Enhanced chemoresistance and tumor sphere formation as a laboratory model for peritoneal micrometastasis in epithelial ovarian cancer.

Abstract

Ovarian cancers are composed of heterogeneous cell populations, including highly proliferative immature precursors and differentiated cells that may belong to different lineages. The main reason why epithelial ovarian cancer is difficult to treat is the unusual mechanism of dissemination that involves local invasion of pelvic and abdominal organs. But, unlike many other carcinomas, initial dissemination rarely requires blood or lymph vessels. Because it has been proven that aggregates of malignant cells within the ascites of patients diagnosed with ovarian cancer represent an impediment to cure such cancers, in the present study we adopted suspension culture combined with anti-cancer regimens as a laboratory strategy for research of the initial process of peritoneal micrometastasis.
MLS human ovarian cancer cells were cultured in serum-free medium. Cells of passage eight were treated in combination with the anticancer agent doxorubicin at different peak plasma concentrations for 24 hours, and then maintained under suspension culture. The acquired increased aggressiveness properties was confirmed by multidrug resistance assays and by their ability to grow in an anchorage-independent manner in vitro as tumor spheroids.
Cells selected after chemotherapy had a increased proliferative potential, eliminated Rhodamine 123 in culture and also formed spheroids in suspension.
Here we present direct evidence that the metastasis of human ovarian cancer may be a result of transformation and dysfunction of immature precursor cells in the ovary. Also, spheroid formation may represent a key component of chemotherapy recurrence and a better understanding of these 3D structures can contribute to the development of new treatments for metastatic carcinoma.

Full text

Page 1

Romanian Journal of Morphology and Embryology 2010, 51(2):259–264

O OR RI IG GI IN NA AL L P PA AP PE ER R
Enhanced chemoresistance and tumor sphere
formation as a laboratory model for peritoneal
micrometastasis in epithelial ovarian cancer
OLGA SORIŢĂU1), C. I. TOMULEASA1), EMÖKE PÁLL1), PIROSKA VIRÁG1),
EVA FISCHER-FODOR1), V. FORIS1), OTILIA BARBOS1),
CORINA TATOMIR1), G. KACSÓ2), A. IRIMIE3)
1)Laboratory of Cell Cultures
2)Department of Brachytherapy
3)Department of Surgical Oncology
“Ion Chiricuţă” Comprehensive Cancer Center, Cluj-Napoca
Abstract
Background and Purpose: Ovarian cancers are composed of heterogeneous cell populations, including highly proliferative immature
precursors and differentiated cells that may belong to different lineages. The main reason why epithelial ovarian cancer is difficult to treat is
the unusual mechanism of dissemination that involves local invasion of pelvic and abdominal organs. But, unlike many other carcinomas,
initial dissemination rarely requires blood or lymph vessels. Because it has been proven that aggregates of malignant cells within the
ascites of patients diagnosed with ovarian cancer represent an impediment to cure such cancers, in the present study we adopted
suspension culture combined with anti-cancer regimens as a laboratory strategy for research of the initial process of peritoneal
micrometastasis. Experimental Design: MLS human ovarian cancer cells were cultured in serum-free medium. Cells of passage eight were
treated in combination with the anticancer agent doxorubicin at different peak plasma concentrations for 24 hours, and then maintained
under suspension culture. The acquired increased aggressiveness properties was confirmed by multidrug resistance assays and by their
ability to grow in an anchorage-independent manner in vitro as tumor spheroids. Results: Cells selected after chemotherapy had a
increased proliferative potential, eliminated Rhodamine 123 in culture and also formed spheroids in suspension. Conclusions: Here we
present direct evidence that the metastasis of human ovarian cancer may be a result of transformation and dysfunction of immature
precursor cells in the ovary. Also, spheroid formation may represent a key component of chemotherapy recurrence and a better
understanding of these 3D structures can contribute to the development of new treatments for metastatic carcinoma.
Keywords: ovarian cancer, spheroid formation, resistance to chemotherapy, micrometastasis.
? Introduction
Epithelial ovarian cancer is the eighth most common
cancer among women and causes more deaths than any
other female reproductive tract cancer, with approx.
25 000 new cases diagnosed and 16 000 deaths per year
in the EU alone [1]. The average lifetime risk is about
one in 70, with a strong family history of ovarian or
breast cancer being the most important risk factor [2].
Patients may report abdominal fullness, dyspepsia,
early satiety or bloating because of increased abdominal
pressure due to ascites or involvement of the omentum
[3]. Other physical examination findings include ascites,
pleural affusions or the Sister’s Mary Joseph nodule [4].
Paraneoplastic signs include hormonally mediated
hypercalcemia, subacute cerebellar degeneration, the
Leser–Trélat sign, migratory superficial thrombophle-
bitis, palmar fasciitis, dermatomyositis or even poly-
arthritis [5–9].
Ovarian epithelial neoplasia arises from the
coelomic epithelium covering the ovarian surface or
from inclusion cysts, but recent evidence has shown that
ovarian cancer may also originate from cells other than
the surface epithelia. The pathology subtypes are classi-
fied based on cell morphology into serous, mucinous,
clear cell or endometrioid. These diverse histological
types can be explained by the hypothesis that ovarian
cancer develops from remains of the Müllerian duct
structures [10–13], but another idea is that of ovarian
surface epithelial cell metaplasia through dedifferent-
iation to adopt the various histology characteristics
during transformation [14].
Ovarian surface epithelial cells are histologically
organized as a single cell layer by a sheet of basement
membrane and it is believed that the contact of ovarian
surface cells with this membrane regulates cell growth
and differentiation. In ovarian cancer, tumor cells often
lose not only their ability to synthesize collagen IV and
laminin, key components of the basement membrane,
but also the apical-basolateral polarity [15]. As the cell-
cell adhesion molecules are remodeled, the newly
formed spheroids disaggregate on the mesothelium of
the peritoneum. This is when the first step of metastasis
occurs.
? Material and Methods
Cell lines
MLS ovarian tumor cell line was kindly gifted by

Page 2

Olga Soriţău et al.

260
Dr Yael Schiffenbauer, from the Drug Hypersensitivity
and Tissue Typing Laboratory, Rabin Medical Center,
Beilinson Campus, Tel-Aviv, Israel. As control groups
for the Rhodamine 123 efflux assay, we used both
the HFL human lung fibroblast cell line (European
Collection of Cell Cultures, Budapest, Hungary) and
human liver cancer stem cells, isolated from a hepato-
cellular carcinoma by our research team [16]. Cells
were grown as monolayers in Dulbecco’s modified
Eagles Medium (DMEM) containing 10% fetal calf
serum (FCS), 2 mM L-glutamine, non-essential amino
acids, 100 U/mL penicillin and 100 µg/mL streptomycin
(all from Sigma Aldrich, St. Louis, USA). For all lines,
the medium was replaced every day with fresh one and
the flasks were kept in a 370C incubator with 93% air
and 7% CO2.
In vitro propagation in serum-free culture
medium
Every two passages, the FCS concentration was
dropped by 2.5%. The cell morphology started to
change by forming small tumor spheres, apparently
suggesting a epithelial to mesenchymal transition. Cells
were cultured in DMEM/F12 containing 20 ng/mL
basic fibroblast growth factor (bFGF), 20 ng/mL
epidermal growth factor (EGF), 10 ng/mL insulin
growth factor (IGF) (all from Sigma Aldrich) and 2%
B27 (Invitrogen, Carlsbad, USA) at a density of 1000
cells/mL. Cells and spheres were passages by enzymatic
dissociation with trypsin to single cells through a gauge
21 needle of a 5 mL syringe every three days and then
reseeded.
Anticancer regimens and Darwinian selection
of tumor cells
Cells at passage 10 were plated in complete serum-
free medium into 24-well plates for three days until
formation of tumor spheres. Then doxorubicin was
administered at 3 µg/mL and 5 µg/mL concentrations.
Each concentration was repeated thrice. After incuba-
tion for 24 hours, cells were centrifuged, mechanically
dissociated into single cells and recultured for seven
days in fresh serum-free medium, in accordance to
Li HZ et al. [17].
Tumor sphere formation as an in vitro model
for in vivo peritoneal metastasis
Tumor cells that have survived the anticancer
regimens screening assay were plated at a density of
105 cell/mL in serum-free culture media, as described
by Casey RC et al. [18]. Because in ovarian carcinoma
cells detach from the surface of the tumor into the
peritoneal cavity, the unvascularized 3-D multicellular
spheroids formed by culture in serum-deprivation
culture represent an in vitro model of peritoneal micro-
metastasis, whose adhesive abilities have yet to be
elucidated. Spheres were observed under an Olympus
CKX 41 inverted light microscope, at 100× and 200×
amplification.
Drug resistance assays
Highly proliferative ovarian cancer cells, obtained
after chemotherapy selection, were compared with MLS
ovarian tumor cells. Exponentially growing cells were
cultured in 96-well plates before adding carboplatin and
doxorubicin. After 24 and 48 hours, the relative cell
number was determined by standard MTT assay.
Post-therapy ovarian cancer cells, hepatic cancer
stem cells, MLS ovarian tumor cells and HFL human
lung fibroblasts were seeded in specific culture media
supplemented with FCS, non-essential amino acids,
L-glutamine and antibiotics, at 20×104 cells/mL. Cells
were stained with 10 µM Rhodamine 123 and then
incubated for three hours at 370C and 7% CO2. After
culture, all cell types were washed three times with PBS
before intracellular fluorescence studies, according to
Donnenberg VS et al. protocols [19].
Statistical analysis
Statistical analysis was done using Prism 5.0 statistics
program for Windows (GraphPad, San Diego, USA).
Data were analyzed using one-way ANOVA with the
Bonferroni multiple comparison test (Kruskal–Wallis
as non-parametric). Statistical significance was set at
p<0.05 and all experiments were performed in triplicate.
? Results
MLS cell growth in serum-free culture media
and enrichment of the subpopulation of putative
ovarian proliferative cancer cells
The goal of this experiment was to test whether
MLS ovarian tumor cells had the capability to divide in
a serum-free culture system. After the FCS concent-
ration was dropped systematically, cells had begun to
show morphologic changes consistent with epithelial-to-
mesenchymal transition (Figure 1).
Primary tumor spheres were initially observed after
seven days of culture in complete serum-free media
(Figure 2). Spheres were then disaggregated into a
single cell suspension and serially passaged at clonal
density of 1000 cells/mL. Secondary tumor spheres
appeared on the second day of culture.
Ovarian proliferative cancer cell sorting by
suspension culture combined with Darwinian
selection after chemotherapy
Cells at the eighth passage under serum-free sus-
pension culture were incubated in combination with
different concentrations of doxorubicin (3 µg/mL and
5 µg/mL) for 24 hours. The cells were afterwards washed
thoroughly with PBS to remove all of the doxorubicin
from the flask and maintained in suspension culture by
gently agitating aggregates as single cells. By centrifu-
gation to exclude all dead cells and debris, fresh culture
media were replaced every two days. After six days of
culture, cell viability was tested using the standard
protocol of Trypan Blue staining. Almost all cells had
underwent apoptosis and only 2–5% of cells were
viable.
Acquired resistance to conventional chemo-
therapy
Our research team assessed the expression of

Page 3

Enhanced chemoresistance and tumor sphere formation as a laboratory model for peritoneal micrometastasis…

261
ABCG2, encoding a membrane efflux transporter
expressed in hematopoietic stem cells and also
associated with chemotherapy resistance. The cellular
transport proteins of the ABC (ATP Binding Cassette)
superfamily are active in the first step of the action of
toxic substances, at the stage of drug penetration
through the cell membrane and its intracellular
accumulation. Because Rhodamine 123 uses the same
pathways to pass through the membrane as conventional
drugs used in oncologic treatments, by measuring the
optical density (OD) of this fluorescent substance, we
were able to determine indirectly whether the cell

population isolated from the MSL cell line expressed
the proteins responsible for multidrug resistance.
Ovarian proliferative tumor cells were compared with
hepatic cancer stem cells, HFL human fibroblasts and to
MLS ovarian tumor cells (Figure 3).
To examine whether the isolated cells possessed
a hypothesized chemoresistant phenotype, we also
assessed the sensitivity of the cells to carboplatin and
doxorubicin cultured under the same conditions.
Compared with HFL fibroblasts and MLS tumor
ovarian cells, both carboplatin and doxorubicin IC50
values were greater (p<0.05) (Figure 4).
(a) (b)
Figure 1 – (a) MLS cells cultured in classic FCS medium. After the serum concentration was dropped, ovarian tumor
cells changes, developing into spindle-shaped cells with loss of polarity and an increased intercellular separation (b).

(a)
(b)
Figure 2 – After gathering in small groups, cells had adopted an early 3D conformation (a). This structure later on
started to form small tumor spheres (b).

Figure 3 – Direct dye efflux assay.

Page 4

Olga Soriţău et al.

262

(a)
(b)
(c)
Figure 4 – Ovarian highly proliferative tumor cells were resistant to conventional chemotherapy. There was no
significant difference between cells sorted by suspension culture with doxorubicin at 3 µg/mL (a, b) and cell sorted
with chemotherapy drugs at 5 µg/mL (c, d).
(d)

? Discussion
The rate of mortality in ovarian cancer has changed
little in the past three decades due to drug resistance.
Early detection is critical and many genes specifically
overexpressed in the context of ovarian cancer provide
potential biomarkers for diagnosis, genes such as
CA 125, osteopontin, MUC1 and HE4 [20]. Although
chemotherapeutics target rapidly proliferating tumor
cells and provide temporary remission, only the bulk
of tumor cells is destroyed and drug-resistant cells
remain.
According to the incessant ovulation hypothesis, the
increased cancer risk is due to genetic mutations that
might occur during proliferation of the surface epithe-
lium in the process of postovulatory repair [21].
In ovarian carcinogenesis, the epithelial surface or
inclusion cysts will become multicell layered and form
adenomas. Subsequently, the epithelial cells can invade
the surrounding stroma or the tumor cells may detach
from the primary cancer bulk and spread in the
peritoneal cavity as ascites. These cells have lost their
basal-apical polarity because of genetic and epigenetic
alterations, allowing them to survive and proliferate,
resisting the positioning rules imposed on normal epi-
thelial cells [22].
Characteristically, initial epithelial ovarian cancer
dissemination is intra-abdominal and involves local
invasion of the pelvic or abdominal organs, rarely
involving blood or lymph vessels. Malignant cells are
shed from the primary tumor into the peritoneal cavity
where they are disseminated throughout the abdominal
cavity by peritoneal fluid or ascites. Cells are shed as
spheroids and later on settle onto the surface of the
peritoneum, where disaggregation and metastatic out-
growth may occur. This process requires the remodeling
of cadherins as the spheroids disaggregate on the
mesothelium of the peritoneum while integrins anchor
the spheroid body to the sub-mesothelial extracellular
matrix. In serum-free culture, our cells confirm this
hypothesis and may be key areas of deficiency in
current treatment of cancer. Tumor spheres are present
in malignant ascites and have a reduced response to
chemotherapeutic drugs both in vivo and in vitro,
representing a significant impediment to efficient
treatment of FIGO stage II and III ovarian cancer.

Page 5

Enhanced chemoresistance and tumor sphere formation as a laboratory model for peritoneal micrometastasis…

263
In clinical practice, chemotherapy alone may lead to
massive cytoreduction but seldom cures the disease.
The majority of patients who respond to primary
chemotherapy will develop recurrent, usually drug-
resistant disease. By acquiring a more aggressive
phenotype, malignant ovarian tumor cells will re-express
high levels of ABC drug transporters. The two ABC
transporter-encoding genes that have been studied
most extensively are ABCB1, which encodes P-glyco-
proteins, and ABCG2, a promiscuous transporter of
both hydrophobic and hydrophilic compounds [23].
These proteins have important role in normal physio-
logy in the transport of drugs across the placenta and
in the retention of drugs in the intestinal lumen, but
also are important components of the blood–brain and
blood–testis barriers. By using the energy of ATP
hydrolysis, these proteic components of the cell mem-
brane actively efflux drugs from cells, serving to protect
them from cytotoxic agents as also proven by our
results. The ovarian tumor cells derived from the MLS
line eliminate Rhodamine 123 from in the surrounding
medium, results confirmed by international data, where
scientists have proven that highly proliferative cells
(both normal and malignant) actively eliminate
Rhodamine 123 and Hoechst 33342 whereas most
mature, differentiated cells accumulate these fluorescent
dyes [24–28].
? Conclusions
Despite advances in surgery and chemotherapy,
most patients diagnosed with ovarian cancer relapse and
become drug-resistant. Tumor spheroids are found in
ascites, have migratory abilities and can establish
metastatic lesions after intraperitoneal injection in
NOD/SCID mice. Spheroids are a very likely source of
recurrent disease as the vast majority of therapies are
ineffective in preventing their growth and dissemina-
tion. Further characterization of these tumorigenic
populations will allow oncologists to identify different
markers and molecules and use them as targeted
therapy. With these targets known, the small fraction of
cells responsible for relapse and tumor progression will
be eliminated. Consequently, defining the unique pro-
perties of ovarian tumor spheres remains one main
priority for future development of early diagnosis and
effective therapies.
Acknowledgements
We are grateful to the excellent assistance of Dr Ion
Dan Postescu regarding statistical analysis and interpre-
tation of the data.
References
[1] ***, National Institute of Health Consensus Conference.
Ovarian cancer. Screening, treatment and follow-up. NIH
Consensus Development Panel on Ovarian Cancer, JAMA,
1995, 273(6):491–497.
[2] VELCULESCU VE, Defining the blueprint of the cancer
genome, Carcinogenesis, 2008, 29(6):1087–1091.
[3] MARKMAN M, Antineoplastic agents in the management of
ovarian cancer: current status and emerging therapeutic
strategies, Trends Pharmacol Sci, 2008, 29(10):515–519.
[4] ALBANO EA, KANTER J, Images in clinical medicine. Sister
Mary Joseph’s nodule, N Engl J Med, 2005, 352(18):1913.
[5] TSUNEMATSU R, SAITO T, IGUCHI H, TSUKAMOTO N, Hyper-
calcemia due to parathyroid hormone-related protein
produced by primary ovarian clear cell adenocarcinoma:
case report, Gynecol Oncol, 2000, 76(2):218–222.
[6] FURNEAUX HM, ROSENBLUM MK, DALMAU J, WONG E,
WOODRUFF P, GRAUS F, POSNER JB, Selective expression of
Purkinje-cell antigens in tumour tissue from patients with
paraneoplastic cerebellar degeneration, N Engl J Med,
1990, 322(26):1844–1851.
[7] RAFFAEL GD, GRAVALLESE EM, SCHWAB P, JOSEPH JT,
CANNISTRA SA, Diagnostic dilemmas in oncology: Case 2.
Dermatomyositis and ovarian cancer, J Clin Oncol, 2001,
19(23):4341–4343.
[8] MEDSGER TA, DIXON JA, GARWOOD VF, Palmar fasciitis and
polyarthritis associated with ovarian carcinoma, Ann Intern
Med, 1982, 96(4):424–431.
[9] CANNISTRA SA, Cancer of the ovary, N Engl J Med, 2004,
351(24):2519–2529.
[10] AOKI Y, KAWADA N, TANAKA K, Early form of ovarian cancer
originating in inclusion cysts. A case report, J Reprod Med,
2000, 45(2):159–161.
[11] FEELEY KM, WELLS M, Precursor lesions of ovarian
epithelial malignancy, Histopathology, 2001, 38(2):87–95.
[12] BOYD J, Whence epithelial malignant carcinoma?, Gynecol
Oncol, 2008, 109(2):161–163.
[13] DUBEAU L, The cell of origin of epithelial tumors and the
ovarian surface epithelium dogma: does the emperor have
no clothes?, Gynecol Oncol, 1999, 72(3):437–442.
[14] HANAHAN D, WEINBERG RA, The hallmarks of cancer, Cell,
2000, 100(1):57–70.
[15] SEIDMAN JD, RUSSEL P, KURMAN RJ, Surface epithelial
tumors of the ovary. In: KURMAN RJ (ed), Blaustein’s
pathology of the female genital tract, 5th edition, Springer
Verlag, New York, 2002, 791–904.
[16] TOMULEASA C, SORITAU O, RUS CIUCA D, POP T, TODEA D,
PINTEA B, MOSNEANU O, FORIS V, SUSMAN S, KACSO G,
IRIMIE A, Isolation and characterization of hepatic cancer
cells with stem cell properties from hepatocellular
carcinoma, J Gastrointest Liver Dis, 2009, in press.
[17] LI HZ, YI TB, WU ZY, Suspension culture combined with
chemotherapeutic agents for sorting of breast cancer stem
cells, BMC Cancer, 2008, 8:135–142.
[18] CASEY RC, BURLESON KM, SKUBITZ KM, PAMBUCCIAN SE,
OEGEMA TR JR, RUFF LE, SKUBITZ APN, β 1-integrins
regulate the formation
carcinoma multicellular spheroids, Am J Pathol, 2001,
159(6):2071–2080.
[19] DONNENBERG
VS,
MEYER
Measurement of multiple drug resistance transporter activity
in putative cancer stem/progenitor cells. In: YU JS (ed),
Cancer stem cells. Methods and protocols, Springer, Los
Angeles, 2009, 261–281.
[20] BAST RC JR, BADGWELL D, LU Z, MARQUEZ R, ROSEN D,
LIU J, BAGGERLY KA, ATKINSON EN, SKATES S, ZHANG Z,
LOKSHIN A, MENON U, JACOBS I, LU K, New tumor
markers: CA 125 and beyond, Int J Gynecol Cancer, 2005,
15(Suppl 3):274–281.
[21] CAPO-CHICHI CD, SMITH ER, YANG DH, ROLAND IH,
VANDERVEER L, COHEN C, HAMILTON TC, GODWIN AK,
XU XX, Dynamic alterations of the extracellular environment
of ovarian surface epithelial cells in premalignant trans-
formation, tumorigenicity, and metastasis, Cancer, 2002,
95(8):1802–1815.
[22] CVETKOVIĆ D, Early events in ovarian oncogenesis, Reprod
Biol Endocrinol, 2003, 1:68.
[23] DEAN M, RZHETSKY A, ALLIKMETS R, The human ATP-binding
cassette (ABC) transporter superfamily, Genome Res, 2001,
11(7):1156–1166.
[24] SUMMER R, KOTTON DN, SUN X, MA B, FITZSIMMONS K,
FINE A, Side population cells and Bcrp1 expression in lung,
Am J Physiol Lung Cell Mol Physiol, 2003, 285(1):L97–194.
[25] ZHOU S, SCHUETZ JD, BUNTING KD, COLAPIETRO AM,
SAMPATH J, MORRIS JJ, LAGUTINA I, GROSVELD GC, OSAWA M,
NAKAUCHI H, SORRENTINO BP, The ABC transporter
Bcrp1/ABCG2 is expressed in a wide variety of stem cells
and is a molecular determinant of the side-population
phenotype, Nat Med, 2001, 7(9):1028–1034.
and adhesion of ovarian
EM,
DONNENBERG
AD,

Page 6

Olga Soriţău et al.

264
[26] STAVROVSKAYA AA, STROMSKAYA TP, Transport proteins of
the ABC family and multidrug resistance of tumor cells,
Biochemistry (Moscow), 2008, 73(5):592–604.
[27] HIRSCHMANN-JAX C, FOSTER AE, WULF GG, NUCHTERN JG,
JAX TW, GOBEL U, GOODELL MA, BRENNER MK, A distinct
“side population” of cells with high drug efflux capacity
in human tumor cells, Proc Natl Acad Sci U S A, 2004,
101(39):14228–14233.






Corresponding author
Ciprian I. Tomuleasa, Laboratory of Cell Cultures, “Ion Chiricuţă” Comprehensive Cancer Center, 34–36
Republicii Street, 400015 Cluj-Napoca, Romania; Phone: +40741–337 480, Fax +40233–235 249, e-mail:
ciprian.tomuleasa@gmail.com






Received: October 12th, 2009
[28] KONDO T, SETOGUCHI T, TAGA T, Persistence of a small
subpopulation of cancer stem-like cells in the C6 glioma cell
line, Proc Natl Acad Sci U S A, 2004, 101(3):781–786.




Accepted: March 10th, 2010