Development of Mouse-Derived Organoid Lines from Fallopian Tube Epithelial Cells for High Grade Serous Ovarian Carcinoma Modeling.

Abstract

Effective modeling of diseases in realistic environments is crucial to improve our understanding of diverse pathologies. In this aspect, organoids offer a more faithful environment than their classical two-dimensional counterparts in vitro. Similarly, syngeneic murine models also allow researchers to investigate more complete tumor-host interactions, such as with the immune system, in vivo. Here we present a complete protocol on extracting fallopian tube epithelial cells, the cell-of-origin of high-grade serous ovarian carcinomas (HGSOC), from a mouse and derive them as primary organoid cultures, as well as their in vitro culture and maintenance. Then, we describe how to use genetic engineering techniques, such as lentiviral or retroviral gene overexpression, as well as CRISPR-Cas9 gene deletion, to modify these lines and transform them into pre-cancerous tumoroids. We also report how to use them for in vitro validation, via the extraction of genetic materials and immunofluorescence staining. Finally, we indicate how to effectively inject these tumoroids in the ovarian bursa, the autochthonous site of HGSOC, for tumor initiation.