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.
