Characterization of Ovarian Cancer Ascites on Cell Invasion,Proliferation, Spheroid Formation, and Gene Expression inan In Vitro Model of Epithelial Ovarian Cancer1,2

Marie-Line Puiffe*, Cecile Le Page*, Abdelali Filali-Mouhim*, Magdalena Zietarska*,3, Veronique Ouellet*,3,Patricia N. Tonin y,z,§, Mario Chevrette§,b, Diane M. Provencher*,# and Anne-Marie Mes-Masson*,**

*Centre de recherche du Centre hospitalier de l’Universite de Montreal/Institut du cancer de Montreal,Montreal, Quebec, Canada; yDepartment of Medicine, McGill University, Montreal, Quebec, Canada;zDepartment of Human Genetics, McGill University, Montreal, Quebec, Canada; §The Research Instituteof McGill University Health Centre, Montreal, Quebec, Canada; bDepartment of Surgery, Urology Division,McGill University, Montreal, Canada; #Division of Gynecologic Oncology/Universite de Montreal, Montreal,Quebec, Canada; **Departement de medicine, Universite de Montreal, Montreal, Quebec, Canada

Abstract

At least one third of all cases of epithelial ovarian

cancer are associated with the production of ascites,

although its effect on tumor cell microenvironment

remains poorly understood. This study addresses the

effect of the heterologous acellular fraction of ovarian

cancer–derived ascites on a cell line (OV-90) derived

from the chemotherapy-naıve ovarian cancer patient.

Ascites were assayed for their effect on cell invasion,

growth, and spheroid formation. When compared to

either no serum or 5% serum, ascites fell into one

of two categories: stimulatory or inhibitory. RNA from

OV-90 cells exposed to selected ascites were arrayed

on an Affymetrix HG-U133A GeneChip. A supervised

analysis identified a number of differentially expressed

genes and quantitative polymerase chain reaction vali-

dation based on OV-90 cells exposed to 54 indepen-

dent ascites demonstrated that stimulatory ascites

affected the expression of ISGF3G, TRIB1, MKP1, RGS4,

PLEC1, and MOSPD1 genes. In addition, TRIB1 ex-

pression was shown to independently correlate with

prognosis when its expression was ascertained in an

independent set of primary cultures established from

ovarian ascites. The data support the validity of the

strategy to uncover molecular events that are associ-

ated with tumor cell behavior and highlight the impact

of ascites on the cellular and molecular parameters of

ovarian cancer.

Neoplasia (2007) 9, 820–829

Keywords: Epithelial ovarian cancer, ovarian ascites, cell behavior, inva-sion, molecular profiling.

Introduction

Ovarian cancer is the fifth leading cause of cancer-related

deaths in the Western world, the second most common

gynecological cancer, and the leading cause of death from

gynecological malignancies. The most common form of cancer

of the ovary is epithelial ovarian cancer (EOC). EOCs originate

either from the normal ovarian surface epithelium itself or from

the crypts and inclusion cysts on the surface epithelium [1].

Ovarian carcinomas can spread by local extension, lymphatic

invasion, intraperitoneal implantation, hematogenous dissem-

ination, and/or transdiaphragmatic passage. In the commonly

observed intraperitoneal dissemination, malignant cells appear

to implant anywhere over peritoneal surfaces though mainly in

sites of stasis along the peritoneal fluid circulation.

At least one third of ovarian cancer patients present with

ascites [2,3], a generally voluminous exudative fluid with a cel-

lular fraction consisting mainly of ovarian cancer cells, lympho-

cytes, and mesothelial cells. The neoplastic cells in the ascites

are present either as single cells, as aggregates, or as spher-

oids, and may contribute to the spread of cancer to secondary

sites. Indeed, it has been demonstrated that ascites spheroids

adhere to extracellular matrix through b1 integrins, indicating

that this process may play a role in the dissemination of the

disease [4]. The acellular fraction of ascites is known to harbor

angiogenic factors such a vascular endothelial growth factor

[5] and growth factors such as the epidermal growth factor [6],

lysophosphatidic acid [7], and transforming growth factor (TGF)

family members [8,9] among others. How this acellular frac-

tion affects the tumor microenvironment, and specifically how it

Address all correspondence to: Anne-Marie Mes-Masson, CR-CHUM/ICM, 1560, rue Sherbrooke

est, Montreal, Quebec, Canada H2L 4M1. E-mail: anne-marie.mes-masson@umontreal.ca1This research was supported by The Cancer Research Society, Inc., Strategic Grant Program

in Genomics and Proteomics of Metastatic Cancer award to A.-M.M-M, P.N.T., M.C., and D.P.

The ovarian tissue bank was supported by the Banque de tissus et de donnees of the Reseau

de recherche sur le cancer of the Fonds de la Recherche en Sante du Quebec, affiliated with the

Canadian Tumor Repository Network.2This article refers to supplementary material, which is designated by ‘‘Figure W1’’ and ‘‘Table

W1’’ and is available online at www.bcdecker.com.3V.O. was supported by studentships from the Canadian Institutes of Health Research, and

V.O. and M.Z. by the Canderel fund of the Institut du cancer de Montreal.

Received 11 June 2007; Revised 21 August 2007; Accepted 22 August 2007.

Copyright D 2007 Neoplasia Press, Inc. All rights reserved 1522-8002/07/$25.00

DOI 10.1593/neo.07472

Neoplasia . Vol. 9, No. 10, October 2007, pp. 820 –829 820

www.neoplasia.com

RESEARCH ARTICLE

affects the cellular and molecular properties of tumor cells per

se is still a matter of debate [10,11]. Indeed, the recent resur-

gence of intraperitoneal-based chemotherapies highlights the

importance of understanding this important component of the

disease [12–14]. Recently, a targeted approach demonstrated

the ability of ascites to modulate the expression of urokinase

plasminogen activator, its receptor, and integrins, and these

modifications in expression were associated with changes in

the cellular behavior of ovarian cancer cell lines [15].

In the present study, we conducted a comprehensive

analysis of the effect of ascites on the growth characteristics

of the OV-90 ovarian cancer cell line, which we have pre-

viously described [16]. This EOC cell line, derived from the

cellular fraction of ascites from a chemotherapy-naıve pa-

tient, has been characterized by morphological, immunohis-

tochemical, cytogenetic, and molecular analyses including

gene expression profiles [16,17] and was shown to har-

bor mutations in genes implicated in ovarian cancer such as

tumor protein p53 (TP53), cyclin-dependent kinase inhibitor

1A (CDNK2A), and TGF�-RII. In addition to characterizing

the cellular effects of ascites, a DNA microarray approach

was used to assess differences in gene expression in OV-90

cell line grown in the presence or absence of serum, as

well as in the presence of ascites without serum. Statistical

analysis was used to identify differentially expressed genes

that correlated with cellular invasion for the OV-90 cell line.

Candidate genes were further validated on both arrayed

RNA and an extended test set using quantitative polymerase

chain reaction (Q-PCR), and their association with survival

was tested in an independent set of primary cultures derived

from patients with ovarian ascites.

Materials and Methods

Cell Culture, Clinical Material, and Patients

The OV-90 cell line was maintained in OSE media con-

sisting of 50:50 medium 199:105 (Sigma-Aldrich, St. Louis,

MO) supplemented with 10% fetal bovine serum (FBS), 2.5 mg/

ml amphotericin B and 50 mg/ml gentamicin [18]. Following

appropriate consent, ascites were collected at the time of

clinical intervention at the Centre Hospitalier de l’Universite de

Montreal (Montreal, QC, Canada). Ascites were centrifuged at

2500 rpm for 5 minutes. The acellular fractions were stored at

�20jC and tested within 6 months of reception. The protein

concentration in ascites fluid was measured by the Bradford

assay (Bio-Rad, Hercules, CA). Histopathology, grade, and

stage of ovarian tumors were assigned according to the Inter-

national Federation of Gynecology and Obstetrics criteria. Of

the 54 ascites included in our study on invasion, two thirds

were from patients diagnosed with papillary serous adeno-

carcinomas and most presented with stage IIIC and grade 3

disease (Table 1). One third of samples were from patients

who had already received chemotherapy prior to surgery. The

presence of neoplastic cells in ascites was determined from

pathology reports. Table 2 describes the 28 ovarian cancer

patients diagnosed with accompanying ascites that were

included in the survival analysis.

In Vitro Invasion Assay

Cellular invasion was assayed by determining the ability

of cells to invade a synthetic basement membrane (Matrigel;

Becton-Dickinson, Bedford, MA). Polycarbonate membranes

(8-mm pore size) of the upper compartment of Transwell

culture chambers were coated with 0.4 mg/ml Matrigel. The

upper compartment was filled with OSE media containing 1%

FBS and the lower compartment was filled with OSE media

either with no serum, with 5% FBS, or with 5% of the indicated

ascites. For inactivation, ascites were heated for 10 minutes at

100jC to denature proteins. Ovarian cancer cells were trypsi-

nized and resuspended in OSE media containing 1% FBS.

The cell suspension (20 � 103 cells/well) was placed in the

upper compartment. Then, cells were incubated at 37jC and

allowed to invade through the Matrigel barrier for 24 hours.

Following incubation, membranes were fixed with methanol

and stained (Giemsa; Sigma-Aldrich). Noninvading cells were

removed using a cotton swab, whereas invading cells on the

underside of the membrane were counted using an inverted

microscope. All experiments were performed at least twice.

Cell Proliferation

Two thousand cells were plated either with no serum,

with 5% FBS, or with 5% of the indicated ascites in six-well

plates and incubated at 37jC. At defined intervals, cells were

trypsinized and cell viability was assessed by a Trypan Blue

exclusion assay. Cell numbers were evaluated using a hemato-

cytometer. Each experiment was performed in triplicate.

Spheroid Formation

Spheroids were formed using a modification of the hang-

ing droplet method [19]. Briefly, 4 � 103 cells were resus-

pended in 15 ml of OSE media supplemented either with 5%

FBS, with 5% of the indicated ascites, or without serum, and

then placed on the cover of a 150-mm tissue culture plate.

The cover was placed over a plate that contained 15 ml of

OSE to prevent dehydration of the hanging droplet. Spheroid

formation was monitored after 4 days and representative

spheroids were photographed.

RNA Extraction

Total RNA was extracted with a reagent (TRIzol; Gibco/

BRL, Life Technologies, Inc., Grand Island, NY) as recom-

mended by the manufacturer. RNA was extracted from tumor

cells grown to 80% confluence in 100-mm Petri dishes. RNA

quality was assessed using a 2100 Bioanalyzer with the RNA

6000 Nano LabChip kit (Agilent Technologies, Mississauga,

ON, Canada) according to the manufacturer’s protocol.

Microarray Analysis

Hybridization assays and data collection were performed

at the McGill University and Genome Quebec Innovation

Centre (Montreal, Canada). Briefly, 20 mg of total RNA from

each sample was reverse-transcribed using an oligo-dT

primer containing a T7 RNA polymerase binding site. In vitro

transcription was performed on this cDNA and the resulting

cRNA was biotinylated through incorporation of biotinylated

Ascites and Ovarian Tumor Cell Interaction Puiffe et al. 821

Neoplasia . Vol. 9, No. 10, 2007

dUTP and dCTP. Samples were fragmented in 40 mM Tris–

acetate, 100 mM potassium acetate, and 30 mM MgCl2 (pH

8.1) at 95jC to reduce secondary structure. A total of 15 mg of

cRNA was hybridized to an Affymetrix HG-U133A GeneChip

array (Santa Clara, CA), washed, stained, and scanned with

a Hewlett Packard Gene Array scanner (Palo Alto, CA) and

.CEL files were normalized based on a quantile method.

Gene expression profiles were analyzed using R (www.

r-project.org), a statistical programming language, and Bio-

conductor [20], an open source software library for the

analyses of genomic data based on R. Background subtrac-

tion, normalization (quantile normalization), and expression

value calculations were performed using the justGCrma

function available as part of Bioconductor’s gcrma pack-

age. Bioconductor’s genefilter package was used to filter

out genes with insufficient variation in expression across all

samples tested. Expression values retained after this filtering

process presented intensities greater than 100 U in at least

Table 1. Clinical Characteristics of Patients from Which Ascites Were Obtained.

Ascites Age Histopathology Grade Stage Neoplastic Cells in Ascites Prior Chemotherapy Clinical Intervention Associated with Ascites Collection

A1185(2)* 82 PSA G3 IIIC Yes Yes Secondary cytoreduction

A1301 49 SA G3 IIIC Yes No Primary biopsy

A1317 60 PSA G3 IV No Yes Primary surgery

A1318 52 PSA G2 IIIC Yes No Primary surgery

A1322 71 PSA G3 IIIC Yes No Primary surgery

A1322(2) 71 PSA G3 IIIC Yes Yes Secondary cytoreduction

A1330 48 SA G3 IIIC Yes No Primary surgery

A1337 45 PSA G3 IIIC Yes No Primary surgery

A1369 60 PSA G3 IIIC Yes No Primary surgery

A1369(2) 60 PSA G3 IIIC Yes Yes Secondary cytoreduction

A1396 54 PSA G2 IIIC Yes No Primary surgery

A1406 49 PSA G2 IIIC N/S Yes Secondary cytoreduction

A1464 63 PSA G3 IIIC Yes No Primary surgery

A1483 74 PSA G3 IIIC Yes No Primary surgery

A1519 73 CCA G3 IV N/S No Primary surgery

A1526 50 PSA G2 IIIC Yes Yes Secondary cytoreduction

A1526(2) 50 PSA G2 IIIC Yes Yes Secondary cytoreduction

A1592 35 MCA G3 IIIC N/S No Primary surgery

A1607 59 SA GB IB No No Primary surgery

A1610 72 PSA G3 IIIC Yes No Primary surgery

A1611 45 MA GB IA No No Primary surgery

A1613 76 SA G3 IIIC No No Primary surgery

A1642 79 SA G3 IIB Yes No Primary surgery

A1717 45 PSA G2 IIIC N/S No Primary surgery

A1739 64 MA N/S N/S N/S No Primary surgery

A1778 87 PSA G3 IV Yes No Primary surgery

A1793 55 SA G3 IIIC No No Primary surgery

A1801 65 PSA G2 IIIC Yes No Primary surgery

A1810 62 PSA G2 IIIC Yes No Primary surgery

A1813 55 PSA G3 IIIC Yes No Primary surgery

A1830 56 PSA G3 IIIC Yes No Primary surgery

A1835 69 PSA G3 IIIC Yes No Primary surgery

A1876 44 PSA G3 IIIC No No Primary surgery

A1884 69 PSA G3 IV Yes No Primary surgery

A1891 62 PSA G3 IIIC No No Primary surgery

A1922 52 PSA G3 IIIC Yes No Primary surgery

A1927 48 PSA G3 IIIC Yes No Primary surgery

A1931 67 PSA G3 IIIC Yes No Primary surgery

A1946 75 PSA G3 IIIC Yes No Primary surgery

A1998 70 UA N/S N/S Yes Yes N/S

A1998(2) 70 UA N/S N/S Yes Yes N/S

A2069 63 MCA G3 IIIC Yes No Primary surgery

A2070 78 SA G3 IIIC Yes No Primary surgery

A2085 65 PSA G3 IIIC N/S Yes Secondary cytoreduction

A2085(2) 65 PSA G3 IIIC N/S Yes Secondary cytoreduction

A2085(3) 65 PSA G3 IIIC N/S Yes Secondary cytoreduction

A2090 76 UA N/S IIIC Yes Yes N/S

A2093 62 PSA G3 IV Yes Yes Secondary cytoreduction

A2093(2) 62 PSA G3 IV Yes Yes Secondary cytoreduction

A2774 42 EA G3 IB No No Primary surgery

A2775 49 PSA G2 IIIC Yes No Primary surgery

A2834 63 PSA G3 IIIC Yes No Primary surgery

A2912 54 PSA G3 IIIC Yes No Primary surgery

A2965 71 MCA G3 IIIC Yes No Primary surgery

PSA, papillary serous adenocarcinoma; SA, serous adenocarcinoma; MA, mucinous adenocarcinoma; MCA, mixed cell adenocarcinoma; CCA, clear cell

adenocarcinoma; UA, undifferentiated adenocarcinoma; EA, endometrioid adenocarcinoma; N/S, not specified.

Ascites further characterized in this study are in bold.

*Denotes second and third ascites collected from patient with the same number.

822 Ascites and Ovarian Tumor Cell Interaction Puiffe et al.

Neoplasia . Vol. 9, No. 10, 2007

two samples and a log base 2 scale of at least 0.2 for

the interquartile range across all tested samples. Differ-

entially expressed genes were identified using the limma

package, which estimates the fold-change between prede-

fined groups by fitting a linear model and using an empirical

Bayes method to moderate standard errors of the estimated

log-fold changes for expression values from each probe set.

Quantitative PCR

cDNA synthesis was prepared using the SuperScript

First-Strand Synthesis System for reverse transcription–

polymerase chain reaction (Invitrogen Life Technologies,

Carlsbad, CA) according to the manufacturer’s instruction.

Reverse transcription–polymerase chain reaction was per-

formed on 2 mg of total RNA using 2.5 ml of the random

hexamer solution. Samples were diluted 1:10 in water prior

to Q-PCR. Positive and negative controls were included in

all experiments.

Q-PCR was performed using the Rotor-gene 3000 (Corbett

Research, Montreal Biotech Inc., Montreal, QC, Canada).

Quantitect SYBR Green PCR (QIAGEN Inc., Mississauga,

ON, Canada) was used for labeling in a final volume of 25 ml

containing 5 ml of sample cDNA and 10 pg of the different

primers and reactions performed as described by the manu-

facturer. Experiments were repeated at least twice. Serial

dilutions (1:5) were performed to generate a standard curve

for each gene tested to define the efficiency of the Q-PCR

reaction and a melt curve was done to confirm the speci-

ficity of the reaction. We used the Pfaffl analysis method to

measure the relative quantity of gene expression [21]. The

algorithm is defined by R = (Etarget)DCp target(control � sample)/

(Eref)DCp ref(control � sample), where R is the relative expression

ratio, E is the efficiency of the PCR reaction, and DCp is the

difference of the Ct (crossing point of the sample at a given

threshold). The reference gene, ActinB, was selected based

on its stable expression in all samples by microarray analysis.

Moreover, Q-PCR confirmed its appropriateness because no

significant statistical differences were noted among the sam-

ples. The first sample (with 5% FBS) served as the reference

sample in each experiment. The mean value of the Ct from

replicates was used to calculate R. Marker expression was

evaluated in the OV-90 cell line either with no serum, with 5%

FBS, or with 5% of the indicated ascites, under the same con-

ditions used to evaluate the invasion potential of the OV-90

cell line. For each marker, a Pearson correlation was calcu-

lated between the scored invasion result (1 < 100% and 2 z100% of invasion) and the scored genes expression (1 <

median and 2 z median).

Statistical Analysis

Univariate Cox proportional hazard regression, Kaplan-

Meier survival plots, and log-rank tests were performed to

determine the significance of markers’ ability to predict the

survival of EOC patients (Table 2). The expression threshold

used in the log-rank test that gave the best sensitivity–

specificity values was established based on the receiver op-

erating characteristics (ROC) curves. We used the ROC and

SURVIVAL packages from R version 2.4.0 (Vienna, Austria).

The Spearman correlation coefficient test (two-tailed)

was used to estimate the correlation between the invasion

rates and clinical data and gene expression. Statistical analy-

ses were performed with SPSS software 11.0 (SPSS Inc.,

Chicago, IL).

Results

Effect of Ascites on OV-90 Invasive Potential

To address the interactions among elements found within

the peritoneal tumor environment, we characterized the

effect of a large panel of ascites (Table 1) on the ability of

an aggressive EOC cell line (OV-90) to stimulate invasion in

an in vitro assay. Media containing 5% of ascites (acellular

fraction) from patients with ovarian cancer was added to the

lower chamber of Transwell plates containing micropore fil-

ters precoated with Matrigel and OV-90 cells were added to

the upper chamber. The potential for ascites to affect OV-90

invasion was scored in comparison to media supplemented

with 5% FBS (Figure 1A). OV-90 is capable of invasion in

Matrigel assay in presence of 5% FBS (Figure 1). A large

number of ascites led to an inhibition of OV-90 cell invasion

compared to cells in the presence of FBS. A lower, but still

important number of ascites was more stimulatory for inva-

sion than the FBS control. We assessed the correlation be-

tween the invasion rates of ascites and clinical parameters

Table 2. Patients’ Cohort with Ovarian Cancer and Accompanying Ascites,

for Survival Analyses.

Patients Death Survival (months) Histopathology

90 Yes 0 UA

513 No 37 SA

595 Yes 11 PSA

665 Yes 32 PSA

747 Yes 9 SA

866 No 9 PSA

889 Yes 51 SA

892 No 67 PSA

893 Yes 46 EA

899 No 5 SA

908 Yes 11 PSA

926 Yes 54 PSA

944 No 60 PSA

960 Yes 16 PSA

962 No 72 EA

976 Yes 0 CCA

980 No 26 PSA

993 No 13 SA

999 No 43 MA

1005 No 12 PSA

1012 Yes 3 EA

1035 No 41 CCA

1127 Yes 45 PSA

1129 Yes 16 CCA

1193 Yes 6 PSA

1330 No 13 SA

1830 No 6 PSA

1946 Yes 0 PSA

UA, undifferentiated adenocarcinoma; SA, serous adenocarcinoma; PSA, papil-

lary serous adenocarcinoma; EA: endometrioid adenocarcinoma; CCA, clear

cell adenocarcinoma; MA, mucinous adenocarcinoma.

Patients died following the progression of the disease, except for patients 976

and 1946, death was due to myocardium infarction and digestive hemorrhage.

Ascites and Ovarian Tumor Cell Interaction Puiffe et al. 823

Neoplasia . Vol. 9, No. 10, 2007

available for patients: age, grade, stage, presence of neo-

plastic cells, and chemotherapy, but no significant correlation

was observed (Table 1).

The acellular fraction of 10 independent EOC ascites

(A1317, A1318, A1322, A1322(2), A1337, A1592, A1835,

A1946, A2085, and A2090; Table 1) was selected for further

characterization based on their ability to invade. The invasive

potential was again compared to a 5% FBS control. In OV-90

cells, it should be noted that there is no difference in the

invasion rate with or without 5% FBS and thus the classifi-

cation of ascites as being either inhibitory or stimulatory is

independent of the effect of serum. Inhibitory ascites reduce

the invasion rate compared to either no serum or 5% FBS,

whereas stimulatory ascites results from a higher number of

cells able to cross a Matrigel barrier in comparison to con-

trols. Ascites A1592, A1946, A2085, and A2090 induced an

invasion rate greater to the one observed in the presence

or the absence of FBS. A1317, A1318, A1322, A1322(2),

A1337, and A1835 diminished OV-90 cell invasion potential

(Figure 1B). These effects were not due to differences in

protein concentration of each ascites because correcting for

the amount of protein did not affect the overall patterns

observed (data not shown).

Effect of Ascites on OV-90 Growth

To determine the effect of ascites on proliferation rates, 10

ascites were selected and tested for their ability to alter the

growth potential of OV-90 cells compared to a 5% FBS

control (Figure 2). OV-90 cells were incubated for 2 days in

media supplemented either with no serum, with 5% FBS, or

with 5% of the indicated ascites and cell growth was evalu-

ated by a Trypan Blue exclusion assay. The highest growth

rates were observed with exposure to FBS, as well as

A1835, A1946 or A2085, whereas the remaining ascites

samples conferred variable but lower growth rates. In partic-

ular, OV-90 exhibited lower growth rate in the presence of the

A1337 and A1322(2) ascites compared to those observed

with no serum. We noted that after 24 hours of incubation,

the time used to monitor invasion, no statistically significant

differences in the growth rate were observed (with one

exception). This suggests that the monitored invasive effects

are not a simple reflection of cell growth.

Effect of Ascites on OV-90 Spheroid Formation

We have previously demonstrated that the OV-90 cells

are able to grow as large compact spheroids [19]. Because

the relation between these three-dimensional structures and

invasive potential remains poorly defined, we determined the

effect of ascites on the formation of in vitro spheroids. For

Figure 1. Effect of ascites on the invasion of the ovarian cancer cell line OV-90. The invasive potential of OV-90 (solid bars) was determined by its ability to invade

a synthetic basement membrane after 24 hours compared to 5% FBS (%Invasion*). (A) Effects of 54 ascites on the invasive potential of the cell line. (B) Invasion

profile of OV-90 with OSE medium in the absence or presence of 5% FBS or with 5% of those ascites selected for gene expression analysis.

Figure 2. Effect of ascites on the proliferation of the OV-90 ovarian cancer

cell line. On day 0, 2 � 105 cells were incubated in media supplemented

either with no serum, with 5% FBS, or with 5% ascites and the cell growth

was evaluated with a Trypan Blue exclusion assay at 24 and 48 hours. Note

that, with one exception, there were no statistically significant differences in

growth after 24 hours in the different tested conditions. The asterisk denotes

a statistical significance (P < .05).

824 Ascites and Ovarian Tumor Cell Interaction Puiffe et al.

Neoplasia . Vol. 9, No. 10, 2007

this purpose, the OV-90 cell line was incubated either in the

absence of serum, in 5% FBS, or in 5% of ascites. The

formation of spheroids was monitored after 4 days. As shown

in Figure 3, the OV-90 cell line formed multiple very small and

nonreproducible spheroids from drop to drop in the absence

of FBS, which is consistent with previous findings [19]. In the

presence of FBS or ascites A1317 and A1592, however, OV-

90 formed large (approximately 500 mm in diameter) and

compact spheroids. This was reproducible from drop to drop

with one unique spheroid of similar size formed in each drop.

Although cell scattering around spheroids was observed, it

was not significantly different between spheroids generated

in the presence of different ascites. The ability for different

ascites to induce large and compact spheroid formation was

also confirmed with ascites A1318, A1322, A1322(2), A1337,

A1835, A1946, A2085, and A2090, which gave similar

results (Figure W1). Therefore, the ability of ascites to

stimulate spheroid formation did not correlate with the inva-

sion potential of the tested ascites.

Ascites Invasive Phenotype Characterization

Ascites that stimulated or inhibited OV-90 cell invasion

were combined to determine which effect is the most pre-

dominant (Figure 4A). We selected one stimulatory (A2090)

and two inhibitory (A1369 and A1526) ascites. When stimula-

tory and inhibitory ascites were added together (5% A2090 +

5% A1369 or 5% A2090 + 5% A1526), we observed that

the invasion rate was not significantly different compared

to the presence of inhibitory ascites alone. This result sug-

gests that the inhibitory ascites have a predominant impact

on invasion of OV-90 cells compared to a stimulatory as-

cites. This effect was not due to an increased concentration

of ascites in the assay as adjusting individually tested as-

cites to 10% had no effect on the invasive phenotype (data

not shown).

To determine if this predominant effect is protein-dependent,

inhibitory ascites were boiled at 100jC for 10 minutes before

being tested on OV-90 cells (Figure 4B). The results showed

that protein inactivation abolished the inhibitory effect of

the two selected ascites (A1369 and A1526). These com-

bined results suggest that inhibition of OV-90 cell invasion is

protein-based and that the inhibitory effect is stronger than

the stimulatory effect.

Modification of Gene Expression Induced By Ascites

in OV-90 Cells

To identify potential molecular players in invasion regu-

lated by ascites, gene profiling was performed using Affy-

metrix HG-U133A GeneChip arrays with RNA extracted from

OV-90 cells after 24 hours of exposure to either no serum, to

5% FBS, or to 5% of 1 of 10 different ascites (A1317, A1318,

A1322, A1322(2), A1337, A1592, A1835, A1946, A2085,

and A2090).

For supervised analysis purposes, two groups were cre-

ated for comparison. The group that stimulated invasion

(referred here as GSTIMUL group) included samples with

no FBS, with 5% FBS, or with 5% of the four ascites that

stimulated OV-90 cell invasion (A1592, A1946, A2085, and

A2090). An inhibitory group (referred here as GINHIB group)

Figure 4. In vitro invasion assay with the ovarian cancer cell line OV-90. (A) Effect of simultaneous exposure to ascites that stimulate or inhibit the invasion of OV-

90. Note that the inhibitory effect appears to be dominant. (B) Proteins of ascites A1369 and A1526 were inactivated by heating and their effect on the invasion of

OV-90 was evaluated. Note that ascites no longer maintain their inhibitory effect.

Figure 3. Effect of ascites on spheroid formation. Spheroids were formed using

a modification of the hanging droplet method. Cells were incubated with OSE

media supplemented either with no serum, with 5% FBS, or with 5% of the

indicated ascites. Spheroid formation was monitored after 4 days. All pictures

were taken at a magnification of �100.

Ascites and Ovarian Tumor Cell Interaction Puiffe et al. 825

Neoplasia . Vol. 9, No. 10, 2007

containing the six ascites that inhibited cell invasion (A1317,

A1318, A1322, A1322(2), A1337, and A1835) was also

defined. Expression analysis identified 243 probe sets to

be differentially expressed (P V .1) between the GSTIMUL

and GINHIB groups (Table W1).

Differential Expression Validation of Selected

Candidates By Q-PCR

As differences in expression are subtle but tended to be

statistically significant, it was important to validate the value

of the cutoff selected. For this purpose, we selected genes

with different P values (Table 3) to test the robustness of their

association with invasion. Table 3 describes the seven can-

didate genes upregulated in the GSTIMUL group [dickkopf

homolog 1 (Xenopus) (DKK1), regulator of G-protein sig-

naling 4 (RGS4), interferon-stimulated transcription factor 3,

gamma 48 kDa (ISGF3G), tribbles homolog 1 (Drosophila)

(TRIB1), MAP kinase phosphatase 1 (MKP1), cyclooxy-

genase 2 (COX2), and motile sperm domain containing 1

(MOSPD1)] and two candidate genes upregulated in the

GINHIB group [plectin 1, intermediate filament binding pro-

tein 500 kDa (PLEC1) and myristoylated alanine-rich protein

kinase C substrate (MARCKS)] that were selected for fur-

ther validation. Q-PCR was used to validate the differential

expression of selected candidates in RNA derived from

OV-90 cells exposed individually to the entire panel of

54 ascites (Table 1). The relative expression ratio (R) of

each gene, based on the Pfaffl method (see Materials and

Methods section for details), was quantified and, for each

experiment, the median ratio was calculated and the results

scored. Pearson correlations were then calculated for each

candidate correlating ascites invasion effects (stimulatory or

inhibitory) and scored gene expression, as shown in Table 4.

The scored gene expression of RGS4, ISGF3G, TRIB1,

MKP1, MOSPD1, and PLEC1 correlated significantly with

the ascites’ invasion effects, but not the expression of DKK1,

COX2, and MARCKS. These results also suggest that an

extensive validation of candidates identified in the super-

vised analysis by Q-PCR is warranted to uncover the rich-

ness of genes implicated in the invasive process.

Survival

To evaluate the prognosis potential of the selected gene

candidates RGS4, ISGF3G, TRIB1, MKP1, MOSPD1, and

PLEC1, we sought to access the expression of these genes

in samples derived from patients with ovarian ascites. There-

fore, we extracted expression values from a microarray

analysis of RNA extracted from 28 primary cultures derived

from the cellular fraction of ascites from ovarian cancer pa-

tients (Table 2). Univariate Cox regression analysis showed

a strong association only between TRIB1 gene expression

and overall survival (P = .0007). Using a threshold deter-

mined by ROC analysis, a Kaplan-Meier curve coupled to a

log-rank test identified the presence of two patient groups

and high TRIB1 expression was associated with a poorer

survival rate (log-rank P = .005) (Figure 5).

Discussion

Although ascites is a common phenotype in ovarian cancer,

the origin of malignant ascitic fluid and its relationship to the

developing tumor is still poorly understood. The observation

that ascites is often associated with the most invasive

malignant tumors indirectly supports the notion that ascites

is involved in the progression of ovarian cancer [22] presum-

ably by favoring the dissemination of malignant cells within

the peritoneal cavity. Contributing to the development of

ascites are soluble factors produced by tumor cells that are

known to increase vascular permeability and induce angio-

genesis [5,23]. Although ascitic soluble factors such as

chemokines, angiogenic factors, and growth factors have

been implicated in ovarian cancer cell invasion [24–26], the

combined effect of these factors in the progression of ovarian

cancer has not been addressed. In this study, we assessed

the effects of ascites on the invasion, proliferation, spheroid

formation, and gene expression of the cell line OV-90 [16].

The overall aim was to determine how ascites alters its

microenvironment and thus the biological characteristics of

ovarian cancer cells.

Using a Matrigel-based invasion assay, 54 individual

ascites showed varying effects on OV-90 cell invasion with

some ascites being either poorly stimulatory or inhibitory

compared to serum. This data is in contrast to other ascites

that strongly stimulated the invasive capacity of this cell line.

The varying invasive effects were not associated with total

protein concentration in ascites, because adjusting for this

factor did not affect the overall effect of invasion (data not

shown). The inhibitory effect of ascites was lost when

samples were heated, suggesting that the effect was due

to protein inactivation rather than other soluble factors.

Moreover, no correlation between the effect of ascites on

invasion and chemotherapy, received prior to ascites collec-

tions, was noted.

Correlation of the invasion assay results with two other

parameters, cellular proliferation and spheroid formation,

was assessed because stimulation of cell growth could lead

to bias in the number of cells counted in the upper chamber

of the Transwell and because formation of compact spher-

oids may interfere with the ability to pass through the

polycarbonate membrane pores. The initial characterization

studies focused on 10 ascites possessing either stimulatory

or inhibitory properties. Although cell growth and spheroid

formation were influenced to some extent by ascites, neither

parameter was strongly associated with OV-90 invasive

capacity. In general, higher doubling times or saturation

densities with ascites in comparison to serum were not

observed. These findings are similar to results obtained by

independent analyses of the PEO.36, OVHS, and SKOV-3

ovarian cancer cell lines [15,27], suggesting that although

ascites might affect the in vitro biological characteristics

(e.g., survival) of tumor cells, it does not necessarily contrib-

ute to the proliferation of tumor cells.

To investigate the molecular events associated with the

invasive effect of ascites, global RNA gene expression from

OV-90 cells in the presence or absence of serum or ascites

was characterized. As expected, differential gene expression

826 Ascites and Ovarian Tumor Cell Interaction Puiffe et al.

Neoplasia . Vol. 9, No. 10, 2007

Tab

le3.

Sele

cte

dG

enes

Diff

ere

ntia

llyE

xpre

ssed

betw

een

the

GS

TIM

UL

Gro

up

and

the

GIN

HIB

Gro

up.

PR

egula

tion

inO

V-9

0C

ells

with

Ascite

s

That

Inhib

ited

the

Invasio

n

Pro

be

Set

HG

-U133a

UniG

ene

Descriptio

nS

ym

bol

Cyto

band

GO

Bio

logic

al

Pro

cess

Descriptio

nG

OM

ole

cula

rF

unctio

nD

escriptio

n

.011

Dow

n204602_at

Hs.4

0499

Dic

kkopf

hom

olo

g1

(Xenopuslaevis

)DKK1

10q11

.2D

evelo

pm

ent

Sig

nal

transducer

activ

ity

Wnt

recepto

rsig

nalin

gpath

way

Pro

tein

bin

din

g

Gro

wth

facto

ractiv

ity

Low

-density

lipopro

tein

recepto

rbin

din

g

.027/.

082*

Dow

n204337_at

Hs.3

86726

Regula

tor

of

G-p

rote

insig

nalin

g4

RGS4

1q23.3

Inactiv

atio

nof

MA

PK

activ

ityS

ignal

transducer

activ

ity

204339_s_at

G-p

rote

in–

couple

dre

cepto

rpro

tein

sig

nalin

gpath

way

GT

Pase

activ

ato

ractiv

ity

Calm

odulin

bin

din

g

.044

Dow

n203882_at

Hs.1

706

Inte

rfero

n-s

timula

ted

transcriptio

n

facto

r3,

gam

ma

48

kD

a

ISGF3G

14q11

.2T

ranscriptio

nT

ranscriptio

nfa

cto

ractiv

ity

Imm

une

response

Pro

tein

bin

din

g

Cell

surf

ace

recepto

r–lin

ked

sig

nal

transductio

n

Meta

lio

nbin

din

g

Response

tovirus

.052

Dow

n202241_at

Hs.4

44947

Tribble

shom

olo

g1

(Drosophila

)TRIB1

8q24.1

3P

rote

inam

ino

acid

phosphory

latio

nP

rote

inserine/t

hre

onin

ekin

ase

activ

ity

Cell

pro

lifera

tion

Regula

tion

of

MA

PK

activ

ityA

TP

bin

din

g

.057

Dow

n201041_s_at

Hs.1

71695

MA

Pkin

ase

phosphata

se

1MKP1

5q34

Pro

tein

am

ino

acid

Non

–m

em

bra

ne-s

pannin

gpro

tein

Dephosphory

latio

nT

yro

sin

ephosphata

se

activ

ity

Response

tooxid

ativ

estr

ess

Pro

tein

bin

din

g

Cell

cycle

Hydro

lase

activ

ity

MA

Pkin

ase

phosphata

se

activ

ity

Pro

tein

tyro

sin

e/s

erine/t

hre

onin

e

phosphata

se

activ

ity

.066

Dow

n204748_at

Hs.1

96384

Cyclo

oxygenase

2COX2

1q25.2

Pro

sta

gla

ndin

bio

synth

esis

Pero

xid

ase

activ

ity

q25.3

Cell

motil

ityP

rosta

gla

ndin

–endopero

xid

esynth

ase

activ

ity

Physio

logic

alpro

cess

Meta

lio

nbin

din

g

Blo

od

pre

ssure

regula

tion

Oxid

ore

ducta

se

activ

ity

Cyclo

oxygenase

path

way

Kera

tinocyte

diff

ere

ntia

tion

Anagen

Lip

idbio

synth

esis

Infla

mm

ato

ryre

sponse

.082

Dow

n218853_s_at

Hs.5

90789

Motil

esperm

dom

ain

conta

inin

g1

MOSPD1

Xq26.3

Str

uctu

ral

mole

cule

activ

ity

.011

Up

216971_s_at

Hs.4

34248

Ple

ctin

1,

inte

rmedia

tefil

am

ent

bin

din

gpro

tein

500

kD

a

PLEC1

8q24

Cyto

skele

talanchoring

Actin

bin

din

g

Str

uctu

ral

mole

cule

activ

ity

.014

Up

213002_at

Hs.5

19909

Myristo

yla

ted

ala

nin

e-r

ich

pro

tein

kin

ase

Csubstr

ate

MARCKS

6q22.2

Cell

motil

ityC

alm

odulin

bin

din

g

Actin

bin

din

g

The

GS

TIM

UL

gro

up

consis

tsofO

V-9

0sam

ple

sw

ithno

FB

S,w

ith5%

FB

S,or

with

5%

ofth

efo

ur

ascite

sth

atstim

ula

ted

cell

invasio

n(A

1592,A

1946,A

2085,and

A2090).

The

GIN

HIB

gro

up

consis

tsofO

V-9

0sam

ple

sw

ith5%

of

the

six

ascite

sth

at

inhib

ited

cell

invasio

n(A

1317,

A1318,

A1322,

A1322(2

),A

1337,

and

A1835).

*Corr

espondin

gto

two

diff

ere

nt

pro

beset

on

the

Affym

etr

ixH

G-U

133A

GeneC

hip

arr

ay.

Ascites and Ovarian Tumor Cell Interaction Puiffe et al. 827

Neoplasia . Vol. 9, No. 10, 2007

patterns occurred in treated cells from which we determined a

subset of differentially expressed genes that correlated with

invasion potential. Interestingly, most of the genes within this

subset have not been previously linked to ovarian cancer in-

vasion. Among these genes, DUSP1/MKP1, TRIB1, ISGF3g/

IRF-9, RGS4, MOSPD1, and PLEC1 overexpression was

validated by Q-PCR in a large set of samples confirming the

potential role of these genes in the cellular invasion process.

The gene signature of ascites-stimulated OV-90 cells

reinforces the role of the mitogen-activated protein kinase

(MAPK) pathway in the invasion process. Extracellular sig-

nal–regulated protein kinase (ERK) MAPKs have already

been shown to be involved in invasion through the activa-

tion of metalloproteinase [28–31] and Snail [32] promoters.

Here we observed altered expression of two genes impli-

cated in kinase regulation, namelyTRIB1andMKP1.TRIB1 is a

serine/threonine kinase protein interacting with the mitogen-

activated protein kinase 4. The effect of this interaction on

MAPK pathway activity is unknown but TRIB1 overexpres-

sion inhibits Ras- and mitogen-activated protein kinase

kinase/extracellular signal–regulated protein kinase kinase

1–mediated activator protein–1 activation whereas ERK

activation is enhanced. On the other hand, MKP1, the MAP

kinase phosphatase 1, is the target of the ERKs. This phos-

phorylation does not modify the intrinsic ability of MKP1 to

dephosphorylate p44MAPK, but leads to the stabilization of

the protein. Although these results suggest that ascites may

regulate the invasion potential of OV-90 cells through TRIB1

overexpression that activates ERKs, the microarray data

do not show Snail and metalloproteinase overexpression in

OV-90 cells stimulated with invasive ascites. This does not

rule out that other downstream targets of the ERK pathway

could be affected. Indeed, RGS4, a member of the regulator

of G protein signaling family, has been involved in the in-

hibition of MAPK and protein kinase B (PKB/AKT) activation

in neuroblastoma cells as well as ERKs activation induced

by angiotensin and endothelin but not by serum stimula-

tion [33,34]. Moreover, RGS4 has also been associated

with invasion and motility of glioma cells [35]. Our results

are consistent with this latter observation because a direct

correlation between RGS4 gene expression and invasion

was determined.

The gene signature of ascites-stimulated OV-90 also re-

vealed an altered regulation of the ISGF3g/IRF9 and

MOSPD1 genes. ISGF3 is an interferon-dependent tran-

scription factor involved in resistance to chemotherapeu-

tic agents [36,37] but not in any known cellular invasion

process. MOSPD1 belongs to the transmembrane MSP-

containing protein family. Its role in mammals is largely un-

known but it is thought to be involved in the formation of

protein–protein networks [38]. These networks have not yet

been associated with cellular invasion so far but our results

suggest a new role for these proteins in cancer progression.

One explanation for the lack of association of the majority

of the genes identified in this study with the invasive potential

of neoplastic cells is perhaps not surprising because few

studies have attempted to determine the direct effect of as-

cites on ovarian cancer cells. In addition, meta-analyses of

gene profiling studies between normal and cancerous cells

are difficult to compare with our study due to various factors

such as differences in model systems, use of different plat-

forms, and a dearth of clinical data accompanying the stud-

ies including whether tumor formation was accompanied

with ascites formation. Consequently, over the long term,

we favor a functional approach, taking advantage of the

model systems we have developed coupled with in vitro

and in vivo assays, to determine the precise role of promising

candidate genes in the process of invasion.

In summary, this study revealed novel candidate genes

that may play an important role in ovarian cancer cell inva-

sion and potentially affect clinical outcomes. This study has

also begun to define the importance and subtleties of as-

cites in modulating the tumor microenvironment and sug-

gests that both positive and negative regulators of tumor

behavior may be present in ascites. Further functional as-

says are required to determine their exact role in this bio-

logical process. Continued evaluation of ascites on EOC

behavior is warranted and a comprehensive systems bi-

ology approach is required to fully understand the complex

Table 4. Correlation between Invasion and Genes Expression.

Genes Pearson Correlation P

DKK1 0.096 .468

RGS4 0.302* .019

ISGF3G 0.377y .003

TRIB1 0.302* .019

MKP1 0.397y .002

COX2 0.206 .114

MOSPD1 0.357y .005

MARCKS 0.226 .082

PLEC1 0.322* .019

Pearson correlations were calculated between scored invasion results (1 <

100% of invasion and 2 z 100% of invasion) and scored genes expression

(1 < median and 2 z median) for the six candidates quantified by Q-PCR.

*Correlation is significant at the level 0.05.yCorrelation is significant at the level 0.01.

Figure 5. Relation between TRIB1 expression and cumulative survival of pa-

tients with ovarian cancer in the context of concomitant ascites. The threshold

was determined by ROC analysis. Kaplan-Meier graphical representation of

survival curve illustrates the poor survival associated with a high expression of

TRIB1 (P < .005).

828 Ascites and Ovarian Tumor Cell Interaction Puiffe et al.

Neoplasia . Vol. 9, No. 10, 2007

interactions within the peritoneum that influence EOC pro-

gression and metastasis.

Acknowledgements

We are grateful to Louise Champoux, Lise Portelance, Manon

de Ladurantaye, Jason Madore, and Marise Roy for technical

assistance. We thank Pierre Drouin, Philippe Sauthier, and

Philippe Gauthier for their assistance in tissue procurement.

We are grateful to Luke Masson for reviewing the manuscript

and Kate Morris for editing.

References[1] Auersperg N, Wong AS, Choi KC, Kang SK, and Leung PC (2001).

Ovarian surface epithelium: biology, endocrinology, and pathology.

Endocr Rev 22, 255 –288.

[2] Auersperg N, Ota T, and Mitchell GW (2002). Early events in ovarian

epithelial carcinogenesis: progress and problems in experimental ap-

proaches. Int J Gynecol Cancer 12, 691 – 703.

[3] Cvetkovic D (2003). Early events in ovarian oncogenesis. Reprod Biol

Endocrinol 1, 68.

[4] Burleson KM, Casey RC, Skubitz KM, Pambuccian SE, Oegema TR Jr,

and Skubitz AP (2004). Ovarian carcinoma ascites spheroids adhere

to extracellular matrix components and mesothelial cell monolayers.

Gynecol Oncol 93, 170 – 181.

[5] Zebrowski BK, Liu W, Ramirez K, Akagi Y, Mills GB, and Ellis LM

(1999). Markedly elevated levels of vascular endothelial growth factor

in malignant ascites. Ann Surg Oncol 6, 373 – 378.

[6] Miyamoto S, Hirata M, Yamazaki A, Kageyama T, Hasuwa H, Mizushima

H, Tanaka Y, Yagi H, Sonoda K, Kai M, et al. (2004). Heparin-binding

EGF-like growth factor is a promising target for ovarian cancer therapy.

Cancer Res 64, 5720–5727.

[7] Mills GB, Eder A, Fang X, Hasegawa Y, Mao M, Lu Y, Tanyi J, Tabassam

FH, Wiener J, Lapushin R, et al. (2002). Critical role of lysophospholipids

in the pathophysiology, diagnosis, and management of ovarian cancer.

Cancer Treat Res 107, 259–283.

[8] Saltzman AK, Hartenbach EM, Carter JR, Contreras DN, Twiggs LB,

Carson LF, and Ramakrishnan S (1999). Transforming growth factor –

alpha levels in the serum and ascites of patients with advanced epithe-

lial ovarian cancer. Gynecol Obstet Invest 47, 200 –204.

[9] Abendstein B, Stadlmann S, Knabbe C, Buck M, Muller-Holzner E,

Zeimet AG, Marth C, Obrist P, Krugmann J, and Offner FA (2000).

Regulation of transforming growth factor – beta secretion by human

peritoneal mesothelial and ovarian carcinoma cells. Cytokine 12,

1115 –1119.

[10] Jandu N, Richardson M, Singh G, Hirte H, and Hatton MW (2006).

Human ovarian cancer ascites fluid contains a mixture of incompletely

degraded soluble products of fibrin that collectively possess an anti-

angiogenic property. Int J Gynecol Cancer 16, 1536 –1544.

[11] Said NA, Najwer I, Socha MJ, Fulton DJ, Mok SC, and Motamed K

(2007). SPARC inhibits LPA-mediated mesothelial –ovarian cancer cell

crosstalk. Neoplasia 9, 23 – 35.

[12] Fung-Kee-Fung M, Provencher D, Rosen B, Hoskins P, Rambout L,

Oliver T, Gotlieb W, and Covens A (2007). Intraperitoneal chemother-

apy for patients with advanced ovarian cancer: a review of the evidence

and standards for the delivery of care. Gynecol Oncol 105, 747 – 756.

[13] Petignat P, du Bois A, Bruchim I, Fink D, and Provencher DM (2007).

Should intraperitoneal chemotherapy be considered as standard first-

line treatment in advanced stage ovarian cancer? Crit Rev Oncol

Hematol 62, 137 –147.

[14] Armstrong DK, Bundy B, Wenzel L, Huang HQ, Baergen R, Lele S,

Copeland LJ, Walker JL, and Burger RA (2006). Intraperitoneal cisplatin

and paclitaxel in ovarian cancer. N Engl J Med 354, 34– 43.

[15] Ahmed N, Riley C, Oliva K, Rice G, and Quinn M (2005). Ascites in-

duces modulation of alpha6beta1 integrin and urokinase plasminogen

activator receptor expression and associated functions in ovarian car-

cinoma. Br J Cancer 92, 1475 –1485.

[16] Provencher DM, Lounis H, Champoux L, Tetrault M, Manderson EN,

Wang JC, Eydoux P, Savoie R, Tonin PN, and Mes-Masson AM (2000).

Characterization of four novel epithelial ovarian cancer cell lines. In Vitro

Cell Dev Biol Anim 36, 357 – 361.

[17] Tonin PN, Hudson TJ, Rodier F, Bossolasco M, Lee PD, Novak J,

Manderson EN, Provencher D, and Mes-Masson AM (2001). Micro-

array analysis of gene expression mirrors the biology of an ovarian

cancer model. Oncogene 20, 6617 –6626.

[18] Kruk PA, Maines-Bandiera SL, and Auersperg N (1990). A simplified

method to culture human ovarian surface epithelium. Lab Invest 63,

132 – 136.

[19] Zietarska M, Maugard CM, Filali-Mouhim A, Alam-Fahmy M, Tonin PN,

Provencher DM, and Mes-Masson AM (2007). Molecular description

of a 3D in vitro model for the study of epithelial ovarian cancer

(EOC). Mol Carcinog 48, 872 – 885.

[20] Dudoit S, Gentleman RC, and Quackenbush J (2003). Open source soft-

ware for the analysis of microarray data. Biotechniques Suppl, 45–51.

[21] Pfaffl MW (2001). A new mathematical model for relative quantification

in real-time RT-PCR. Nucleic Acids Res 29, e45.

[22] Shen-Gunther J and Mannel RS (2002). Ascites as a predictor of ovar-

ian malignancy. Gynecol Oncol 87, 77– 83.

[23] Richardson M, Gunawan J, Hatton MW, Seidlitz E, Hirte HW, and

Singh G (2002). Malignant ascites fluid (MAF), including ovarian-

cancer-associated MAF, contains angiostatin and other factor(s) which

inhibit angiogenesis. Gynecol Oncol 86, 279 – 287.

[24] Westermann AM, Beijnen JH, Moolenaar WH, and Rodenhuis S (1997).

Growth factors in human ovarian cancer. Cancer Treat Rev 23, 113–131.

[25] Brown MR, Blanchette JO, and Kohn EC (2000). Angiogenesis in ovarian

cancer. Baillieres Best Pract Res Clin Obstet Gynaecol 14, 901–918.

[26] Schutyser E, Struyf S, Proost P, Opdenakker G, Laureys G, Verhasselt

B, Peperstraete L, Van de Putte I, Saccani A, Allavena P, et al. (2002).

Identification of biologically active chemokine isoforms from ascitic fluid

and elevated levels of CCL18/pulmonary and activation-regulated che-

mokine in ovarian carcinoma. J Biol Chem 277, 24584 – 24593.

[27] Yabushita H, Shimazu M, Noguchi M, Kishida T, Narumiya H, and

Sawaguchi K (2003). Vascular endothelial growth factor activating ma-

trix metalloproteinase in ascitic fluid during peritoneal dissemination of

ovarian cancer. Oncol Rep 10, 89– 95.

[28] Montesano R, Soriano JV, Hosseini G, Pepper MS, and Schramek H

(1999). Constitutively active mitogen-activated protein kinase kinase

MEK1 disrupts morphogenesis and induces an invasive phenotype

in Madin-Darby canine kidney epithelial cells. Cell Growth Differ 10,

317 – 332.

[29] Lakka SS, Jasti SL, Kyritsis AP, Yung WK, Ali-Osman F, Nicolson GL,

and Rao JS (2000). Regulation of MMP-9 (type IV collagenase) produc-

tion and invasiveness in gliomas by the extracellular signal – regulated

kinase and jun amino-terminal kinase signaling cascades. Clin Exp

Metastasis 18, 245 –252.

[30] Tan X, Egami H, Abe M, Nozawa F, Hirota M, and Ogawa M (2005).

Involvement of MMP-7 in invasion of pancreatic cancer cells through

activation of the EGFR mediated MEK –ERK signal transduction path-

way. J Clin Pathol 58, 1242 – 1248.

[31] Ahmed N, Oliva K, Wang Y, Quinn M, and Rice G (2003). Downregu-

lation of urokinase plasminogen activator receptor expression inhibits

Erk signalling with concomitant suppression of invasiveness due to loss

of uPAR –beta1 integrin complex in colon cancer cells. Br J Cancer 89,

374 – 384.

[32] Barbera MJ, Puig I, Dominguez D, Julien-Grille S, Guaita-Esteruelas S,

Peiro S, Baulida J, Franci C, Dedhar S, Larue L, et al. (2004). Regula-

tion of Snail transcription during epithelial to mesenchymal transition of

tumor cells. Oncogene 23, 7345 –7354.

[33] Leone AM, Errico M, Lin SL, and Cowen DS (2000). Activation of ex-

tracellular signal – regulated kinase (ERK) and Akt by human serotonin

5-HT(1B) receptors in transfected BE(2)-C neuroblastoma cells is in-

hibited by RGS4. J Neurochem 75, 934 – 938.

[34] Albig AR and Schiemann WP (2005). Identification and characteri-

zation of regulator of G protein signaling 4 (RGS4) as a novel inhibitor

of tubulogenesis: RGS4 inhibits mitogen-activated protein kinases and

vascular endothelial growth factor signaling. Mol Biol Cell 16, 609 – 625.

[35] Tatenhorst L, Senner V, Puttmann S, and Paulus W (2004). Regulators

of G-protein signaling 3 and 4 (RGS3, RGS4) are associated with glioma

cell motility. J Neuropathol Exp Neurol 63, 210 – 222.

[36] Luker KE, Pica CM, Schreiber RD, and Piwnica-Worms D (2001). Over-

expression of IRF9 confers resistance to antimicrotubule agents in

breast cancer cells. Cancer Res 61, 6540 – 6547.

[37] Schmidt M, Schler G, Gruensfelder P, and Hoppe F (2006). Differential

gene expression in a paclitaxel-resistant clone of a head and neck

cancer cell line. Eur Arch Otorhinolaryngol 263, 127 –134.

[38] Laurent F, Labesse G, and de Wit P (2000). Molecular cloning and

partial characterization of a plant VAP33 homologue with a major sperm

protein domain. Biochem Biophys Res Commun 270, 286 –292.

Ascites and Ovarian Tumor Cell Interaction Puiffe et al. 829

Neoplasia . Vol. 9, No. 10, 2007

Figure W1. Effect of ascites on spheroid formation. Spheroids were formed using a modification of the hanging droplet method. Cells were incubated with OSE

media supplemented either with no serum, with 5% FBS, or with 5% ascites. Spheroid formation was monitored after 4 days.

Tab

leW

1.

Genes

Diff

ere

ntia

llyE

xpre

ssed

betw

een

the

GS

TIM

UL

Gro

up

and

the

GIN

HIB

Gro

up.

P

Regula

tion

inO

V-9

0C

ells

with

Ascite

sT

hat

Inhib

ited

the

Invasio

nP

robe

Set

HG

-U133a

UniG

ene

Descriptio

nS

ym

bol

Cyto

band

.009069738

Dow

n205505_at

Hs.5

21568

Glu

cosam

inyl

(N-a

cety

l)tr

ansfe

rase

1,

core

2

(beta

-1,6

-N-a

cety

lglu

cosam

inyltr

ansfe

rase)

GCNT1

9q13

.010986586

Do

wn

204602_at

Hs.4

0499

Dic

kko

pf

ho

mo

log

1(X

en

op

us

laevis

)DKK1

10q

11.2

.011

494572

Dow

n208960_s_at

Hs.4

055

Kru

ppel-lik

efa

cto

r6

KLF6

10p15

.012167277

Dow

n201565_s_at

Hs.1

80919

Inhib

itor

of

DN

Abin

din

g2,

dom

inant

negativ

ehelix

-loop-h

elix

pro

tein

ID2

2p25

.012194066

Dow

n202378_s_at

Hs.2

3581

Leptin

recepto

roverlappin

gtr

anscript

LEPROT

1p31.3

.013785587

Dow

n210297_s_at

Hs.2

55462

Mic

rosem

inopro

tein

beta

MSMB

10q11

.2

.014105088

Dow

n213982_s_at

Hs.5

85378

RA

BG

TP

ase-a

ctiv

atin

gpro

tein

1–

like

RABGAP1L

1q24

Hs.5

91475

Hs.6

15081

.015017693

Dow

n209568_s_at

Hs.4

97148

Ralguanin

enucle

otid

edis

socia

tion

stim

ula

tor–

like

1RGL1

1q25.3

.015955247

Dow

n201844_s_at

Hs.6

42635

RIN

G1

and

YY

1bin

din

gpro

tein

RYBP

3p13

.019102681

Dow

n203053_at

Hs.2

2960

Bre

ast

carc

inom

a–

am

plif

ied

sequence

2BCAS2

1p21

–p13.3

.019591227

Dow

n213668_s_at

Hs.3

57901

SR

Y(s

ex-d

ete

rmin

ing

regio

nY

)-box

4SOX4

6p22.3

.019677541

Dow

n202068_s_at

Hs.2

13289

Low

-density

lipopro

tein

recepto

r(f

am

ilialhyperc

hole

ste

role

mia

)LDLR

19p13.3

.020450451

Dow

n212993_at

.020537623

Dow

n203910_at

Hs.4

83238

Rho

GT

Pase-a

ctiv

atin

gpro

tein

29

ARHGAP29

1p22.1

.021916807

Dow

n205849_s_at

Hs.1

31255

Ubiq

uin

ol–

cyto

chro

mec

reducta

se

bin

din

gpro

tein

UQCRB

8q22

.022344932

Dow

n204224_s_at

Hs.8

6724

GT

Pcyclo

hydro

lase

1(D

OP

A-r

esponsiv

edysto

nia

)GCH1

14q22.1

–q22.2

.02250785

Dow

n218859_s_at

Hs.3

69284

Chro

mosom

e20

open

readin

gfr

am

e6

C20orf6

20p12.1

.023487203

Dow

n219230_at

Hs.1

73233

Tra

nsm

em

bra

ne

pro

tein

100

TMEM100

17q22

.024086813

Dow

n221046_s_at

Hs.1

27496

GT

P-b

indin

gpro

tein

8(p

uta

tive)

GTPBP8

3q13.2

.024674823

Dow

n212155_at

Hs.3

56377

Rin

gfin

ger

pro

tein

187

RNF187

1q42.1

3

.02542067

Dow

n203893_at

Hs.2

48941

TA

F9

RN

Apoly

mera

se

II,

TA

TA

box

bin

din

gpro

tein

(TB

P)–

associa

ted

facto

r,32

kD

a

TAF9

5q11

.2–

q13.1

.025748062

Dow

n21911

9_at

Hs.5

92275

LS

M8

hom

olo

g,

U6

sm

all

nucle

ar

RN

A-a

ssocia

ted

(Saccharomyces

cerevisiae

)

LSM8

7q31.1

–q31.3

.026383595

Dow

n202049_s_at

Hs.2

69211

Zin

cfin

ger,

MY

M-t

ype

4ZMYM4

1p32

–p34

.026879501

Dow

n204717_s_at

Hs.5

69017

Solu

tecarr

ier

fam

ily29

(nucle

osid

etr

ansport

ers

),m

em

ber

2SLC29A2

11q13

.027130811

Do

wn

204337_at

Hs.3

86726

Reg

ula

tor

of

G-p

rote

insig

nali

ng

4RGS4

1q

23.3

.02816821

Dow

n221989_at

Hs.5

34404

Rib

osom

al

pro

tein

L10

RPL10

Xq28

.02911

9959

Dow

n211

475_s_at

Hs.3

77484

BC

L2-a

ssocia

ted

ath

anogene

BAG1

9p12

.031204448

Dow

n202371_at

Hs.1

94329

Tra

nscriptio

nelo

ngatio

nfa

cto

rA

(SII

)–lik

e4

TCEAL4

Xq22.2

.033222043

Dow

n209066_x_at

Hs.1

31255

Ubiq

uin

ol–

cyto

chro

mec

reducta

se

bin

din

gpro

tein

UQCRB

8q22

.033426388

Dow

n203261_at

Hs.1

58427

Dynactin

6DCTN6

8p12

–p11

.03413633

Dow

n202536_at

Hs.4

76930

Chro

matin

-modifyin

gpro

tein

2B

CHMP2B

3p11

.2

.03460712

Dow

n218546_at

Hs.5

19839

Chro

mosom

e1

open

readin

gfr

am

e11

5C1orf115

1q41

.036049912

Dow

n209194_at

Hs.8

2794

Centr

in,

EF

-hand

pro

tein

,2

CETN2

Xq28

.036137594

Dow

n218263_s_at

Hs.1

83684

Zin

cfin

ger,

BE

D-t

ype

conta

inin

g5

ZBED5

11p15.3

.036963092

Dow

n218718_at

Hs.5

70855

Pla

tele

t-derived

gro

wth

facto

rC

PDGFC

4q32

.037577573

Dow

n213346_at

Hs.3

9811

1C

hro

mosom

e13

open

readin

gfr

am

e27

C13orf27

13q33.1

.03797463

Dow

n217971_at

Hs.5

17438

Mito

gen-a

ctiv

ate

dpro

tein

kin

ase

kin

ase

1in

tera

ctin

gpro

tein

1MAP2K1IP1

4q23

Hs.6

42730

.03885642

Dow

n203008_x_at

Hs.5

36122

Thio

redoxin

dom

ain

conta

inin

g9

TXNDC9

2q11

.2

.039788327

Dow

n209577_at

Hs.5

85089

Phosphate

cytid

yly

ltransfe

rase

2,

eth

anola

min

ePCYT2

17q25.3

Hs.6

40301

.04068575

Dow

n212990_at

Hs.4

73632

Synapto

janin

1SYNJ1

21q22.2

.040856296

Dow

n206340_at

Hs.2

82735

Nucle

ar

recepto

rsubfa

mily

1,

gro

up

H,

mem

ber

4NR1H4

12q23.1

.043263551

Dow

n203095_at

Hs.1

49894

Mito

chondrial

transla

tional

initi

atio

nfa

cto

r2

MTIF2

2p14

–p16

.043675477

Dow

n213365_at

.043675478

Dow

n213853_at

Hs.1

87269

Zin

cfin

ger,

CS

L-t

ype

conta

inin

g3

ZCSL3

11p13

.044556918

Dow

n204833_at

Hs.2

64482

AT

G12

auto

phagy

–re

late

d12

hom

olo

g(S

.cerevisiae

)ATG12

5q21

–q22

.044832106

Do

wn

203882_at

Hs.1

706

Inte

rfero

n-s

tim

ula

ted

tran

scri

pti

on

facto

r3,

gam

ma

48

kD

aISGF3G

14q

11.2

.045284015

Dow

n201362_at

Hs.4

97183

Influ

enza

virus

NS

1A

bin

din

gpro

tein

IVNS1ABP

1q25.1

–q31.1

.045488235

Dow

n204905_s_at

Hs.6

31818

Eukary

otic

transla

tion

elo

ngatio

nfa

cto

r1

epsilo

n1

EEF1E1

6p24.3

–p25.1

.048564587

Dow

n217523_at

Hs.5

02328

CD

44

mole

cule

(India

nblo

od

gro

up)

CD44

11p13

.049825918

Dow

n221838_at

Hs.5

17419

Kelc

h-lik

e22

(Drosophila

)KLHL22

22q11

.21

.051012172

Dow

n210130_s_at

Hs.3

1130

Tra

nsm

em

bra

ne

7superf

am

ilym

em

ber

2TM7SF2

11q13

.051573086

Dow

n205070_at

Hs.4

89811

Inhib

itor

of

gro

wth

fam

ily,

mem

ber

3ING3

7q31

.052089067

Dow

n220046_s_at

Hs.4

859

Cyclin

L1

CCNL1

3q25.3

2

.052837334

Do

wn

202241_at

Hs.4

44947

Tri

bb

les

ho

mo

log

1(D

rosophila

)TRIB1

8q

24.1

3

.053014196

Dow

n203219_s_at

Hs.2

8914

Adenin

ephosphoribosyltr

ansfe

rase

APRT

16q24

.053104357

Dow

n205476_at

Hs.7

5498

Chem

okin

e(C

–C

motif

)lig

and

20

CCL20

2q33

–q37

.055079649

Dow

n212077_at

Hs.4

90203

Cald

esm

on

1CALD1

7q33

.055257276

Dow

n221803_s_at

Hs.4

49628

Nucle

ar

recepto

rbin

din

gfa

cto

r2

NRBF2

10q21.2

.056358813

Dow

n201719_s_at

Hs.4

86470

Ery

thro

cyte

mem

bra

ne

pro

tein

band

4.1

–lik

e2

EPB41L2

6q23

.056740375

Dow

n203469_s_at

Hs.1

09

Cyclin

-dependent

kin

ase

(CD

C2-lik

e)

10

CDK10

16q24

.057018399

Dow

n204093_at

Hs.2

92524

Cyclin

HCCNH

5q13.3

–q14

.057376594

Dow

n218640_s_at

Hs.2

9724

Ple

ckstr

inhom

olo

gy

dom

ain

conta

inin

g,

fam

ilyF

(with

FY

VE

dom

ain

)

mem

ber

2

PLEKHF2

8q22.1

.057726739

Do

wn

201041_s_at

Hs.1

71695

MA

Pkin

ase

ph

osp

hata

se

1MKP1

5q

34

.05848611

1D

ow

n212852_s_at

Hs.2

88178

TR

OV

Edom

ain

fam

ily,

mem

ber

2TROVE2

1q31

.058922152

Dow

n204518_s_at

Hs.1

10364

Peptid

ylp

roly

lis

om

era

se

C(c

yclo

phili

nC

)PPIC

5q23.2

.060798058

Dow

n209090_s_at

Hs.1

36309

SH

3-d

om

ain

GR

B2-lik

eendophili

nB

1SH3GLB1

1p22

.060979735

Dow

n218669_at

Hs.1

19889

RA

P2C

,m

em

ber

of

RA

Soncogene

fam

ilyRAP2C

Xq25

.0611

51359

Dow

n203351_s_at

Hs.5

58364

Origin

recogniti

on

com

ple

x,

subunit

4–

like

(yeast)

ORC4L

2q22

–q23

.062158872

Dow

n218618_s_at

Hs.1

59430

Fib

ronectin

type

III

dom

ain

conta

inin

g3B

FNDC3B

3q26.3

1

.063122027

Dow

n216262_s_at

Hs.6

32264

TG

Fb-

induced

facto

r2

(TA

LE

fam

ilyhom

eobox)

TGIF2

20q11

.2–

q12

.063521509

Dow

n208714_at

Hs.7

744

NA

DH

dehydro

genase

(ubiq

uin

one)

flavopro

tein

1,

51

kD

aNDUFV1

11q13

.064380244

Dow

n202829_s_at

Hs.2

4167

Synapto

bre

vin

-lik

e1

SYBL1

Xq28

and

Yq12

.064471815

Dow

n204064_at

Hs.5

92342

TH

Ocom

ple

x1

THOC1

18p11

.32

.064815795

Dow

n218932_at

Hs.5

111

Chro

mosom

e1

open

readin

gfr

am

e181

C1orf181

1p22.3

.065594194

Dow

n209892_at

Hs.3

90420

Fucosyltr

ansfe

rase

4(a

lpha

(1.3

)fu

cosyltr

ansfe

rase,

myelo

id-s

pecifi

c)

FUT4

11q21

Hs.5

72064

Hs.6

23098

.066601688

Dow

n221580_s_at

Hs.3

55750

Josephin

dom

ain

conta

inin

g3

JOSD3

11q21

.066737657

Dow

n215136_s_at

Hs.2

94041

Exosom

ecom

ponent

8EXOSC8

13q13.1

.066741405

Do

wn

204748_at

Hs.1

96384

Cyclo

oxyg

en

ase

2COX2

1q

25.2

–q

25.3

.066935084

Dow

n204731_at

Hs.4

82390

Tra

nsfo

rmin

ggro

wth

facto

r,beta

recepto

rII

I(b

eta

gly

can,

300

kD

a)

TGF�R3

1p33

–p32

.070237276

Dow

n212451_at

Hs.9

997

KIAA0256

15q21.1

.070395379

Dow

n2211

93_s_at

Hs.2

9700

Zin

cfin

ger,

CC

HC

dom

ain

conta

inin

g10

ZCCHC10

5q31.1

.070491097

Dow

n205555_s_at

Hs.8

9404

msh

hom

eobox

hom

olo

g2

(Drosophila

)MSX2

5q34

–q35

.071242737

Dow

n204605_at

Hs.5

9106

Cell

gro

wth

regula

tor

with

ring

finger

dom

ain

1CGRRF1

14q22.2

.071332992

Dow

n202486_at

Hs.5

28996

AF

G3

AT

Pase

fam

ilygene

3–

like

2(y

east)

AFG3L2

18p11

.071381219

Dow

n211

297_s_at

Hs.1

84298

Cyclin

-dependent

kin

ase

7(M

O15

hom

olo

g;Xenopuslaevis

)CDK7

5q12.1

.072368959

Dow

n202268_s_at

Hs.4

60978

Am

ylo

idbeta

pre

curs

or

pro

tein

bin

din

gpro

tein

1APPBP1

16q22

.072874958

Dow

n212036_s_at

Hs.4

09965

Pin

in,

desm

osom

e-a

ssocia

ted

pro

tein

PNN

14q21.1

.073704052

Dow

n218889_at

Hs.7

4899

Nucle

ola

rcom

ple

x–

associa

ted

3hom

olo

g(S.cerevisiae)

NOC3L

10q23.3

3

.07411

839

Dow

n209180_at

Hs.7

8948

Rab

gera

nylg

era

nyltr

ansfe

rase,

beta

subunit

RABGGTB

1p31

.074852081

Dow

n201993_x_at

Hs.5

27105

Hete

rogeneous

nucle

ar

ribonucle

opro

tein

D–

like

HNRPDL

4q13

–q21

.07580573

Dow

n208788_at

Hs.5

20189

ELO

VL

fam

ilym

em

ber

5,

elo

ngatio

nof

long

chain

fatt

yacid

s(F

EN

1/E

lo2,

SU

R4/E

lo3

–lik

e,

yeast)

ELO

VL5

6p21.1

–p12.1

.076258333

Dow

n210609_s_at

Hs.5

0649

Tum

or

pro

tein

p53

–in

ducib

lepro

tein

3TP53I3

2p23.3

Tab

leW

1.

(contin

ued

)

P

Regula

tion

inO

V-9

0C

ells

with

Ascite

sT

hat

Inhib

ited

the

Invasio

nP

robe

Set

HG

-U133a

UniG

ene

Descriptio

nS

ym

bol

Cyto

band

.076478131

Dow

n202133_at

Hs.4

77921

WW

dom

ain

conta

inin

gtr

anscriptio

nre

gula

tor

1WWTR1

3q23

–q24

.076884129

Dow

n204108_at

Hs.1

0441

Nucle

ar

transcriptio

nfa

cto

rY,

alp

ha

NFYA

6p21.3

.077281863

Dow

n219603_s_at

Hs.1

45956

Zin

cfin

ger

pro

tein

226

ZNF226

19q13.2

.077559522

Dow

n2011

97_at

Hs.1

5911

8A

denosylm

eth

ionin

edecarb

oxyla

se

1AMD1

6q21

–q22

.077668773

Dow

n210260_s_at

Hs.2

71955

Tum

or

necro

sis

facto

ralp

ha

–in

duced

pro

tein

8TNFAIP8

5q23.1

.077919339

Dow

n214429_at

Hs.5

07536

Myotu

bula

rin-r

ela

ted

pro

tein

6MTMR6

13q12

.077956649

Dow

n218395_at

Hs.1

15088

AR

P6

actin

-rela

ted

pro

tein

6hom

olo

g(y

east)

ACTR6

12q23.1

.078491847

Dow

n212591_at

Hs.5

35224

RN

Abin

din

gm

otif

pro

tein

34

RBM34

1q42.3

.0787004

Dow

n203341_at

Hs.1

35406

CC

AA

T/e

nhancer

bin

din

gpro

tein

zeta

CEBPZ

2p22.2

.078787012

Dow

n222257_s_at

Hs.1

78098

Angio

tensin

I–convert

ing

enzym

e(p

eptid

yl-dip

eptid

ase

A)

2ACE2

Xp22

.079315679

Dow

n32069_at

Hs.5

11839

N4BP1

16q12.1

.079593909

Dow

n201418_s_at

Hs.3

57901

SR

Y(s

ex-d

ete

rmin

ing

regio

nY

)-box

4SOX4

6p22.3

.079820109

Dow

n203038_at

Hs.1

55919

Pro

tein

tyro

sin

ephosphata

se,

recepto

rty

pe,

KPTPRK

6q22.2

–23.1

.079933064

Dow

n2011

96_s_at

Hs.1

5911

8A

denosylm

eth

ionin

edecarb

oxyla

se

1AMD1

6q21

–q22

.079943784

Dow

n212944_at

Hs.3

02742

Solu

tecarr

ier

fam

ily5

(inosito

ltr

ansport

ers

),m

em

ber

3SLC5A3

21q22.1

2

.080193374

Dow

n202125_s_at

Hs.1

52774

Tra

ffic

kin

gpro

tein

,kin

esin

bin

din

g2

TRAK2

2q33

.081057755

Dow

n203883_s_at

Hs.1

73656

RA

B11

fam

ilyin

tera

ctin

gpro

tein

2(c

lass

I)RAB11FIP2

10q26.1

1

.081093659

Dow

n203138_at

Hs.6

32532

His

tone

acety

ltransfe

rase

1HAT1

2q31.2

–q33.1

.081709641

Dow

n209096_at

Hs.4

91695

Ubiq

uiti

n-c

onju

gating

enzym

eE

2variant

2UBE2V2

8q11

.21

.081976932

Do

wn

218853_s_at

Hs.5

90789

Mo

tile

sp

erm

do

main

co

nta

inin

g1

MOSPD1

Xq

26.3

.082278907

Dow

n204354_at

Hs.3

1968

PO

T1

pro

tectio

nof

telo

mere

s1

hom

olo

g(Schizosaccharomycespombe)

POT1

7q31.3

3

.082506541

Dow

n202919_at

Hs.1

197

Pre

impla

nta

tion

pro

tein

3PREI3

2q33.1

.082610888

Dow

n210592_s_at

Hs.2

8491

Sperm

idin

e/s

pe

rmin

eN

1-a

cety

ltransfe

rase

SAT

Xp22.1

.082927418

Dow

n203120_at

Hs.5

23968

Tum

or

pro

tein

p53

bin

din

gpro

tein

,2

TP53BP2

1q42.1

.082938504

Dow

n210896_s_at

Hs.3

32422

Aspart

ate

beta

-hydro

xyla

se

ASPH

8q12.1

.083201635

Do

wn

204339_s_at

Hs.3

86726

Reg

ula

tor

of

G-p

rote

insig

nali

ng

4RGS4

1q

23.3

.083611

292

Dow

n202010_s_at

Hs.2

70869

Zin

cfin

ger

pro

tein

410

ZNF410

14q24.3

.084208435

Dow

n217945_at

Hs.4

59149

BT

B(P

OZ

)dom

ain

conta

inin

g1

BTBD1

15q24

.084303999

Dow

n209814_at

Hs.1

20766

Zin

cfin

ger

pro

tein

330

ZNF330

4q31.1

–q31.2

.084378582

Dow

n202294_at

Hs.4

12586

Str

om

al

antig

en

1STAG1

3q22.3

.084439365

Dow

n217997_at

Hs.4

84885

Ple

ckstr

inhom

olo

gy

–lik

edom

ain

,fa

mily

A,

mem

ber

1PHLDA1

12q15

Hs.6

17254

.085120438

Dow

n201220_x_at

Hs.5

01345

C-t

erm

inal

bin

din

gpro

tein

2CTBP2

10q26.1

3

.085808072

Dow

n209286_at

Hs.3

69574

CD

C42

effecto

rpro

tein

(Rho

GT

Pase

bin

din

g)

3CDC42EP3

2p21

.08645717

Dow

n221884_at

Hs.5

54762

Ecotr

opic

viralin

tegra

tion

site

1EVI1

3q24

–q28

.086514089

Dow

n202511

_s_at

Hs.4

86063

AT

G5

auto

phagy

–re

late

d5

hom

olo

g(S

.cerevisiae

)ATG5

6q21

.087498297

Dow

n213164_at

Hs.3

02742

Mito

chondrial

ribosom

al

pro

tein

S6

MRPS6

21q21.3

–q22.1

.088913121

Dow

n202324_s_at

Hs.5

20207

Acyl-coenzym

eA

bin

din

gdom

ain

conta

inin

g3

ACBD3

1q42.1

2

.089365717

Dow

n216028_at

Hs.5

96944

DKFZP564C152

.09000975

Dow

n211

758_x_at

Hs.5

36122

Thio

redoxin

dom

ain

conta

inin

g9

TXNDC9

2q11

.2

.091009742

Dow

n203607_at

Hs.3

69755

Inosito

lpoly

phosphate

-5-p

hosphata

se

FINPP5F

10q26.1

1

.091289556

Dow

n208772_at

Hs.4

34219

Ankyrin

repeat

and

KH

dom

ain

conta

inin

g1

ANKHD1

5q31.3

.09141519

Dow

n211

975_at

Hs.4

36204

Zin

cfin

ger

pro

tein

289,

ID1

regula

ted

ZNF289

11p11

.2–

p11

.12

.091466436

Dow

n219962_at

Hs.1

78098

Angio

tensin

I–convert

ing

enzym

e(p

eptid

yl-dip

eptid

ase

A)

2ACE2

Xp22

.091539936

Dow

n208093_s_at

Hs.1

6355

nudE

nucle

ar

dis

trib

utio

ngene

Ehom

olo

glik

e1

(Aspergillusnidulans)

NDEL1

17p13.1

Hs.3

72123

.092699795

Dow

n210907_s_at

Hs.4

78150

Pro

gra

mm

ed

cell

death

10

PDCD10

3q26.1

.093669107

Dow

n221970_s_at

Hs.4

63936

Nucle

ola

rpro

tein

11NOL11

17q24.2

.094101077

Dow

n212589_at

Hs.5

02004

Rela

ted

RA

Sviral(r

-ras)

oncogene

hom

olo

g2

RRAS2

11p15.2

.094697368

Dow

n219974_x_at

Hs.4

86410

Enoylcoenzym

eA

hydra

tase

dom

ain

conta

inin

g1

ECHDC1

6q22.3

3

.094809596

Dow

n222138_s_at

Hs.5

21973

WD

repeat

dom

ain

13

WDR13

Xp11

.23

.095015393

Dow

n200961_at

Hs.1

18725

Sele

nophosphate

synth

eta

se

2SEPHS2

16p11

.2

.095206438

Dow

n201427_s_at

Hs.2

75775

Sele

nopro

tein

P,

pla

sm

a,

1SEPP1

5q31

Hs.6

43494

.095716917

Dow

n221786_at

Hs.4

35933

Chro

mosom

e6

open

readin

gfr

am

e120

C6orf120

6q27

Hs.5

91375

.096224309

Dow

n221547_at

Hs.1

611

81

PR

P18

pre

-mR

NA

pro

cessin

gfa

cto

r18

hom

olo

g(S.cerevisiae

)PRPF18

10p13

.096395443

Dow

n208961_s_at

Hs.4

055

Kru

ppel-lik

efa

cto

r6

KLF6

10p15

.096791953

Dow

n205354_at

Hs.8

1131

Guanid

inoaceta

teN

-meth

yltr

ansfe

rase

GAMT

19p13.3

.097014688

Dow

n218839_at

Hs.2

34434

Hairy/e

nhancer-

of-

split

rela

ted

with

YR

PW

motif

1HEY1

8q21

.09727472

Dow

n219137_s_at

Hs.4

71528

Chro

mosom

e2

open

readin

gfr

am

e33

C2orf33

2q36.3

.098024766

Dow

n211

506_s_at

Hs.6

24

Inte

rleukin

8IL8

4q13

–q21

Hs.4

43948

Hs.4

49585

Hs.5

51925

Hs.5

61078

.099035679

Dow

n201791_s_at

Hs.5

03134

7-D

ehydro

chole

ste

rolre

ducta

se

DHCR7

11q13.2

–q13.5

.099690261

Dow

n204881_s_at

Hs.3

04249

UD

P–

glu

cose

cera

mid

eglu

cosyltr

ansfe

rase

UGCG

9q31

Hs.5

93014

.099764699

Dow

n202542_s_at

Hs.5

91680

Sm

all

inducib

lecyto

kin

esubfa

mily

E,

mem

ber

1(e

ndoth

elia

l

monocyte

-activ

atin

g)

SCYE1

4q24

.099913337

Dow

n210559_s_at

Hs.3

34562

Cell

div

isio

ncycle

2,

G1

toS

and

G2

toM

CDC2

10q21.1

.099922386

Dow

n220547_s_at

Hs.5

00419

Fam

ilyw

ithsequence

sim

ilarity

35,

mem

ber

AFAM35A

10q23.2

.0011

26617

Up

206561_s_at

Hs.1

16724

Ald

o–

keto

reducta

se

fam

ily1,

mem

ber

B10

(ald

ose

reducta

se)

AKR1B10

7q33

.00261037

Up

201224_s_at

Hs.1

8192

Serine/a

rgin

ine

repetit

ive

matr

ix1

SRRM1

1p36.1

1

.003250785

Up

219161_s_at

Hs.1

5159

Chem

okin

e-lik

efa

cto

rCKLF

16q21

.004857387

Up

209160_at

Hs.7

8183

Ald

o–

keto

reducta

se

fam

ily1,

mem

ber

C3

(3-a

lpha

hydro

xyste

roid

dehydro

genase,

type

II)

AKR1C3

10p15

–p14

.007192628

Up

202595_s_at

Hs.1

46585

Leptin

recepto

roverlappin

gtr

anscript–

like

1LEPROTL1

8p21.2

–p21.1

.007559525

Up

218755_at

Hs.7

3625

Kin

esin

fam

ilym

em

ber

20A

KIF20A

5q31

.008187471

Up

214711

_at

Hs.3

69624

15E1.2

12q24.3

1

.010930189

Up

208699_x_at

Hs.8

9643

Tra

nsketo

lase

(Wern

icke-K

ors

akoff

syndro

me)

TKT

3p14.3

.011257353

Up

216971_s_at

Hs.4

34248

Ple

cti

n1,

inte

rmed

iate

fila

men

tb

ind

ing

pro

tein

500

kD

aPLEC1

8q

24

.011

376131

Up

202589_at

Hs.5

92338

Thym

idyla

tesynth

eta

se

TYMS

18p11

.32

.014883308

Up

213002_at

Hs.5

19909

Myri

sto

yla

ted

ala

nin

e-r

ich

pro

tein

kin

ase

Csu

bstr

ate

MARCKS

6q

22.2

.015913145

Up

212804_s_at

Hs.4

95134

GT

Pase-a

ctiv

ating

pro

tein

and

VP

S9

dom

ain

s1

GAPVD1

9q33.3

.018202268

Up

201328_at

Hs.5

17296

v-e

tsery

thro

bla

sto

sis

virus

E26

oncogene

hom

olo

g2

(avia

n)

ETS2

21q22.3

.018385256

Up

214101_s_at

Hs.4

43837

Am

inopeptid

ase

puro

mycin

-sensiti

ve

NPEPPS

17q21

.022757889

Up

204363_at

Hs.6

2192

Coagula

tion

facto

rII

I(t

hro

mbopla

stin

,tis

sue

facto

r)F3

1p22

–p21

.023172458

Up

201508_at

Hs.4

62998

Insulin

-lik

egro

wth

facto

rbin

din

gpro

tein

4IG

FBP4

17q12

–q21.1

.023408518

Up

201540_at

Hs.4

35369

Four

and

ahalf

LIM

dom

ain

s1

FHL1

Xq26

.023845409

Up

396_f_

at

Hs.3

75142

Ery

thro

poie

tinre

cepto

rEPOR

19p13.3

–p13.2

Hs.6

31624

.024106098

Up

212009_s_at

Hs.3

37295

Str

ess-induced

phosphopro

tein

1(H

sp70/H

sp90-o

rganiz

ing

pro

tein

)STIP1

11q13

.025879993

Up

217294_s_at

Hs.5

17145

Enola

se

1,

(alp

ha)

ENO1

1p36.3

–p36.2

.030350275

Up

209120_at

Hs.3

47991

Nucle

ar

recepto

rsubfa

mily

2,

gro

up

F,

mem

ber

2NR2F2

15q26

.030563305

Up

211

159_s_at

Hs.5

33308

Pro

tein

phosphata

se

2,

regula

tory

subunit

B(B

56),

delta

isofo

rmPPP2R5D

6p21.1

.031970709

Up

203925_at

Hs.3

15562

Glu

tam

ate

–cyste

ine

ligase,

modifi

er

subunit

GCLM

1p22.1

.032952351

Up

201946_s_at

Hs.1

89772

Chapero

nin

conta

inin

gT

CP

1,

subunit

2(b

eta

)CCT2

12q15

.033839191

Up

216348_at

Hs.4

33427

Rib

osom

al

pro

tein

S17

RPS17

15q

Hs.5

911

17

.034984241

Up

201715_s_at

Hs.1

24490

Apopto

ticchro

matin

condensatio

nin

ducer

1ACIN1

14q11

.2

.036651891

Up

217445_s_at

Hs.4

73648

Phosphoribosylg

lycin

am

ide

form

yltr

ansfe

rase

GART

21q22.1

Tab

leW

1.

(contin

ued

)

P

Regula

tion

inO

V-9

0C

ells

with

Ascite

sT

hat

Inhib

ited

the

Invasio

nP

robe

Set

HG

-U133a

UniG

ene

Descriptio

nS

ym

bol

Cyto

band

Phosphoribosylg

lycin

am

ide

synth

eta

se

Phosphoribosyla

min

oim

idazole

synth

eta

se

.03907979

Up

218516_s_at

Hs.5

91872.

Inosito

lm

onophosphata

se

dom

ain

conta

inin

g1

IMPAD1

8q12.1

Hs.5

95255

.040109269

Up

218726_at

Hs.5

32968

DKFZp762E1312

2q37.1

.042122483

Up

211

657_at

Hs.4

66814

Carc

inoem

bry

onic

antig

en

–re

late

dcell

adhesio

nm

ole

cule

6(n

onspecifi

c

cro

ss-r

eactin

gantig

en)

CEACAM6

19q13.2

.044612439

Up

210567_s_at

Hs.2

3348

S-p

hase

kin

ase-a

ssocia

ted

pro

tein

2(p

45)

SKP2

5p13

.045719671

Up

205729_at

Hs.1

20658

Oncosta

tinM

recepto

rOSMR

5p13.1

.048046212

Up

216603_at

Hs.6

32348

Solu

tecarr

ier

fam

ily7

(catio

nic

am

ino

acid

transport

er,

y+

syste

m),

mem

ber

8

SLC7A8

14q11

.2

.048541567

Up

221520_s_at

Hs.5

24571

Cell

div

isio

ncycle

–associa

ted

8CDCA8

1p34.3

.050464722

Up

201555_at

Hs.1

79565

MC

M3

min

ichro

mosom

em

ain

tenance-d

efic

ient

3(S.cerevisiae

)MCM3

6p12

.050727362

Up

206055_s_at

Hs.5

28763

Sm

all

nucle

ar

ribonucle

opro

tein

poly

peptid

eAV

SNRPA1

15q26.3

.051366041

Up

204962_s_at

Hs.1

594

Centr

om

ere

pro

tein

ACENPA

2p24

–p21

.056793421

Up

209339_at

Hs.4

77959

Seven

inabsentia

hom

olo

g2

(Drosophila

)SIAH2

3q25

.056873703

Up

209142_s_at

Hs.4

62035

Ubiq

uiti

n-c

onju

gating

enzym

eE

2G

1(U

BC

7hom

olo

g,

yeast)

UBE2G1

1q42

.058777804

Up

215073_s_at

Hs.3

47991

Nucle

ar

recepto

rsubfa

mily

2,

gro

up

F,

mem

ber

2NR2F2

15q26

.060517092

Up

201978_s_at

Hs.2

10532

KIA

A0141

KIAA0141

5q31.3

.060749639

Up

213642_at

Hs.1

99739

Rib

osom

al

pro

tein

L27

RPL27

17q21.1

–q21.2

Hs.5

14196

.061262381

Up

204900_x_at

Hs.5

91715

Sin

3A

-associa

ted

pro

tein

,30

kD

aSAP30

4q34.1

.061541582

Up

213093_at

Hs.5

31704

Pro

tein

kin

ase

C,

alp

ha

PRKCA

17q22

–q23.2

Hs.6

37776

.062594025

Up

212135_s_at

Hs.3

43522

AT

Pase,

Ca

2+

transport

ing,

pla

sm

am

em

bra

ne

4ATP2B4

1q32.1

.062847997

Up

205008_s_at

Hs.1

29867

Calc

ium

and

inte

grin

bin

din

gfa

mily

mem

ber

2CIB2

15q24

.064320779

Up

220633_s_at

Hs.1

42442

Hete

rochro

matin

pro

tein

1,

bin

din

gpro

tein

3HP1BP3

1p36.1

2

.065638186

Up

203581_at

Hs.2

96169

RA

B4A

,m

em

ber

RA

Soncogene

fam

ilyRAB4A

1q42

–q43

.066286963

Up

218753_at

Hs.5

5024

XK

,K

ell

blo

od

gro

up

com

ple

xsubunit

–re

late

dfa

mily

,m

em

ber

8XKR8

1p35.3

.0666011

08

Up

201912_s_at

Hs.5

28780

G1

toS

phase

transiti

on

1GSPT1

16p13.1

.067445733

Up

209101_at

Hs.5

91346

Connectiv

etis

sue

gro

wth

facto

rCTGF

6q23.1

.067510121

Up

217144_at

Hs.3

56190

Ubiq

uiti

nB

UBB

17p12

–p11

.2

.06804711

3U

p208781_x_at

Hs.1

2102

Sort

ing

nexin

3SNX3

6q21

.068892541

Up

205673_s_at

Hs.1

9404

Ankyrin

repeat

and

SO

CS

box

–conta

inin

g9

ASB9

.068897609

Up

200653_s_at

Hs.2

82410

Calm

odulin

1(p

hosphory

lase

kin

ase,

delta

)CALM1

14q24

–q31

.068938006

Up

211

410_x_at

Hs.6

43606

Kill

er

cell

imm

unoglo

bulin

–lik

ere

cepto

r,tw

odom

ain

s,

long

cyto

pla

sm

ic

tail,

5A

KIR2DL5A

19p13.3

.06902663

Up

203744_at

Hs.1

911

4H

igh-m

obili

tygro

up

box

3HMGB3

Xq28

.070332508

Up

213803_at

Hs.5

32793

Kary

opherin

(im

port

in)

beta

1KPNB1

17q21.3

2

.072861566

Up

201088_at

Hs.5

94238

Kary

opherin

alp

ha

2(R

AG

cohort

1,

import

inalp

ha

1)

KPNA2

17q23.1

–q23.3

.073174705

Up

219336_s_at

Hs.5

00007

Activ

atin

gsig

nalcoin

tegra

tor

1com

ple

xsubunit

1ASCC1

10pte

r–q25.3

.073421992

Up

204174_at

Hs.5

07658

Ara

chid

onate

5-lip

oxygenase

–activ

atin

gpro

tein

ALOX5AP

13q12

.074525606

Up

2011

82_s_at

Hs.1

62233

Chro

modom

ain

helic

ase

DN

Abin

din

gpro

tein

4CHD4

12p13

.075211

814

Up

200709_at

Hs.4

71933

FK

506

bin

din

gpro

tein

1A

,12

kD

aFKBP1A

20p13

.075504884

Up

210052_s_at

Hs.2

44580

TP

X2

mic

rotu

bule

-associa

ted

hom

olo

g(Xenopuslaevis

)TPX2

20q11

.2

.07569381

Up

20811

3_x_at

Hs.4

58280

Poly

(A)

bin

din

gpro

tein

,cyto

pla

sm

ic3

PABPC3

13q12

–q13

.077198877

Up

212016_s_at

Hs.1

72550

Poly

pyrim

idin

etr

act

bin

din

gpro

tein

1PTBP1

19p13.3

.079810692

Up

212074_at

Hs.4

38072

Unc-8

4hom

olo

gA

(Caenorhabditiselegans)

UNC84A

7p22.3

.0828011

03

Up

210754_s_at

Hs.4

91767

v-y

es-1

Yam

aguchi

sarc

om

aviral–

rela

ted

oncogene

hom

olo

gLYN

8q13

Hs.6

14775

.08285677

Up

206155_at

Hs.3

68243

AT

P-b

indin

gcassett

e,

subfa

mily

C(C

FT

R/M

RP

),m

em

ber

2ABCC2

10q24

.083154934

Up

213671_s_at

Hs.6

32707

Meth

ionin

e-t

RN

Asynth

eta

se

MARS

12q13.2

.084684756

Up

212064_x_at

Hs.2

3650

MY

C-a

ssocia

ted

zin

cfin

ger

pro

tein

(purine-b

indin

gtr

anscriptio

nfa

cto

r)MAZ

16p11

.2

.085140368

Up

202974_at

Hs.4

96984

Mem

bra

ne

pro

tein

,palm

itoyla

ted

1,

55

kD

aMPP1

Xq28

.08611

8323

Up

222231_s_at

Hs.3

70927

Leucin

e-r

ich

repeat

conta

inin

g59

LRRC59

17q21.3

3

.08648983

Up

208624_s_at

Hs.4

33750

Eukary

otic

transla

tion

initi

atio

nfa

cto

r4

gam

ma,

1EIF4G1

3q27

–qte

r

.088237636

Up

205905_s_at

Hs.7

7961

MH

Ccla

ss

Ipoly

peptid

e–

rela

ted

sequence

AMICA

6p21.3

Hs.5

49053

.089037251

Up

211

653_x_at

Hs.4

60260

Ald

o–

keto

reducta

se

fam

ily1,

mem

ber

C2

(dih

ydro

dio

ldehydro

genase

2;

bile

acid

bin

din

gpro

tein

;3-a

lpha

hydro

xyste

roid

dehydro

genase,

type

III)

AKR1C2

10p15

–p14

Hs.5

67256

.089916902

Up

212379_at

Hs.4

73648

Phosphoribosylg

lycin

am

ide

form

yltr

ansfe

rase

GART

21q22.1

Phosphoribosylg

lycin

am

ide

synth

eta

se

Phosphoribosyla

min

oim

idazole

synth

eta

se

.090303455

Up

220363_s_at

Hs.2

10469

Engulfm

ent

and

cell

motil

ity2

ELMO2

20q13

.091459911

Up

211

984_at

Hs.2

82410

Calm

odulin

1(p

hosphory

lase

kin

ase,

delta

)CALM1

14q24

–q31

.091528808

Up

202032_s_at

Hs.1

16459

Mannosid

ase,

alp

ha,

cla

ss

2A

,m

em

ber

2MAN2A2

15q26.1

.093413146

Up

206665_s_at

Hs.5

16966

BC

L2-lik

e1

BCL2L1

20q11

.21

.093798599

Up

204030_s_at

Hs.1

34665

Schw

annom

inin

tera

ctin

gpro

tein

1SCHIP1

3q25.3

3

.094856181

Up

205687_at

Hs.3

459

UBPH

16p12

.096611

542

Up

202275_at

Hs.4

61047

Glu

cose-6

-phosphate

dehydro

genase

G6PD

Xq28

.097379811

Up

217356_s_at

Hs.7

8771

Phosphogly

cera

tekin

ase

1PGK1

Xq13

.0974342

Up

202122_s_at

Hs.1

40452

Mannose-6

-phosphate

recepto

rbin

din

gpro

tein

1M6PRBP1

19p13.3

.098406916

Up

40093_at

Hs.1

10675

Basalcell

adhesio

nm

ole

cule

(Luth

era

nblo

od

gro

up)

BCAM

19q13.2

The

GS

TIM

UL

gro

up

consis

tsofO

V-9

0sam

ple

sw

ithno

FB

S,w

ith5%

FB

S,or

with

5%

ofth

efo

ur

ascite

sth

atstim

ula

ted

cell

invasio

n(A

1592,A

1946,A

2085,and

A2090).

The

GIN

HIB

gro

up

consis

tsofO

V-9

0sam

ple

sw

ith5%

of

the

six

ascite

sth

at

inhib

ited

cell

invasio

n(A

1317,

A1318,

A1322,

A1322(2

),A

1337,

and

A1835).

Genes

furt

her

chara

cte

rized

inth

isstu

dy

are

inb

old

.