Tumour cells are subjected to evolutionary selection pressures during progression from initiation to metastasis. We analysed the clonal evolution of squamous skin carcinomas induced by DMBA/TPA treatment using the K5CreER-Confetti mouse and stage-specific lineage tracing. We show that benign tumours are polyclonal, but only one population contains the Hras driver mutation. Thus, benign papillomas are monoclonal in origin but recruit neighbouring epithelial cells during growth. Papillomas that never progress to malignancy retain several distinct clones, whereas progression to carcinoma is associated with a clonal sweep. Newly generated clones within carcinomas demonstrate intratumoural invasion and clonal intermixing, often giving rise to metastases containing two or more distinct clones derived from the matched primary tumour. These data demonstrate that late-stage tumour progression and dissemination are governed by evolutionary selection pressures that operate at a multicellular level and, therefore, differ from the clonal events that drive initiation and the benign–malignant transition.
This is a preview of subscription content, access via your institution
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Prices vary by article type
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Nowell, P. C. The clonal evolution of tumor cell populations. Science 194, 23–28 (1976).
Greaves, M. & Maley, C. C. Clonal evolution in cancer. Nature 481, 306–313 (2012).
Calbo, J. et al. A functional role for tumor cell heterogeneity in a mouse model of small cell lung cancer. Cancer Cell 19, 244–256 (2011).
Cleary, A. S., Leonard, T. L., Gestl, S. A. & Gunther, E. J. Tumour cell heterogeneity maintained by cooperating subclones in Wnt-driven mammary cancers. Nature 508, 113–117 (2014).
Dotto, G. P. Multifocal epithelial tumors and field cancerization: stroma as a primary determinant. J. Clin. Invest. 124, 1446–1453 (2014).
Jonason, A. S. et al. Frequent clones of p53-mutated keratinocytes in normal human skin. Proc. Natl Acad. Sci. USA 93, 14025–14029 (1996).
Martincorena, I. et al. High burden and pervasive positive selection of somatic mutations in normal human skin. Science 348, 880–886 (2015).
Reddy, A. L. & Fialkow, P. J. Influence of dose of initiator on two-stage skin carcinogenesis in BALB/c mice with cellular mosaicism. Carcinogenesis 9, 751–754 (1988).
Winton, D. J., Blount, M. A. & Ponder, B. A. Polyclonal origin of mouse skin papillomas. Br. J. Cancer 60, 59–63 (1989).
Li, S. et al. A keratin 15 containing stem cell population from the hair follicle contributes to squamous papilloma development in the mouse. Mol. Carcinog. 52, 751–759 (2013).
Driessens, G., Beck, B., Caauwe, A., Simons, B. D. & Blanpain, C. Defining the mode of tumour growth by clonal analysis. Nature 488, 527–531 (2012).
Huang, P. Y. et al. Leucine-rich G-protein coupled receptor 6 (Lgr6) is a cancer stem cell marker in mouse squamous carcinomas. Nat. Genet. (in press).
Harney, A. S. et al. Real-time imaging reveals local, transient vascular permeability, and tumor cell intravasation stimulated by TIE2hi macrophage-derived VEGFA. Cancer Discov. 5, 932–943 (2015).
Cheung, K. J., Gabrielson, E., Werb, Z. & Ewald, A. J. Collective invasion in breast cancer requires a conserved basal epithelial program. Cell 155, 1639–1651 (2013).
Aceto, N. et al. Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis. Cell 158, 1110–1122 (2014).
Maddipati, R. & Stanger, B. Z. Pancreatic cancer metastases harbor evidence of polyclonality. Cancer Discov. 5, 1086–1097 (2015).
Gundem, G. et al. The evolutionary history of lethal metastatic prostate cancer. Nature 520, 353–357 (2015).
Sanborn, J. Z. et al. Phylogenetic analyses of melanoma reveal complex patterns of metastatic dissemination. Proc. Natl Acad. Sci. USA 112, 10995–11000 (2015).
Snippert, H. J. et al. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell 143, 134–144 (2010).
Swanton, C. Intratumor heterogeneity: evolution through space and time. Cancer Res. 72, 4875–4882 (2012).
Westcott, P. M. K. et al. The mutational landscapes of genetic and chemical models of Kras-driven lung cancer. Nature 517, 489–492 (2014).
McCreery, M. Q. et al. Evolution of metastasis revealed by mutational landscapes of chemically induced skin cancers. Nat. Med. 21, 1514–1520 (2015).
McFadden, D. G. et al. Genetic and clonal dissection of murine small cell lung carcinoma progression by genome sequencing. Cell 156, 1298–1311 (2014).
Schepers, A. G. et al. Lineage tracing reveals Lgr5+ stem cell activity in mouse intestinal adenomas. Science 337, 730–735 (2012).
Wong, C. E. et al. Inflammation and Hras signaling control epithelial–mesenchymal transition during skin tumor progression. Genes Dev. 27, 670–682 (2013).
Quintanilla, M., Brown, K., Ramsden, M. & Balmain, A. Carcinogen-specific mutation and amplification of Ha-ras during mouse skin carcinogenesis. Nature 322, 78–80 (1986).
Bigger, C. A., Sawicki, J. T., Blake, D. M., Raymond, L. G. & Dipple, A. Products of binding of 7,12-dimethylbenz(a)anthracene to DNA in mouse skin. Cancer Res. 43, 5647–5651 (1983).
Nassar, D., Latil, M., Boeckx, B., Lambrechts, D. & Blanpain, C.Genomic landscape of carcinogen-induced and genetically induced mouse skin squamous cell carcinoma. Nat. Med. 21, 946–954 (2015).
Aldaz, C., Trono, D., Larcher, F., Slaga, T. & Conti, C. Sequential trisomization of chromosomes 6 and 7 in mouse skin premalignant lesions. Mol. Carcinog. 2, 22–26 (1989).
Vogelstein, B. et al. Cancer genome landscapes. Science 339, 1546–1558 (2013).
Stransky, N. et al. The mutational landscape of head and neck squamous cell carcinoma. Science 333, 1157–1160 (2011).
Pickering, C. R. et al. Mutational landscape of aggressive cutaneous squamous cell carcinoma. Clin. Cancer Res. 20, 6582–6592 (2014).
Pham, T. T., Angus, S. P. & Johnson, G. L. MAP3K1: genomic alterations in cancer and function in promoting cell survival or apoptosis. Genes Cancer 4, 419–426 (2013).
Novelli, M. R. et al. Polyclonal origin of colonic adenomas in an XO/XY patient with FAP. Science 272, 1187–1190 (1996).
Parsons, B. L. Many different tumor types have polyclonal tumor origin: evidence and implications. Mutat. Res. 659, 232–247 (2008).
Ito, M. et al. Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epidermis. Nat. Med. 11, 1351–1354 (2005).
Dvorak, H. F. Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing. N. Engl. J. Med. 315, 1650–1659 (1986).
Plikus, M. V. et al. Epithelial stem cells and implications for wound repair. Semin. Cell Dev. Biol. 23, 946–953 (2012).
Good, B. H., McDonald, M. J., Barrick, J. E., Lenski, R. E. & Desai, M. M. The dynamics of molecular evolution over 60,000 generations. Nature 551, 45–50 (2017).
McGranahan, N. & Swanton, C. Biological and therapeutic impact of intratumor heterogeneity in cancer evolution. Cancer Cell 27, 15–26 (2015).
Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 2009).
McKenna, A. et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297–1303 (2010).
DePristo, M. A. et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat. Genet. 43, 491–498 (2011).
Cibulskis, K. et al. Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples. Nat. Biotechnol. 31, 213–219 (2013).
Wang, K., Li, M. & Hakonarson, H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 38, e164(2010).
Talevich, E., Shain, A. H., Botton, T., & Bastian, B. C. CNVkit: genome-wide copy number detection and visualization from targeted DNA sequencing. PLoS Comput. Biol. 12, e1004873 (2016).
This work was supported by US National Cancer Institute (NCI) grants RO1CA184510, UO1 CA176287 and R35CA210018 and the Barbara Bass Bakar Professorship of Cancer Genetics. M.Q.R. is supported by NCI F31 NRSA award CA206459. We are greatly appreciative of help and comments from our colleagues in refining this study and manuscript, and also thank T. Nystul and R. Akhurst for providing the Confetti mouse, S. Vlachos and D. Laird for assistance with whole-mount fluorescent imaging, D. Larsen and the Nikon Imaging Center for microscopy training and making the Nikon 6D microscope available, and to S. Elmes and the Laboratory for Cell Analysis core for flow cytometry training.
M.Q.R. designed the study, carried out most of the in vivo and tumour analysis studies and wrote the manuscript, with contributions from the other co-authors. E.K. carried out the tumour analysis, immunohistochemistry and fluorescent imaging. S.H. carried out the fluorescent imaging analysis. R.D.R. carried out the mouse breeding and tumour induction experiments. A.B. conceived and designed the study and wrote the manuscript together with contributions from the other co-authors.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated Supplementary Information
(A, B) Individual color channels for a streaked papilloma. Panels show individual color channels, overlaid on DAPI (white), for tumor regions shown in Fig. 2e (A) and Fig. 2g (B). (C) Representative FACS plots showing an uncolored control tumor, and an RFP+ tumor with a CFP+ population. As is typical of the Confetti cassette, CFP was notably weaker than the other fluorophores.
Supplementary Figure 2 Colored clones in papillomas and at the border of carcinomas for tumors labeled at 8 weeks.
(A-D) Benign papillomas, showing histologically indistinguishable clones within the benign tumor. Top panels, papilloma with a GFP clone adjacent to an uncolored clone (nuclei marked in DAPI), demonstrating limited intermixing. Serial sections show fluorescent colors (A) and H&E staining (B), 20x. Bottom panels, papilloma with a YFP clone adjacent to an uncolored clone (nuclei marked in DAPI), again demonstrating limited intermixing. Serial sections show fluorescent colors (C) and H&E staining (D), 40x. In both cases the two adjacent clones cannot be distinguished by H&E (2 tumors). (E) The edge of a single-colored RFP+ malignant carcinoma, showing the tumor growing under normal interfollicular epidermis. The epidermis here, as elsewhere across the entire back skin, contains patches of all Confetti colors; however these are morphologically distinguishable from the carcinoma itself. Tumor is characteristic of carcinomas in 8-week-labeling cohort (13 tumors).
Ki67 labeling of a multi-color carcinoma from the 24-week labeling experiment, showing differential levels of Ki67 in different color clones. Tumor was selected as a case study (3 sections stained). (A-C) Confetti labeling in each GFP/YFP (A), CFP (B), and RFP (C) channels. (D) Ki67 staining. (E) Merge, with Confetti colors muted to improve visualization of Ki67 localization.
Supplementary Figure 5 Speckled populations in 24-week-labeled carcinomas are K14+ and do not cluster near blood vessels or lymphatics.
(A) Blood vessels and lymphatics in speckled carcinomas. Speckled carcinomas stained for LYVE-1 and CD31 (4 tumors stained, 5 sections each). Tumor 524-A is uncolored with GFP+ speckles, 524-B is uncolored with RFP+ speckles, and 855-B is uncolored with YFP+ and GFP+ speckles. No trends in localization are observed between speckles and blood vessels (CD31) or lymphatic vessels (LYVE-1). (B) Representative K14 staining of a speckled carcinoma (3 tumor speckled regions stained). Carcinoma shown has a dominant uncolored population and localized YFP+ speckle population. Both uncolored and YFP+ cell populations stain positive for K14.
Case study. (A) Cross-section of lymph node (left) and K14 staining (right) (B) FACS plot showing fractions collected for analysis of red and uncolored cells. (C) Copy number plot of chromosome 7, with proximal end of the chromosome at left and distal end at right. A focal copy number gain at the proximal end is seen in both the RFP+ (left) and uncolored (right) fractions. Contamination of the uncolored cell fraction with lymphocytes results in dilution of signal and lower resolution; however the identical breakpoint (dashed line) is seen in both samples. Copy number analysis based on exome sequencing of FACS-isolated populations shown in (B).
About this article
Cite this article
Reeves, M.Q., Kandyba, E., Harris, S. et al. Multicolour lineage tracing reveals clonal dynamics of squamous carcinoma evolution from initiation to metastasis. Nat Cell Biol 20, 699–709 (2018). https://doi.org/10.1038/s41556-018-0109-0
This article is cited by
British Journal of Cancer (2023)
Nature Ecology & Evolution (2023)
Nature Reviews Cancer (2023)
Nature Reviews Cancer (2023)
Cellular and Molecular Life Sciences (2022)