Gynecologic Oncology 132 (2014) 322327

Contents lists available at ScienceDirect

Gynecologic Oncology
journal homepage: www.elsevier.com/locate/ygyno

Exposure of fallopian tube epithelium to follicular uid mimics

carcinogenic changes in precursor lesions of serous papillary carcinoma
K. Bahar-Shany a,1, H. Brand a,b,1, S. Sapoznik a, J. Jacob-Hirsch a, Y. Yung c, J. Korach d, T. Perri d, Y. Cohen e,
A. Hourvitz b,c,1, K. Levanon a,f,,1
a

Sheba Cancer Research Center, Chaim Sheba Medical Center, Ramat Gan 52621, Israel
Sackler Faculty of Medicine, Tel-Aviv University, Ramat Aviv 69978, Israel
IVF Unit and Reproduction Lab, Department of Obstetrics and Gynecology, Chaim Sheba Medical Center, Ramat-Gan 52621, Israel
d
Department of Gynecologic Oncology, Chaim Sheba Medical Center, Ramat Gan 52621, Israel
e
Institutional Tumor Banks, Chaim Sheba Medical Center, Ramat Gan 52621, Israel
f
The Dr. Pinchas Borenstein Talpiot Medical Leadership Program 2012, Institute of Oncology, Chaim Sheba Medical Center, Ramat Gan 52621, Israel
b
c

H I G H L I G H T S
An ex-vivo model of the inuence of follicular uid on fallopian tube epithelium
FT epithelial cells present pre-neoplastic changes upon exposure to follicular uid.

a r t i c l e

i n f o

Article history:
Received 18 September 2013
Accepted 6 December 2013
Available online 17 December 2013
Keywords:
Ovarian cancer
Fallopian tube
Ovulation
Carcinogenesis
Biomarkers
Follicular uid

a b s t r a c t
Objectives. Ovulation-related inammation is suspected to have a causal role in ovarian carcinogenesis, but
there are no human models to study the molecular pathways. Our aim is to develop such an ex-vivo model
based on human fallopian tube (FT) epithelium exposed to human follicular uid (FF).
Methods. FT epithelium was dissociated from normal surgical specimens. FF was obtained from donors undergoing in-vitro fertilization. The cells were cultured on collagen-coated Transwells and incubated with FF for various periods of time. The transcriptomic changes resulting from FF treatment were proled using Affymetrix
expression arrays. Specic characteristics of the FT pre-cancerous lesions were studied using immunohistochemistry, immunouorescence, RT-PCR and XTT assay.
Results. We show that FF exposure causes up-regulation of inammatory and DNA repair pathways. Double
stranded DNA breaks are induced. There is a minor increase in cell proliferation. TP53, which is the hallmark of
the precursor lesion in-vivo, is accumulated. Levels of expression and secretion of Interleukin-8 are signicantly
increased.
Conclusions. Our model addresses the main non-genetic risk factor for ovarian cancer, namely the impact of
ovulation. This study demonstrates the biological implications of in-vitro exposure of human FT epithelial cells
to FF. The model replicates elements characterizing the precursor lesions of ovarian cancer, and warrants further
investigation of the linkage between repeated exposure to ovulation-related damage and accumulation of neoplastic changes.
2013 Elsevier Inc. All rights reserved.

Introduction
Ovarian epithelial, fallopian tube (FT) and primary peritoneal cancers jointly represent the fth most common cause of death from cancer
among women in Western countries and the most lethal gynecological
malignancy, with a mean 5-year survival rate of 44% [1]. Due to the lack
Corresponding author at: Sheba Cancer Research Center, Sheba Medical Center, Ramat
Gan 52621, Israel. Fax: +972 3 5305942.
E-mail address: Keren.Levanon@sheba.health.gov.il (K. Levanon).
1
These authors contributed equally to this work.
0090-8258/$ see front matter 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.ygyno.2013.12.015

of sensitive and specic screening methods, 75% of all cases are diagnosed as advanced-stage disease [2]. Factors associated with reproductive history are known risk factors, specically nulliparity [3,4]. Fathalla
and others have presented the incessant ovulation theory claiming that
frequent and uninterrupted ovulation increases the risk for ovarian cancer through repetitive minor trauma to the covering epithelium [5,6]. It
has been postulated that the increased cellular proliferation of the ovarian surface epithelium after ovulation may form mitotic gures, crypts,
papillae or cysts, consequently leading to neoplastic changes. Over time,
epidemiologic data supporting this hypothesis has accumulated:
Established ovulation-related risk factors include infertility, nulliparity,

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323

late menopause and early menarche. Protective factors are, in general,

factors that reduce ovulation cycles throughout a woman's life:
multiparity, use of oral contraceptive, and lactation. Hysterectomy and
tubal ligation also have protective inuence, with no seemingly direct
effect on the ovulatory activity [79].
Only recently the cell-of-origin of most high-grade serous ovarian
carcinomas and the precursor lesions has been identied as the
fallopian tube secretory epithelial cell (FTSEC) [10,11], and not the ovarian surface epithelial cell, as previously thought. This discovery is a
major breakthrough in this eld, which has focused the attention on
the molecular and genetic changes occurring in the mbria of the
fallopian tube (FT) early in the course of the disease. The incessant ovulation hypothesis now requires adaptation to the emerging knowledge
regarding the true origin of this malignancy. Taking into account the FT
mbria position in close proximity to the ovary, it is reasonable to assume that carcinogenic factors released during ovulation affect the
FTSEC of the mbria. These factors can reach the FT mbria either via
the circulation or through direct exposure to the follicular uid (FF)
and follicular cells. The repeated exposure of the epithelial cells to
such active biological effectors may result in DNA damage and inammatory changes [12,13].
Human FF is a complex body uid that constitutes the microenvironment of the developing follicles in the ovary. FF is a product of both the
transport of the circulating plasma proteins and the secretory activity of
ovarian cells [14]. FF contains factors that modulate oocyte maturation
and ovulation including, among others, steroid hormones, growth factors, cytokines and interleukins, reactive oxygen species (ROS),
prostanoids and proteolytic enzymes [15,16]. Therefore, ovulation is
considered to be comparable to an inammatory reaction, the relation
of which to cancer is well-established [9,17,18].
Studying the possible molecular pathways that mediate the link
between ovulation-related inammation and carcinogenesis requires
a model of the FT epithelium. Levanon et al. reported a reproducible
ex-vivo culture system of primary benign FT cells that is capable of recapitulating the histology and function of the normal human FT mbria
epithelium [19]. This model is amenable to experimental manipulations
including introduction of typical genetic alterations [20] and exposure
to carcinogenic factors in-vitro. Currently, interest in the biology of the
FF as a stimulating reagent comes from the eld of assisted reproduction
and fertility only and focuses on options to improve oocyte quality. This
study is aimed to interrogate, model, and delineate the fundamental
ovulation-induced changes and their potential contribution to the
carcinogenic process. Upon understanding of these common, basic
events, chemo-preventive strategies can be devised. Furthermore, it
provides directions for investigation of early-detection approaches and
biomarkers.

(Biological Industries, Beit Ha'emek, Israel). Cells were cultured on

24-well plates covered with collagen IV from human placenta
(Sigma-Aldrich, St. Louis, MO, USA) and incubated at 37 C in a humidied 5% CO2 incubator.
FF was obtained from women undergoing oocyte retrieval who provided written informed consent. Ten FF samples were used in this study
as a pool. Four of the women were undergoing in-vitro fertilization
treatments due to mechanical or hormonal infertility (endometriosis
and polycystic ovary syndrome) and the other six for various other reasons (pre-natal genetic diagnosis, male factor or unexplained reason).
The mean age of the FF donors was 36.5 (range 2641). FF was centrifuged to remove blood cells and frozen in aliquots. Before use it was
heated to 56 C for 30 min. Serum-containing culture medium was
used as control for FF, hence an equivalent concentration of 1% Ultroser
G was added to the FF.

Materials and methods

Immunohistochemistry

Samples preparation

After exposure to FF, cells were trypsinized, xed in ethanol/

formaldehyde and the cell pellet was parafn-embedded. 4 m sections were cut and immunostained with anti-TP53 mouse monoclonal
antibody (clone DO1, EMD-Millipore, Billerica, MA, USA at 1:70 dilution
for 1 h at RT). To quantify the differences between FF-treated and control cells, we counted a total of N200 cells per experiment, from 3 different experiments performed on FT cells from 3 donors.

Fresh benign FT mbriae were obtained from the Chaim Sheba

Medical Center Institutional tissue banks upon approval of the institutional review board. Eighteen fresh mbriae were extracted from patients with gynecological conditions other than ovarian cancer: eight
with endometrial carcinoma, ve with benign ovarian cysts, two with
appendiceal neoplasm, and three with uterine leiomyomata. Mean age
was 61.2 years (range 4478 years). The mbria tissues were incubated
in dissociation medium (DMEM, Biological Industries, Beit Ha'emek,
Israel) supplemented with 1.4 mg/ml Pronase (Roche Applied Science,
Indianapolis, IN, USA) and 0.1 mg/ml DNase (Sigma-Aldrich, St. Louis,
MO, USA) for 48 h at 4 C with constant mild agitation. The dissociated
epithelial cells were harvested by centrifugation and re-suspended in
DMEM/Ham's F12 1:1 (Biological Industries, Beit Ha'emek, Israel)
supplemented with 2% serum substitute Ultroser G (PALL Life Sciences,
Cergy-Saint-Christophe, France) and 1% penicillin/streptomycin

Microarray analysis
For microarray experiments we used: two commercially-available
ovarian cancer cell lines (SKOV3, OVCA432); one normal human breast
epithelial cell line (hMEC); 2 lines of immortalized FT epithelial cell
(designated FT190 and FT194, primary cells infected with hTERT- and
SV40 T-antigen-expressing retroviruses, kindly provided by the Drapkin
lab, Dana-Farber Cancer Institute, Boston, MA, USA); and primary FT epithelial cells from a single donor. The cells were incubated with FF for
three time periods: 4 h, 24 h, and 48 h. Total RNA was extracted using
QIAzol reagent (Qiagen, Valencia, CA, USA) followed by RNeasy cleanup kit (Qiagen) according to manufacturer's protocol. It was then subjected to hybridization with Affymetrix Human Genome U133 Plus 2.0
oligonucleotide arrays (Affymetrix, Santa Clara, CA, USA), according to
the manufacturer's standard protocols. Treated and control samples
were compared. The comparison generated a list of active genes
representing probe sets changed by at least 2-fold. Genes were classied
into functional groups using the Ingenuity software and the GO annotation tool.
Cell proliferation assay
The effect of FF on epithelial cells' viability and proliferation was
assessed using an XTT assay (Biological Industries, Beit Ha'emek,
Israel). Epithelial cells were dissociated from the FT mbria as described
above, plated and treated with either FF or culture medium (as control)
for 24 h followed by addition of XTT reagent to the medium. The cells
were incubated for additional 3 h in the CO2 incubator at 37 C. Absorbance was determined using PowerWave X 340 Microplate Reader
(Bio-Tek, Winooski, VT, USA) at a wavelength of 450 nm.

Immunouorescence staining
Epithelial cells, pre-treated with FF for either 4 or 24 h, were xated
with 2% paraformaldehyde (Electron Microscopy Sciences, Hateld, PA,
USA) for 15 min, permeabilized with 0.2% Triton x-100 (Sigma-Aldrich)
for 20 min and blocked with 5% fetal bovine serum (Biological Industries, Israel) for 1 h at RT. Cells were incubated for 2 h at 37 C with
anti-phospho-histone H2A.X (anti-H2A.X, Millipore), followed by incubation with secondary antibody, Peroxidase-conjugated AfniPure

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K. Bahar-Shany et al. / Gynecologic Oncology 132 (2014) 322327

Goat Anti-Mouse (Jackson ImmunoResearch laboratories, West Grove,

PA, USA) for 30 min at 37 C. A minimum of 100 cells were evaluated
for H2A.X staining for each experiment. Positivity is dened as at
least 5 specic nuclear foci.
ELISA
FT epithelial cells were cultured on Transwells (Corning, Corning,
NY, USA) pre-coated with human collagen IV (Sigma-Aldrich). The
cells were treated with FF for 4 h and then washed several times with
PBS to avoid traces of IL8 originating from the FF, and incubated with
PBS to allow accumulation of secreted proteins [19]. Eighteen to 24 h
later, PBS was collected and IL8 secretion was quantied using ELISA
assay. Interleukin-8 (IL8) concentration in the conditioned PBS was determined using human IL8 immunoassay (R&D Systems, Minneapolis,
MN USA) according to manufacturer's protocol.
qRT-PCR
IL-8 mRNA levels were assessed using FastStart Universal SYBR
Green Master (ROX) (Roche, Indianapolis, IN, USA) with the following
primers: (FWD) 5-GTGCAGTTTTGCCAAGGAGT-3 and (REV) 5-CTCT
GCACCCAGTTTTCCTT-3 (Sigma-Aldrich). qRT-PCR was performed on
the ABI PRISM 7900HT Real Time PCR System (Applied Biosystems,
Grand Island, NY, USA).
Statistical analysis
Measurement data are presented as mean + SEM. The Wilcoxon
test was used to determine the signicance of differences between the
treated and non-treated groups. A p-value of less than or equal to 0.05
was considered as statistically signicant.
Results
Expression proling following exposure to follicular uid
In order to obtain a comprehensive prole of the biological effects of
FF on epithelial cells in culture, we compared gene expression proles of
two immortalized FT cell lines (FT190 and FT194), primary FT epithelial
cells, and three cell lines (SKOV3, OVCA432 and hMEC), treated with FF
for 4 h, 24 h and 48 h. The cut-off was set to twofold change in expression. Changes were seen at all three time points, however in most cases,
the largest number of changes was observed after 4 h of incubation with
FF, with reduced number of up-/down-regulated genes at 48 h (Fig. 1A).
Functional analysis of the differentially expressed genes after 4 h of incubation that were common to at least 3 experimental cell types revealed over-representation of genes related to cell cycle and cell
proliferation (Fig. 1B).

Fig. 1. Overview of transcriptional changes resulting from incubation of human cell lines with
FF. Six different cell lines (primary FT epithelium (Fim Epith), FT190, FT194, OVCA432,
SKOV3, and hMEC) were incubated with FF samples for 4, 24 and 48 h. Expression proling
was performed using HG-U133Plus2.0 microarrays. (A) Number of differentially expressed
genes (either upregulated or down regulated) in comparison to untreated cells is plotted
in the y-axis. Changes were most signicant after 4 h of incubation. (B) Ingenuity functional
analysis of differentially expressed genes in at least 3 of the cell lines. The bar length indicates
the p-value for enrichment of the particular functional group. Most signicant changes are related to cell cycle and proliferation.

IL8 transcription and translation induction in FT epithelial cells

Interlueikin-8 (IL8) is one of the genes that were most signicantly
up-regulated in all 6 cell lines as a result of incubation with FF in our microarray experiments. This chemokine is known as a pro-inammatory
agent which plays a key role in host defense mechanism by effecting
neutrophils activation [21] and as a pro-angiogenic and pro-mitogenic
factor [22,23] implicated in several cancer types such as breast cancer
and uveal melanoma [24,25]. Therefore, taking this knowledge into account, we wished to validate IL8 up-regulation in primary FT epithelial
cells upon FF exposure with RT-PCR. To address this issue, the cells were
treated with FF or with culture medium (control) for either 4 or 24 h. As
shown in Fig. 3A, 1.7-fold increase in IL8 mRNA transcription is observed by RT-PCR as a result of 4 h treatment (p = 0.002, n = 6). An
even more pronounced induction of IL8 transcription (2.3-fold change)
is achieved upon 24 h exposure to FF (p = 0.021, n = 4).

FT epithelial cells proliferate upon follicular uid treatment

The gene expression proling experiments showed changes in
121 genes associated with cellular development, growth and proliferation (p-value = 9.97E 20) and 87 genes related to cell cycle
(p-value = 1.11E 15). We therefore wanted to test the inuence
of FF on viability and proliferation of FT epithelial cells. To do so
we used ex-vivo cultures of FT mbria. Isolated epithelial cells
were treated with either FF or culture medium (as control) for
24 h followed by XTT assay in order to evaluate the viability of
cells following each treatment. As shown in Fig. 2, the FF treatment
did not induce cell death, but rather a 24% increase in the colorimetric signal was observed following the treatment (p = 0.021,
n = 4), indicating a mild proliferative effect.

Fig. 2. Increase in FT epithelial cell proliferation upon FF exposure. FT epithelial cells were
incubated with FF (or with culture medium as control) for 24 h, followed by XTT viability
testing. 24% increase in proliferation was observed. Bars represent mean + SEM.

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325

with FF (p-value = 2.10E11). To explore whether FF indeed induces

DNA damage, immunouorescent staining specic for H2A.X, an
established indicator for DNA damage, specically double stranded
breaks [26], was performed after 24 h treatment with FF. The experiment was repeated 5 times (with cells from 5 different donors). We detected an average of 1.7-fold increase in the proportion of H2A.X
positive-cells following FF treatment in 3 of the repeats, as seen in the
representative Fig. 4, and no difference in the other 2 repeats. The
range of percentage of positive cells was 1829% vs. 1119% in FFtreated and control cells, respectively.
TP53 accumulation following follicular uid exposure

Fig. 3. IL8 is induced in FT epithelium upon FF exposure. FT epithelial cells were incubated
with FF (or with culture medium as control). (A): IL8 mRNA levels following 4 or 24 h incubation were increased by 1.7-fold (p = 0.002) and 2.3-fold (p = 0.021), respectively.
(B): IL8 secretion levels following 4 h of treatment were increased by 1.87-fold. Bars represent mean + SEM.

The majority of high-grade serous papillary carcinoma is associated

with TP53 mutations [27]. The pre-malignant lesions of the disease are
characterized by positive immunostaining for TP53, representing aberrant accumulation of the protein, and the latent precursor is dened
based on TP53 immunostaining [2830]. Therefore, we tested the hypothesis that incubation of FT cells with FF results in accumulation of
TP53. Epithelial cell were incubated for 24 h with either FF or culture
medium (as control) and then parafn-embedded and stained with
anti-p53 monoclonal antibody. While absolutely negative in the control
cells, treated cells had clear positivity to TP53 by IHC in an average of 5%
of the cells after single, transient exposure (Fig. 5). This data suggests
that TP53 accumulation is a very early event in the course of carcinogenic changes in the FT epithelium.
Discussion

Next, we tested the induction of IL8 at the protein level. As described

above, PBS incubated with the apical side of the ex-vivo cultures was analyzed using an IL8 ELISA. However not statistically signicant, a 1.87fold increase in IL8 secretion was seen following 4 h of incubation
with FF (n = 4, Fig. 3B). Overall, our data suggest that FF induces IL8
up-regulation in FT mbria epithelial cells, with potential implications
to a carcinogenic effect on the cells-of-origin.

Follicular uid induces DNA damage

Looking back at the microarray experiments, 43 genes related to
DNA repair were differentially expressed in response to incubation

The main obstacle to improving survival of ovarian cancer patients is

the ineffective screening and unsuccessful early detection. Not much
progress has been achieved in the clinical arena, and the lack of models
of early disease hinders basic research as well. The existing models of
ovarian cancer recapitulate advanced disease, as they involve either xenografting of primary ascites cells or cell lines or articial genetic mutations. To date, there is no in-vivo or in-vitro model that addresses the
known gynecologic and physiologic risk factors.
In this study we used a unique model to study the effects of FF on
FT epithelial cells. Although previously described [19,20], this model
has never been implicated in the context of research of the endogenous carcinogenic stimuli in FF exposure. This specic model is

Fig. 4. Enhanced DNA damage in FT epithelium exposed to FF. FT epithelial cells were stained for phosphorylated-histone H2AX (H2AX) after 24 h of incubation with FF or with culture
medium (control). H2AX foci are seen (green) on a background of cell nuclei (counterstained with DAPI, blue). Bar1 m.

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K. Bahar-Shany et al. / Gynecologic Oncology 132 (2014) 322327

Fig. 5. TP53 accumulation in FF treated FT epithelial cells. FT epithelial cells were incubated with FF (or with culture medium as control) for 24 h and stained for TP53. Positive staining
(brown) in the nuclei (marked by arrow) was observed only in FT epithelial cells which were espoused to FF. Bar100 m.

novel and distinct in several aspects, the most signicant one being
that all of the FT epithelial cells and the FF samples used were obtained from annotated human donors, in contrast to animal models [31],
retaining the authenticity of these complex interactions. Moreover,
multiple repeats with specimens from different donors highlighted
the common molecular events. It has been a technical challenge to
dene the exact conditions at which FF can be experimented with.
For example, in our experience native FF induces signicant toxicity
in-vitro, a difculty that has been circumvented by heat-inactivation
of complement proteins [32,33]. On the other hand, there is an inevitable obvious limitation, which is the short term exposures of the
cells to FF. Primary human FT epithelial cells have limited ability to
proliferate in-vitro. Therefore, unlike the repeated exposures of the
cells in the female reproductive system monthly over 4050 years
on average, in this ex-vivo model the FT epithelial cells were exposed
only once for a relatively short period of time to highly concentrated
FF. Although only aberrations that evade the repair and homeostasis
mechanisms and are still present at the time of the next insult may
accumulate into meaningful neoplastic changes, the cellular events
observed in this study replicate those in the latent precursor p53
signature.
Moreover, this study demonstrates the full extent of the transcriptional changes resulting from exposure of human cells to FF. Clearly
the human FF contains many genotoxic factors, which may have either
additive, synergistic or perhaps even opposing effects on cellular signaling. These effects are yet to be fully discovered.
As reported by Jarboe et al. [28], p53 signature lesions found within
benign FT mbria in up to 35% of general population of women undergoing salpingectomy are presumed to be the precursors for high-grade
serous papillary carcinoma. These lesions are characterized by accumulation of TP53 in the nuclei, double stranded DNA breaks, but relatively
low proliferation index, all of which are shown in this study to result
from the exposure of FT epithelial cells to FF, as well as mutations in
TP53 in some cases. The mechanisms responsible for their formation
are not yet clear and warrant further investigation.
The incessant ovulation hypothesis links multiple ovulatory cycles
to development of ovarian cancer. The ovulatory process resembles an
inammatory process [9]. Therefore, one of the goals of this study was
to raise a possible link between the ovulation-related inammation
and the carcinogenic changes occurring in FT epithelial cells which
lead to ovarian cancer development. IL8 is a pro-inammatory chemokine and was proven to be both pro-angiogenic and pro-mitogenic factor [22,23]. It was also shown that IL8 secretion by ovarian cancer cells
increases proliferation, adhesion and invasion of the cells [34]. Specically, IL8 is also known to stimulate epithelial cell proliferation in
other organ systems, such as prostatic epithelial cells [35]. Here we
show an up-regulation of IL8 transcription and secretion in FT epithelial
cells in response to FF. These results suggest that IL8 is one of the
possible links between ovulation-related inammation and serous
carcinogenesis.

This work paves the road for future studies focusing on various aspects of the carcinogenic role of FF, a road that will hopefully provide
clues to prevention among high-risk patients.
Conict of interest statement
No conict of interests has been declared by the authors.

Acknowledgments
This work was supported by research grants from the AACR-George
and Patricia Sehl Fellowship for Cancer Genetics Research, the Israel Science Foundation Legacy Heritage Clinical Research Initiative, the Israel
Cancer Research Fund Clinical Research Career Development Award,
The Israeli Cancer Association, and the Chaim Sheba Medical Center
Dr. Pinchas Bornstein Talpiot Medical Leadership Program. None of the
funding agencies had any inuence on the design of this research or
the content of this manuscript. We thank Dr. Ronny Drapkin for kindly
sharing with us immortalized FT epithelial cell lines.
This work would not have been possible without the kind assistance
of the team of the Department of Gynecologic Oncology, the staff of the
immunohistochemistry lab at the Department of Pathology, the team of
the Affymetrix unit at the Sheba Cancer Research Center, headed by
Prof. Gidi Rechavi, and the staff of the Institutional Tissue Banks (supported by the Flight Attendants Medical Research Institute (FAMRI)),
all at the Chaim Sheba Medical Center, Ramat Gan, Israel.
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