to form tumors in vivo irrespective of their surface expression of the CSC marker CD133, and CD133 -isolated subpopulations of these cells re-acquired CD133 expression during culture [70].…”

“…However, several studies have challenged the nature of true CSCs within tumors by providing evidence that these CSC-like subpopulations of cells dynamically express CSC-associated antigens [70,71]. Notably, low-passage cell lines from human metastatic melanoma patients were recently shown to form tumors in vivo irrespective of their surface expression of the CSC marker CD133, and CD133 -isolated subpopulations of these cells re-acquired CD133 expression during culture [70]. Indeed, this phenomenon has also been demonstrated in normal HSCs, where the expression of characteristic surface antigens was found to fluctuate over time [72].…”