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

Abstract

Multiple cancers may arise from within a clonal region of preneoplastic epithelium, a phenomenon termed 'field change'. 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 cancers. 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.

Figure 1. Notch inhibition by DNM leads to clonal expansion

a: Dominant negative mastermind (DNM) mouse. The Notch binding domain of Maml1 (orange) is fused to GFP (green) and targeted to the Rosa26 locus downstream of a ‘stop’ cassette (red). Following cre induction the stop cassette is excised and DNM protein expressed in a small proportion of oesophageal basal cells. In the control strain, Yellow Fluorescent Protein (YFP) is similarly targeted to the Rosa26 locus. b: Protocol: Clonal labelling was induced in Ahcre^ERTR26^flDNM/wt (DNM) and control Ahcre^ERTR26^flEYFP/wt (YFP) mice, and samples taken from 24 hours to 1 year post induction (green arrows). c: Quantification of area recombined epithelium over time. Three images were analysed from each of three mice at each time point for each mouse strain. d: Rendered confocal z stacks of OE showing ‘top down’ views of typical areas of wholemounts at times indicated, green indicates DNM or YFP, blue is Dapi. Scale bars, 50μm in 24 hour panels and 300μm in other panels. Arrows indicate single recombined cells. e,f: QRT-PCR of Notch regulated transcripts in OE, relative to Gapdh mRNA. e: Flow sorted DNM expressing basal cells compared with control DNM negative basal cells at 15 days post induction. f: Unsorted epithelium from induced DNM mice compared with age matched, uninduced, DNM control mice 1 year post induction. Values are means of 5 (control) or 6 (DNM) independent biological repeats at each time point, normalized to control (=1). Error bars are SEM *, p< 0.05; **, p < 0.01; ***, p< 0.001 by t test.

Figure 2. DNM cell fate is tilted towards proliferation at early time points

a: Confocal images of the basal layer showing typical clones at times indicated. Green, DNM or YFP, blue Dapi, scale bars 10μm. b: Basal cells/clone at 10 days post-induction. Points are data for frequency of clones, YFP (blue, 215 clones), DNM (black, 250 clones), imaged from three animals of each strain in one experiment. Lines show predictions of models shown in g (DNM) and Supplementary Figure 1b (YFP). Shading indicates 95% confidence interval of the model prediction (see Supplementary Note). c: Three-dimensional reconstructions showing typical 10 day clones in DNM and YFP mice, arrows indicate suprabasal cells. Note floating YFP clone with no basal cells. Green, DNM or YFP; white, basal cell marker α6 integrin; scale bars 10μm. d: Proportion of ‘basal’ clones, containing one or more basal cells, imaged 10 days post-induction in YFP (346 clones, open circles) and DNM mice (320 clones, solid circles). Values are means/mouse from four mice per strain in one experiment, ***, p< 0.001 by 2 way Anova. e, f: Size distribution of clones categorized by the number of basal and suprabasal cells, 10 days after induction in YFP (e, 215 clones) and DNM (f, 250 clones) mice from a single experiment. Green bars are data from cell lineage tracing and points are predictions of the models in g (DNM) and Supplementary Fig. 1b (YFP control). Error bars indicate 95% confidence intervals of the model predictions. g: Model of DNM cell behaviour at early time points (see Supplementary Note). Division rate of DNM progenitors (blue with green edge) is 6/week (acceptable parameter range 5.3-6.4, YFP control 2/week, Supplementary Fig. 1b). Stratification rate (arrow) of differentiated DNM cells (red with green edge) is 0.8/week, (0.6-1.1/week, YFP 3.5/week). The proportion of divisions with DD outcome is negligible, 0% (0-0.7%, YFP 10%), 89% of divisions are PD (85-91%, YFP 80%) and 11% are PP (8.5-15%, YFP 10%). Clone is set amid wild type progenitors (blue) and differentiated cells (red). h,i: Validation of model by EdU lineage tracing. 7 days post induction, DNM mice were administered a single dose of EdU and sampled after 24, 48 and 72 hours. h: Rendered confocal Z stacks of typical clones at the times indicated, red is EdU, green DNM, grey indicates clone boundary, scale bars 10μm. i: Mean EdU positive cells per clone in each of three mice per time point in one experiment. Total number of clones imaged was 57 (24 hours), 60 (48 hours) and 57 (72 hours). Red dots indicate predictions of model shown in g (see Supplementary Note).

Figure 3. DNM accelerates neighbouring wild type cell stratification in a Notch dependent manner

a: Protocol: DNM mice were induced, and treated with EdU 48 hours prior to culling at 3 months post induction. b: Ratio of EdU positive suprabasal: basal cells at the edge and distant from clones. Values are means from each animal in one experiment. At least 1100 EdU positive cells from three animals were analysed per group. **, p < 0.01 by t test. c: Rendered confocal z stacks showing typical location of EdU labelled cells (red) at the DNM clone edge (green), and distant from clone. Dapi is blue, scale bars 10μm. Arrows indicate suprabasal, EdU positive wild type cells adjacent to clone. d: Ratio of EdU positive suprabasal:basal cells at clone edges in DNM animals treated with DBZ or vehicle control. Values are means from each animal in one experiment. At least 1100 EdU positive cells from three animals were analysed per group. ** p<0.01 by t test.

Figure 4. DNM epithelium 1 year post induction

a: Typical lateral views of Z stacks stained for the membrane marker Cdh1 (E-cadherin, red), showing basal cell crowding in one year induced DNM animals (lower panels), compared to aged-matched un-induced controls (upper panels). DNM is green, Dapi in blue, basal cell marker Itga6 (α6 integrin), white. Scale bars 10μm. b: Mean basal cell density in DNM animals induced for 3 months and 1 year (solid circles), or un-induced controls (open circles). Data are from at least four fields per mouse in 3 animals at 3 months and 4 animals at 1 year. ***, p< 0.001 by t test. c: Model of cell behaviour 1 year post induction, derived from EdU lineage tracing (d,e). DNM progenitor cell division rate is 4.3/week (acceptable parameter range 3.7-4.9), stratification rate of mutant differentiated cells 5.4/week (5.0-5.8) and proportion of PP and DD divisions 8% (5-11%). Analysis is based on a total of 86 (control, 2 mice) and 78 (induced, 3 mice) EdU doublets, in one experiment. d,e: Clonal EdU lineage tracing. EdU was administered at 6pm, resulting in sparse labelling. Epithelial wholemounts were imaged after 48 hours in DNM mice or age matched uninduced DNM controls. EdU (red), DNM (green); DAPI (blue). Scale bars 10μm. d: Confocal optical slices of the basal layer showing representative EdU+ doublets and triplets e: Confocal Z stack reconstructions of typical ‘floating’ EdU positive suprabasal cell doublets (arrows), indicating restoration of DD outcome of cell division (Supplementary Note). ‘c’ indicates non specific EdU staining in cornified layer.

Figure 5. DNM expression expands p53 mutant clones

a: Protocol: DNM mice were treated with Diethylnitrosamine (DEN, purple) for 15 days, induced (blue arrow) and wholemounts prepared 1 and 5 months post induction (green arrows). b: Scatter and Box plot showing area of clones staining for p53 alone (n=75 clones, open circles), both p53 and DNM (n=11, open squares), or DNM alone (n=129, closed triangles). Oesophagi from 6 mice were imaged one month post DNM induction in one experiment. Whiskers indicate 2.5-97.5 percentiles, ***p< 10⁻⁶ by Mann-Whitney U test. c-f: Representative z-stack projections one month post induction. Typical clones positive for p53 (red) alone (arrows indicate clone positive for DNM alone, c, d), or for both DNM (green) and p53 (e, f) are shown, scale bars 10μm. g, h Area of double positive epithelium, in DEN-treated DNM mouse 5 months after induction, scale bar 50μm. i: Protocol to investigate the effect of subsequent carcinogen exposure on DNM expressing epithelium: DNM mice were induced (blue arrow) (DNM+DEN) or kept un-induced (DEN) and after a 3 month interval treated with DEN for 2 months. The area of tumours in epithelial wholemounts was recorded 10 months later (j). Results are from one experiment with 6 animals per group, * p= 0.014 by Mann-Whitney U test. k: Protocol to investigate the effect of long-term DNM on DEN treated epithelium. DNM mice were treated with Diethylnitrosamine (DEN, purple) for 2 months, followed by DNM induction (blue arrow). Frequency and area of tumours was analyzed 9 months after DNM induction (green arrow, l,m). l: Number of lesions per unit area in DNM positive (open circles) and negative areas (closed circles) in each of 7 mice in one experiment, * P<0.05 by t test. m: Area of individual lesions positive (closed circles) and negative (open circles) for DNM (** P <0.005 by Mann Whitney U test).