Identification of fallopian tube stem cells using patient-derived organoids

Ovarian Cancer is the fifth most common cancer in women. High Grade Serous Ovarian Cancer (HGSOC) presents as the most aggressive histotype, accounting for over 70% of cases. Survival from HGSOC is amongst the lowest for any cancer. Unfortunately, over 70% of patients present at late stages, when 5-year survival verges at a dismal 10-25%. Although the tissue-of-origin of HGSOC is now known to be the Fallopian Tube (FT), the cell-of-origin remains obscure. HGSOC precursor lesions almost always arise in the distal FT, a site reported to be enriched in stem-like cells. Interrogation of HGSOC initiation has been hampered by the lack of appropriate, physiologically relevant models of the FT. Investigation of FT stem cells as the cell-of-origin of HGSOC could not be undertaken, as FT stem cells remain to be identified.
In this thesis, I show that widely-used FT-derived 2D culture is not a suitable FT model. I employ organoids as a superior model, and show that organoids emerge from a single cell to give rise to large FT-like structures composed of all expected cell types. I confirm novel cell subtypes identified by single cell RNA-seq in hFT tissue, and show that organoids impressively reproduce these findings in an in vitro setting, presenting an opportunity for further functional characterization.
Furthermore, I utilize organoids as a model to re-construct the stem cell niche in vitro. I show that organoids are propagated by rare self-renewing cells, for which Wnt activation and TGF-β inhibition are obligate requirements. For further characterization, I use lentiviral-based techniques to genetically integrate a fluorescent-based reporter of Wnt signaling into hFT organoids. Using functional and single cell transcriptomic approaches, I show that a WNT7A-FZD5 signaling axis is critical for renewal of stem cells, with WNT7A protein acting in very close range, possibly through direct physical interaction between donor and recipient cells. I find that RSPO signaling through LGR5 receptor is essential for maintenance of the WNT7A-FZD5 signaling axis. Single cell profiling shows that Wnt-active cells form a cluster of cells that are proliferative and marked by CA125, a clinical marker of HGSOC progression and response to therapy. Single cell data also show that organoids express Estrogen Receptor α. I note that Estrogen suppresses WNT7A in FT organoids, leading to pronounced differentiation and ciliogenesis. Finally, the thesis negates all of these findings for mouse oviduct stem cells, which regenerate using entirely different mechanisms, at least within the organoid setting.
Overall, this thesis proposes a first description of FT stem cells and a basic characterization of their molecular niche requirements. It lays the foundation for further studies and in vivo confirmation of FT stem cells, conditional on addressing inter-species differences. Findings in this thesis open the door to investigating the role of FT stem cells in HGSOC initiation.

Keywords: high grade serous
WNT7A
WNT5A
adult stem cells
FZD5
Mullerian
tissue-resident stem cells
ovarian cancer
fallopian tube
Oestrogen
organoids
wnt signaling
stem cells
LGR5
Estrogen
Advisor: Ahmed, AA