Versican regulates metastasis of epithelial ovarian carcinoma cells and spheroids
© Desjardins et al.; licensee BioMed Central Ltd. 2014
Received: 20 February 2014
Accepted: 19 June 2014
Published: 26 June 2014
Epithelial ovarian carcinoma is a deadly disease characterized by overt peritoneal metastasis. Individual cells and multicellular aggregates, or spheroids, seed these metastases, both commonly found in ascites. Mechanisms that foster spheroid attachment to the peritoneal tissues preceding formation of secondary lesions are largely unknown.
Cell culture models of SKOV-3, OVCAR3, OVCAR4, Caov-3, IGROV-1, and A2780 were used. In this report the role of versican was examined in adhesion of EOC spheroids and cells to peritoneal mesothelial cell monolayers in vitro as well as in formation of peritoneal tumors using an in vivo xenograft mouse model.
The data demonstrate that versican is instrumental in facilitating cell and spheroid adhesion to the mesothelial cell monolayers, as its reduction with specific shRNAs led to decreased adhesion. Furthermore, spheroids with reduced expression of versican failed to disaggregate to complete monolayers when seeded atop monolayers of peritoneal mesothelial cells. Failure of spheroids lacking versican to disaggregate as efficiently as controls could be attributed to a reduced cell migration that was observed in the absence of versican expression. Importantly, both spheroids and cells with reduced expression of versican demonstrated significantly impaired ability to generate peritoneal tumors when injected intraperitoneally into athymic nude mice.
Taken together these data suggest that versican regulates the development of peritoneal metastasis originating from cells and spheroids.
KeywordsOvarian carcinoma Metastasis Versican Adhesion Migration
Epithelial ovarian carcinoma (EOC) is a leading cause of death from gynecologic malignancies and the fifth leading cause of death in women . Nearly 90% of all ovarian cancer cases are epithelial in origin and the majority of those belong to a serous histotype. Metastatic disease is highly lethal, and less than 20% of the affected patients survive over a 5 year interval [2, 3]. Metastatic progression of EOC is very unique, as metastases that cause death spread locoregionally, in the peritoneal cavity . Malignant cells are shed off of the primary tumor and are carried by the intraperitoneal ascitic fluid, which is followed by implantation at the organs and tissues of the peritoneal cavity, anchorage in submesothelial ECM and establishment of metastases [4, 5]. Shed EOC cells may exist as single cells and multicellular aggregates, or spheroids, and both are capable of attaching to the mesothelial layer and transmigrating through outlining peritoneal tissues and organs [6, 7].
Successful colonization of the abdomen by EOC cells and spheroids to a large extent depends on their ability to attach to mesothelial surfaces of the peritoneal organs and tissues. EOC cell adhesion to omental “milky spots” composed of various cell types including mesothelial has also been reported . Mesothelial cells are specialized cells that outline the entire surface of the peritoneal cavity. These cells, among other functions, provide a protective barrier against invading pathogens and secrete surfactant molecules to provide a non-adhesive surface. Multiple interactions between EOC cells and mesothelial cells have been reported to contribute to peritoneal adhesion, including CD44-hyaluronan, α5β1-integrin-fibronectin, L1-neuropilin-1, CA125-mesothelin, and CX3CL1-CX3CR1 [9–16]. Information regarding the mechanisms of EOC spheroid adhesion to the mesothelium is scarce and limited to a single report suggesting the role of β1-integrins in this process . Inhibition of spheroid adhesion could be essential in preventing secondary lesions, as these multicellular aggregates can efficiently escape chemotherapy and radiation, as shown using in vitro models, which might contribute to recurrence of EOC in treated patients [4, 17–19].
Versican (VCAN) is a secreted proteoglycan protein with multiple functions that can promote tumor metastasis [20, 21]. Versican can be expressed in at least 5 different splice variants that were reported to affect cell-cell and cell-matrix adhesion [22–24], migration , proliferation, apoptosis , and a mesenchymal-epithelial transition . Versican contains several domains  that define its binding partners: hyaluronan, integrin, CD44, selectins, EGFR, chemokines, and many others (reviewed in ). The exact composition of versican domains varies in each isoform, however the N-terminal hyaluronan-binding and the C-terminal domains are present in all isoforms. Spatial and temporal regulation of versican expression is regulated by very diverse pathways, such as the canonical Wnt/β-catenin signaling [29, 30], androgen receptor signaling , transcription factor AP-1 , microRNA miR-143 , and others (reviewed in ). Importantly, 50% of tested primary EOC (n = 299) expressed versican . Moreover, overexpression of versican in malignant ovarian stroma is associated with increased invasive potential . Versican could stabilize pericellular matrix and enable stronger adhesion of EOC cells to the mesothelial cells via a CD44-dependent mechanism [15, 36]. Furthermore, our previous data demonstrated upregulation of versican in spheroids , prompting further studies into the role of this ECM-associated protein in the biology of EOC spheroids and EOC. In this report, we have investigated the role of versican in individual cell and spheroid adhesion, migration and disaggregation in vitro, and peritoneal carcinomatosis in vivo.
Matrigel and rat tail collagen type I was obtained from BD Biosciences (Bedford, MA). Human versican shRNA constructs in retroviral GFP vector were obtained from Origene (Rockville, MD). Human versican (pool of 3 proprietary 19 – 25 nt sequences) and control siRNAs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). DharmaFECT1 was obtained from Dharmacon (Lafayette, CO). Mouse anti-human versican antibody clone 12C5 and mouse anti-human-β-tubulin were obtained from Iowa Developmental Studies Hybridoma Bank (Iowa City, Iowa).
Human ovarian carcinoma cell lines of serous histotype originating from malignant cells in ascites, OVCAR4, SKOV-3 and A2780, were obtained from the NCI Tumor Cell Repository (Detrick, MD). These cell lines were cultured as suggested by the manufacturer for no longer than twenty consecutive passages. The human ovarian carcinoma cell line of serous histotype originating from malignant cells in ascites, Caov-3, was obtained from Dr. M.S. Stack (University of Notre Dame, ID) and propagated in minimal essential media supplemented with 10% fetal bovine serum (FBS) for no longer than fifteen consecutive passages. OVCAR3 was obtained from ATCC and cultured as recommended. The human ovarian carcinoma cell line of serous histotype originating from a primary tumor, IGROV-1, was obtained from the NCI Tumor Cell Repository (Detrick, MD) and cultured as suggested by the manufacturer for no longer then twenty consecutive passages. The human immortalized peritoneal mesothelial cell line LP-3 was obtained from the Coriell Aging Cell Repository (Camden, NJ) and cultured as indicated by the manufacturer for 5–8 passages. All cell lines were routinely assessed for cellular morphology and average doubling time. All cell lines were propagated from stocks originally obtained from cell banks and individual investigators and have been stored in aliquots for future use. Each aliquot was further propagated for no longer than 20 consecutive passages or 4 months, whichever came first.
Athymic nude – FOXN1NU mice were obtained from Harlan Laboratories (Madison, WI) and from Charles River Laboratories (Chicago, IL). All experimental procedures were performed according to the Institutional Animal Care and Use Committee protocol (#10-060) approved by the Animal Care Committee of UIC. Animals were fed ad libitum and maintained in Association for Assessment and Accreditation of Laboratory Animal Care International approved facilities on a 12 h light 12 h dark cycle.
EOC cells were cultured to 80% confluence and transfected with siRNAs using DharmaFECT1 according to the manufacturer’s instructions.
Creating stable clones with reduced VCAN expression
SKOV-3 were transfected with VCAN shRNAs following manufacturer’s suggestions. To create VCAN shRNA-silenced sub cell lines we have used the following four different shRNA sequences designed against multiple splice variants at VCAN gene locus: TGTGACTATGGCTGGCACAAATTCCAAGG, GGATACAGCGGAGACCAGTGTGAACTTGA, GGAAATATCAAGATTGGTCAGGACTACAA, TGGTCATCCAATAGATTCAGAATCTAAAG (Origene Technologies). Several clones have been selected based on their resistance to puromycin and expression of GFP. Residual expression of 5 known isoforms of VCAN has been determined using qPCR resulting in selection of several clones for further experiments.
Quantitative real-time PCR
Real-time PCR was performed using MyiQ (Bio-Rad) according to the manufacturer’s instructions. Primers for detection of V0, V1, V2, and V3 isoforms of VCAN and a housekeeping gene control eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) have been synthesized using previously reported sequences . The primers for mRNA detection of V4 isoform of VCAN were constructed according to requirements for oligonucleotide primers for quantitative real-time PCR using the Primer3 software. Primer specificity was determined using serial dilutions of the template and by examination of the product melting curves. SYBR Green was used for quantitative PCR as a double-stranded DNA-specific fluorophore. PCR was conducted by initial denaturation for 10 min at 95°C followed by 40 cycles of 94°C for 15 sec and 60°C for 30 sec using the iTaq SYBR Green Supermix (BIO-RAD). To determine the specificity of the PCR primers, the melting curves were collected by denaturing the products at 95°C, then cooling to 65°C, and then slowly melting at 0.5°/sec up to 95°C.
Spheroids were generated using an agarose overlay method described previously . Briefly, non-adhesive agarose plates were prepared by solidifying agarose solution (0.5% in complete culture media) in cell culture plates. EOC cells were released from the monolayers with 0.05% trypsin/EDTA solution, suspended in media containing 2% fetal bovine serum at the concentration of 125,000 cells/ml. 2 ml of this solution was added atop of the solidified agarose and incubated for 48 h at 37°C and 5% CO2. Spheroids formed in the suspension and were visualized using bright field microscopy and their diameters measured using AxioVision software (Zeiss). Spheroids were collected from the media with gentle centrifugation for 1 min at 30 × g.
Cell-cell adhesion and spheroid disaggregation
A human-derived peritoneal mesothelial cell-line LP-3 was cultured in 96-well plates to near confluence. SKOV-3 cells were cultured in monolayers and labeled with fluorescent DiI (Invitrogen) according to the manufacturer’s instructions prior to the adhesion assays. SKOV-3 were subsequently released from monolayers with 0.05% trypsin/EDTA solution and resuspended in serum-free cell culture media. To study adhesion and disaggregation of spheroids, SKOV-3 in monolayers were labeled with DiI and subjected to the spheroid formation assay followed by plating atop of the confluent monolayer of LP-3 (in triplicate per condition) in serum-free media. When needed, SKOV-3 cells were transiently transfected with either control or VCAN-specific siRNAs and used in adhesion assays between 48 and 72 h from the start of transfection. Several clones of SKOV-3 stably transfected with VCAN shRNAs were also used along with the controls stably transfected with scrambled shRNA. Subsequently, the monolayers were washed two times with PBS and fixed in a methanol-containing cell fixative. Adherent cells showing round cell morphology were visualized by red fluorescent signals using a Zeiss fluorescent microscope. The adherent DiI-labeled cells were counted, averaged, and characterized as a percentage from the total. To analyze the role of VCAN in spheroid disaggregation, adherent spheroids were allowed to disaggregate for 24 h followed by outlining the outer perimeter and quantifying the total area taken by the disaggregated spheroid using AxioVision software (Zeiss). Area fold change was calculated by dividing the total area of disaggregated spheroid by that of the spheroid at time zero.
Cell-ECM adhesion and spheroid disaggregation
Tissue culture-treated 48-well plates were pre-coated with 10 μg/ml human collagen type I and Matrigel (diluted 1:100), or PBS (designated “culture plate”) for 1 h at 37°C. The plates were subsequently rinsed with PBS and air dried. Next, 10 000 SKOV-3 cells were seeded (in triplicate for each condition) in serum-free media in coated wells and allowed to adhere for 5 h at 37°C and 5% CO2. This seeding was followed by two washes with PBS, fixation in a methanol-containing cell fixative, and staining. Cells were counted, averaged, and plotted. Spheroids were seeded in serum-free media and allowed to disaggregate for 24 h followed by data analysis including calculation of the area fold change as described above for spheroid disaggregation on LP-3.
Wound healing assay
To study cell migration using wound healing assays, cells were cultured to complete monolayers in complete media containing 10% FBS followed by 24 h incubation in a media containing no FBS or other growth factors. Wounds were introduced with a plastic pipette tip. Cells cultured in serum-free media were monitored for up to 10 h and photographed using Zeiss AxioVision software. Wound healing was calculated based on the widths of the initial wounds (0 time point) and those at 5 and 10 h and derived as a percentage from the initial. Measurements of the gaps were taken in 10 random places along the wounds, averaged, and percentage of wound healing was calculated based on the lengths of gaps at the initial wounding and at 5 and 10 h, respectively.
Transwell cell migration
Inserts with 0.8-micron porous membranes were bottom-coated with 1:100 diluted Matrigel for 1 h at 37°C, rinsed, and air-dried. SKOV-3 and IGROV-1 cells (5,000/transwell) in a final volume of 300 μl were seeded in the inserts, which were then placed into 24-well plates filled with serum-free minimal essential media. The cells were allowed to migrate for 5 h at 37°C and 5% CO2. Migration was stopped by removing the non-migrated cells from the inside of the inserts. Cells that had migrated through the membranes were fixed, stained, and counted.
The monolayer cells (1×106 per tube) were harvested with trypsin/EDTA and spheroids were harvested with centrifugation and brought to the individual cell state with trypsin/EDTA. Further cells were resuspended in 100 μl of ice cold PBS supplemented with 10% fetal calf serum and 1% sodium azide, fixed in a 1% paraformaldehyde solution in PBS for 15 min on ice, and permeabilized using methanol for 1 h at −20°C. 2 mg of mouse anti-human versican antibody (clone 12C5, Iowa Developmental Studies Hybridoma Bank) was added to the cells. For negative controls, the cells were incubated with either 2 μg of anti-mouse IgG antibody or no primary antibody. Cells were incubated for 1 h on ice in the dark with agitation following washing and resuspension in 400 μl ice cold PBS. 2 μg of goat anti-mouse FITC-conjugated IgG (Millipore) was added to the cells and incubated for 1 h on ice in the dark with agitation. The cells were washed and resuspended in 400 μl of ice cold PBS supplemented with 2% BSA and 1% sodium azide. Labeled cells were analyzed using an Accuri C6 flow cytometer on the same day.
The cells were cultured on glass coverslips to nearly full confluence, fixed, and blocked in goat serum. Mouse anti-human-versican (clone 12C5) antibodies were used at a 1:100 dilution and incubated with cells for 1 h at 22°C. Secondary anti-mouse Alexa433- or anti-mouse Alexa594-conjugated antibodies were used at 1:500 and incubated with cells for 1 h at RT in the dark. 4',6-Diamidino-2-phenylindole (DAPI) was added to the secondary antibody solution to a final concentration of 10 μg/ml 10 min prior to the end of the incubation period. The cells were washed, air dried, and mounted on glass slides using ProlongGold (Invitrogen, Carlsbad, CA). Fluorescent imaging was performed using a Zeiss AxioObserverD.1 fluorescence microscope.
Western blotting analysis was used to detect the expression of versican and β-tubulin in SKOV-3 cells and spheroids. This procedure was performed as previously described [40–42]. Antibodies were used at the following dilutions: 1:100 mouse anti-human-versican (clone 12C5) in 3% BSA in a solution of 50 mM tris-buffered saline, pH 7.4, 150 mM NaCl, and 0.05% Tween-20 (TBST) (Sigma; St. Louis, MO) and 1:200 mouse anti-human-β-tubulin in 3% BSA in TBST. Immunoreactive bands were visualized with an anti-(mouse-IgG)-peroxidase (Sigma, St. Louis, MO) (1:1000 in 3% BSA in TBST), and enhanced chemiluminescence was read using Chemidoc (Bio-Rad) and Bio-Rad Chemidoc ImageReader software.
In vivo tumor formation
For generation of intraperitoneal tumors 3 × 106 cells/mouse of parental SKOV-3 and SKOV-3 stably expressing VCAN shRNA (2 clones) were used to generate spheroids. Spheroids were injected intraperitoneally (i.p.) into athymic nude mice (n = 6) and animals were monitored three times weekly for tumor formation, ascites development, and survival up to 38 days. To generate intraperitoneal tumors from individual cells, 3 × 106 SKOV-3/mouse were i.p. injected into athymic nude mice (n = 6) and animals were monitored three times weekly for tumor formation, ascites development, and survival for up to 10 weeks. At the end of the experiments animals were sacrificed, dissected, ascites were aspirated, and the abdominal region was examined for tumors. Data analysis was performed as “yes” in case when tumor were visible and “no” when no nodules were seen regardless of the size found at a specific abdominal organ or tissue and plotted as a bar graph depicting the number of animals bearing metastasis at the indicated tissues and organs. Tumors were collected and paraffin-preserved as described earlier .
Statistical analysis of the results of in vivo experiments
The data were treated as coded values for the presence or absence of tumor and compared between the control (SKOV-3) and the SKOV-3 versican shRNA clones 5 and 6 (spheroids). A Chi-square analysis was performed and identified that VCANsh clone6 and VCANsh clone5 were significantly different than the control for all sites in the spheroid groups and VCANsh clone6 was significantly different than the control for all sites in the single cell group (Pearson Chi-square = 13.846, p = 0.001, with a Fisher's exact correction at 0.002).
Expression of versican is upregulated in EOC spheroids
Reduction of versican expression affects EOC spheroid and cell adhesion to peritoneal mesothelial cell monolayer and ECM
Versican regulates spheroid disaggregation on mesothelial cell monolayers
Loss of versican in EOC spheroids reduces formation of peritoneal tumors
EOC, the deadliest gynecologic cancer, has many unique features that set this malignancy apart from others and make it difficult to treat clinically. The pattern of metastatic spread involves peritoneal organs and occurs via dissemination of the malignant cells from the primary tumor, while lymphagenous and hematogenous metastasis are rare. Peritoneal spread of EOC metastasis is characterized by numerous lesions seeded on various peritoneal tissues and organs, leading to obstruction of bowel, malnutrition, and death. Disseminated EOC cells can exist in the ascites as individual cells and multicellular aggregates, which is another unique feature of this malignancy. Although both are capable of forming metastasis, it has been proposed that spheroids may possess increased invasive ability . Cells forming spheroids are less susceptible to the harmful effects of chemotherapy and radiation in vitro, which may allow them to escape treatment and proceed with peritoneal metastases [17–19, 39]. Follow-up chemotherapy in EOC becomes significantly less efficient eventually leading to development of incurable metastases. Thus, it is especially important to understand the biology of these potentially more aggressive aggregates in order to prevent or hamper formation of deadly peritoneal metastases. Therapies preventing initial seeding from malignant spheroids could lead to less metastatic lesions, better treatment outcomes, and longer survival. New therapies could be used after surgical resection of primary EOC in early stage patients, as well as for reduction of further spread in patients with advanced disease.
The data presented in this report indicate that a reduction of all forms of versican may abrogate formation of peritoneal lesions seeded by EOC spheroids and it impedes tumor formation by individual cells. Of note, tested cellular properties that ensure success of metastasis, such as peritoneal adhesion, migration, and spheroid disaggregation, were all reduced by only about 30% when versican expression was silenced according to the in vitro results. Tumor lesions following intraperitoneal injection of spheroids that were deficient for versican expression demonstrated a delay in seeding such that no lesions were present at five and a half weeks for both clones while tumors could be identified although at a much lower level after ten weeks. These results might lead to a speculation that in some instances it may be sufficient to abrogate multiple metastatic abilities, but not necessarily to completely eliminate them in order to prevent and or delay the formation of metastasis. Alternatively, in the in vitro situation, spheroids may precipitate more easily due to gravitational forces, which would allow for their tighter connection with the mesothelial layer. In vivo, it is likely that completely different forces may contribute to peritoneal adhesion of spheroids resulting in less successful implantation. Other unknown factors could potentially explain why no lesions after i.p. spheroid injection were formed in our in vivo experiments at five and a half weeks, even though they were able to form after 10 weeks and in the in vitro studies they were reduced each only by a third. Importantly, spheroids expressing versican (parental SKOV-3 group) were able to form tumors, indicating that the presence of versican is essential for seeding and development of peritoneal lesions. Furthermore, our data suggest that expression of endogenous versican could be important for metastatic progression of EOC along with the stromal versican previously reported by Ghosh . Lastly, in the conditions ensuring that animals injected with parental SKOV-3 become moribund, individual cells lacking versican expression were more successful in forming visible tumors, perhaps, because they had more time to develop visible tumors. These data suggest that versican is a key protein that regulates peritoneal carcinomatosis by cells and spheroids in a xenograft model of EOC.
It remains to be tested in more detail how abrogation of versican in already existent metastasis might result in better outcomes. It is important to mention that versican can promote EOC cell proliferation . However, our data do not fully support that changes in proliferation contribute to formation of tumors in our in vivo experiments, as cell proliferation in one of the clones with reduced versican expression was similar to the controls.
At present there are no specific small molecule drugs directed at reduction of versican expression. Nevertheless, other approaches that target the expression and function of versican could be feasible in the future. Our experiments show that versican-specific siRNA and shRNA are effective against formation of EOC peritoneal metastases. These data provide feasibility of this approach provided that future technological discoveries will make it possible to use small RNAs to treat diseases. Another approach that could be attempted in preclinical models is the use of bio-neutralizing antibodies against versican. As versican is a secreted protein associated with the extracellular matrix, use of antibodies, if successful, could target many stages of EOC dissemination starting from peritoneal seeding to the latest stages characterized by expansion of terminal metastasis. While some of these approaches remain to be tested, our data presented here emphasize the potential importance of versican in formation and development of peritoneal metastases of EOC.
Our data elucidate the expression and role of versican in the formation and development of peritoneal metastases of EOC from both individual cells and spheroids. Our results may also suggest that multiple pro-metastatic cellular functions, such as adhesion and migration, play a significant role in development of metastasis from ovarian carcinoma.
Epithelial ovarian carcinoma
Short hairpin ribonucleic acid
Epidermal growth factor receptor
Green fluorescent protein
Polymerase chain reaction
Eukaryotic translation elongation factor 1 alpha1
Fetal bovine serum
Phosphate buffered saline
Bovine serum albumin
Small inhibitory ribonucleic acid.
The authors thank Mr. Daniel Lantvit for his outstanding technical assistance. The authors gratefully acknowledge financial support by Hans and Ella McCollum Vahlteich Endowment Fund based at the University of Illinois College of Pharmacy (to MVB), American Cancer Society, Illinois Division Grant #198484 (to MVB), National Cancer Institute (grant # CA160917 to MVB), and Ovarian Cancer Research Foundation Liz Tilberis Scholar Award (to MVB and JEB).
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