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

Summary

This protocol details the development of high-grade serous ovarian carcinoma (HGSOC) mouse models in vitro and in vivo. From the derivation of murine fallopian tube organoids, their genetic modification to recapitulate human HGSOC genetics, and their introduction in the ovarian bursa of syngeneic mice for in vivo development of tumors.

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.

Introduction

High-grade serous ovarian carcinoma (HGSOC) remains a poorly understood and highly lethal disease. Historically, the lack of faithful in vitro and in vivo models that represent HGSOC biology and therapy response has been a major barrier to progress. For example, multiple (if not most) studies have used cell lines that fail to share key genomic/genetic features of HGSOC, rendering questionable the relevance of their findings^1,2,3,4. For example, the common ID8 syngeneic mouse model is derived from ovarian surface epithelium ....

Protocol

All animal tissue collection and procedures were performed ethically under the approval of the Institutional Animal Care and Use Committee (IACUC) at Alexandria Center for Life Sciences and the Perlmutter Cancer Center at NYU Langone Health, New York, New York.

1. Preparation of materials

1. Murine organoid media (MOM) preparation
   1. Prepare the basal medium by combining the items on Table 1.
      NOTE: Media is stable for up to a month at 4 °C.
2. 2D media preparation
   1. Combine recombinant murine EGF 50 ng/mL, Insulin-Transferrin-Selenium (final conc....

Results

A schematic summarizing the workflow for generating and validating HGSOC ovarian cancer is shown in Figure 1. This process begins with the extraction of fallopian tube tissue from a female C57BL/6J wild-type mouse (Figure 2A). It is recommended to start with a generous piece of tissue to ensure proper collection. Using a dissecting microscope allows for the precise isolation of the distal fallopian tube while removing surrounding tissues such as fat and the ovar.......

Discussion

Despite significant advancements in oncology research, HGSOC remains the most aggressive type of gynecologic malignancy and the leading cause of mortality amongst all ovarian cancers^23,24. As gynecologic oncology research has evolved, so should the models used in it. Indeed, many historical ovarian cancer model cell lines have been discovered to be suboptimal to study this disease for several reasons, including genomic relevance, cell-of-origin discrepancies or l.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.25% Trypsin, 0.1% EDTA in HBSS w/o Calcium, Magnesium and Sodium Bicarbonate	Corning	25053CI
A83-01	Tocris Bioscience	2939
Advanced DMEM/F12	Gibco	12-634-028
Aged C57BL/6J (female)	The Jackson Laboratory	000664
AllPrep DNA/RNA Mini Kit	QIAGEN	80204	For DNA/RNA extraction
Alt-R S.p. Cas9-GFP V3	Integrated DNA Technologies	10008100
Anti-Ki67 antibody	Abcam	ab15580
AutoClip System	Fine Science Tools	12020-00	For wound closure post-IBI
B-27 Supplement (50x), serum free	Gibco	17-504-001
Blasticidin HCl (10 mg/mL) 50 mg	InvivoGen	ant-bl-05
Bovine Serum Albumin	Sigma Aldrich	A9647
Buffer RLT Plus	QIAGEN	1053393	To lyse cells before DNA/RNA extraction
Cell Recovery Solution	Corning	354253
Collagenase/Hyaluronidase	STEMCELL Technologies	07912
CoraLite 594-Phalloidin (red)	Proteintech	PF00003
Cover glass 15CIR-1	Fisherbrand	12-545-83
Cover glass 24 mm ´ 40 mm-1	Fisherbrand	12-544-12
CTS (Cell Therapy Systems) N-2 Supplement	Gibco	A1370701
DAPI	Roche	10236276001
Deoxyribonuclease I (DNAse I) from bovine pancreas	Sigma Aldrich	D5025
Difco Skim Milk	Becton Dickinson	232100
Dispase	STEMCELL Technologies	07913
DMEM with L-Glutamine, 4.5 g/L Glucose and Sodium Pyruvate	Corning	10013CV
DNase I recombinant, RNase-free	Roche	04716728001
Dulbecco's Phosphate-Buffered Solution	Sigma Aldrich	D8537
Dumont #5 - Ceramic Coated Forceps	Fine Science Tools	11252-50	For fallopian tube dissection
Fetal Bovine Serum	Corning	35-010-CV	Heat-Inactivate at 56 °C for 60 min.
Fine Scissors - ToughCut	Fine Science Tools	14058-09	For fallopian tube collection and IBI incision
Geneticin Selective Antibiotic (G418 Sulfate) (50 mg/mL)	Gibco	10131027
GlutaMAX Supplement (100x)	Gibco	35-050-061
Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Cyanine3	Invitrogen	A10520
Graefe Forceps	Fine Science Tools	11053-10	For tissue handling
HEPES buffer	Corning	25060CI
Human R-Spondin 1 Recombinant Protein	PeproTech	120-38
Insulin Syringe, U-100 0.5 mL 0.36 mm (28 G)x 12.7 mm (1/2")	BD	BD329461
Insulin-Transferrin-Selenium (ITS -G) (100x)	Gibco	41-400-045
L-Glutamine 200 mM (100x)	Gibco	25-030-081
Matrigel GFR Basement Membrane Matrix	Corning	356231	BMM
Mouse EGF Recombinant Protein	PeproTech	315-09
Mouse FGF-basic Recombinant Protein	PeproTech	450-33
Mouse Noggin Recombinant Protein	PeproTech	250-38
Mouse Wnt-3a Recombinant Protein	PeproTech	315-20
N2 Supplement	Gibco	17502048
N-acetylcysteine amide	Sigma Aldrich	A0737
Neon NxT Electroporation System	Invitrogen	NEON1S	For CRISPR-Cas9 electroporation
Neon NxT Electroporation System 10-μL Kit with 1-Channel Tubes	Invitrogen	N1025	Consumables for electroporation, including genome editing buffer
Nicotinamide	Sigma Aldrich	N3376
Normal Mouse Serum	Invitrogen	10410
Normal Rat Serum	STEMCELL Technologies	13551
Opti-MEM I Reduced Serum Medium	Gibco	31985070
PAX8 Polyclonal antibody	Proteintech	10336-1-AP
Penicillin-Streptomycin (10,000 U/mL)	Gibco	15-140-122
Puromycin	Sigma Aldrich	P8833
Selectfrost 25 mm ´ 75 mm ´ 1.0 mm	Fisherbrand	12-550-003
Triton X-100	Fisher Bioreagents	BP151
TrypLE Express Enzyme (1x), no phenol red	Gibco	12-604-013
Vannas Spring Scissors - 4 mm Cutting Edge	Fine Science Tools	15019-10	For fallopian tube dissection
Y27632	STEMCELL Technologies	72304	Rho-kinase (ROCK) Inhibitor