Patterns of Proliferative Activity in the Colonic Crypt Determine Crypt Stability and Rates of Somatic Evolution

Epithelial cells in the colon are arranged in cylindrical structures called crypts in which cellular proliferation and migration are tightly regulated. We hypothesized that the proliferation patterns of cells may determine the stability of crypts as well as the rates of somatic evolution towards colorectal tumorigenesis. Here, we propose a linear process model of colonic epithelial cells that explicitly takes into account the proliferation kinetics of cells as a function of cell position within the crypt. Our results indicate that proliferation kinetics has significant influence on the speed of cell movement, kinetics of mutation propagation, and sensitivity of the system to selective effects of mutated cells. We found that, of all proliferation curves tested, those with mitotic activities concentrated near the stem cell, including the actual proliferation kinetics determined in in vivo labeling experiments, have a greater ability of delaying the rate of mutation accumulation in colonic stem cells compared to hypothetical proliferation curves with mitotic activities focused near the top of the crypt column. Our model can be used to investigate the dynamics of proliferation and mutation accumulation in spatially arranged tissues

Colorectal Cancer Through Simulation and Experiment

Colorectal cancer has continued to generate a huge amount of research interest over several decades, forming a canonical example of tumourigenesis since its use in Fearon and Vogelstein’s linear model of genetic mutation. Over time, the field has witnessed a transition from solely experimental work to the inclusion of mathematical biology and computer-based modelling. The fusion of these disciplines has the potential to provide valuable insights into oncologic processes, but also presents the challenge of uniting many diverse perspectives. Furthermore, the cancer cell phenotype defined by the ‘Hallmarks of Cancer’ has been extended in recent times and provides an excellent basis for future research. We present a timely summary of the literature relating to colorectal cancer, addressing the traditional experimental findings, summarising the key mathematical and computational approaches, and emphasising the role of the Hallmarks in current and future developments. We conclude with a discussion of interdisciplinary work, outlining areas of experimental interest which would benefit from the insight that mathematical and computational modelling can provide

A theoretical investigation of the effect of proliferation & adhesion on monoclonal conversion in the colonic crypt

The surface epithelium lining the intestinal tract renews itself rapidly by a coordinated programme of cell proliferation, migration and differentiation events that is initiated in the crypts of Lieberkühn. It is generally believed that colorectal cancer arises due to mutations that disrupt the normal cellular dynamics of the crypts. Using a spatially structured cell-based model of a colonic crypt, we investigate the likelihood that the progeny of a mutated cell will dominate, or be sloughed out of, a crypt. Our approach is to perform multiple simulations, varying the spatial location of the initial mutation, and the proliferative and adhesive properties of the mutant cells, to obtain statistical distributions for the probability of their domination. Our simulations lead us to make a number of predictions. The process of monoclonal conversion always occurs, and does not require that the cell which initially gave rise to the population remains in the crypt. Mutations occurring more than one to two cells from the base of the crypt are unlikely to become the dominant clone. The probability of a mutant clone persisting in the crypt is sensitive to dysregulation of adhesion. By comparing simulation results with those from a simple one-dimensional stochastic model of population dynamics at the base of the crypt, we infer that this sensitivity is due to direct competition between wild-type and mutant cells at the base of the crypt. We also predict that increases in the extent of the spatial domain in which the mutant cells proliferate can give rise to counter-intuitive, non-linear changes to the probability of their fixation, due to effects that cannot be captured in simpler models

Stem cell biology and clonal expansion in normal and adenomatous human intestinal crypts

Gastrointestinal cancer is thought to be primarily a disease of stem cells, whereby a tumorigenic stem cell clone can expand within an individual colonic crypt and then within the epithelium to form an adenoma - the pre-malignant lesion of the colon. However, data demonstrating stem cell populations and the dynamics of clonal expansion in human intestinal crypts is lacking. Naturally occurring, somatic clonal mutations in mitochondrial DNA were used to identify the progeny of a putative single stem cell lineage within crypts; this allowed the visualization of putative stem cell clones arising and expanding within human colon and small bowel crypts. Immunohistochemistry for lineage specific markers, to confirm multi-potentiality, and in-situ hybridisation for stem cell markers was then performed to phenotype clones further and to demonstrate a single stem cell lineage and their direct progeny within the human crypt. By combining clonal somatic mutations in mitochondrial DNA with methylation signatures, the dynamics of clonal expansion within normal colon crypts and the epithelium was studied, and using mathematical modeling the time course of these events was able to be estimated. By applying the same techniques, in addition to studying genomic mutations, the dynamics of human adenoma growth was investigated; this suggested that adenomas appear to grow in a punctuated manner, with episodes of rapid clonal expansion being followed by periods of relative quiescence

Exploring stem cell dynamics, clonal expansion and pseudopolyps in inflammatory bowel disease.

PhDInflammatory bowel disease (IBD) confers a high risk of development of colitis-associated colorectal cancer in patients with extensive colitis. Crypt fission is a mechanism of clonal expansion in the intestinal epithelium. Although fission is rare in the normal colon, many crypts in IBD patients are in the process of fission. Protumourigenic mutations can spread through the entire inflamed colon relatively quickly indicating that stem cell dynamics are altered in IBD. Some patients with IBD develop pseudopolyps as a result of mucosal ulceration and epithelial regeneration. The aim of this PhD was to investigate the effect of inflammation on niche succession, the crypt cycle and the expansion of clones in the IBD intestine. Pseudopolyps were examined as potential sites for clonal expansion by determining the frequency of mutated pseudopolyps and proliferative potential, and examining their microRNA (miRNA) profile relative to inactive, active and dysplastic mucosa, and adenoma and cancerous tissue. This thesis will show that crypt fission cycles in inflammatory bowel diseased colon are protracted and that each stage of crypt fission appears to be slow. Overall, clonally related adjacent IBD crypts seem to share a more recent common ancestor than non-related IBD crypts, supporting increased crypt fission rates in IBD. The proliferative drive induced by continuous inflammation and mucosal repair in ulcerative colitis (UC) appears to promote the expansion of CCO-deficient patches. Furthermore, niche succession appears to be faster in active IBD. Pseudopolyps are a source of regeneration within the epithelium and, as shown here, have a faster proliferative drive than background mucosa in IBD patients. Pseudopolyps are not genetically inert and are a potential source of protumourigenic mutations in UC. Hence, pseudopolyps are a potential reservoir within the inflamed epithelium where mutations are harboured and where there is no competition from neighbouring epithelium, as it has been denuded following previous inflammation. MiRNA expression in pseudopolyps differs from that of UC-dysplasia and mucosa. In particular, the MiR-29 family was downregulated in pseudopolyps, a miRNA family that has been implicated in intestinal fibrosis formation in stricturing Crohn’s disease. Pseudopolyps have been traditionally thought of as benign, genetically inert and incidental findings characteristic of chronic inflammation. My research runs counter to this view indicating an exciting paradigm shift in the way we consider pseudopolyps, which may eventually alter the endoscopic management of these lesions in the future.Bart’s and The London Charity; Medical Research Counci

Accumulation of Somatic Mutations in Normal Human Colonic Epithelium

I investigated stem cell dynamics in normal human colon by detecting somatic variants affecting X-linked and autosomal genes using immunohistochemistry. Applying neutral clonal marks to a large cohort of patients to interpret age-related trends in clone frequencies has established the baseline stem cell dynamics of the tissue. Analysis of a number of new clonal marks showing biased behaviours suggests that different gene-specific mutations can subvert constraints resulting from the tissue architecture in different ways. In respect of intra-gland competition of stem cell derived clones, a disadvantage is observed for the histone modifier HDAC6, while at the other end of the spectrum, loss of the cohesin member STAG2 strongly advantages affected stem cells. Subsequent clonal expansion beyond the boundary of a single crypt is recognised by clones occupying multi-crypt patches. Quantification of such events allows the rate of lateral expansion for different mutations to be measured. Moderate effects were found for PTEN, p53 and STAG2, while mutations in the histone demethylase KDM6A generate very large areas of mutant epithelium in aged humans. Further, targeted sequencing revealed dramatic expansion of KRAS-mutant clones in histologically normal colon. Patches may arise from crypt fission and fusion events. Using a clonal mark based on mild periodic acid-Schiff staining, the neutral crypt fission and fusion rates were quantified. Against this baseline, it was found that KDM6A-mutant clones expand mainly by fission, while fusion remains at homeostatic levels. The emerging picture is that of the aged human colon consisting of a mosaic of different mutations. The work presented in this thesis offers detailed insights into the rates at which different gene-specific mutations arising in colonic stem cells can become fixed within individual crypts and undergo subsequent lateral expansion through crypt fission and fusion events. This defines the timeframe taken for cancer drivers to achieve high mutant allele burden within the tissue, which can serve as a basis for cancer prevention strategies.Wellcome Trus

Mathematical modeling of Lynch syndrome carcinogenesis

Cancer is one of the leading causes of disease-related death worldwide. In recent years, large amounts of data on cancer genetics and molecular characteristics have become available and accumulated with increasing speed. However, the current understanding of cancer as a disease is still limited by the lack of suitable models that allow interpreting these data in proper ways. Thus, the highly interdisciplinary research field of mathematical oncology has evolved to use mathematics, modeling, and simulations to study cancer with the overall goal to improve clinical patient care. This dissertation aims at developing mathematical models and tools for different spatial scales of cancer development at the example of colorectal cancer in Lynch syndrome, the most common inherited colorectal cancer predisposition syndrome. We derive model-driven approaches for carcinogenesis at the DNA, cell, and crypt level, as well as data-driven methods for cancer-immune interactions at the DNA level and for the evaluation of diagnostic procedures at the Lynch syndrome population level. The developed models present an important step toward an improved understanding of hereditary cancer as a disease aiming at rapid implementation into clinical management guidelines and into the development of novel, innovative approaches for prevention and treatment

Determining the impact of mitochondrial dysfunction on stem cell dynamics and proliferation within the colon

PhD ThesisClonally expanded mitochondrial DNA (mtDNA) point mutations have been shown to cause mitochondrial dysfunction in the form of cytochrome c oxidase deficiency (COX deficiency) within ageing human colonic epithelium. Currently, there are a lack of robust stem cell markers within human colonic epithelium, however the detection of mitochondrial dysfunction has been shown to be a useful stem cell lineage marker, enabling the investigation of stem cell dynamics within colonic crypts. Using the most robust data set of COX deficiency frequency and mtDNA mutation number, a computational model that simulates stem cell dynamics within human colonic crypts was constructed. In silico stem cell modelling suggests: there are approximately 5 stem cells within human colonic crypts, stem cell divisions occur asymmetrically the majority of the time, and that infrequent symmetric stem cell division permits niche succession of individual stem cell clones. However, the in silico data was unable to match the biological data when a model simulating neutral drift stem cell dynamics was used, suggesting a change in stem cell biology when mitochondrial dysfunction was present. In order to assess the impact of mitochondrial dysfunction within colonic stem cells in vivo, a mouse model of mitochondrial dysfunction was crossed with a mouse model enabling the visualisation of cells expressing Lgr5 (a well-accepted stem cell marker). Double thymidine analogue labelling was used to identify cells traversing through the cell cycle, together with a marker of proliferation. This data suggests that stem cells with mitochondrial dysfunction have a small but significant increase in cell cycle progression rate compared to normal stem cells. When these data were included in the model, a better fit to the biological data was achieved. These findings suggest that mitochondrial dysfunction does significantly impact on stem cell homeostasis. As stem cells with mitochondrial dysfunction are more likely to out-compete normal stem cells over time, this may have potential implications for an increased risk of cancer propagation within the colon

Plasticity in Colorectal Cancer: Why Cancer Cells Differentiate

The cancer stem cell hypothesis poses that the bulk of differentiated cells are non-tumorigenic and only a subset of cells with self-renewal capabilities drive tumor initiation and progression. This means that differentiation could have a tumor-suppressive effect. Accumulating evidence shows, however, that in some solid tumors, like colorectal cancer, such a hierarchical organization is necessary. The identification of Lgr5 as a reliable marker of normal intestinal epithelial stem cells, together with strategies to trace cell lineages within tumors and the possibility to selectively ablate these cells, have proven the relevance of Lgr5+ cells for cancer progression. On the contrary, the role of Lgr5− cells during this process remains largely unknown. In this review, we explore available evidence pointing towards possible selective advantages of cancer cells organized hierarchically and its resulting cell heterogeneity. Clear evidence of plasticity between cell states, in which loss of Lgr5+ cells can be replenished by dedifferentiation of Lgr5− cells, shows that cell hierarchies could grant adaptive traits to tumors upon changing selective pressures, including those derived from anticancer therapy, as well as during tumor progression to metastasis