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Differentiation imbalance in single oesophageal progenitor cells causes clonal immortalization and field change

Nature Cell Biology volume 16, pages 612–619 (2014)Cite this article

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Abstract

Multiple cancers may arise from within a clonal region of preneoplastic epithelium, a phenomenon termed ‘field change’1,2. However, it is not known how field change develops. Here we investigate this question using lineage tracing to track the behaviour of scattered single oesophageal epithelial progenitor cells expressing a mutation that inhibits the Notch signalling pathway. Notch is frequently subject to inactivating mutation in squamous cancers3,4,5,6. Quantitative analysis reveals that cell divisions that produce two differentiated daughters are absent from mutant progenitors. As a result, mutant clones are no longer lost by differentiation and become functionally immortal. Furthermore, mutant cells promote the differentiation of neighbouring wild-type cells, which are then lost from the tissue. These effects lead to clonal expansion, with mutant cells eventually replacing the entire epithelium. Notch inhibition in progenitors carrying p53 stabilizing mutations creates large confluent regions of doubly mutant epithelium. Field change is thus a consequence of imbalanced differentiation in individual progenitor cells.

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Figure 1: Notch inhibition by a dominant negative mutant of Maml1 (DNM) leads to clonal expansion.
Figure 2: DNM cell fate is tilted towards proliferation at early times.
Figure 3: DNM accelerates neighbouring wild-type cell stratification in a Notch dependent manner.
Figure 4: DNM epithelium 1 year post induction.
Figure 5: DNM expression expands p53 mutant clones.

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Acknowledgements

We thank Warren Pear (University of Pennsylvania) for the DNMaml mouse strain, E. Choolun, K. Murai, J. Fowler and the staff at ARES (Cambridge) for support and technical assistance and R. Kopan for informative discussions. We acknowledge the support of the MRC, the NC3Rs (National Centre for the Replacement, Refinement and Reduction of Animals in Research), the Wellcome Trust (grant numbers WT090334MA, P.H.J., and 098357/Z/12/Z, B.D.S.), a Deutsche Forschungsgemeinschaft Research Fellowship (P.G.) and European Union Marie Curie Fellowship PIEF-LIF-2007-220016 (M.P.A.).

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Authors and Affiliations

  1. MRC Cancer Unit, University of Cambridge, Hutchison-MRC Research Centre, Box 197, Cambridge Biomedical Campus, Cambridge CB2 0XZ, UK

    Maria P. Alcolea, Agnieszka Wabik, Julia Frede & Philip H. Jones

  2. Department of Physics, Cavendish Laboratory, University of Cambridge, J.J. Thomson Avenue, Cambridge CB3 0HE, UK

    Philip Greulich & Benjamin D. Simons

  3. The Wellcome Trust-Cancer Research UK Gurdon Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QN, UK

    Benjamin D. Simons

  4. Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, Cambridge CB2 1QN, UK

    Benjamin D. Simons

Authors
  1. Maria P. Alcolea
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  4. Julia Frede
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Contributions

P.H.J. envisaged the project. M.P.A., P.G., B.D.S. and P.H.J. were involved with experimental design. M.P.A., A.W. and J.F. carried out experiments. P.G. and B.D.S. carried out quantitative analysis. M.P.A. and P.H.J. wrote the manuscript, P.G. and B.D.S. contributing quantitative sections.

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Correspondence to Philip H. Jones.

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Integrated supplementary information

Supplementary Figure 1 Cell behaviour in normal mouse oesophageal epithelium.

a, Section of mouse oesophageal epithelium. Scale bar, 200 μm. Inset: Structure of epithelium. Proliferation is confined to the basal layer b, differentiating cells enter the suprabasal layers (sb), migrate to the tissue surface, and are then shed into the oesophageal lumen (l). Inset scale bar, 50 μm. b, Cell fate in basal layer of normal homeostatic mouse oesophageal epithelium. Progenitor cells (P, blue) divide at a rate of 1.9/week. Each division can have one of three outcomes: two progenitor daughters (PP), two differentiating daughters (red, DD) or one cell of each (PD). Differentiating cells stratify out of the basal layer (arrow) at a rate of 3.5/week. The outcome of a specific division is unpredictable but the probabilities of each outcome (shown as %) are balanced to achieve homeostasis across the progenitor population. c, Consequences of stochastic cell fate of normal progenitors for clonal survival. Two progenitor cells following the dynamics in b are illustrated. By chance, the progeny of left hand cell clone all differentiate due to a PD division followed by a DD division. Such events are frequent with 90% of clones being lost by differentiation within 3 months7. Progeny of the right hand cell undergo early PP and PD divisions, by the fourth generation the odds of all three progenitor cells undergoing DD division and the clone differentiating are 1 in 1,000. Normal progenitors generate few such persistent clones7. d, Tiled images showing OE wholemounts, immunostained for GFP (green), 1 year after clonal induction of DNM mice. Appearances shown are typical of 17 induced DNM mice and 6 control uninduced DNM mice, 1 year post induction. Control is age matched un-induced DNM animal. Dapi is blue, scale bar, 500 μm.

Supplementary Figure 2 Clone size distributions: data and fit to model.

a,b, Size distribution of clones categorized by the number of basal and suprabasal cells/clone, 7 days after induction. a, YFP control (n = 103 clones), b DNM clones (n = 300 clones), imaged from three mice per group in one experiment . Green bars are data from three-dimensional clone imaging, points indicate model predictions, error bars are 95% confidence intervals of the predictions of the models in Fig. 2g (DNM) and Supplementary Fig. 1b (YFP control) (Supplementary Note). c, Basal clone size distribution at 7 days post induction. Points are data, indicating the percentage of clones with a given number of basal cells in clones from YFP control (blue, n = 103 clones) or DNM induced mice (black, n = 300 clones). Solid lines show predictions of models shown in Fig. 2g (DNM) and Supplementary Fig. 1b (YFP controls), shading indicates 95% confidence intervals of the model predictions (Supplementary Note). d, Basal clone size distribution 15 days after induction of DNM mice using the best fit parameters shown in Fig. 2g. Data is shown in a logarithmic binning, where each bin grows by a factor of two from left to right, n = 60 clones imaged from three mice in one experiment, indicating the percentage of clones with a given number of basal cells. Solid line shows model prediction, shading indicates 95% confidence interval of model prediction (Supplementary Note). e, Full data set fitting of basal clone size distributions in DNM mice 10 days after induction (Supplementary Note). The resulting fit parameters were λ = 5.5/week, r = 0.065, γ = 0.85/week. Lines are model predictions, points show data and shading 95% confidence range of model predictions. f, Clonal area prediction over time (Supplementary Note) measurements of clonal areas in DNM induced mice are compared with model predictions. The best fit model parameters were used for this prediction. Taking into account the calculated labelling frequency of 1/526, for model extrapolation we assume that each basal cell corresponds to a total area fraction of 0.19%. g, Two parameter fit of total clone size distribution 10 days post induction (Supplementary Note). Data (black squares) together with model predictions of the simplified model from Supplementary Note, which has only two parameters λ and r (black line, shaded area is 95% confidence range). In this model cell loss is neglected (σ = 0) and cell fate is fully tilted (δ = 1). The parameters are λ = 5.4/week and r = 0.065 resulting from a maximum likelihood fit of this model.

Supplementary Figure 3 Expression of Sox9, Krt6 and Itgb4 in DNM and wild type cells.

Confocal images of cryosections of oesophageal epithelium in DNM mice 3 months post induction. Green is DNM, Blue, Dapi, Red is the basal cell marker Itga6 (which partners Itgb4 in the hemidesmosome) and white is Sox9 a,b, Krt6, c,d and Itgb4 e,f. Scale bar, 20 μm. Each image is typical of at least 8 sections from each of three mice in one experiment.

Supplementary Figure 4 Immune effects of clonal DNM induction.

a, Serum Tslp levels at 10 days, 3 months and 1 year following clonal induction of DNM mice (DNM) and in aged-matched un-induced controls. Data are from n = 5 mice (10d) and 3 mice (3m) per condition. At 1 year, n = 5 mice (control), 11 mice (DNM) and 8 mice (DNM-High). As a positive control, DNM animals were treated with high dose β napthoflavone and tamoxifen, to induce epidermal DNM expression (inset) which resulted in increased serum Tslp. **P < 0.01 by t test. Inset shows epidermal DNM expression in typical wholemounts of tail epidermis from DNM and DNM-High groups at 6 months post induction; Dapi is blue, DNM green, scale bar, 100 μm, images typical of 3 mice/group. b,c, Epithelial leukocytes are excluded from DNM epithelium. OE wholemounts were stained for the pan-leukocyte marker Cd45 (red) and the T lymphocyte marker Cd3 (white) in induced DNM mice and age-matched un-induced controls. DNM is green, Dapi blue, scale bars, 50 μm, except low magnification views 100 μm, images shown typical of at least 4 sections per mouse in 3 mice/group. b, Typical images from 6 months induced DNM mice and age-matched un-induced controls, note the exclusion of immune cells from the DNM mutant areas. c, Typical fields from control and DNM epithelium one year post induction. d, Number of Cd45 positive cells per field, one year post induction. At least 200 Cd45 positive cells were scored per condition. n = 3 mice per group in one experiment. *P < 0.05 by t test e, Epithelial Cd3:Cd45 ratio, one year post induction. At least 250 Cd45 positive cells were scored, n = 3 mice per group in one experiment. f, Leukocytes in oesophageal submucosa in six months induced DNM mice and age-matched un-induced controls. Cryosections were stained for the pan-leukocyte marker Cd45 (red) and DNM (green), Dapi is blue, scale bar, 20 μm. No significant difference in leukocyte density was observed, images shown typical of at least 4 sections per mouse in 3 mice/group. g, Oesophageal submucosal wholemounts stained for activated fibroblast marker α-smooth muscle actin (αSMA, red) in one year induced DNM mice and age-matched un-induced controls. Dapi is blue, scale bar, 20 μm. No αSMA staining was detected, images shown typical of at least 4 sections per mouse in 3 mice/group. h, Typical sections of epithelium in induced and age-matched uninduced control DNM mice 1 year post induction, stained for activated Caspase3 (red). UV irradiated sample is a positive control, exposed to ultraviolet radiation and maintained as explant culture, positive cells are indicated by arrowheads. No positive cells were detected in 10 sections/mouse from 3 DNM or 3 control mice. DNM is green and Dapi blue, scale bar, 20 μm.

Supplementary Figure 5 Representative lesions in uninduced and induced DNM mice subsequently treated with DEN.

a–d, Representative images of epithelial wholemounts showing typical tumours found in animals treated in protocol shown in Fig. 5i. a,b, DEN treated uninduced DNM controls (low level green is auto-fluorescence). c,d, induced DNM mice. DNM is stained green, Dapi is blue, scale bar, 200 μm.

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Alcolea, M., Greulich, P., Wabik, A. et al. Differentiation imbalance in single oesophageal progenitor cells causes clonal immortalization and field change. Nat Cell Biol 16, 612–619 (2014). https://doi.org/10.1038/ncb2963

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