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



We 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.



We 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.



We 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.
Malaghan Institute of Medical Research, P.O. Box 7060, Wellington, 6242, New Zealand.
Carole
C
Grasso
Ludwig Institute for Cancer Research, Olivia Newton-John Cancer & Wellness Centre, Austin Hospital, Heidelberg, VIC, 3084, Australia.
Matthew
M
Anaka
Harvard T.H. Chan School of Public Health, 677 Huntington Avenue, Boston, MA, 02115, USA.|Harvard Stem Cell Institute, Holyoke Center, Suite 727W, 1350 Massachusetts Avenue, Cambridge, MA, 02138, USA.
Oliver
O
Hofmann
Harvard T.H. Chan School of Public Health, 677 Huntington Avenue, Boston, MA, 02115, USA.
Ramakrishna
R
Sompallae
Malaghan Institute of Medical Research, P.O. Box 7060, Wellington, 6242, New Zealand.
Kate
K
Broadley
Harvard T.H. Chan School of Public Health, 677 Huntington Avenue, Boston, MA, 02115, USA.|Sheffield Institute for Translational Neuroscience, The University of Sheffield, 385a Glossop Road, Sheffield, S10 2HQ, UK.
Winston
W
Hide
Malaghan Institute of Medical Research, P.O. Box 7060, Wellington, 6242, New Zealand.
Michael V
MV
Berridge
Ludwig Institute for Cancer Research, Olivia Newton-John Cancer & Wellness Centre, Austin Hospital, Heidelberg, VIC, 3084, Australia.
Jonathan
J
Cebon
Ludwig Institute for Cancer Research, Olivia Newton-John Cancer & Wellness Centre, Austin Hospital, Heidelberg, VIC, 3084, Australia.
Andreas
A
Behren
Malaghan Institute of Medical Research, P.O. Box 7060, Wellington, 6242, New Zealand. Melanie.McConnell@vuw.ac.nz.
Melanie J
MJ
McConnell
D000071916:AC133 Antigen / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D045744:Cell Line, Tumor; D019070:Cell Lineage; D049109:Cell Proliferation; D002469:Cell Separation / Q000379:methods*; D006801:Humans; D008545:Melanoma / Q000276:immunology / Q000473:pathology*; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D009362:Neoplasm Metastasis; D009368:Neoplasm Transplantation; D014411:Neoplastic Stem Cells / Q000276:immunology* / Q000473:pathology; D010641:Phenotype; D059016:Tumor Microenvironment
D000071916:AC133 Antigen; C000606023:PROM1 protein, human