Biased competition between Lgr5 intestinal stem cells driven by oncogenic mutation induces clonal expansion

Abstract

The concept of 'field cancerization' describes the clonal expansion of genetically altered, but morphologically normal cells that predisposes a tissue to cancer development. Here, we demonstrate that biased stem cell competition in the mouse small intestine can initiate the expansion of such clones. We quantitatively analyze how the activation of oncogenic K-ras in individual Lgr5(+) stem cells accelerates their cell division rate and creates a biased drift towards crypt clonality. K-ras mutant crypts then clonally expand within the epithelium through enhanced crypt fission, which distributes the existing Paneth cell niche over the two new crypts. Thus, an unequal competition between wild-type and mutant intestinal stem cells initiates a biased drift that leads to the clonal expansion of crypts carrying oncogenic mutations.

Figure 1. Clonal expansion of sporadically induced K-ras^G12D in Lgr5^hi cells

1. Confocal scanning of the bottom of small intestinal crypts at indicated time points after sporadic activation of K-ras^G12D mutation in intestinal stem cells (bottom panels) or in WT controls (top panels). Lgr5 stem cells are marked with EGFP (green). Clones are randomly marked with YFP (pseudo color white), RFP (red) or membrane tagged CFP (blue), driven from the R26R-Confetti locus. K-ras^G12D clones expand faster over time than their WT counterparts, many crypts being fixated within 14 days of tracing. Scale bars; 50 μm.

2. Expansion of Lgr5^hi cell numbers over time within clones that contain at least one Lgr5^hi cell. The numbers represent the percentage of clones with a certain number of Lgr5^hi cells for each time point. As the average size of clones gradually increases in WT, the K-ras^G12D activated clones colonize the stem cell compartment much faster. Blue hues represent the relative frequency of Lgr5^hi cell numbers per time point, 50% is blue; 0% is white. Left matrix is for WT clones, right matrix is for K-ras^G12D clones.

Data information: Data from WT clones is reproduced from .

Figure 2. K-ras^G12D mutated Lgr5^hi cells follow a pattern of biased drift due to a faster cell cycle

1. Average size of K-ras clones within the Lgr5^hi stem cell compartment over time, with WT data shown for comparison. Open and blue squares show experimental data from the K-ras mutant and WT clones, respectively, at day 2, 3, 7 and 14 post-induction (data are represented as mean ± SEM.) Black and blue lines show a modeled fit of the biased drift dynamics to the data. The parameter δ controls the degree of imbalance; with δ = 0.45±0.05 (biased) δ = 0 (unbiased) (supplementary theory). In both cases, we separated the crypt in octants, translating the effective stem cell number, n = 8.

2. Extrapolation of the growth curves from (A) over a longer period of time showing the speed with which the biased drift dynamics converges towards monoclonality of the crypt.

3. Clone size distribution of the marked clones in the WT background shows an excellent fit of the experimental data (bars) to the neutral drift model (squares) in (A).

4. Clone size distribution of the K-ras mutant clones shows an excellent fit of the experimental data (bars) to the biased drift model when the bias is set to δ = 0.45 (squares).

5. Confocal image of proximal small intestinal epithelium; either WT or K-ras^G12D activated, illustrate similar EdU (red) incorporation and no abnormal morphology after activation of K-ras^G12D. Paneth cells are stained for lysozyme (green). Arrows point to EdU⁺ intestinal CBC stem cells. Scale bars; 50 μm.

6. Quantification of EdU⁺ CBC stem cells and Paneth cells in whole crypts, and EdU⁺ TA cells per cross-section, in WT and K-ras^G12D mice. More CBC stem cells per crypt enter the S-phase when mutant for K-ras^G12D, indicating a faster cell cycle (> 100 crypts; 3 mice per group).

Data information: In (A), (C), (D), error bars denote SEM and in (F), error bars denote STDEV.

Figure 3. K-ras^G12D mutant clones expand through enhanced crypt fission

1. A  Small intestinal crypts with sporadically activated Lgr5^hi stem cells. Lgr5 stem cells are in green. R26R-Confetti clones are visualized and marked with YFP (pseudo color white), RFP (red) or membrane tagged CFP (blue). After 8 weeks, the majority of surviving WT clones dominated their crypt as the outcome of neutral drift dynamics. Adjacent labeled crypts either had the same color ‘XX’ (ellipse), or different colors ‘XY’ (dashed ellipse).

2. B  As in (A) but for K-ras mutant clones.

3. C  Quantification of labeled crypt clusters for WT and K-ras mice 8 weeks after labeling. The number of XX crypt pairs is significantly higher than in the WT situation.

4. D–F  As in (A–C) but after 16 weeks of tracing. Patches of multiple adjacent labeled crypts appeared. Patches were larger and more frequent in K-ras mutant crypts.

5. G  Probability of obtaining two neighboring monoclonal crypts of the same (P_XX) or different (P_XY) color in WT normalized by the chance that an isolated crypt is fully labeled, P_x. P_XXX represents the probability to finding a cluster of three crypts with the same color, normalized by the probability P_x. Points represent experimental data derived from B, and the lines represent the modeled fit of the crypt fission dynamics with induction frequency p₀^WT = 0.006 ± 0.001 per crypt and a crypt fission rate of f^WT=0.01±0.002 per 8 weeks per crypt (main text and supplementary theory).

6. H  As in (G), but for K-ras mutant clones. In this case, a fit of the data leads to an induction frequency of p₀^Kras = 0.004 ± 0.001 per crypt and a crypt fission rate of f^Kras=0.3±0.04 per 8 weeks per crypt.

Data information: In (G) and (H), error bars denote SEM. Scale bars, 100 μm.

Figure 4. Existing Paneth cell niche is divided over both new crypts

1. Schematic representation of experimental setting. Due to fast colonization and enhanced crypt fission rate of K-ras mutant cells, we captured crypts undergoing fission (physical connected between two halves higher up in the crypt) that contained Paneth cells that already existed prior to clonal marking (non-labeled). Distribution of pre-existing Paneth cells over both halves suggests that fission involved splitting of the existing stem cell population and its niche, rather than single stem cell derived clones that branches off the existing crypt.

2. Left panels, bottom view of crypt undergoing fission. Lgr5^hi cells (green) are all clonally marked by Confetti-YFP (pseudo color white). Pre-existing Paneth cells, not marked by Confetti-YFP, are divided over both new crypts (yellow arrows). Arrowhead points to newly derived clonal Paneth cell. Right panel, 3D rendering of the crypt fission to illustrate the connection still present between the two young crypts (yellow dashed line).

3. Results using the experimental setting outlined in (A), but the clone is marked by Confetti-RFP (red) rather than Confetti-YFP.