Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

Summary

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

Abstract

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

Introduction

Ovarian cancer is the deadliest gynecologic malignancy¹. The majority of patients are diagnosed after the cancer has disseminated throughout the peritoneal cavity. Once the cancer has spread throughout the peritoneal cavity, cytoreductive surgery and chemotherapy are often not sufficient treatment to prevent cancer recurrence and chemoresistance, resulting in a less than 30% 5-year survival rate. Ovarian cancer metastasis is predominantly limited to the peritoneal cavity, and several other cancer types, including gastric, pancreatic, and colon cancers, metastasize to the same anatomic sites in the peritoneal cavity. In general, ovarian cancer cells detach f....

Protocol

All research protocols described have been reviewed by the University of Chicago Institutional Review Board (IRB). Informed consent was obtained from each patient before surgery and the study was approved by the University of Chicago IRB. A biological safety cabinet type 2 and gloves should be used when handling human tissue for protection and to reduce risk of contaminating cells.

1. Isolation and Culture of Primary Untransformed Stromal Cells

1. Human tissue collection and preparation.
   1. Obtain specimens of human omentum, 2 cm³, removed during an abdominal surgery, and immediately immerse tissue in RT phospha....

Results

The organotypic culture was assembled by first mixing primary human fibroblasts with collagen type I and then overlaying this culture with 5 times the number of mesothelial cells. The culture was incubated for at least 18 hr before ovarian cancer cells were added to study adhesion, invasion or proliferation. Each assay was repeated with multiple (n=3-5) 3D cultures obtained from different patients and numerous wells were tested in each condition for the adhesion (n=5), proliferation (n=5) and invasion assays (n=3). Ovari.......

Discussion

An organotypic model of the peritoneal microenvironment was established to assess the individual and collective function(s) of both the cellular and innate components of the microenvironment in ovarian cancer dissemination. The specific protocols for plating and customizing the 3D organotypic culture to investigate ovarian cancer cell adhesion, proliferation, and invasion are provided. Primary human omental mesothelial cells and fibroblasts were isolated from patients and used at an early passage to preserve normal morph.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1. Isolation and culture of primary cells
PBS	Fisher Scientific	SH3001304
Single-edged razor blades	Fisher Scientific	12-640
15 cm culture dishes	BD Biosciences	353025
Glass flask	?	?
Fetal Bovine Serum (FBS)	Life Technologies	16000044_3616914956
DMEM with L-Glutamine	Corning	10-013-CV
MEM Vitamins	Corning	25-020-Cl
MEM Nonessential amino acids	Corning	25-025-CI
Penicillin-Streptomycin	Corning	30-002-CI
Shaker	Thermo-Fisher	MaxQ 4450
Centrifuge	Eppendorf	5702
Incubator	Thermo-Fisher	Forma Series II Water Jacketed CO2 Incubator Model 3100
Trypsin EDTA, 1x (0.25%)	Corning	25-053-CI
Hyaluronidase	Worthington Biochemical	LS002592
T-75 Flasks	BD Biosciences	353136
T-175 Flasks	BD Biosciences	353112
Pipet tips	Rainin	P2, P10, P20, P200 and P1000
Pipet tips	Corning	Filtered tips P2, P10, P20, P200 and P1000
Name of Reagent/ Equipment	Company	Catalog Number	Comments/Description
2. Plating 3D culture
Cell Counter	Invitrogen	Countess
Countess Cell Counting Chamber Slides	Invitrogen	C10313
Trypan Blue Stain (0.4%)	Gibco	15250-061
Collagen Type I (Rat Tail)	BD Biosciences	354236
96 well plate, clear bottom, black	BD Biosciences	353219
Name of Reagent/ Equipment	Company	Catalog Number	Comments/Description
3. Adhesion assay
Multichannel pipet	Eppendorf	Xplorer 300
Paraformaldehyde solution 4% in PBS	Santa Cruz Biotechnology	sc-281692
Plate reader	Molecular Devices	Minimax
Name of Reagent/ Equipment	Company	Catalog Number	Comments/Description
5. Invasion assay
Cell Culture Inserts (8um, 24-well)	BD Biosciences	353097
Cotton swabs	Q-tips	cotton swabs
Microscope	Zeiss	Axiovert 200m
Cell Profiler	public domain
24 well plate	BD Biosciences	353047
Name of Reagent/ Equipment	Company	Catalog Number	Comments/Description
6. Antibodies
Anti-Integrin αVβ3 Antibody, clone LM609	EMD Millipore	MAB1976
Beta 1	Oncosynergy	OS2966
Alpha 5 [CD49e]	ID Pharmingen	555615
Beta 4 [CD104]	EMD Millipore	MAB 2058