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

The Interplay between Wnt Mediated Expansion and Negative Regulation of Growth Promotes Robust Intestinal Crypt Structure and Homeostasis

The epithelium of the small intestinal crypt, which has a vital role in protecting the underlying tissue from the harsh intestinal environment, is completely renewed every 4–5 days by a small pool of stem cells at the base of each crypt. How is this renewal controlled and homeostasis maintained, particularly given the rapid nature of this process? Here, based on the recent observations from in vitro “mini gut” studies, we use a hybrid stochastic model of the crypt to investigate how exogenous niche signaling (from Wnt and BMP) combines with auto-regulation to promote homeostasis. This model builds on the sub-cellular element method to account for the three-dimensional structure of the crypt, external regulation by Wnt and BMP, internal regulation by Notch signaling, as well as regulation by internally generated diffusible signals. Results show that Paneth cell derived Wnt signals, which have been observed experimentally to sustain crypts in cultured organs, have a dramatically different influence on niche dynamics than does mesenchyme derived Wnt. While this signaling can indeed act as a redundant backup to the exogenous gradient, it introduces a positive feedback that destabilizes the niche and causes its uncontrolled expansion. We find that in this setting, BMP has a critical role in constraining this expansion, consistent with observations that its removal leads to crypt fission. Further results also point to a new hypothesis for the role of Ephrin mediated motility of Paneth cells, specifically that it is required to constrain niche expansion and maintain the crypt’s spatial structure. Combined, these provide an alternative view of crypt homeostasis where the niche is in a constant state of expansion and the spatial structure of the crypt arises as a balance between this expansion and the action of various sources of negative regulation that hold it in check

Elucidating the Complex Signaling Events Driving Intestinal Stem Cell Plasticity Following Injury

Signaling events governing intestinal stem cell (ISC) homeostasis maintain the delicate balance of active self-renewal and passive differentiation to replenish intestinal epithelial cells (IEC) every 3-5 days. However, under certain contexts, ISC function is irreversibly compromised—requiring committed IEC lineages to dedifferentiate and regain “stemness”. In the current studies, we examine the signaling events driving epithelial cell responses to injury to expose pathologic failures in the healing response. Our specific goal is to tease out the cellular contexts that promote dedifferentiation to design effective therapeutics for disease conditions compromising ISC function. First, we generated a novel transgenic animal using the epithelial-specific Villin promoter to constitutively ablate the critical negative regulator in the Phosphotidyl-inositol 3 Kinase (PI3K) signaling pathway, p85α, in IECs. At baseline, removal of p85α led to increased activation of PI3K/Akt, as seen through elevations in phosphorylated PTEN, Akt, and GSK3β. The consequence of the persistent activation was a shifting of cells out of the stem cell niche, into more differentiated the proliferative progenitor pool, as seen by a decrease in Lgr5+ cells per crypt and an increase in Axin2+ transcripts by in situ hybridization. Further investigations revealed PI3K activation led to a bolstered secretory lineage, increased total number and size of Paneth cells within the crypt through reduced Notch signaling. Subsequent studies mechanistically clarify mitochondrial bioenergentics in p85-deficient epithelial cells demonstrate enhanced State III respiration to drive production of ROS. ROS activates p38-MAPK to mount observed differentiation pressure. Using whole-body irradiation to target ISCs, we revealed that p85-deficient crypts underwent restitution at a faster pace than their control counterparts. The consequence of the enhanced regeneration was an increased organism survival after lethal irradiation. Transcript flux revealed that p85-deficient crypts increased Wnt and PI3K signaling targets, specifically Lgr5, Axin2 and Survivin earlier in the regenerative process, suggesting the increased secretory progenitor pool could enact dedifferentiation mechanisms to replenish ISCs. Using human samples of radiation-induced intestinal injury, we demonstrate the requirement of Wnt-target Survivin protein expression in IEC survival, suggesting a conserved mechanism and possible avenue for future therapeutic intervention. Next, using isolated crypt epithelial cells from patients with inflammatory bowel disease (IBD), we establish that steroid-treated (prednisone) IBD patients harbor aberrant Wnt/β-catenin and NFκB signaling in IECs despite clinical improvement. At the molecular level, human IECs had significantly blunted cytosolic accumulation of Axin2 protein and subsequently decreased nuclear localization of downstream transcriptional activator, p-β-cateninSer552. Using a validated murine model of IBD, Dextran Sodium Sulfate-induced colitis (DSS colitis), we appreciated conserved blunting of inflammation-induced Wnt activation following dexamethasone administration. Studies in isolated murine ISC cultures revealed that the blunting of Wnt activation occurred in the absence of inflammatory stimulus—suggesting steroids directly affect ISC activation by interfering with β-catenin transcriptional activity. By tracking β-catenin-directed TCF/LEF transcriptional activity with a stably-transfected luciferase construct, we identified the signaling disruption occurred downstream of the β-catenin destruction complex, and resulted in limited ISC activation and mobilization in response to injury. Appropriately, primary ISC cultures from mice demonstrated that at high doses, steroid therapy inappropriately limits ISC activation sufficient Wnt ligands in culture. This leads to a depleted ISC pool and preventing mucosal restitution after injury

Investigation of Inflammation and Tissue Patterning in the Gut Using a Spatially Explicit General-Purpose Model of Enteric Tissue (SEGMEnT)

The mucosa of the intestinal tract represents a finely tuned system where tissue structure strongly influences, and is turn influenced by, its function as both an absorptive surface and a defensive barrier. Mucosal architecture and histology plays a key role in the diagnosis, characterization and pathophysiology of a host of gastrointestinal diseases. Inflammation is a significant factor in the pathogenesis in many gastrointestinal diseases, and is perhaps the most clinically significant control factor governing the maintenance of the mucosal architecture by morphogenic pathways. We propose that appropriate characterization of the role of inflammation as a controller of enteric mucosal tissue patterning requires understanding the underlying cellular and molecular dynamics that determine the epithelial crypt-villus architecture across a range of conditions from health to disease. Towards this end we have developed the Spatially Explicit General-purpose Model of Enteric Tissue (SEGMEnT) to dynamically represent existing knowledge of the behavior of enteric epithelial tissue as influenced by inflammation with the ability to generate a variety of pathophysiological processes within a common platform and from a common knowledge base. In addition to reproducing healthy ileal mucosal dynamics as well as a series of morphogen knock-out/inhibition experiments, SEGMEnT provides insight into a range of clinically relevant cellular-molecular mechanisms, such as a putative role for Phosphotase and tensin homolog/phosphoinositide 3-kinase (PTEN/PI3K) as a key point of crosstalk between inflammation and morphogenesis, the protective role of enterocyte sloughing in enteric ischemia-reperfusion and chronic low level inflammation as a driver for colonic metaplasia. These results suggest that SEGMEnT can serve as an integrating platform for the study of inflammation in gastrointestinal disease.</p

Paracrine and autocrine regulation of gene expression by Wnt-inhibitor Dickkopf in wild-type and mutant hepatocytes

BACKGROUND: Cells are able to communicate and coordinate their function within tissues via secreted factors. Aberrant secretion by cancer cells can modulate this intercellular communication, in particular in highly organised tissues such as the liver. Hepatocytes, the major cell type of the liver, secrete Dickkopf (Dkk), which inhibits Wnt/β-catenin signalling in an autocrine and paracrine manner. Consequently, Dkk modulates the expression of Wnt/β-catenin target genes. We present a mathematical model that describes the autocrine and paracrine regulation of hepatic gene expression by Dkk under wild-type conditions as well as in the presence of mutant cells. RESULTS: Our spatial model describes the competition of Dkk and Wnt at receptor level, intra-cellular Wnt/β-catenin signalling, and the regulation of target gene expression for 21 individual hepatocytes. Autocrine and paracrine regulation is mediated through a feedback mechanism via Dkk and Dkk diffusion along the porto-central axis. Along this axis an APC concentration gradient is modelled as experimentally detected in liver. Simulations of mutant cells demonstrate that already a single mutant cell increases overall Dkk concentration. The influence of the mutant cell on gene expression of surrounding wild-type hepatocytes is limited in magnitude and restricted to hepatocytes in close proximity. To explore the underlying molecular mechanisms, we perform a comprehensive analysis of the model parameters such as diffusion coefficient, mutation strength and feedback strength. CONCLUSIONS: Our simulations show that Dkk concentration is elevated in the presence of a mutant cell. However, the impact of these elevated Dkk levels on wild-type hepatocytes is confined in space and magnitude. The combination of inter- and intracellular processes, such as Dkk feedback, diffusion and Wnt/β-catenin signal transduction, allow wild-type hepatocytes to largely maintain their gene expression

A mathematical-biological joint effort to investigate the tumor-initiating ability of cancer stem cells.

The involvement of Cancer Stem Cells (CSCs) in tumor progression and tumor recurrence is one of the most studied subjects in current cancer research. The CSC hypothesis states that cancer cell populations are characterized by a hierarchical structure that affects cancer progression. Due to the complex dynamics involving CSCs and the other cancer cell subpopulations, a robust theory explaining their action has not been established yet. Some indications can be obtained by combining mathematical modeling and experimental data to understand tumor dynamics and to generate new experimental hypotheses. Here, we present a model describing the initial phase of ErbB2(+) mammary cancer progression, which arises from a joint effort combing mathematical modeling and cancer biology. The proposed model represents a new approach to investigate the CSC-driven tumorigenesis and to analyze the relations among crucial events involving cancer cell subpopulations. Using in vivo and in vitro data we tuned the model to reproduce the initial dynamics of cancer growth, and we used its solution to characterize observed cancer progression with respect to mutual CSC and progenitor cell variation. The model was also used to investigate which association occurs among cell phenotypes when specific cell markers are considered. Finally, we found various correlations among model parameters which cannot be directly inferred from the available biological data and these dependencies were used to characterize the dynamics of cancer subpopulations during the initial phase of ErbB2+ mammary cancer progression

Investigating Clonal Interactions and Field Effects in Colorectal Adenomas

PhD thesisAccording to the somatic mutation theory of carcinogenesis, tumours are derived from a single mutated cell that clonally expands into a neoplasm. However, studies on familial adenomatous polyposis (FAP) colonic adenomas and some sporadic microadenomas have revealed tumours that are polyclonal in origin – they are derived from more than one clone. This has questioned the current dogma of how colonic tumours are initiated; however the mechanisms of how polyclonality is generated are unknown. Studies using chimeric mice have suggested that polyclonal adenomas arise through crosstalk between unique clones in close physical proximity. In this project, the local microenvironment surrounding human adenomas was characterised by investigating the non-dysplastic crypts in close proximity to adenomas, in particular their mutation burden, DNA damage status, crypt stem cell dynamics and the cellular makeup of the stroma. Immunohistochemistry was used to quantify cell proliferation (Ki67), DNA damage (γH2AX) and Wnt signalling status (nuclear β-catenin) in non-dysplastic crypts, stratified according to their physical distance from the nearest dysplastic crypt. Normal crypts within 250μm of an adenoma displayed increased cell proliferation, DNA damage and Wnt signalling. These effects were associated with an increase in T cell, macrophage and fibroblast infiltrate in the non-dysplastic stroma, however the concentration of intraepithelial CD8 T cells in dysplastic crypts showed a significant decrease. Furthermore, cytochrome c oxidase histochemistry (a marker of mitochondrial DNA (mtDNA) mutations - a proxy for mutation pressure on crypts) was used to demonstrate that crypts neighbouring an adenoma contained a higher mutation burden. Furthermore, the proximity of a crypt to an adenoma also affected stem cell dynamics: using somatic mtDNA mutations to trace clonal lineages, it was found that human intestinal stem cell evolution in adenomas and surrounding normal crypts followed neutral drift dynamics. The effects of an adenoma on gene expression in normal epithelium were investigated using murine organoid cultures. Wild type (WT) organoids when grown in the presence of fibroblasts previously exposed to mutant Apc1322/+ organoids demonstrated a significant upregulation of the MAPK, JAK/STAT and Wnt pathway when compared to WT only. Moreover, Tnf-α, MMP9 and collagen genes were found to be upregulated in exposed WT. To conclude, clonal interactions between dysplastic and non-dysplastic epithelium driving clonal expansion were demonstrated: adenomas create a field effect, dysplastic crypts exert mutagenic pressure, and crypt-to-crypt crosstalk between adenomatous and immune cells takes place leading to a pro-tumourigenic environment. This work has made a significant contribution to the understanding of the initiation of cancer in the human colon.Cancer Research UK studentshi

The role of cell location and spatial gradients in the evolutionary dynamics of colon and intestinal crypts

BACKGROUND: Colon and intestinal crypts serve as an important model system for adult stem cell proliferation and differentiation. We develop a spatial stochastic model to study the rate of somatic evolution in a normal crypt, focusing on the production of two-hit mutants that inactivate a tumor suppressor gene. We investigate the effect of cell division pattern along the crypt on mutant production, assuming that the division rate of each cell depends on its location. RESULTS: We find that higher probability of division at the bottom of the crypt, where the stem cells are located, leads to a higher rate of double-hit mutant production. The optimal case for delaying mutations occurs when most of the cell divisions happen at the top of the crypt. We further consider an optimization problem where the “evolutionary” penalty for double-hit mutant generation is complemented with a “functional” penalty that assures that fully differentiated cells at the top of the crypt cannot divide. CONCLUSION: The trade-off between the two types of objectives leads to the selection of an intermediate division pattern, where the cells in the middle of the crypt divide with the highest rate. This matches the pattern of cell divisions obtained experimentally in murine crypts. REVIEWERS: This article was reviewed by David Axelrod (nominated by an Editorial Board member, Marek Kimmel), Yang Kuang and Anna Marciniak-Czochra. For the full reviews, please go to the Reviewers’ comments section. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13062-016-0141-6) contains supplementary material, which is available to authorized users