Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1

Summary

Ovarian cancer stem cells (OCSC) are responsible for cancer initiation, recurrence, therapeutic resistance, and metastasis. The OCSC vascular niche is considered to promote self-renewal of OCSCs, leading to chemoresistance. This protocol provides the basis for establishing a reproducible OCSC vascular niche model in vitro.

Abstract

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular matrix. The ability of CSCs to participate in the development of endothelium constitutes an important characteristic that directly contributes to the general understanding of the mechanisms of tumorigenesis and tumor metastasis. The purpose of this work is to establish a reproducible methodology to investigate the tumor-initiation capability of ovarian cancer stem cells (OCSCs). Herein, we examined the neovascularization mechanism between endothelial cells and OCSCs along with the morphological changes of endothelial cells using the in vitro co-culture model NICO-1. This protocol allows visualization of the neovascularization step surrounding the OCSCs in a time course manner. The technique can provide insight regarding the angiogenetic properties of OCSCs in tumor metastasis.

Introduction

Ovarian cancer is the eighth most common malignancy in women worldwide, with approximately 300,000 new diagnoses and an estimated 180,000 deaths annually¹. At initial diagnosis, ovarian cancer often presents with severe symptoms, with about 75% of patients already at stage III-IV. Accordingly, the 5-year survival rate is <30% and the mortality rate is the highest among gynecological cancers², with the efficiency of treatment for ovarian cancer being highly dependent on clinical factors such as the successful accomplishment of debulking surgery, resistance to chemotherapy, and recurrence after the initial therapy.

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Protocol

All procedures were performed under the protocol approved by the Ethics Committee for human welfare. All patients provided written informed consent for the research use of their samples, and the collection and use of tissues for this study were approved by the Human Genome, Gene Analysis Research Ethics Committee at Teikyo University.

1. Isolation and culture of ovarian cancer stem cells (OCSCs) from Patients with ovarian cancer and ascites in a level 2 biosafety cabinet

1. Isolate ca.......

Discussion

The presented protocol describes how to mimic the tumor microenvironment of OCSCs in an in vitro setting. The primary component of the method constitutes the highly reproducible coculture model obtained using the NICO-1 system, an indirect Transwell co-culture system. Many of the currently available coculture models examine the effects of direct cell-cell contact on cocultured cell populations^12,13,14,

Materials

Name	Company	Catalog Number	Comments
0.025% Trypsin	Thermo	R001100
10 mL Pipet	Thermo	170356N
1250 µL Pipet tip	QSP	T112XLRS-Q
15 mL tube	Nunc	339650
200 µL Pipet tip	QSP	T110RS-NEW
2-Mercaptoethanol	Thermo (Gibco)	21985023
5 mL Pipet	Thermo	170366N
50 mL tube	Corning	430290
AccuMAX	Innovative Cell Technologies	AM105
BioCoatTM Collagen I 60mm Dish	Corning	356401
Centrifuge	KUBOTA	2800
Costar 6 Well Clear Flat Bottom Ultra Low Attachment Multiple Well Plates	Corning	3471
Endothelial Cell Growth Medium 2	PromoCell	C-22011
Ethanol	WAKO	057-00456
FGF-Basic	Thermo (Gibco)	PHG0021
Histodenz	SIGMA	D2158
HUEhT-1 cell	JCRB Cell Bank	JCRB1458
ICCP Filter 0.6 µm	Ginrei Lab.	2525-06
Insulin, human	SIGMA (Roche)	11376497001
Luminometer	PerkinElmer	ARVO MX-flad
Matrigel Matrix	Corning	356234
Microscope	Yokogawa	CQ-1
NICO-1	Ginrei Lab.	2501-02
OptiPlate-96	PerkinElmer	6005290
P1000 Pipet	Gilson	F123602
P200 Pipet	Gilson	F123601
PBS	Thermo (Gibco)	14190-144
StemPro hESC SFM	Thermo (Gibco)	A1000701
Transfer Pipet	FALCON	357575
Y-27632	WAKO	253-00513