Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

Summary

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

Abstract

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called “cancer stem cells” (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.

Introduction

There is increasing evidence that ovarian carcinomas are comprised of heterogeneous mixtures of cells and harbor so-called “cancer stem cells” (CSCs) responsible for disease initiation, maintenance and relapse after conventional cytotoxic therapies^1-3. Therefore, the development of molecular strategies targeting ovarian CSCs is an important goal and promises to improve the therapy of ovarian cancer patients.

A pre-requisite for the understanding of the molecular features of CSCs is their reliable isolation from the non-CSCs. However, identification of ovarian CSCs appears challenging. While CD133 expression and aldehy....

Protocol

1. Generation of OVCAR-3 Human ovarian Carcinoma Cells Stably Transduced with Lentiviruses Containing the SOX2 Regulatory Region Reporter Construct

1. Generate lentiviral particles by transfecting the HEK 293T-packaging cell line with a reporter construct recognizing a SOX2 regulatory region as described^10,21.
   NOTE: The reporter construct further contains a destabilization domain of the ProteoTuner Shield System ahead of the tdTomato fluorescence protein. Shield1 binds to the destabilization domain thereby preventing the proteasome to degrade the fluorescence protein²².
2. Transduce OVCAR-3 cells with lent....

Results

In conventional spheres assays, nearly 40% of RFP+ OVCAR-3 cells vs. 20% of RFP- cells gave rise to an individual tumor sphere in the primary spheres assay (Figure 4A). Moreover, spheres formed by RFP+ cells were larger in size than those formed by RFP- cells.

When plated in single cell-based assays, RFP+ cells also formed more spheres than RFP- cells, confirming the results above. However, there was a tendency towards generation of fewer spheres per plated in the single versu.......

Discussion

Spheres cultures are a widely used method to assay cancer stem cell potential and enrich for stem-like cells in a wide range of human tumor cells^15,25,26. Under these culture conditions, cancer cells that lack self-renewal ability are expected to differentiate and eventually undergo cell death. Although they may initially form cell clusters or even tumor spheres especially in primary assays, they are not able to sustain sphere-forming ability upon serial replating due to lack of self-renewing properties. Spher.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Low-attachment plate	Corning	3474
MEGM	Lonza	CC-3151
Insulin	Lonza	CC-4136	SingleQuots™ Kit
Hydrocortisone	Lonza	CC-4136	SingleQuots™ Kit
EGF	Lonza	CC-4136	SingleQuots™ Kit
EGF	Sigma	E9644	end concentration: 20 ng/ml
FGF	PeproTech	100-18B	end concentration: 20 ng/ml
B-27	Invitrogen/ Gibco	17504-044	end concentration: 1X
Heparin-Natrium-25000 IE	Ratiopharm	N68542.02	dilution 1:1,000
Pen/Strep	Gibco	15140-122
FCS	Gibco	10500-064
RPMI 1640	Gibco	21875-034
Trypsin-EDTA	Gibco	25300-054
Dulbecco’s PBS (1X)	Gibco	14190-094
Shield1	Clontech	632189	dilution 1:1,000
DMEM/F12	Gibco	21041-025
DMEM/F12 (powder)	Gibco	42400-010
Methyl cellulose	Sigma	M0387
Puromycin dihydrochloride	Applichem	A2856
Cell sorter	BD		Aria III cell sorter
FACS analyser	BD		Accuri c6 flow cytometer
Microscope	Olympus		IX50 Osiris