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

Nature Cell Biology volume 20, pages 699–709 (2018)Cite this article

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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.

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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.

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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.

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  1. Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, CA, USA

    Melissa Q. Reeves, Eve Kandyba, Sophie Harris, Reyno Del Rosario & Allan Balmain

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  1. Melissa Q. Reeves
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Correspondence to Melissa Q. Reeves or Allan Balmain.

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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).

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Supplementary Table 1

Bulk population mutations

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Streak population mutations

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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

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