Defining the clonal dynamics leading to mouse skin tumour initiation

Abstract

The changes in cell dynamics after oncogenic mutation that lead to the development of tumours are currently unknown. Here, using skin epidermis as a model, we assessed the effect of oncogenic hedgehog signalling in distinct cell populations and their capacity to induce basal cell carcinoma, the most frequent cancer in humans. We found that only stem cells, and not progenitors, initiated tumour formation upon oncogenic hedgehog signalling. This difference was due to the hierarchical organization of tumour growth in oncogene-targeted stem cells, characterized by an increase in symmetric self-renewing divisions and a higher p53-dependent resistance to apoptosis, leading to rapid clonal expansion and progression into invasive tumours. Our work reveals that the capacity of oncogene-targeted cells to induce tumour formation is dependent not only on their long-term survival and expansion, but also on the specific clonal dynamics of the cancer cell of origin.

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Figure 1: SCs but not CPs initiate BCC formation upon HH activation.
Figure 2: Homeostatic renewal of mouse tail epidermis.
Figure 3: SmoM2 expression in CPs induces clonal expansion that does not progress into BCC.
Figure 4: SmoM2 expression in SCs induces tumour SCs that lead to BCC formation.
Figure 5: p53 deletion in CPs leads to BCC formation.

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Acknowledgements

We would like to thank J.-M. Vanderwinden and the LiMiF for the help with confocal microscopy. C.B. is an investigator of WELBIO. A.S.-D. and J.C.L. are supported by a fellowship of the FNRS and FRIA respectively. B.D.S. and E.H. are supported by the Wellcome Trust (grant numbers 098357/Z/12/Z and 110326/Z/15/Z). E.H. is supported by a fellowship from Trinity College, Cambridge. This work was supported by the FNRS, the IUAP program, the Fondation contre le Cancer, the ULB fondation, the foundation Bettencourt Schueller, the foundation Baillet Latour, a consolidator grant of the European Research Council.

Author information

A.S.-D., C.B., E.H. and B.D.S. designed the experiments, performed data analysis and wrote the manuscript; A.S.-D. performed all of the biological experiments; E.H. performed all the mathematical modelling. J.C.L. and M.L. provided technical support. K.K.Y. made initial observations pertinent to the study.

Correspondence to Benjamin D. Simons or Cédric Blanpain.

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Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information

Nature thanks S. Benitah, F. de Sauvage, L. Vermeulen and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 The fate of oncogene-targeted clones is determined by the initial targeted cell (SC or CP) and their location in scale or interscale regions.

a, Orthogonal view used to quantify the number of clones, cells stained with β4-integrin and SmoM2. (left). Quantification of the number of clones induced 1 week after tamoxifen administration in scale and interscale regions in K14-CreER/Rosa-SmoM2 (n = 4 animals, 0.1 mg tamoxifen) and Inv-CreER/Rosa-SmoM2 (n = 3 animals, 2.5 mg tamoxifen) (right). b, Immunostaining for β4-integrin and SmoM2 in K14-CreER/Rosa-SmoM2 and Inv-CreER/Rosa-SmoM2 clones located in the scale and interscale regions, 8 weeks after oncogene activation. c, Immunostaining for the differentiation marker keratin-10, K10, and SmoM2 in K14-CreER/Rosa-SmoM2 and Inv-CreER/Rosa-SmoM2 clones 8 weeks after oncogene activation, showing absence of differentiated cells in K14-CreER/Rosa-SmoM2 clones and alteration of the differentiation in Inv-CreER/Rosa-SmoM2 clones. Hoechst nuclear staining is represented in blue; scale bars, 10 μm.

Extended Data Figure 2 Evolution of K14-CreER/Rosa-YFP and Inv-CreER/Rosa-YFP clones in scale and interscale regions.

a, Whole-mount immunostaining for YFP/K31 in K14-CreER/Rosa-YFP mice and Inv-CreER/Rosa-YFP mice upon tamoxifen administration. b, Scheme representing the area of tail epidermis (area comprised by 6 groups of triplets of hair follicles, highlighted in black) that is used to quantify the clone number and persistence. c, Distribution of K14-CreER/Rosa-YFP and Inv-CreER/Rosa-YFP total clone sizes as measured by total cell content of surviving clones, imaged by confocal microscopy on whole-mount tail epidermis from 1 to 24 weeks after tamoxifen administration. The number of analysed clones is indicated for each time point. Hoechst nuclear staining is represented in blue; scale bars, 100 μm. Histograms and error bars represent the mean and the standard error of the mean (s.e.m.).

Extended Data Figure 3 The interscale is maintained by two cell populations during homeostasis.

a, Evolution in time of the total labelled cell fraction under three hypotheses. For a perfect single population of equipotent balanced progenitors, the labelled cell fraction remains constant. For a single population of equipotent balanced progenitors displaying short-term priming, the labelled cell fraction increases transiently for the cells primed to divide, and decreases transiently for the cells primed to differentiate, but after the priming period, both fractions remain constant at different values. For two populations organized in a hierarchy, the labelled fraction of the progenitors decreases continuously to zero, while the labelled fraction of the stem cells continuously increases to reach a steady state value, corresponding to its average progeny size. b, Cumulative basal clone size distribution of Inv-CreER/Rosa-YFP clones at homeostasis in the interscale upon tamoxifen administration. c, Cumulative basal clone size distribution of K14-CreER/Rosa-YFP clones at homeostasis in the interscale upon tamoxifen administration. Clonal distributions are plotted in log-plot, error bars indicate s.d., thick lines are the model prediction and shaded areas indicate 95% confidence intervals in the model prediction.

Extended Data Figure 4 The scale is maintained by a single population during homeostasis.

a, Evolution of mean surviving basal (top) and suprabasal (bottom) clone size in the scale for K14-CreER/Rosa-YFP (red) and Inv-CreER/Rosa-YFP (blue). In contrast to the interscale, in the scale K14-CreER and Inv-CreER clones behave identically, indicative of a single progenitor pool. The lines are the fit from the model from which we extract the fate choices of progenitors displayed in b. b, Fate choices of the equipotent progenitor pool in the scale, as extracted from the fits. c, Clonal persistence (top) and labelled cell fraction (bottom) in the scale for K14-CreER/Rosa-YFP (red) and Inv-CreER/Rosa-YFP (blue). The blue and red lines are the predictions of the model (see Supplementary Notes for details) using only the parameters extracted in b. K14- and Inv-CreER clones behave similarly and display near-perfect long-term balance. For the clonal persistence data, we examined in each mouse a randomly chosen area shown in Extended Data Fig. 2b. Error bars represent the s.e.m. d, Cumulative basal clone size distribution of K14-CreER/Rosa-YFP clones at homeostasis in the scale upon Tamoxifen administration. One should note that there were too few Involucrin clones in the scale to plot meaningful distributions. Clonal distributions are plotted in log-plot, error bars indicate s.d., thick lines are the model prediction and shaded area indicate 95% confidence intervals in the model prediction.

Extended Data Figure 5 Clonal dynamics of interscale Inv-SmoM2 clones is consistent with a single imbalanced population of progenitors slowing down in time.

a, Distribution of Inv-CreER/Rosa-YFP (black) and Inv-CreER/Rosa-SmoM2 (red) clone sizes as measured by total cell content, imaged by confocal microscopy on whole-mount tail epidermis from 1 weeks to 24 weeks following tamoxifen administration. The number of clones analysed in Inv-CreER/Rosa-SmoM2 is indicated in Fig. 3b. The number of clones counted in Inv-CreER/Rosa-YFP is as indicated in Fig. 2b. b, Evolution of the clonal persistence for interscale Inv-CreER/Rosa-SmoM2 clones. c, Labelled cell fraction for interscale Inv-CreER/Rosa-SmoM2 clones. d, Fraction of EdU–BrdU double-labelled cells as a function of basal clone size at 8 weeks for Inv-CreER/Rosa-SmoM2 clones, for 2 (left), 4 (centre) and 6 (right) days of continuous BrdU incorporation. e, Immunostaining for β4-integrin, SmoM2 and active-caspase-3 in Inv-CreER/Rosa-SmoM2 clones at 8 weeks after induction. f, Percentage of dysplastic, hyperplastic and normally differentiating Inv-CreER/Rosa-SmoM2 clones presenting at least one active-caspase positive cell within the clone at 8 weeks after induction (n = 73 clones analysed from 4 independent experiments). g, Quantification of the number (%) of basal and suprabasal apoptotic cells in dysplastic, hyperplastic and normally differentiating Inv-CreER/Rosa-SmoM2 clones 8 weeks after SmoM2 activation. h, Percentage of dysplastic, hyperplastic and normally differentiating Inv-CreER/Rosa-SmoM2 clones presenting apoptosis in basal and suprabasal compartments 8 w after oncogenic activation. i, Cumulative distribution of the fraction of basal apoptosis as a function of basal cell number in an Inv-CreER/Rosa-SmoM2 clone at 8 weeks (data in blue). The green line is the expected theoretical distribution of apoptotic fraction if apoptosis occurred randomly (following a Poisson process), in any clone with the same probability. The data are statistically different from the random theory, showing that apoptosis clusters in certain clones at a given time point. j, Short-term fate outcome of progenitors in Inv-CreER/Rosa-SmoM2 clones at 8 weeks, as assessed by using EdU as a clonal marker. We count only cell doublets and classify them as either basal–basal, basal–suprabasal, or suprabasal–suprabasal (n = 47 clones from 3 independent experiments). Immunostaining for β4-integrin, EdU and SmoM2 showing the different type of cell fate outcomes found in Inv-CreER/Rosa-SmoM2 clones. Hoechst nuclear staining is represented in blue; scale bars, 10 μm. Histograms and error bars represent the mean and the s.e.m.

Extended Data Figure 6 Clonal dynamics of Inv-CreER/Rosa-SmoM2 and K14-CreER/Rosa-SmoM2 clones in the scale are similar.

a, Evolution of mean surviving basal clone sizes (top) and labelled cell fraction (bottom), for K14-CreER/Rosa-SmoM2, in the scale. b, Evolution of mean surviving basal clone sizes (top) and labelled cell fraction (bottom), for Inv-CreER/Rosa-SmoM2, in the scale. Whereas the interscale clones show net expansion, scale clones, both Inv-CreER and K14-CreER, show near balance at the population level. c, Evolution of the persistence of K14-CreER/Rosa-SmoM2 (green) and Inv-CreER/Rosa-SmoM2 (purple) clones in the scale. Notably, and in contrast to the interscale, both K14 and Involucrin clones have the same persistence. d, Mean basal clone size, normalized by the mean clone size at 1 week for both Inv-CreER and K14-CreER clones. Even though one can see on a and b that the final clone size is higher in K14, this is fully explained by short-term differences in fate during the first week indicative of short-term priming for K14. Correspondingly, the evolution of the labelling fraction is very similar for K14 and Involucrin in scale. Therefore, K14-CreER/Rosa-SmoM2 and Inv-CreER/Rosa-SmoM2 in scale display the same long-term kinetics upon oncogenic activation, consistent with the one-population model uncovered at homeostasis. Error bars represent the s.e.m.

Extended Data Figure 7 Clonal dynamics of interscale K14-CreER/Rosa-SmoM2 clones is consistent with two populations.

a, Distribution of K14-CreER/Rosa-YFP (black) and K14-CreER/Rosa-SmoM2 (red) clone sizes as measured by total cell content, imaged by confocal microscopy on whole mount tail epidermis from 1 week to 24 weeks after induction. The number of clones analysed for K14-CreER/Rosa-SmoM2 is indicated in Fig. 4b; the number of clones counted in K14-CreER/Rosa-YFP is as indicated in Fig. 2a. b, c, Evolution of the clonal persistence (b) and labelled cell fraction (c) for K14-CreER/Rosa-SmoM2 clones in the interscale. d, Fraction of EdU–BrdU double-labelled cells as a function of basal clone size at 8 weeks for K14-CreER/Rosa-SmoM2 clones, for 2 (left), 4 (centre) and 6 (right) days of continuous BrdU incorporation. e, Immunostaining for β4-integrin, SmoM2 and active-caspase-3 in K14-CreER/Rosa-SmoM2 clones 8 weeks after SmoM2 activation. f, Percentage of BCC, dysplastic, hyperplastic and normally differentiating clones presenting at least one active-caspase-3 positive cell at 8 weeks after induction (n = 117 clones analysed from 4 independent experiments). g, Quantification of the number (%) of basal and suprabasal apoptotic cells in dysplastic, hyperplastic and normally differentiating Inv-CreER/Rosa-SmoM2 clones 8 weeks after SmoM2-activation. h, Percentage of dysplastic, hyperplastic and normally differentiating Inv-CreER/Rosa-SmoM2 clones presenting basal and suprabasal apoptosis 8 weeks after oncogenic activation. i, Cumulative distribution of the fraction of basal apoptosis as a function of basal cell number in a K14-CreER/Rosa-SmoM2 clone at 8 weeks (data in red). The green line is the expected theoretical distribution of apoptotic fraction if apoptosis occurred randomly (following a Poisson process), in any clone with the same probability. The data are statistically different from the random theory, showing that apoptosis clusters in certain clones at a given time point. j, Quantification of EdU–BrdU double-labelled cells as a function of the period of continuous BrdU incorporation for large K14 clones at 4 weeks (black), 8 weeks (orange) and 12 weeks (red) after clonal induction. The dashed lines represent the model fit (Supplementary Theory). k, Whisker plot of the suprabasal clone size in the interscale. The boxes delineate the first and third quartiles of the data, and the whiskers delineate the first and last deciles of the data at a given time point. The thick continuous line is the best fit from the model from which we extract the probability of fate choices in tumour SC and progenitors, displayed in Fig. 4g. The thin lines represent the mean clone sizes of SC- (top curve) and CP- (bottom curve) derived clones if they were alone. l, Short-term fate outcome of progenitors in K14-CreER/Rosa-SmoM2 clones at 8 weeks, as assessed by using EdU as a clonal marker. We count only cell doublets and classify them as either basal–basal, basal–suprabasal, or suprabasal–suprabasal (n = 49 clones from 3 independent experiments). Immunostaining for β4-integrin, EdU and SmoM2 in K14-CreER/Rosa-SmoM2 hyperplastic/dysplastic clones (top) and in BCC (bottom panel). SB, suprabasal. Hoechst nuclear staining is represented in blue; scale bars, 10 μm. Error bars represent the s.e.m.

Extended Data Figure 8 Effect of p53 deletion in the cellular dynamics of CPs and SCs.

a, Immunohistochemistry staining for p53 in Inv-CreER/Rosa-SmoM2 and K14-CreER/Rosa-SmoM2 clones 12 weeks after induction. b, Quantification of normal, hyperplastic, dysplastic and BCC clones in scale region of K14CreER/Rosa-SmoM2/p53fl/fl and Inv-CreER/Rosa-SmoM2/p53fl/fl mice. Description of number of counted clones is found in the Methods section. c, Distribution of clone sizes as measured by total cell content, imaged by confocal microscopy on whole mount tail epidermis. The number of clones analysed is indicated in Fig. 5d. Clone merger events were observed after 12 weeks following oncogenic activation in K14-CreER/Rosa-SmoM2/p53fl/fl preventing the accurate quantification of clonal persistence and clone size at longer times. d, Comparison of basal clone size distribution of Inv-CreER/Rosa-SmoM2/p53fl/fl versus Inv-CreER/Rosa-SmoM2 and K14-CreER/Rosa-SmoM2/p53fl/fl versus K14-CreER/Rosa-SmoM2 at 8 weeks and 12 weeks upon tamoxifen administration. e, Evolution of the clonal persistence of Inv-CreER/Rosa-SmoM2/p53fl/fl and K14-CreER/Rosa-SmoM2/p53fl/fl clones. f, Immunostaining of active-caspase-3 and SmoM2 8 weeks after induction in Inv-CreER/Rosa-SmoM2/p53fl/fl. g, Quantification of the proportion of apoptotic clones in Inv-CreER/Rosa-SmoM2/p53fl/fl (n = 90 clones from 3 independent experiments), and K14-CreER/Rosa-SmoM2/p53fl/fl (n = 82 animals from 3 independent experiments) 8 weeks after induction. Hoechst nuclear staining is represented in blue; scale bars, 10 μm. Error bars represent the s.e.m.

Extended Data Figure 9 Model of BCC initiation.

Activation of SmoM2 in SCs leads to the generation of BCC owing to an increase in cell proliferation and resistance to apoptosis. However, activation of p53 in SmoM2-expressing CPs restricts the progression of dysplastic clones to BCC by promoting apoptosis and cell-cycle arrest. Deletion of p53 in CPs allows them to progress into BCC.

Supplementary information

Supplementary Information

This file contains the Supplementary Theory, which provides further details on the modeling approach to study cell fate in the mouse tail epidermis, as well as on the data analysis and statistics used. It starts by describing how quantitative analysis of clonal date data can be used to elucidate the lineage hierarchy and progenitor fate behavior under conditions of normal homeostasis. This modeling scheme and its analysis provides a platform to then consider how stem and progenitor cell fates are perturbed in the progression to basal cell carcinoma (BCC) following smoothened activation. (PDF 523 kb)

Supplementary Data 1

This file contains persistence data (corresponding to data shown in Figure 2 and Extended Data Figures 4, 5, 6, 7 & 8). (XLSX 42 kb)

Supplementary Data 2

This file contains clone size data (corresponding to data shown in Figures 2, 3, 4 & 5 and Extended Data Figures 2, 3, 4, 5, 6, 7 & 8). (XLSX 147 kb)

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Sánchez-Danés, A., Hannezo, E., Larsimont, J. et al. Defining the clonal dynamics leading to mouse skin tumour initiation. Nature 536, 298–303 (2016). https://doi.org/10.1038/nature19069

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