Specific aims:
1. Identification and characterization of oviduct epithelial stem cells
Using immunostaining and FACS analysis we have showed the existence of Lgr6 and Troy positive cell populations in mouse oviduct epithelium. BrdU experiments and Ki67 staining revealed the low turn-over nature of oviduct epithelium. Accordingly, only few cells were traced after long-term (6 months) lineage tracing experiments.
We have established culture system that supports long term expansion of mouse oviduct organoids (more than 20 passages). These organoids are comprised of both ciliated and secretory cells (demonstrated by gene expression analysis, immunostaining and time-lapse movies showing beating cilia), and therefore faithfully recapitulate their tissue of origin. Using this platform, we have demonstrated that Troy positive cells have high organoid forming capacity unlike Troy negative cells. Moreover, by sorting and plating Troy positive cells we could show that a single cell can grow into organoinds, which are comprised of both ciliated and secretory cells supporting their stem/progenitor cell identity.
We have successfully adapted CRISPR/Cas9 technology to oviduct organoids and targeted the tumor driver genes: Tp53, Brca1 and Pten. Metaphase spread analysis of the different clones demonstrated that triple mutated clones acquire genetic instability. Moreover, preliminary orthotropic transplantations to the mouse bursa demonstrated that these clones have tumorigenic potential.
2. Establish in-vitro human model for ovarian cancer
We have established normal fallopian tube organoid cultures from germline mutation carrier patients (BRCA1/2) that went through prophylactic bilateral salpingo-oophorectomy (pBSO) and non-carriers. These organoids are comprised of both ciliated and secretory cells (demonstrated by gene expression analysis, immunostaining, scanning electron microscopy and time-lapse microscopy showing beating cilia), and therefore faithfully recapitulate their tissue of origin.
We have successfully derived organoids from ovarian cancer tissue. These organoids can be maintained and expand in culture for long term (more than 15 passages). Histologic characterization of these organoids demonstrated that they retain the expression of ovarian cancer markers such as, PAX8 and P53, and show high Ki67 index. Moreover, H&E staining demonstrated tumor characteristics such as nuclear and cellular atypia, papillary like structures and multinucleated cells. Metaphase spread analysis corroborated histological analysis and showed aberrant chromosomal numbers. Next generation whole genome sequencing analysis showed high correlation between tumor and organoid couples.
We have adapted electroporation protocol to normal fallopian tube organoids in order to target tumor driver genes. Thus, we were able to target both alleles of TP53 in clonally expanded organoids.
Finally, we have adapted mid-scale drug screening assay that enables to demonstrate organoid line sensitivity to different drugs commonly used in the clinics.
