The tumour initiation project

Thesis

High-grade serous ovarian cancer (HGSOC) is the most lethal gynaecological malignancy – an imminently required screening tool may be developed through the study of tumour initiation. It is believed that mutagens from follicular fluid (FF) released during ovulations collaborate with components of retrograde menstruation to transform multipotent human fallopian tube (hFT) stem cells. In-house works recently illustrated that the WNT7A-FZD5 axis is fundamental to hFT stem cells in vitro as organoids. In this thesis, in hFT organoids, it was corroborated that FF reactive oxygen species (ROS), delivered as H2O2, are transduced intracellularly through NOX wherein they potently induce dsDNA breaks. Their lower toxicity compared to previous monolayer studies is likely insidious; since cells better escape death, they accrue DNA damage – sitting closer to the transformative ‘Goldilocks Zone’. Curiously, FF transferrin was only genotoxic in collaboration with H2O2, perhaps through the former triggering an intracellular Fenton reaction. Similarly, FF Fe3+ was only toxic when paired with FF transferrin, suggesting that the latter shuttles in the former, perhaps driving lipid peroxidation (LPO). Fascinatingly, mutagens appeared to preferentially target already-proliferating stem cells. Surrogate Wnt and CHIR99021, synthetic factors, were shown to activate Wnt/β-Catenin signalling, whereas FF IGF2 and FF insulin did not. Regardless, these all promoted survival of stressed and unstressed organoids; corresponding pathway manipulators in FF likely enable ROS-damaged stem cells to expand in vivo, a carcinogenetic process. FF progesterone impressively abrogated this, inhibiting organoid growth ~30-fold more potently than monolayer studies predict, indicating therapeutic value in HGSOC prevention. FF estradiol also slightly antagonised organoid formation, and both hormones induced differentiation. Lastly, using aforementioned survival factors, media were successfully established to enable the recovery of organoids post-lentiviral genetic manipulation. Methods to purify mutant populations were then refined, and a highly cost-effective protocol for organoid clonal selection was developed, facilitating the next research stages.

Authors: Aggarwal, A

• Alternative title: Developing organoid models to study tumour initiation in high-grade serous ovarian carcinoma
• DOI: 10.5287/ora-pyj29xxk9
• Type of award: MSc by Research
• Awarding institution: University of Oxford
• Language: English
• Keywords: fallopian tube; follicular fluid; retrograde menstruation; DNA damage; organoids; genomics; mutagenesis; ovarian cancer
• Subjects: Reproductive health; p53 protein; Cancer; Mutation (Biology); DNA damage; BRCA genes