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 (0.1–0.0001%) form tumours when transplanted into non-obese diabetic/severe combined immunodeficiency (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 interleukin-2 receptor gamma chain null (Il2rg-/-) 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 tumours under these more permissive conditions. In single-cell transplants, an average of 27% of unselected melanoma cells from four different patients formed tumours. Modifications to xenotransplantation assays can therefore dramatically increase the detectable frequency of tumorigenic cells, demonstrating that they are common in some human cancers.
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This work was supported by the Howard Hughes Medical Institute and by the Allen H. Blondy Research Fellowship. The University of Michigan Melanoma Bank was supported by a gift from Lewis and Lillian Becker. Flow cytometry was partly supported by the University of Michigan Comprehensive Cancer Center grant from the National Institutes of Health CA46592. We thank: D. Adams, M. White and the University of Michigan Flow Cytometry Core Facility for support; N. McAnsh and the University of Michigan Cancer Centre Histology Core for histological studies; G. K. Smyth for assistance with statistics; and Z. Azizan for support with tissue collection. Antibody production was supported in part by the National Institute of Diabetes, Digestive, and Kidney Diseases, grant NIH5P60-DK20572 to the Michigan Diabetes Research and Training Center. Some antibodies were provided by Caltag or by eBioscience to screen for cancer stem-cell markers. Human primary melanocyte cultures were provided by M. Soengas. Human mesenchymal stem cells were provided by Z. Wang and P. Krebsbach. E.Q. was supported by the Spanish Ministry of Education and the Marie Curie Outgoing International Fellowship from the European Commission. M.S. was supported by the Australian National Health and Medical Research Council, the Human Frontiers Science Program and Australia Post.
Author Contributions E.Q., M.S. and S.J.M. planned the project. E.Q. and M.S. performed the experiments, and analysed data with S.J.M. M.S.S. and T.M.J. obtained consent from the patients and surgically obtained many of the melanoma specimens. D.R.F. performed all pathology and diagnosed the tumours with T.M.J. T.M.J. banked the melanomas, and provided clinical information. E.Q., M.S. and S.J.M. wrote the paper.
This file contains Supplementary Tables 1, 2 and Supplementary Figures 1-6 with legends.