Modeling Ovarian Cancer Multicellular Spheroid Behavior in a Dynamic 3D Peritoneal Microdevice

Summary

To study ovarian tumor progression in a physiologically relevant model, multicellular spheroids were cultured in a microdevice under simulated fluid flow. This dynamic 3D model emulates the intraperitoneal environment with the cellular and mechanical components where ovarian cancer metastasis occurs.

Abstract

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with poor clinical outcomes and currently lacks effective treatment. Both the three-dimensional (3D) environment and the dynamic mechanical forces are very important factors in this metastatic cascade. However, traditional cell cultures fail to recapitulate this natural tumor microenvironment. Thus, in vivo-like models that can emulate the intraperitoneal environment are of obvious importance. In this study, a new microfluidic platform of the peritoneum was set up to mimic the situation of ovarian cancer spheroids in the peritoneal cavity during metastasis. Ovarian cancer spheroids generated under a non-adherent condition were cultured in microfluidic channels coated with peritoneal mesothelial cells subjected to physiologically relevant shear stress. In summary, this dynamic 3D ovarian cancer-mesothelium microfluidic platform can provide new knowledge on basic cancer biology and serve as a platform for potential drug screening and development.

Introduction

Ovarian cancer is the most lethal gynecological cancer and is characterized by widespread peritoneal dissemination and the formation of malignant ascites¹. This extensive peritoneal metastasis represents a major clinical challenge and is associated with poor clinical outcomes. Unlike most solid carcinomas that metastasize via the blood, ovarian cancer primarily disseminates within the peritoneal cavity. Tumor cells exist as multicellular aggregates/spheroids during the process of metastasis². The fact that suspension culture can enrich ovarian cancer stem/tumor-initiating cells further suggests that these spheroids may b....

Protocol

1. Microfluidic Device Design and Fabrication

1. Microfluidic master design
   1. Design and draw the microfluidic channel pattern with any computer-aided design (CAD) software.
      NOTE: Normally, the CAD drawing can be sent to a photomask company to produce the photomask. The microfluidic design consists of three identical parallel channels, each with the following dimensions: 4 mm × 25 mm × 250 µm (width × length × height) and set 2 mm apart. Both channel ends were designed with 127° angles to facilitate liquid entrance and exit (Figure 1B). The channel used in this protocol was reported in a previous publication^1....

Results

Using this protocol, a microfluidic platform was set up to model ovarian cancer spheroids with mesothelial cells under hydrodynamic conditions. Primary human peritoneal mesothelial cells were cultured in the microdevice for 16 h and observed under a bright-field microscope. As shown in Figure 2A, the channel bottom was successfully covered with a monolayer of HPMCs. It is important to note that bubble formation during fibronectin or HPMC patterning will lead to channel co.......

Discussion

This assay offers a flexible and physiologically relevant model that can be incorporated with various biochemical and cell-based assays, including, but not limited to, adhesion assays, mesothelial clearance assays, and drug screening. It can be applied to the evaluation of the effect of the intraperitoneal microenvironment on cancer progression. However, several experimental conditions might need to be optimized, depending on the aims of the project (e.g., the number of HPMCs and cancer spheroids seeded per chan.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Silicon wafer	University wafer	#1196	100 mm
SU-8 2075 photoresist	Microchem
SU-8 developer	Microchem	108-65-6
Trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane	Sigma	448931
Sylgard 184	Dow Corning	1673921	Polydimethylsiloxane (PDMS) + curing agent kit
Biopsy punch	Miltex	33-31AA	1 mm diameter
Plasma cleaner	Harrick Plasma	PDC-002
Polyethylene tubing	SCI	BB31695-PE/5	0.86 mm (inner diameter)
Syringe	Terumo
Syringe pump	Longer precision pump	LSP01-2A
Medium 199	Invitrogen	31100-035	Add 2.2 g/L sodium bicarbonate
MCDB 105 Medium	Sigma	M6395
Fetal bovine serum (FBS)	Hyclone	SH30068.02
Penicillin/streptomycin	Invitrogen	15070-063
Trypsin EDTA solution	Gibco	25300-054	0.05% Trypsin - 0.01% EDTA, phenol red
Fibronectin human	BD	354008
Agarose	Invitrogen	15510-027
5-chloromethylfluorescein diacetate	Life technologies	C7025	Green CMFDA
CO2 incubator	SANYO	MCO-18AIC
Centrifuge	Hitachi	CT15RE
Fluorescent microscope	Nikon		Model: 80i or ECLIPSE Ti; software: SPOT
SKOV-3			Gift from Dr. N Auersperg (University of British Columbia)