Lineage tracing reveals multipotent stem cells maintain human adenomas and the pattern of clonal expansion in tumor evolution

Adam Humphries, Biancastella Cereser, Laura J. Gay, Daniel S. J. Miller, Bibek Das, Alice Gutteridge, George Elia, Emma Nye, Rosemary Jeffery, Richard Poulsom, Marco R. Novelli, Manuel Rodriguez-Justo, Stuart A. C. McDonald, Nicholas A. Wright, and Trevor A. Graham
PNAS published ahead of print June 13, 2013 https://doi.org/10.1073/pnas.1220353110

  1. Edited by Michael Wigler, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, and approved May 3, 2013 (received for review November 27, 2012)

Significance

The organization of cells within human colorectal adenomas, and specifically whether the tumors are maintained by stem cells, is unclear. Furthermore, the patterns of clonal evolution leading to the development of a malignant tumor have not been determined. We performed lineage tracing in human adenomas using a combination of nuclear and mitochondrial DNA lesions and epigenetic markers. Our data identify a stem cell population within adenomas and suggest that new growth of intratumor clones occurs infrequently, not as a steady continual process as often is assumed. Our work offers a unique insight into human cancer development.

Abstract

The genetic and morphological development of colorectal cancer is a paradigm for tumorigenesis. However, the dynamics of clonal evolution underpinning carcinogenesis remain poorly understood. Here we identify multipotential stem cells within human colorectal adenomas and use methylation patterns of nonexpressed genes to characterize clonal evolution. Numerous individual crypts from six colonic adenomas and a hyperplastic polyp were microdissected and characterized for genetic lesions. Clones deficient in cytochrome c oxidase (CCO−) were identified by histochemical staining followed by mtDNA sequencing. Topographical maps of clone locations were constructed using a combination of these data. Multilineage differentiation within clones was demonstrated by immunofluorescence. Methylation patterns of adenomatous crypts were determined by clonal bisulphite sequencing; methylation pattern diversity was compared with a mathematical model to infer to clonal dynamics. Individual adenomatous crypts were clonal for mtDNA mutations and contained both mucin-secreting and neuroendocrine cells, demonstrating that the crypt contained a multipotent stem cell. The intracrypt methylation pattern was consistent with the crypts containing multiple competing stem cells. Adenomas were epigenetically diverse populations, suggesting that they were relatively mitotically old populations. Intratumor clones typically showed less diversity in methylation pattern than the tumor as a whole. Mathematical modeling suggested that recent clonal sweeps encompassing the whole adenoma had not occurred. Adenomatous crypts within human tumors contain actively dividing stem cells. Adenomas appeared to be relatively mitotically old populations, pocketed with occasional newly generated subclones that were the result of recent rapid clonal expansion. Relative stasis and occasional rapid subclone growth may characterize colorectal tumorigenesis.

Footnotes

  • ↵1To whom correspondence may be addressed. E-mail: n.a.wright{at}qmul.ac.uk or trevor.graham{at}ucsfmedctr.org.

  • Author contributions: A.H., S.A.C.M., N.A.W., and T.A.G. designed research; A.H., B.C., L.J.G., D.S.J.M., B.D., A.G., G.E., E.N., R.J., R.P., and T.A.G. performed research; A.H., M.R.N., M.R.-J., S.A.C.M., N.A.W., and T.A.G. analyzed data; and A.H., N.A.W., and T.A.G. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1220353110/-/DCSupplemental.

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Stem cell clone evolution in human adenomas
Adam Humphries, Biancastella Cereser, Laura J. Gay, Daniel S. J. Miller, Bibek Das, Alice Gutteridge, George Elia, Emma Nye, Rosemary Jeffery, Richard Poulsom, Marco R. Novelli, Manuel Rodriguez-Justo, Stuart A. C. McDonald, Nicholas A. Wright, Trevor A. Graham
Proceedings of the National Academy of Sciences Jun 2013, 201220353; DOI: 10.1073/pnas.1220353110
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