Heterogeneity in clone dynamics within and adjacent to intestinal tumours identified by Dre-mediated lineage tracing.

Somatic models of tissue pathology commonly use induction of gene-specific mutations in mice mediated by spatiotemporal regulation of Cre recombinase. Subsequent investigation of the onset and development of disease can be limited by the inability to track changing cellular behaviours over time. Here, a lineage-tracing approach based on ligand-dependent activation of Dre recombinase that can be employed independently of Cre is described. The clonal biology of the intestinal epithelium following Cre-mediated stabilisation of ß-catenin reveals that, within tumours, many new clones rapidly become extinct. Surviving clones show accelerated population of tumour glands compared to normal intestinal crypts but in a non-uniform manner, indicating that intra-tumour glands follow heterogeneous dynamics. In tumour-adjacent epithelia, clone sizes are smaller than in the background epithelia, as a whole. This suggests a zone of ∼seven crypt diameters within which clone expansion is inhibited by tumours and that may facilitate their growth.

Quantifying Microsatellite Mutation Rates from Intestinal Stem Cell Dynamics in Msh2-Deficient Murine Epithelium.

Microsatellite sequences have an enhanced susceptibility to mutation, and can act as sentinels indicating elevated mutation rates and increased risk of cancer. The probability of mutant fixation within the intestinal epithelium is dictated by a combination of stem cell dynamics and mutation rate. Here, we exploit this relationship to infer microsatellite mutation rates. First a sensitive, multiplexed, and quantitative method for detecting somatic changes in microsatellite length was developed that allowed the parallel detection of mutant [CA]n sequences from hundreds of low-input tissue samples at up to 14 loci. The method was applied to colonic crypts in Mus musculus, and enabled detection of mutant subclones down to 20% of the cellularity of the crypt (∼50 of 250 cells). By quantifying age-related increases in clone frequencies for multiple loci, microsatellite mutation rates in wild-type and Msh2-deficient epithelium were established. An average 388-fold increase in mutation per mitosis rate was observed in Msh2-deficient epithelium (2.4 × 10-2) compared to wild-type epithelium (6.2 × 10-5).

Fixation and Spread of Somatic Mutations in Adult Human Colonic Epithelium.

We investigated the means and timing by which mutations become fixed in the human colonic epithelium by visualizing somatic clones and mathematical inference. Fixation requires two sequential steps. First, one of approximately seven active stem cells residing within each colonic crypt has to be mutated. Second, the mutated stem cell has to replace neighbors to populate the entire crypt in a process that takes several years. Subsequent clonal expansion due to crypt fission is infrequent for neutral mutations (around 0.7% of all crypts undergo fission in a single year). Pro-oncogenic mutations subvert both stem cell replacement to accelerate fixation and clonal expansion by crypt fission to achieve high mutant allele frequencies with age. The benchmarking of these behaviors allows the advantage associated with different gene-specific mutations to be compared irrespective of the cellular mechanisms by which they are conferred.