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A novel integrative strategy to prevent colorectal cancer within the diet-host-microbiota triangle: from organoids to human in vivo reality

Periodic Reporting for period 1 - TRIANGLE (A novel integrative strategy to prevent colorectal cancer within the diet-host-microbiota triangle: from organoids to human in vivo reality)

Reporting period: 2018-07-01 to 2020-06-30

Colorectal cancer (CRC) is the second cause of death in the EU, with one million cases of CRC seen annually worldwide, and a mortality rate of 50%. In the light of these figures, and associated costs, there is an overwhelming need to prioritize and integrate primary prevention measures in existing healthcare plans. The forecasts show that the global burden of CRC is expected to increase by 60%, to over 2.2 million new cases and 1.1 million deaths by 2030, due to an aging population and western dietary patterns.
Dietary patterns, or the food we eat, are the sum of a multitude of small molecules foreign to the body. After being ingested and digested, nutrients are altered by the trillions of microorganisms that inhabit our gastrointestinal (GI) tract, shaping the chemical structures of such compounds and thus modifying the lifespan, bioavailability, and biological effects. In this context, dietary patterns modulate the gut microbiome and alter its functions by modulating the production of gut microbial metabolites (GMMs). Lastly, these GMMs are capable of regulating homeostasis and the risk of GI disease. TRIANGLE has studied the mechanisms by which GMMs may prevent CRC and promote gut health by modelling diet-microbiome-host interactions. The first objective was to establish 3D intestinal models closely recapitulating homeostasis and carcinogenesis. Secondly, diet-gut microbiota interactions were evaluated using a GI track model. Lastly, human colon organoid and tumoroids responses to GMMs deriving from flavan-3-ols were evaluated by 3D imaging techniques and multi-omics approaches.
The main aim of the WP1 was to establish human colon organoid and tumoroids cultures, to reach this goal, two collaborators assisted me. First, I carried out a secondment at BRIC (University of Copenhagen). This research stay has established a fruitful collaboration creating knowledge, skills, and values to develop a biobank of human colon organoids and tumoroids. Secondly, all samples used for deriving human colon organoids and tumoroids were shipped from the “Casa Sollievo della Sofferenza” Hospital, San Giovanni Rotondo (Italy).
WP2 consists of research activities directed towards gut microbial metabolites released by lean and obese gut microbiotas. Renetta Canada and control samples (cellulose, pectin, and blank) were digested following in vitro gastric and duodenal digestion protocols. After the digestion of fruit and vegetables, up to 90-95% of dietary polyphenols, such as flavan-3-ols are not absorbed by the small intestine, and together with non-digestible polysaccharides (fibre) reach the colon almost intact, where they interact with gut microbiota. At this point, proanthocyanidins (PACs), and fibre undergo extensive microbial bioconversion, producing phenyl-γ-valerolactones (PVLs), their derived hydroxy-phenylvaleric acids (PVAs), small phenolic acids, and short-chain fatty acids (SCFAs). The latter group of GMMs, SCFAs, has demonstrated an extraordinary potential to influence cell behavior in homeostasis and CRC disease. By contrast, intestinal epithelial cell responses to PVLs, PVAs, and phenolic acids are still unknown.
Work Packages 3 and 4 investigated the impact of GMMs on the gut epithelium in homeostasis and CRC disease. Cell images were first acquired using an imageXpress’Micro confocal (Molecular Devices). The system quantified several features, such as number, shape, texture, size of organoids, as well as proliferation, DNA content, and apoptosis, among others. Human colon organoid and tumorid responses to two native polyphenols (procyanidin B1 and epicatechin) and GMMs (butyric acid, one PVL and one PVA) were investigated. Strikingly, the PVL, which is an epicatechin metabolite, provided a superior apoptotic area compared to non-treated and treated tumoroids. Likewise, butyric acid, a well-known GMM involved in homeostasis and cancer cell behavior, raised the apoptotic region. In this direction, treated-tumoroids with PVL, and butyric acid were smaller, and the number of proliferating cells substantially decreased. These promising and exciting results have confirmed that human colon organoids/tumoroids retain essential physiological functions, and respond as intestinal epithelium cells. Hence, the model is suitable for investigating responses to GMMs. Secondly, GMMs, such as PVLs may promote apoptosis and inhibits proliferation on human colon tumoroids, supporting the idea that GMMs may play a pivotal role in improving gut health.
To understand the mechanisms that underscore the interactions with and responses in intestinal epithelial cells, a combination of multi-omics approaches and intestinal organoids is crucial. Focusing on small molecules, the metabolome of human colon organoid/tumoroids can elucidate significant metabolic processes affecting organoid and tumoroids, as metabolites represent both the downstream output of the genome and the upstream input from the environment. TRIANGLE project has optimized a metabolomics protocol on human colon organoids and tumoroids. The most significant markers were tentatively identified and biologically interpreted. As a result, human colon organoids and tumoroids metabolically behave as in vivo cells. Therefore, the metabolomics workflow proposed has provided a standardized protocol to evaluate the chemical signatures of human colon organoids and tumoroids.
Gut microbiota and diet play a pivotal role in the production of bioactive metabolites. In this context, I have designed hypothesis-driven analysis, combining a set of smart approaches that have unique properties, without the inherent shortcomings of the traditional methods, to tackle the challenges associated with GMMs and the prevention of CRC. This strategy is of paramount importance not only to broaden our knowledge of these small molecules produced by the gut microbiota but also to design new preventive actions that will help us to counteract CRC. In this frame, TRIANGLE has opened new horizons, leading to determine how the GMMs affect the distribution of cell types and metabolome changes in human intestinal organoids. The outcome of this project has significantly extended our knowledge of GMMs, and in the long term, will allow personalized nutrition/medicine to be employed for the prevention of CRC. In the future, I believe that this research line will substantially decrease associated healthcare costs and mitigate health risks by implementing nutritional recommendations and promoting precision pre- and pro-biotics.
TRIANGLE has concerned the fields of personalized nutrition/medicine, gut microbiota, and biochemistry, by bringing to light GMMs that (i) directly inhibit proliferation and promote apoptosis of CRC cells, and (ii) can stimulate the gut epithelium to improve gut health. Bridging these three separate fields have been the main general achievement of this project. This project will have a significant impact on the research of many biologists, nutritionists, (bio)chemists and microbiologists, who will be able to study personalized nutrition and medicine better. For the metabolomics community, TRIANGLE has provided a metabolomics workflow. The nutrimetabolomics community will benefit in terms of a full understanding of the role of GMMs in the prevention of CRC, which may lead to hypotheses regarding host-microbiome interaction and pro- and pre-biotic intervention studies.