(View plain language abstract)
A high fibre diet is associated with a protective effect against colorectal cancer (CRC). The mechanism for this effect remains unclear as the majority of research on CRC has focused on the roles of the key genetic/epigenetic mutations that drive it. The mechanism is likely to involve components of the metabolome derived from the gut bacteria which digest the fibre. Certain commensal gut bacteria have been shown to protect against CRC while others are hypothesised to initiate or progress CRC. Understanding these effects will require investigating the complex interactions between the diet, microbiome, metabolome, inflammatory/immune system, intestinal stem cell (ISC) and the proposed CRC ISC. Recently much progress has been made in identifying cancer related components of the gut microbiome/metabolome and the development of in vivo and ex vivo models for studying the normal and malignant ISCs. We propose to use these new tools to directly investigate the effects of the microbiome/metabolome on the ISCs to further our understanding of the aetiology of CRC.
The aims of the research is to identify whether specific gut microbes and metabolites, associated with CRC, alter the ability of normal or malignant ISCs to grow intestinal organoids ex vivo and either maintain intestinal homeostasis or alter tumour progression in vivo. By correlating this data we will be able to identify if specific components of the microbiome/metabolome can alter the behaviour of the ISC and potentially the CRC phenotype.
We have state of the art mouse models in which the ISCs can be sorted based on their expression of GFP and used to establish ex-vivo organoid cultures. Further Apc can be conditionally deleted in these ISCs using cre/lox technology. The Apc gene is part of the canonical Wnt signalling which is the key pathway in maintaining intestinal epithelial homeostasis, the inactivation of this gene is the earliest and most common event in CRC. The normal ISCs can be grown ex vivo to form intestinal crypts/villus structures containing all differentiated cell types. Whereas Apc deleted ISCs generate aberrant structures akin to early adenomas in patients. These organoids can be passaged to permit a determination of self-renewal capacity (one key aspect of 'stemness'). We aim to take these normal ISCs or malignant ISCs and observe their ability to grown intestinal organoids in the presence of specific relevant CRC related gut metabolites. This will allow us to identify if these metabolites affect the ability of these cells to form organoids and further investigate any alterations in expression or protein levels of the pathways associated with intestinal homeostasis. We also aim is to deliver relevant doses of specific CRC associated bacteria or metabolites to mice in which we have deleted the Apc gene in the ISC in vivo. In such an in vivo setting we will be able to investigate the interaction between the microbiome/metabolome, ISC or malignant ISC and the immune system in situ. The mice will be treated with CRC associated agents pre- and post-deletion of the Apc gene and the rate of tumour formation and progression observed. Tumours will then histologically characterised and undergo expression and protein profiling to identify alterations in the pathways important in intestinal epithelial homeostasis and CRC.
This proposed investigation is pioneering as the tools to perform a critical assessment of the role played by the intestinal microbiota and metabolome on normal and malignant ISCs have only recently been developed. It will establish for the first time specific microbiome/metabolome agents which plays on an ISC at the very earliest stage of CRC formation. It is predicted that the research will identify specific microbial species and components of the metabolome associated with a positive effect on CRC that may have potential as therapeutics, and vice versa, indicators of a negative effect which could have use as biomarkers of CRC.
In 2011 the World Cancer Research Fund upgraded the evidence for a protective effect of dietary fibre on colorectal cancer (CRC) from 'probable' to 'convincing'. One reason suggested for this effect is that the fibre is known to promote the growth of certain types of bacteria commonly found in the intestine. These bacteria digest the fibre and produce chemicals which interact with the cells that line the intestine. These types of bacteria are often referred to as probiotics and have been shown to suppress immune inflammatory responses. Inflammation of the intestine is linked to the onset of CRC, as it can cause damage to the DNA of intestinal stem cells, which maintain the surface of the intestine. In contrast, other types of bacteria produce chemicals which are known to mutate DNA and could potentially cause the DNA mutations in the stem cells that initiate and drive CRC. Thus the interactions between the diet, bacteria, immune system and the intestinal stem cells that promote or prevent CRC is highly complex. Our limited understanding of this effect is currently a significant barrier to the obvious potential for identifying possible treatments for CRC, the fourth most commonly diagnosed cancer in the world.
Our overall aim is to identify if certain bacteria that live in the intestine can affect the behaviour of a normal and/or cancerous intestinal stem cell and establish how they do it. We will determine this in two ways. Firstly by extracting the normal and cancerous stem cells from a mouse and growing them in the laboratory in the presence of different bacteria or the chemicals that they produce. Secondly by introducing different types of bacteria to the intestine of mice in which we can create cancerous stem cells in a controlled fashion. This will allow us to directly investigate the effect that each bacteria has on the stem cells in the absence or presence of normal bacteria and immune responses.
We will perform this work using state of the art mouse models of human CRC in which the intestinal stem cells have been labelled. This label allows us to identify them and isolate them for growth in the laboratory. We can use these stem cells to grow the normal structures of the lining of the intestine. We can also take these stem cells and in a controlled fashion mutate a gene called Apc. Mutation of the Apc gene is the earliest and most common event in CRC patients. After mutation, the stem cells then grow to form structures similar to the tumours observed in CRC patients. We will grow these stem cells together with different bacteria that have been associated with pro- or anti- CRC effects. Using this method we will screen for bacteria that have an effect on the normal and/or cancerous stem cells. Any bacteria we identify will then be introduced to the intestines of mice in which we can also replicate human cancer by deleting the Apc gene. Using these tools, we propose to grow normal and cancerous stem cells in the presence of different bacterial species, to identify which types alter their behaviour and which chemicals are responsible for the effect.
At the end of this study, we will have identified types of bacteria which affect the behaviour of the normal and cancerous intestinal stem cell and which chemicals they produce are responsible. The project could have two key impacts. It will create an explanation of how diet via the bacteria in our intestine can have a negative or positive impact on CRC, thereby helping to promote the benefits of a good diet. Secondly our increased understanding of the relationships between bacteria and the normal or cancerous stem cell may identify new treatments for CRC and/or it may deliver a set of bacterial markers that will permit a much better risk assessment for patients with CRC or identify patients who are susceptible to CRC.