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Dissecting microbiome and immune interactions in human intestinal (cancer) organoids

Periodic Reporting for period 3 - Organoid (Dissecting microbiome and immune interactions in human intestinal (cancer) organoids)

Reporting period: 2018-11-01 to 2020-04-30

Our discovery of human and murine stem cells of the small intestine, colon, stomach, fallopian tube, salivary gland, mammary gland, kidney, liver, pancreas, oesophagus, ovarian and prostate has allowed us, using tissue specific mix of (growth) factors, to develop an unique 3D culture system in which stem cells of these organs can be expanded and differentiate. The cultured organoids (mini-organs) retained their original organ identity, were genetically stable and could be expanded long term. This 3D culture system has also been successfully applied to diseased epithelia, including cancer. Organoid technology can therefore be used to model human organ development and various human pathologies ‘in a dish.” Additionally, patient-derived organoids hold promise to predict drug response in a personalized fashion. Organoids open up new avenues for regenerative medicine and, in combination with editing technology, for gene therapy. The many potential applications of this technology are only beginning to be explored.
In this ERC grant I proposed to use the organoid technology to dissect interactions of the gut microbiome with healthy and diseased 'mini-guts' organoids and of Tumor Infiltrating Lymphocytes (TILs) with colorectal cancer (CRC) organoids.
To this end, we will describe/study: 1) All immune receptors, -regulators and -effectors in the individual epithelial cell types, 2) 'Mini-guts' recombined with individual bacterial species, 3) CRC tumoroids recombined with their cultured TILs and subjected to immune checkpoint manipulation. Using advanced molecular and imaging technologies, we will chart the molecular mechanisms that underlie the interactions from the ‘epithelial perspective’.
Our discovery of organ specific stem cells has allowed us to develop a unique culture system in which the stem cells of these organs can grow into 3-dimensional (3D), ever-expanding epithelial organoids that retain their original organ identity. This 3D culture system has also been successfully applied to diseased epithelia, including cancer. Organoid technology can therefore be used to model human organ development and various human pathologies ‘in a dish’.
In this ERC grant I proposed to use the organoid technology to dissect interactions of the gut microbiome with healthy and diseased organoids and assess immunoreactivity of Tumour Infiltrating Lymphocytes (TILs) with colorectal cancer (CRC) organoids.
The colon epithelium is constantly exposed to a great variety of gut bacteria, which help in digestion of food and regulation of immune responses. In contrast to these beneficial effects, some species of gut microbiota show associations to diseases such as colorectal cancer. Our aim is to move these findings beyond mere associations and investigate direct effects of potentially carcinogenic bacteria on human colonic epithelial cells using organoids. Fusobacterium nucleatum (Fn), which is normally found in the oral cavity, is the bacterial species found most highly enriched in colorectal cancer. We showed the presence of Fusobacteria in colon tumor organoids. We are currently testing the implications of this invasion on bacterial and epithelial cell behaviour. In addition, we have characterized transcriptional response to Fusobacterium exposure in organoid lines, which were engineered to contain subsets of mutations in the adenoma-to-carcinoma cancer cascade.
Additionally, we are focusing on another group of bacteria, namely genotoxic Escherichia coli. These bacteria have been strongly associated with human colorectal cancer. In addition, they induces colorectal cancers in susceptible mouse models. However, the potential of these bacteria to transform human wild-type epithelium is unexplored. Our main goal is to establish whether the reported DNA damaging effects of these bacteria result in a characteristic mutational signature and relevant driver mutations in colorectal cancer, which would position these bacteria as potential carcinogens. To test this hypothesis, we have set up a long-term co-culture system, which allows exposure of human wild-type epithelium to the genotoxic bacteria for several passages. We have validated for the first time the DNA damaging properties of a panel of these bacteria in organoids. As the next step, we will also analyse the mutational patterns arising after a long-term co-culture with genotoxic and non-genotoxic E. coli by whole genome sequencing and investigate the nature of the arising DNA damage using genetically engineered human colon organoids.
We have established conditions for culturing human small intestinal and colonic organoids in 2D, both on transwells and on a custom-made organ-on-a-chip device. With these, we are currently investigating the effects of IBD-associated bacteria on epithelial barrier integrity and cell type-specific interactions on organoids derived from IBD patients as well as healthy control donors.
We have made a complete inventory of immune checkpoint regulators and their roles in the (lack of) immunoreactivity of TILs towards the human CRC tumours. We have successfully established methods to isolate TILs and have established co-cultures of both normal colon and CRC organoids with allogeneic T-cells as well as an imaging system for visualizing direct organoid cytolysis. We showed that T cells can kill organoids in an antigen specific manner and that this can be quantified by imaging.
To identify relevant neo-antigens, we have subjected organoid lines from tumour and corresponding healthy tissue to HLA-A2-restricted peptidome analysis. HLA-A2 restricted peptides have therefore been eluted from organoids and were analysed using mass-spectrometry. Currently we
This program will ultimately provide molecular detail to the effects of the microbiome and immune system on our gut, in health and disease