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 aldehyde dehydrogenase (ALDH) activity^4,5 have been reported to mark ovarian CSCs, some data indicate that these markers are unstable⁶. Consistently, in ovarian cancer, other than for example in breast carcinoma⁷, expression of ALDH1 associates with favorable outcome⁸ and expression of the proposed stem cell marker CD44 variant has no prognostic value⁹. More recently, we have shown that expression of the embryonic stem cell protein SOX2 confers stemness to ovarian carcinoma cells¹⁰ and high SOX2 expression associates with clinically aggressive ovarian and breast carcinomas^11,12. Therefore, in this report we use a lentiviral reporter construct containing a red fluorescence protein (RFP) whose expression is controlled by a SOX2 regulatory region, as a method to isolate putative ovarian CSCs.

By definition, CSCs can both self-renew and differentiate, giving rise to all tumor cell types. Putative CSC populations need to be analyzed in functional assays performed in vivo. For obvious reasons, in human cells such functional tests are confined to xenograft assays, comprising mostly transplantation of human tumor cells into immuno-compromised mice^10,13.

An alternative in vitro method was offered by Brent Reynolds and Sam Weiss who firstly reported the so-called neurosphere assay as a surrogate assay evaluating stem potential in neural cells¹⁴. Dontu and colleagues later confirmed the use of this assay for evaluation of stem cell potential in breast cells^15,16. Here, human mammary cells were plated in different numbers in serum-free medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), B-27 and heparin and cultured under non-adherent conditions for seven to ten days before sphere formation was scored by microscopy. Following this protocol with some adjustments in cell numbers, growth medium and supplements, several groups have explored in vitro stem cell potential from several cancer types such as breast¹⁷, brain¹⁸, pancreas¹⁹ and colon²⁰ tumors. In ovarian carcinoma, we have recently reported feasibility of the spheres assay and compared its results to those collected in in vivo murine xenograft models¹⁰. We found that overexpression of the stem cell protein SOX2 enhanced both in vitro sphere formation as well as in vivo tumorigenicity of human ovarian carcinoma cells¹⁰. However, the frequency of sphere-initiating cells was higher than the frequency of tumor-initiating cells measured in vivo¹⁰ suggesting that either the sphere assay may lead to false positive results due to technical reasons or, alternatively, the in vivo assay may be inefficient and result in false negative results.

In this report, we analyze multi cell-based ovarian spheres assays in more detail, review the different protocols available in the literature and compare them to a single cell-based assay. We show that the single cell-based assay provides more accurate and reproducible results than multi cell-based assays, which can be highly influenced by the density of plated cells unless methylcellulose is added to the cultures to immobilize cells. However, also in single cell-based assays, in vitro sphere-initiating potential is observed at higher frequency than in vivo tumor-initiating potential.

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 lentiviral particles over a time period of 24 hr. Afterwards, remove the viral supernatant and wash the cells with phosphate buffered saline (PBS) and cultured in complete medium (RPMI supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin).
3. 48 hr later, 10 μg/ml puromycin were added to the cultures and maintained for 5 days to allow selection of properly transduced cells.

2. Preparation of Cell Sorting and Plating

1. Add Shield1 at 1:1,000 dilution 24 hr prior to cell sorting. Use stably transduced OVCAR-3 cells without Shield1 treatment as negative controls (Figure 1). Aspirate media from flask, wash cells with 1x PBS and trypsinize cells with 0.05% Trypsin-EDTA for 3 min.
2. Stop trypsin by using complete medium (see above), count cell numbers, centrifuge cells at 300 x g at RT (15 - 25 °C) for 5 min.
3. Decant supernatant and resuspend cells carefully in 0.5 - 1 ml sterile PBS.
4. Use 40 µm cell strainer cap filter to obtain single-cell suspension.
5. Adjust cell count to 5 million cells per ml.
6. Prepare ultra low-attachment 96-well plates with 100 μl spheres medium (MEGM supplemented with growth factors, cytokines, and supplements, B-27, heparine-sodium; or DMEM/F12 supplemented with growth factors, cytokines, and supplements, B-27, heparine-sodium with or without addition of 1% methylcellulose, see also Table 1). Optionally add antibiotics to the medium at a concentration of 100 U/ml penicillin and 100 μg/ml streptomycin to minimize the risk of possible contamination.
7. Sort RFP+ and RFP- cells into prepared 96-well plates from above, 1 cell per well (single cell-based spheres assay) and 100 cells per well (multi cell-based spheres assay), respectively. Perform sort on commercially available cell sorter (see Materials) using single cell mode, Sort setup: 100 μ nozzle, sheath pressure 20 psi, and yield mask 0, purity mask 32, phase mask 16.
8. Assess plating efficiency by microscopically scoring wells containing cells (for the single cell-based assay) and by counting cell numbers in individual wells (for the multi cell-based assay; Figure 2).
9. Incubate cells under standard conditions in spheres medium (for composition see step 2.6) at 37 °C and 5% CO₂. Supplement daily bFGF (20 ng/ml) and EGF (20 ng/ml).
10. After one week, count numbers of emerging tumor spheres using a standard microscope with 4X or 10X magnification and a fluorescence microscope to detect fluorescence signal from the integrated reporter system. Count spheres with a diameter exceeding 100 μm as “large” spheres, and spheres with a diameter 50 - 100 μm as “small” spheres (Figure 3). Be sure that you count real spheres and not cell clusters.
    NOTE: In single cell-based assays sphere formation is easier to score microscopically after 10 (versus 7) days of culture.
11. Calculate the proportion of sphere-forming cells in RFP+ and respectively RFP- cells in single cell-based assays (one 96-well plate for each individual experiment) or multi cell-based spheres assays (one well for each individual experiment) as presented by Shaw et al.¹⁶
    NOTE: Proportion of sphere forming cells (%) = (number of spheres) / (number of seeded cells) x 100

3. Serial Passaging of Spheres

1. Place the content of each well in an appropriate sterile tube and centrifuge at 300 x g for 10 min at RT. For multi cell-based spheres assays, collect together the spheres from one well. Wash the well 3 - 5 times with PBS and centrifuge 2 min longer. For single cell-based spheres assays, collect individual spheres. Due to the low numbers of cells, use 1.5 ml tubes for centrifugation and washing steps.
2. Remove supernatant and resuspend pellet in 200 µl of 0.05% Trypsin-EDTA.
3. In order to achieve optimal cell separation, incubate the cell suspension at 37 °C for 5 min in a soft shaker. Optimize trypsinization time for your cell line to lower cell death rate: if no large spheres is visible triturate gently using a 100 μl pipette tip. In case large spheres are still present, incubate with trypsin another 3 min and then proceed to the trituration step. In case of single cell-based assays make sure to optimize the time for optimal cell yield of living cells during the trypsinization step.
4. To inactivate the trypsin, add 500 µl complete medium and centrifuge at 300 x g for 10 min, in the case of single cell assays, centrifuge for additional 2 min.
5. Remove supernatant and resuspend cells carefully in spheres medium.
6. Use a 40 µm cell strainer cap filter to obtain a single-cell suspension.
7. In the case of using RFP+ and RFP- cells in multi cell-bassed assays, assess the percentage of fluorescent cells in each well after resuspension via flow cytometer analysis.
8. For serial replating assays of single cells, seed 1 cell per well into a new ultra low-attachment 96-well plate prepared as detailed above. From one individual sphere, seed approximately 20 individual wells. For replating assays of multi cell-based primary spheres, seed cells obtained from one well of primary spheres into a new 96-well plate and count the cell numbers next day by microscopy.
9. Assess the proportion of sphere-forming cells in secondary spheres assays using the formula described in step 2.11.

4. Result Analysis

1. Analyze results from experiments performed in independent triplicates and use two-sided Student’s t-Test to analyze normally distributed values and otherwise Mann-Whitney-Tests for statistical analysis.

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