Kashfi, Seyed Mohammad Hossein
(2020)
Studying colon cancer using intestinal organoid models for anticancer drug and gene functional analysis.
PhD thesis, University of Nottingham.
Abstract
Colorectal tumours demonstrate broad genetic and phenotypic heterogeneity in comparison to their matched normal tissue. To shed light into this diversity in depth, an advanced in-vitro 3D model capable of self-renewal, self-organisation and exhibit organ functionality is required. Self-renewal of the small intestine and colonic epithelium is mediated by the proliferation of stem cells and their progenitors in the crypt base. Recently, a long-term and stable 3D culture system maintaining basic crypt physiology known as organoid became available from both normal and matched-tumour tissues of the intestine. Organoids faithfully reproduce functional genetic, pharmacological and morphological alterations exist in their parental tissue. Patient-derived organoids (PDO) can be generated and expanded indefinitely in-vitro from a small biopsy sample of CRC patients (from both tumour and adjacent normal tissue), providing a promising platform for personalised medicine, drug discovery and targeted gene editing research studies. Therefore, in this study, we have initially adapted patient-derived organoids culture from 17 CRC patients in our laboratory and further investigated the phenotypical alteration of both tumour, and normal adjacent derived organoids. Based on our findings, tumour organoids displayed different phenotypical traits from one patient to another, highlighting their distinct molecular subtyping. The growth and morphological alterations of tumour organoids also significantly differed from normal derived organoids in culture.
We further evaluated the effect of Noggin, a BMP antagonist on PDO and colon cancer cell lines. Our findings demonstrate that despite the significant role of Noggin in the homeostasis of normal colonic derived organoids, the administration of Noggin does not influence colon cancer cells and PDOs behaviour in culture.
Furthermore, organoids have opened a new window toward drug discovery and personalised medicine. To address this, the anticancer therapeutic value of Rifabutin antibiotic, recently screened on our laboratory were also assessed in PDO and colon cancer cell lines. Rifabutin drastically hindered the growth of PDOs and colon cancer cell lines in culture and impeded Wnt/β-catenin signalling mediated cancer cell growth.
With CRISPR/Cas9 induced-accurate mutations and 3D organoids cultures, it is now possible to model how individual genes may function in multiple tissue types and without the cost and ethical considerations of in-vivo alternatives. Hence, we aimed to combine the organoid system with genome editing technology, CRISPR/Cas9, to generate knockout organoids for rapid identification of genes with unknown biological function in the intestinal epithelium. Here, we evaluated the role of F-box genes as substrate recognition subunit of Skp, Cullin, F-box containing complex (SCF) E3-ligases in intestinal organoids. Knockout F-box intestinal organoids demonstrated differential phenotypical alterations that were associated with aberrant proliferation and differentiation and led the identification of different F-box genes including Fbxl5, Fbxo31, Fbxl18, Fbxo17 and Fbxl17. In conclusion, we have shown that organoids are invaluable and flexible tools to investigate multiple aspects of cellular events for homeostasis and disease condition and they may represent a significant step forward in reducing and replacing the use of animal models in research.
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