Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Multicolour lineage tracing reveals clonal dynamics of squamous carcinoma evolution from initiation to metastasis

Abstract

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 options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Tamoxifen-induced Confetti labelling of the skin.
Fig. 2: The streaked appearance of papillomas arising from Confetti-labelled skin.
Fig. 3: Genetic analysis of bulk and streak papilloma populations.
Fig. 4: Tumour evolution following Confetti labelling at 8 weeks.
Fig. 5: Carcinoma evolution following Confetti labelling at 24 weeks.
Fig. 6: Speckle populations observed in carcinomas.
Fig. 7: Evidence for polyclonal seeding of metastasis.

Similar content being viewed by others

References

  1. Nowell, P. C. The clonal evolution of tumor cell populations. Science 194, 23–28 (1976).

    Article  PubMed  CAS  Google Scholar 

  2. Greaves, M. & Maley, C. C. Clonal evolution in cancer. Nature 481, 306–313 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  3. 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).

    Article  PubMed  CAS  Google Scholar 

  4. 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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  5. Dotto, G. P. Multifocal epithelial tumors and field cancerization: stroma as a primary determinant. J. Clin. Invest. 124, 1446–1453 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. Jonason, A. S. et al. Frequent clones of p53-mutated keratinocytes in normal human skin. Proc. Natl Acad. Sci. USA 93, 14025–14029 (1996).

    Article  PubMed  CAS  Google Scholar 

  7. Martincorena, I. et al. High burden and pervasive positive selection of somatic mutations in normal human skin. Science 348, 880–886 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. 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).

    Article  PubMed  CAS  Google Scholar 

  9. Winton, D. J., Blount, M. A. & Ponder, B. A. Polyclonal origin of mouse skin papillomas. Br. J. Cancer 60, 59–63 (1989).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. 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).

    PubMed  CAS  Google Scholar 

  11. 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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. 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).

  13. 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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  14. 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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Aceto, N. et al. Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis. Cell 158, 1110–1122 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Maddipati, R. & Stanger, B. Z. Pancreatic cancer metastases harbor evidence of polyclonality. Cancer Discov. 5, 1086–1097 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  17. Gundem, G. et al. The evolutionary history of lethal metastatic prostate cancer. Nature 520, 353–357 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  18. Sanborn, J. Z. et al. Phylogenetic analyses of melanoma reveal complex patterns of metastatic dissemination. Proc. Natl Acad. Sci. USA 112, 10995–11000 (2015).

    Article  PubMed  CAS  Google Scholar 

  19. Snippert, H. J. et al. Intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Cell 143, 134–144 (2010).

    Article  PubMed  CAS  Google Scholar 

  20. Swanton, C. Intratumor heterogeneity: evolution through space and time. Cancer Res. 72, 4875–4882 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  21. Westcott, P. M. K. et al. The mutational landscapes of genetic and chemical models of Kras-driven lung cancer. Nature 517, 489–492 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. McCreery, M. Q. et al. Evolution of metastasis revealed by mutational landscapes of chemically induced skin cancers. Nat. Med. 21, 1514–1520 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. McFadden, D. G. et al. Genetic and clonal dissection of murine small cell lung carcinoma progression by genome sequencing. Cell 156, 1298–1311 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. Schepers, A. G. et al. Lineage tracing reveals Lgr5+ stem cell activity in mouse intestinal adenomas. Science 337, 730–735 (2012).

    Article  PubMed  CAS  Google Scholar 

  25. Wong, C. E. et al. Inflammation and Hras signaling control epithelial–mesenchymal transition during skin tumor progression. Genes Dev. 27, 670–682 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  26. 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).

    Article  PubMed  CAS  Google Scholar 

  27. 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).

    PubMed  CAS  Google Scholar 

  28. 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).

    Article  PubMed  CAS  Google Scholar 

  29. 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).

    Article  PubMed  CAS  Google Scholar 

  30. Vogelstein, B. et al. Cancer genome landscapes. Science 339, 1546–1558 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Stransky, N. et al. The mutational landscape of head and neck squamous cell carcinoma. Science 333, 1157–1160 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Pickering, C. R. et al. Mutational landscape of aggressive cutaneous squamous cell carcinoma. Clin. Cancer Res. 20, 6582–6592 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. 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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Novelli, M. R. et al. Polyclonal origin of colonic adenomas in an XO/XY patient with FAP. Science 272, 1187–1190 (1996).

    Article  PubMed  CAS  Google Scholar 

  35. Parsons, B. L. Many different tumor types have polyclonal tumor origin: evidence and implications. Mutat. Res. 659, 232–247 (2008).

    Article  PubMed  CAS  Google Scholar 

  36. 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).

    Article  PubMed  CAS  Google Scholar 

  37. 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).

    Article  PubMed  CAS  Google Scholar 

  38. Plikus, M. V. et al. Epithelial stem cells and implications for wound repair. Semin. Cell Dev. Biol. 23, 946–953 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. 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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. McGranahan, N. & Swanton, C. Biological and therapeutic impact of intratumor heterogeneity in cancer evolution. Cancer Cell 27, 15–26 (2015).

    Article  PubMed  CAS  Google Scholar 

  41. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  42. McKenna, A. et al. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 20, 1297–1303 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  43. DePristo, M. A. et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat. Genet. 43, 491–498 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  44. Cibulskis, K. et al. Sensitive detection of somatic point mutations in impure and heterogeneous cancer samples. Nat. Biotechnol. 31, 213–219 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. Wang, K., Li, M. & Hakonarson, H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 38, e164(2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. 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).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

Download references

Acknowledgements

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.

Author contributions

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.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Melissa Q. Reeves or Allan Balmain.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated Supplementary Information

Supplementary Figure 1 Streaked papillomas contain distinctly colored subpopulations.

(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).

Supplementary Figure 3 Ki67 localization in relation to Confetti colored clones.

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 4 Individual color channels for carcinomas showing intermixing and speckled populations.

Panels show individual color channels, overlaid on DAPI (white), for tumor regions shown in Fig. 5f (A) and Fig. 6b (B).

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.

Supplementary Figure 6 Analysis of a mixed uncolored and RFP+ lymph node metastasis.

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).

Supplementary information

Supplementary Information

Supplementary Figures 1–6 and Supplementary Table 1

Reporting Summary

Supplementary Table 1

Bulk population mutations

Supplementary Table 2

Streak population mutations

Supplementary Table 3

Statistics source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41556-018-0109-0

This article is cited by

Search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer