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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - HGSOC (A novel approach into modeling origin and development of ovarian cancer)

Teaser

Summary

High grade serous ovarian cancer (HGSOC) is the most common and lethal subtype of ovarian tumors, and is the 5th leading cause of cancer related deaths among women in the western world. Recent studies have pointed at the fimbriae of the fallopian tube (FT) as one of HGSOCâ€™s main origins. The existence of FT stem cells (SCs) has been suggested, however, their isolation and characterization have been so far elusive. Given the potential of SCs to accumulate mutations, these cells may represent one of the cellular origins of HGSOC.
Xenografts and cell lines are the main platforms for ovarian cancer research. These models have many limitation including low derivation success rate and pure representation of ovarian cancer heterogeneity and hallmarks.
This project is aimed at identifying SCs of the FT, evaluating their tumorigenic capacity and establishing a novel in-vitro human based platform for ovarian cancer research.
A successful conclusion of this research will provide scientists an innovative tool for the study of ovarian cancer and contribute to the worldwide effort being invested in developing early detection methodologies and new drugs for ovarian cancer.

Conclusions-

â€¢ We have established culture system that supports long term expansion of mouse oviduct organoids. These organoids are comprised of both ciliated and secretory cells and recapitulate the tissue of origin.
â€¢ Troy positive cells have high organoid forming capacity. Moreover, organoid derived from single Troy positive cells can give rise to both ciliated and secretory cells supporting their stem/progenitor identity.
â€¢ We have targeted tumor driver genes using CRISPR/Cas9 technology and established new model for studding tumor development.
â€¢ We have established culture system that supports long term expansion of fallopian tube and ovarian cancer organoids.
These organoids recapitulate their tissue of origin can be can be genetically manipulated and serve as a platform for drug sensitivity assays.

Work performed

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.

Final results

Specific aims:
1. Identification and characterization of oviduct epithelial stem cells

We are now analyzing clones in which we have introduced mutations into different genes (by analyzing their gene expression, histology and genetic stability). Moreover, we have performed additional orthotropic transplantations to the bursa of immunodeficient mice. Our goal is to perform comprehensive analysis to prospective tumors and thus, establishing a novel murine ovarian cancer developmental model. Such a platform can be used in order to test potential tumor driver genes and study early tumor development.

2. Establish in-vitro human model for ovarian cancer
We have successfully established a platform that supports the growth of ovarian cancer organoids. We intend to expand our ovarian cancer organoid biobank and to use it as platform to study tumor resistance mechanisms and drug development. Moreover, such a platform could be easily be used by the scientific community and facilitate ovarian cancer research. To that end we are now finalizing a manuscript that give a comprehensive description of this novel system.