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(American Journal of Pathology. 2001;159:2071-2080.)
© 2001 American Society for Investigative Pathology

Regular Article

ß1-Integrins Regulate the Formation and Adhesion of Ovarian Carcinoma Multicellular Spheroids

Rachael C. Casey*, Kathryn M. Burleson*, Keith M. Skubitz{dagger}, Stefan E. Pambuccian*, Theodore R. Oegema, Jr.{ddagger}, Laura E. Ruff* and Amy P. N. Skubitz*

From the Departments of Laboratory Medicine and Pathology,*
Medicine,{dagger}
and Orthopaedic Surgery,{ddagger}
University of Minnesota, Minneapolis, Minnesota


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Ovarian carcinoma multicellular spheroids are an in vitro model of micrometastasis whose adhesive abilities have not been elucidated. In this study, we identified adhesion molecules that mediate the formation of ovarian carcinoma spheroids and their subsequent adhesion to extracellular matrix proteins. The NIH:OVCAR5, but not the SKOV3, ovarian carcinoma cell line formed spheroids similar to multicellular aggregates isolated from patient ascitic fluid. NIH:OVCAR5 spheroid formation was augmented by a ß1-integrin-stimulating monoclonal antibody or exogenous fibronectin, but was inhibited by blocking monoclonal antibodies against the {alpha}5- or ß1-integrin subunits. By immunohistochemical staining, {alpha}2-, {alpha}3-, {alpha}5-, {alpha}6-, and ß1-integrin subunits, CD44, and fibronectin were detected in NIH:OVCAR5 spheroids. NIH:OVCAR5 spheroids adhered to fibronectin, laminin, and type IV collagen, and this adhesion was partially inhibited by blocking antibodies against the {alpha}5-, {alpha}6-, and {alpha}2-integrin subunits, respectively. A blocking monoclonal antibody against the ß1-integrin subunit completely inhibited adhesion of the spheroids to all three proteins. These results suggest that interactions between the {alpha}5ß1-integrin and fibronectin mediate the formation of ovarian carcinoma spheroids and that their adhesion to extracellular matrix proteins at sites of secondary tumor growth may be mediated by a complex interaction between multiple integrins and their ligands.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Ovarian cancer is the leading cause of gynecological malignancy and the fifth leading cause of cancer death among women in the United States.1 In ovarian carcinoma, cancer cells detach from the surface of the tumor into the peritoneal cavity. Subsequent peritoneal implants are characterized by the adhesion, migration, and invasion of the tumor cells into the peritoneum and underlying organs. Free-floating tumor cells are found in the peritoneal cavity both as both single cells and as multicellular aggregates.2 However, because they are difficult to study and manipulate compared to single-cell suspensions, ovarian carcinoma multicellular aggregates have been primarily ignored in most studies.

Many human tumor cells and cell lines can be cultured as multicellular aggregates, which are spherical, mechanically stable, and viable.3 Early in vitro studies indicated that ovarian carcinoma tumor cells and cell lines were unable to form spheroids, but remained exclusively as free-floating single cells or formed monolayers in tissue culture.4 More recently, spheroids have been successfully generated from some ovarian cancer cells and cell lines, and used as three-dimensional in vitro models to study the efficacy of therapeutic strategies.5-7 Cells in ovarian carcinoma spheroids exhibit changes in their position in the cell cycle and are protected from radiation-induced5,6 and taxol-induced8,9 apoptosis, compared to cells cultured as monolayers. However, the phenomena that mediate the formation of ovarian carcinoma multicellular spheroids and their subsequent abilities to adhere, migrate, invade, and proliferate at secondary growth sites have not been investigated, and their contributions to secondary tumor growth, if any, have not been assessed. It still remains unclear whether the floating multicellular aggregates found in patients’ ascites fluid are capable of adhering to the extracellular matrix (ECM) of mesothelial cells or whether they are merely nonadhesive, and therefore noninvasive or benign, counterparts to the malignant ovarian carcinoma cells that adhere to the peritoneal lining.

Many cell-cell and cell-matrix interactions are regulated by integrins, a family of heterodimeric transmembrane receptors.10 ECM proteins, which include fibronectin, type IV collagen, and laminin, affect the in vitro growth, morphology, survival, and differentiation of normal and malignant cells via their interactions with integrins.11 In ovarian carcinoma, integrins have been shown to mediate the organization of ECM,12 adhesion to ECM components,13-15 and cell motility.16-18 Integrins have also been shown to mediate interactions between ovarian carcinoma cells and the mesothelial cells that line the abdominal organs.15 CD44, another cell surface receptor found on ovarian carcinoma cells,15,18,19 binds the ECM glycosaminoglycan hyaluronan with high affinity20 and also has a weak affinity for fibronectin, type IV collagen, and laminin.21 Interactions between CD44 and hyaluronan affect cell adhesion,15 migration,18,19 and tumor growth22 in ovarian carcinoma cells.

The purpose of this study was to elucidate the biological properties of ovarian carcinoma spheroids by developing an in vitro model from established ovarian carcinoma cell lines. We examined the roles of integrins, CD44, and ECM proteins in the formation of ovarian carcinoma spheroids. The proliferative ability and viability of ovarian carcinoma cells cultured as spheroids were also determined and compared to that of the same cell lines cultured as monolayers. We examined the expression of adhesion molecules in NIH:OVCAR5 spheroids. We assessed the ability of ovarian carcinoma spheroids to adhere to ECM molecules and identified integrin subunits that mediated these interactions. The results from this study identify adhesion molecules that participate in the formation of ovarian carcinoma spheroids and suggest that their subsequent adhesion to secondary sites of tumor growth may be integrin-dependent events. Our findings suggest that ovarian carcinoma cells present as free-floating multicellular aggregates may exhibit markedly different behavior than ovarian carcinoma single cells or monolayers. It is possible that these differences may then translate into different metastatic abilities in vivo and/or responses to treatments.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Unless otherwise stated, all standard reagents and materials were obtained from Sigma Chemical Company (St. Louis, MO), all pictures were photographed with a Nikon Coolpix 950 camera (Melville, NY), and all experiments were performed in triplicate and repeated a minimum of three times.

Cell Culture

The human ovarian carcinoma cell lines NIH:OVCAR5 and SKOV3 were chosen for their ability to mimic the progression of ovarian carcinoma when injected into in vivo mouse models.23 These cells have also been shown to adhere to peritoneal mesothelial cells in in vitro models.24,25 The ovarian carcinoma cell line SKOV3 was obtained from Dr. Robert Bast, Jr., M.D. Anderson Cancer Center, University of Texas, Houston, TX. These cells were maintained in McCoy’s 5A medium supplemented with 15% fetal bovine serum, 2 mmol/L L-glutamine, and 50 U/ml penicillin G/streptomycin (Life Technologies, Grand Island, NY). The ovarian carcinoma cell line NIH:OVCAR5 was originally established by Dr. Thomas Hamilton (Fox Chase Cancer Center, Philadelphia, PA)26 and obtained from Dr. Judah Folkman, Harvard Medical School, Boston, MA. This cell line was maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mmol/L L-glutamine, 0.2 U/ml insulin, and 50 U/ml penicillin G/streptomycin. Both cell lines were maintained in 75-mm2 tissue culture flasks in a humidified incubator with 5% CO2 at 37°C.

Purification of Primary Ovarian Carcinoma Cells

Primary ascites fluid samples from six patients diagnosed with serous ovarian adenocarcinoma were obtained with the approval of the University of Minnesota Institutional Review Board from the University of Minnesota Cancer Center Tissue Procurement Facility. Tumor cells were collected by centrifugation (1000 x g, 10 minutes). To lyse erythrocytes, the cells were resuspended in 10 mmol/L potassium bicarbonate, 155 mmol/L ammonium chloride, 0.1 mmol/L ethylenediaminetetraacetic acid, pH 7.4, for 5 minutes. The remaining cells were collected by centrifugation (1000 x g, 10 minutes). The tumor cells were layered on Ficoll-Paque Plus (Pharmacia Biotech, Uppsala, Sweden) and centrifuged at 2000 x g for 15 minutes. The tumor cells were removed from the top of the Ficoll layer and washed with RPMI 1640 medium.

Spheroid Culture

The method used to generate spheroids was based on the liquid overlay technique.27 To prohibit cell adhesion to a substratum, the wells of 24-well tissue culture plates (Becton Dickinson, Franklin Lakes, NJ) were coated with 200 µl of 0.5% SeaKem LE agarose (BioWittaker Molecular Applications, Rockland, ME) in serum-free media, and allowed to solidify for 30 minutes at 20°C. NIH:OVCAR5 or SKOV3 cells were grown in monolayer cultures, released with 0.5% trypsin, 2 mmol/L ethylenediaminetetraacetic acid as described previously,28 and resuspended in complete cell culture media at 5000 to 50,000 cells/ml. Cell suspensions were layered onto the top of the solid agarose-coated plates at a volume of 1 ml/well, and then incubated for 48 hours at 37°C. By this technique, cells remained afloat in the media, and did not become incorporated or implanted in the solidified agarose that coats the wells of the 24-well plates. After 48 hours, the resulting spheroids were gently removed from the surface of the solidified agarose and centrifuged at 300 x g for 3 minutes to remove single cells before use in subsequent experiments.

Antibodies

Purified monoclonal antibodies (mAbs) that block the adhesive activity of human integrin subunits {alpha}1 (clone FB12), {alpha}2 (clone P1E6), {alpha}3 (clone P1B5), {alpha}4 (clone P1H4), {alpha}5 (clone P1D6), {alpha}6 (clone GoH3), and {alpha}vß3 (clone LM609) were purchased from Chemicon International (Temecula, CA). A purified mAb against the human ß1-integrin subunit that stimulates cell adhesion to ECM proteins (clone 21C8) was also purchased from Chemicon International. Purified immunoglobulin (IgG) of mouse mAb P5D2, which blocks the adhesive activity of human ß1-integrin subunits, was provided by Dr. Leo Furcht (University of Minnesota). Affinity-purified IgG of mAb IM7, which blocks the hyaluronan-binding site of CD44, was purchased from Pharmingen (San Diego, CA). Polyclonal rabbit IgG against human fibronectin was purchased from Calbiochem-Novabiochem Corporation (San Diego, CA). Normal mouse IgG and normal goat serum were purchased from Sigma.

ECM Molecules

Type IV collagen, isolated from mouse Engelbreth-Holm-Swarm tumor, was purchased from Life Technologies. Mouse Engelbreth-Holm-Swarm laminin, prepared as previously described,29 was provided by Dr. Leo Furcht, University of Minnesota. Human plasma fibronectin, purified as described,30 was provided by Dr. James McCarthy, University of Minnesota.

Proliferation Assays

Single-cell suspensions of NIH:OVCAR5 or SKOV3 cells were added to 24-well tissue culture plates to form monolayers, or to agarose-coated 24-well plates to form spheroids, at a density of 500 cells/200 µl. The cells were cultured in complete cell culture media for up to 21 days. At various time points, 2 mg/ml WST-1 (Boehringer-Mannheim Corporation, Indianapolis, IN) was added to each well and incubated for 2 hours. WST-1 is a tetrazolium salt that is reduced by mitochondrial dehydrogenases to form a formazan dye. The formazan product was quantitated by a SpectaMax 250 scanning multiwell spectrophotometer (Molecular Devices Corporation, Sunnyvale, CA) by measuring absorbance at 450 nm. These experiments were performed in quadruplicate.

Role of Adhesion Molecules in Multicellular Aggregation

To examine the role of cell surface receptors in ovarian carcinoma spheroid formation, single-cell suspensions of NIH:OVCAR5 or SKOV3 cells in serum-free media were added to agarose-coated 24-well plates at a density of 5000 cells/200 µl in the presence of 10 µg/ml of normal mouse IgG or mAbs against integrin subunits or CD44. To examine the effect ECM proteins on spheroid formation, the cells were incubated in the presence of 25 µg/ml of fibronectin, laminin, type IV collagen, or ovalbumin. The cells were incubated at 37°C for up to 24 hours in a humidified incubator, examined under a light microscope, and photographed.

Expression of Cell Surface Receptors and ECM Molecules in Spheroids

NIH:OVCAR5 spheroids were collected by centrifugation at 300 x g for 3 minutes and resuspended in 80 µl of expired human plasma (American Red Cross, Minneapolis, MN). To suspend the spheroids in a semisolid clot suitable for embedding, 40 µl of 10 U/ml of human thrombin was added to the suspension. Alternately, NIH:OVCAR5 spheroids were suspended in 1% agarose. After polymerization, the clots were embedded in OCT frozen embedding material (Fisher Scientific, Pittsburgh, PA) on dry-ice. Six µm-thick sections were cut on a cryostat and collected on poly-L-lysine-coated slides. The sections were washed with phosphate-buffered saline (PBS), pH 7.6, and blocked with normal goat serum for 30 minutes. The samples were then incubated with 1 µg/ml of the primary mouse IgG for 1 hour, followed by incubation with the secondary goat anti-mouse biotinylated antibody for 30 minutes. Endogenous peroxidase was quenched by incubating the sections in 0.3% H2O2 in PBS for 30 minutes. The sections were incubated for 30 minutes with Vectastain ABC reagent (Vector Laboratories, Burlingame, CA) and developed with the peroxidase substrate solution to obtain optimal color. The enzymatic reaction was quenched with excess PBS, and the sections were fixed in 2% formaldehyde, followed by mounting with Cytoseal (Fisher Scientific), and examination under a light microscope. In some cases, the samples were counterstained with methyl green stain (Vector Laboratories) according to the manufacturer’s instructions.

Adhesion Assays

Glass chamber slides (Nalge Nunc International, Naperville, IL) were coated with 50 µg/ml of fibronectin, laminin, type IV collagen, or ovalbumin in PBS for 16 hours at 37°C. The slides were blocked with 2 mg/ml ovalbumin in PBS for 1 hour at 37°C. Approximately 50 to 80 NIH:OVCAR5 spheroids in 200 µl of serum-free medium were added to the slides and incubated for up to 4 hours at 37°C. The total number of spheroids in each sample was manually counted, and then nonadherent cells were gently rinsed off with PBS. Adherent cells were fixed with 2% formaldehyde in PBS, stained with Diff-Quik (Dade Behring Inc., Newark, DE), sealed with Cytoseal, and manually counted under a light microscope.

Inhibition of Spheroid Adhesion

NIH:OVCAR5 spheroids were allowed to adhere to glass chamber slides as described above, except that the spheroids were incubated in the presence of 10 µg/ml of normal mouse IgG or mAbs against the {alpha}2-, {alpha}5-, {alpha}6-, or ß1-integrin subunits, or CD44. After a 2-hour incubation, the total number of spheroids that were present in each chamber was manually counted before the nonadherent cells were removed as described above. The adherent spheroids were fixed, stained, manually counted, and expressed as a percentage of the total spheroids initially added to each chamber.

Statistical Analysis

Student’s t-test was performed as a test of significance with the use of Microsoft Excel 1997 (Microsoft Co., Redmond, WA). P values of <0.01 were considered to indicate statistically significant differences.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Formation of Ovarian Carcinoma Spheroids

Multicellular aggregates of ovarian carcinoma cells were observed in the ascitic fluid obtained from six different patients diagnosed with stage III or stage IV ovarian carcinoma. The sizes of the multicellular aggregates in the patient samples ranged from 50 to 750 µm in diameter; a representative sample is shown in Figure 1a . All of the patients’ samples contained spheroids of varying sizes, and the range of sizes of the spheroids varied from patient to patient. Typically, however, the spheroids ranged in size from a score of - to ++ (see Table 1 ), with the majority of the spheroids scoring as ++. It was not possible to disaggregate these spheroids into a single-cell suspension, even when the spheroids were physically manipulated (via repetitive pipetting) and treated with trypsin (not shown).



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  		Figure 1. Ovarian carcinoma spheroid formation. Ovarian carcinoma cells obtained from patients’ ascitic fluid, the NIH:OVCAR5 ovarian carcinoma cell line, or the SKOV3 ovarian carcinoma cell line were cultured in 0.5% agarose-coated 24-well plates at a density of 20,000 cells/well for 48 hours then photographed. These pictures were representatives of ovarian carcinoma cells obtained from one of the six different patients diagnosed with serous ovarian carcinoma (a), the NIH:OVCAR5 cell line (b), and the SKOV3 cell line (c). Scale bar, 250 µm.

 

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  		Table 1. Quantitation of Spheroid Formation

 
Ovarian carcinoma cell lines were cultured in agarose-coated plates to determine whether spheroid phenotypes could be generated in the absence of an adhesive substratum. After 48 hours, the NIH:OVCAR5 ovarian carcinoma cell line formed spheroids (Figure 1b) that appeared similar to the multicellular aggregates found in ovarian carcinoma patients’ ascites samples (Figure 1a) and were similarly resistant to physical manipulation and trypsin treatment. In addition, like the multicellular aggregates obtained from the patients’ ascites samples, NIH:OVCAR cells formed spheroids of varying sizes that ranged from 60 to 400 µm in diameter, with a score of ++ in size.

In contrast, the SKOV3 ovarian carcinoma cell line formed irregular multicellular aggregates (Figure 1c) that dispersed when subjected to mild agitation. These SKOV3 cell aggregates were significantly larger than those found in patients’ ascites samples; SKOV3 spheroids contained hundreds of cells and scored as +++ by our criteria (Table 1) . In all cases, the cells were viable, as determined by trypan blue staining (data not shown).

Aggregation into Multicellular Spheroids Decreases the Proliferative Abilities of Ovarian Carcinoma Cell Lines

To more fully examine the effects of multicellular aggregation on the viability and proliferative ability of the cells, NIH:OVCAR5 and SKOV3 cells were cultured as monolayers or multicellular aggregates for up to 21 days (Figure 2) . When cultured in tissue culture-treated plates as monolayers, the proliferation rates of the NIH:OVCAR5 cells (Figure 2a , filled squares) and SKOV3 cells (Figure 2b , filled squares) increased until 4 days, when confluence was achieved. In striking contrast, the proliferative rates of both NIH:OVCAR5 cells (Figure 2a , open circles) and SKOV3 cells (Figure 2b , open circles) cultured in agarose-treated 24-well plates, which prohibited the adhesion of the cells to a substratum, were initially much lower. The proliferative rates of the NIH:OVCAR5 spheroids and the SKOV3 multicellular aggregates slowly increased until they approached that of the monolayers by 21 days. The multicellular aggregates and monolayers that formed were viable, as determined by trypan blue and propidium iodide staining, which identify dead cells, and immunohistochemical staining for annexin V, which identifies apoptotic cells (data not shown). Furthermore, samples were inspected under a light microscope to ensure nearly complete incorporation of cells into spheroids and that monolayers had not formed either beneath or atop the agarose applied to the wells (not shown).



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  		Figure 2. Proliferative index of ovarian carcinoma cells grown as monolayers or spheroids. NIH:OVCAR5 (a) and SKOV3 cells (b) were added to agarose-coated wells at a concentration of 500 cells/well and cultured as spheroids (open circles) or as monolayers (filled squares) for up to 21 days. The level of proliferation was quantitated as described in the Material and Methods. Data are expressed are mean ± SD.

 
Spheroid Formation Is Mediated by {alpha}5- and ß1-Integrin Subunits

We have previously shown that ß1-integrin subunits mediate the adhesion of single-cell suspensions of ovarian carcinoma cells to ECM molecules and mesothelial cells.15 We therefore hypothesized that ß1-integrins also play a role in ovarian carcinoma spheroid formation. Single-cell suspensions of NIH:OVCAR5 cells were incubated in serum-free medium in the presence or absence of mAbs that blocked or stimulated the ß1-integrin subunit for up to 24 hours (Figure 3) . At 8 hours, spheroids began to form in serum-free medium (Figure 3a) , scored as ++. Spheroid formation was accelerated by a mAb that stimulates the adhesive abilities of human ß1-integrin subunits (Figure 3b) , scored as +++. Spheroid formation was inhibited by a mAb that blocks the binding site of the ß1-integrin subunit (Figure 3c) , scored as +/-. At 24 hours, large spheroids, scored as +++, had formed in serum-free medium (Figure 3d) and in the presence of the ß1-integrin-stimulating mAb (Figure 3e) . The ß1-integrin-blocking mAb continued to partially retard spheroid formation (Figure 3f) , scored as ++/+++. These mAbs had similar effects on the aggregation of SKOV3 cells into multicellular aggregates (not shown). These data suggest that ß1-integrin subunits may mediate the initial formation of ovarian carcinoma spheroids.



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  		Figure 3. Formation of ovarian carcinoma spheroids is mediated by ß1-integrins. Single-cell suspensions of NIH:OVCAR5 cells at a density of 5000 cells/200 µl were added to agarose-coated wells for 8 hours (a–c) or 24 hours (d–f). The cells were incubated in serum-free medium (a and d) or in the presence of 10 µg/ml of a mAb that stimulates ß1-integrin subunits (b and e), or 10 µg/ml of a mAb that blocks ß1-integrin subunits (c and f). Scale bar, 250 µm.

 
We have previously shown that {alpha}-integrin subunits and CD44 also mediate the adhesion of ovarian carcinoma cells to ECM molecules and mesothelial cells.15 To determine the role of these cell surface receptors in ovarian carcinoma spheroid formation, single-cell suspensions of NIH:OVCAR5 cells were incubated in serum-free medium in the presence of normal mouse IgG or blocking mAbs against {alpha}-integrin subunits, integrin {alpha}vß3, or CD44 (Figure 4) . Spheroid formation was inhibited by a mAb against the {alpha}5-integrin subunit (Figure 4f) and scored as +/-. In contrast, large spheroids, scored as +++, formed in the presence of normal mouse IgG (Figure 4a) or mAbs against the {alpha}1-integrin subunit (Figure 4b) , {alpha}2-integrin subunit (Figure 4c) , {alpha}3-integrin subunit (Figure 4d) , {alpha}4-integrin subunit (Figure 4e) , {alpha}6-integrin subunit (Figure 4g) , integrin {alpha}vß3 (Figure 4h) , or CD44 (Figure 4i) . These mAbs had similar effects on the aggregation of SKOV3 cells into multicellular aggregates (not shown). This suggests that the {alpha}5ß1-integrin may mediate ovarian carcinoma spheroid formation.



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  		Figure 4. Formation of ovarian carcinoma spheroids is mediated by the {alpha}5-integrin subunit. NIH:OVCAR5 cells at a density of 5000 cells/200 µl were added to agarose-coated wells in serum-free medium in the presence of 10 µg/ml of mouse IgG (a) or 10 µg/ml of blocking mAbs against the {alpha}1-integrin subunit (b), {alpha}2-integrin subunit (c), {alpha}3-integrin subunit (d), {alpha}4-integrin subunit (e), {alpha}5-integrin subunit (f), {alpha}6-integrin subunit (g), integrin {alpha}vß3 (h), or CD44 (i) for 16 hours. Scale bar, 500 µm.

 
To examine the effect of ECM proteins, the ligands of integrins, on spheroid formation, single-cell suspensions of NIH:OVCAR5 cells were cultured at a density of 5000 cells/well in agarose-coated wells in serum-free media in the presence of 25 µg/ml fibronectin, laminin, or type IV collagen (Figure 5) . The addition of exogenous fibronectin enhanced spheroid formation (Figure 5b) , scored as +++, compared to the ovalbumin control (Figure 5a) , scored as +/++. Enhanced spheroid formation was also observed in the presence of laminin (Figure 5c) , scored as +++, but not in the presence of type IV collagen (Figure 5d) , scored as +/++. These results suggest that ECM proteins affect ovarian carcinoma spheroid formation.



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  		Figure 5. Addition of exogenous ECM proteins alters the formation of ovarian carcinoma spheroids. A single-cell suspension of NIH:OVCAR5 cells at a density of 5000 cells/200 µl was cultured in agarose-coated plates in serum-free medium with 25 µg/ml of ovalbumin (a), fibronectin (b), laminin (c), or type IV collagen (d) for 16 hours and then photographed. Scale bar, 250 µm.

 
Immunolocalization of Adhesion Molecules in Spheroids

The expression of integrin subunits and CD44 on NIH:OVCAR5 spheroids was analyzed by immunohistochemistry (Figure 6) . The ovarian carcinoma spheroids, which were embedded in thrombin clots, stained positively for integrin subunits {alpha}2 (Figure 6c) , {alpha}3 (Figure 6d) , {alpha}5 (Figure 6f) , {alpha}6 (Figure 6g) , and ß1 (Figure 6h) . In addition, the ovarian carcinoma spheroids stained positively for CD44 (Figure 6i) . In contrast, the integrin subunits {alpha}1 (Figure 6b) and {alpha}4 (Figure 6e) were not detected in the spheroids.



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  		Figure 6. Spheroids express adhesion molecules. NIH:OVCAR5 spheroids were suspended in thrombin clots, embedded in OCT, sectioned at 6 µm thick, and stained with mouse IgG (a) or mAbs against the {alpha}1-integrin subunit (b), {alpha}2-integrin subunit (c), {alpha}3-integrin subunit (d), {alpha}4-integrin subunit (e), {alpha}5-integrin subunit (f), {alpha}6-integrin subunit (g), ß1-integrin subunit (h), or CD44 (i). Scale bar, 1 mm.

 
The potential interaction of the {alpha}5ß1-integrin and its ligand, fibronectin, in fully formed NIH:OVCAR5 ovarian carcinoma spheroids was also assessed by immunohistochemical staining (Figure 7) . Because the human plasma used to make thrombin clots contains fibronectin, the spheroids in this set of experiments were embedded in agarose clots. Also, the samples were counterstained with methyl green stain after immunostaining. The {alpha}5- and ß1-integrin subunits were detected on the surface of individual NIH:OVCAR5 cells in the spheroids (Figure 7, b and c , respectively). Fibronectin was also detected on the surface of the cells and in the ECM surrounding them (Figure 7d) . This suggests that interactions between the {alpha}5ß1-integrin and fibronectin may continue to mediate early adhesion events in ovarian carcinoma spheroids. No staining was observed on the surface of cells incubated in the presence of normal mouse IgG (Figure 7a) .



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  		Figure 7. Localization of the {alpha}5- and ß1-integrin subunits and fibronectin in spheroids. NIH:OVCAR5 spheroids were suspended in agarose clots, embedded in OCT, sectioned at 6 µm thick, and stained with normal mouse IgG (a), a mAb against the {alpha}5-integrin subunit (b), a mAb against the ß1-integrin subunit (c), or a polyclonal antibody against fibronectin (d). The slides were then counterstained with methylene green. The {alpha}5- and ß1-integrin subunits, as well as fibronectin, localized to the cell surface and were concentrated at points of cellular contact. Scale bar, 100 µm.

 
Ovarian Carcinoma Spheroid Adhesion to ECM Proteins Is Mediated by Integrins

The ability of NIH:OVCAR5 spheroids to adhere to ECM components was assessed as a model to determine whether spheroids are inherently unable to adhere, which would explain the free-floating multicellular aggregates found in ovarian carcinoma patients’ ascites fluid. Only NIH:OVCAR5 cells were used in the following series of experiments, because of their phenotypic resemblance to patient ascites cells, in that the NIH:OVCAR5 spheroids remained intact after manipulations, unlike the SKOV3 multicellular aggregates, which dispersed with even minimal manipulation. To determine the ability of ovarian carcinoma spheroids to adhere to ECM proteins, NIH:OVCAR5 spheroids were allowed to adhere to glass chamber slides coated with 50 µg/ml of fibronectin, laminin, or type IV collagen for up to 4 hours (Figure 8) . Spheroid adhesion to all three ECM proteins occurred in a time-dependent manner, with maximum adhesion attained by 4 hours. Approximately 90% of the spheroids adhered to fibronectin (Figure 8 , squares) and laminin (Figure 8 , triangles) at 4 hours, and ~80% of the spheroids adhered to type IV collagen (Figure 8 , circles) at 3 hours. The spheroids failed to adhere to chamber slides coated with ovalbumin (Figure 8 , diamonds). These data suggest that ovarian carcinoma spheroids have the ability to adhere to ECM glycoproteins, despite the fact that many ovarian carcinoma multicellular aggregates are found floating in the ascitic fluid of patients and seemingly fail to adhere in vivo.



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  		Figure 8. Adhesion of NIH:OVCAR5 spheroids to ECM proteins. NIH:OVCAR5 spheroids were incubated on glass chamber slides coated with 50 µg/ml of ovalbumin (diamonds), fibronectin (squares), laminin (triangles), or type IV collagen (circles) for up to 4 hours. Nonadherent cells were washed away, and the remaining adherent cells were fixed, stained, and photographed. Scale bar, 250 µm. Data are expressed as mean ±SD.

 
The role of integrin subunits in NIH:OVCAR5 spheroid adhesion toward fibronectin, laminin, and type IV collagen was investigated (Table 2) . Spheroids were allowed to adhere to the ECM proteins or ovalbumin for 2 hours in the presence of blocking mAbs against integrin subunits. Spheroid adhesion to all three ECM proteins was almost completely inhibited in the presence of a blocking mAb against the ß1-integrin subunit (P < 0.001). In addition, a mAb against the {alpha}5-integrin subunit inhibited spheroid adhesion to fibronectin by 60% (P < 0.005), a mAb against the {alpha}6-integrin subunit decreased spheroid adhesion to laminin by 40% (P < 0.01), and a mAb against the {alpha}2-integrin subunit decreased spheroid adhesion to type IV collagen by 55% (P < 0.01). Spheroid adhesion to all three ECM proteins was slightly increased in the presence of a ß1-integrin-stimulating mAb, but was not significantly affected by blocking mAbs against the {alpha}3- or {alpha}4-integrin subunits, integrin {alpha}vß3, or CD44 (data not shown). These results suggest that spheroid adhesion to secondary growth sites is a complex, multivalent phenomenon that is mediated by multiple cell-matrix interactions between integrins and ECM components.


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  		Table 2. Spheroid Adhesion to ECM Proteins Is Mediated by Integrins

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In ovarian carcinoma, both single cells and multicellular aggregates are found in patients’ ascitic fluid.2 Extensive research has been performed using single-cell suspensions of cell lines derived from primary ovarian carcinoma tumors and ascites cells. However, because multicellular aggregates of ovarian carcinoma cells are not suited for assays that require single-cell suspensions, they have been primarily overlooked by the scientific community. In this study, we generated ovarian carcinoma cell spheroids, which are intermediate in complexity between monolayers and solid tumors, and more closely approximate the in vivo conditions of ovarian carcinoma patients than single-cell suspensions or monolayers. We used this model to identify adhesion molecules that mediate the formation of ovarian carcinoma spheroids and their subsequent adhesion to ECM proteins.

Early attempts to create spheroids from ovarian cancer cells were unsuccessful.4 Ovarian carcinoma spheroids were eventually created; however, some primary tumor cells and cell lines required weeks to form stable spheroids, whereas others did not form multicellular aggregates at all.2,4-9 In this study, we used the liquid overlay method27 to show that the NIH:OVCAR5 ovarian carcinoma cell line formed stable spheroids within 48 hours. These spheroids appeared similar to those present in vivo in patients’ ascites samples. In contrast, the SKOV3 ovarian carcinoma cell line failed to form stable spheroids unless incubated for more than 14 days (not shown). Another group was able to generate SKOV3 spheroids in 10 days using an alternate method.8 This disparity may reflect the heterogeneity of ovarian carcinoma cells, even within well-defined cell lines. It is also important to note that cell lines, which are selected for their ability to proliferate and adhere, may form spheroids at a much faster rate than ovarian carcinoma cells in vivo.

Condensation into spheroids or multicellular aggregates decreased the proliferative abilities of the NIH:OVCAR5 and SKOV3 ovarian carcinoma cell lines when compared to the same cells cultured as monolayers. Both cell lines underwent a marked decrease in proliferation in the absence of adhesion to a substratum. However, the proliferative rates of the spheroids eventually approached those of the confluent monolayers. The cells in the spheroids and monolayers remained viable, as determined by staining for markers of cell death and apoptosis. Previous reports show that increased percentages of ovarian carcinoma monolayer cells accumulate in G2/M phase compared to spheroid cells when exposed to Taxol.9 The spheroid cells may be arrested at an earlier step of the cell cycle, which may inhibit both cell proliferation and apoptosis. The slower proliferative rate of ovarian carcinoma spheroids may protect them from therapies directed against fast-growing tumor cells.

Although several groups have generated spheroids from ovarian carcinoma cells,5-9 the biological mechanisms by which the spheroids formed have not been defined. The NIH:OVCAR5 and SKOV3 ovarian carcinoma cell lines express a variety of adhesion molecules on their surfaces, including integrins, ICAM-1, and CD44.15,18 In this study, we report that a functional blocking mAb against the ß1-integrin subunit inhibited the formation of spheroids by NIH:OVCAR5 cells at an 8-hour time point, whereas a mAb that stimulates ß1-integrin-mediated cell adhesion hastened the phenomenon. These data suggest that ß1-integrin subunits mediate the initial formation of ovarian carcinoma spheroids. The incomplete inhibition of the ß1-integrin subunits at the 24-hour time point suggests that if the ß1-integrin subunits are inactivated, the ovarian carcinoma cells may possess a compensatory mechanism to facilitate spheroid condensation. However, it is also possible that the mAbs against the ß1-integrin subunits may simply be internalized by 24 hours, eventually allowing the ovarian carcinoma cells to condense into spheroids. We also report that a functional blocking mAb against the {alpha}5-integrin subunit inhibited the formation of spheroids by NIH:OVCAR5 cells. Monoclonal antibodies that blocked the functional sites of the {alpha}1-, {alpha}2-, {alpha}3-, {alpha}4-, and {alpha}6-integrin subunits, integrin {alpha}vß3, or CD44 had no effect on spheroid formation. This suggests that ovarian carcinoma spheroid formation may be regulated by the integrin {alpha}5ß1.

The chief ligand of the {alpha}5ß1-integrin is fibronectin.31 Fibronectin has been reported to promote the adhesion of ovarian carcinoma cells32,33 and may crosslink these cells via their {alpha}5ß1-integrin receptors. In our hands, the addition of exogenous fibronectin promoted ovarian carcinoma spheroid formation, which supports our theory that ovarian carcinoma spheroid formation may be mediated by the {alpha}5ß1-integrin. Interestingly, the addition of exogenous laminin also enhanced spheroid formation, whereas type IV collagen inhibited spheroid formation. It is possible that these adhesion molecules may indirectly alter spheroid formation, perhaps by signal transduction mechanisms.

By immunohistochemistry, we detected {alpha}5-integrin subunits, ß1-integrin subunits, and fibronectin in regions of cell-cell contact on the surface of NIH:OVCAR5 spheroids generated after 48 hours in tissue culture. These results are consistent with our finding that the {alpha}5ß1-integrin mediates NIH:OVCAR5 spheroid formation. Although we observed fibronectin on the surface of ovarian carcinoma spheroids, we did not determine whether the fibronectin was secreted by the ovarian carcinoma cells themselves or whether it was incorporated into their pericellular matrix from the complete medium in which they were initially cultured. Fibronectin has been detected in peritoneal fluids obtained from both normal and ovarian cancer patients, and elevated expression of fibronectin has been measured by others in malignant ascites fluid.34 Therefore, we decided to perform the spheroid formation portion of this assay in the presence of sera, because this would more closely approximate in vivo conditions. Interestingly, NIH:OVCAR5 cells grown in fibronectin-free serum substitute media for 48 hours did form spheroids when cultured in agarose-coated wells (data not shown). Altered ECM composition has been reported in glioma cell spheroids, which were found to contain fibronectin and a small proteoglycan not detected when the cells were cultured in monolayers.35 Such alterations may facilitate spheroid formation, suggesting that the cells themselves can manufacture the additional ECM needed or incorporate it from exogenous sources. These results suggest that cell surface {alpha}5ß1-integrins may mediate ovarian carcinoma cell aggregation via interactions with fibronectin that the cells have synthesized and retained on their surfaces or sequestered from their environment.

The {alpha}5ß1-fibronectin interaction may later be augmented or replaced by other cell-cell interactions, including the gap junctions, tight junctions, and desmosomes detected in mature spheroids.36-38 Squamous epithelial cells grown as spheroids express significantly less epidermal growth factor receptors than squamous cell monolayers.39 Altered levels of expression of ICAM-1, CD44, and LFA-1 have been reported in cancer cells grown as spheroids compared to monolayers.40 By flow cytometric analysis, we previously detected {alpha}1-, {alpha}2-, {alpha}3-, {alpha}5-, {alpha}6-, and ß1-integrin subunits on the surface of single-cell suspensions of the NIH:OVCAR5 cell line.18 In this study, we report that the {alpha}2-, {alpha}3-, {alpha}5-, {alpha}6-, and ß1-integrin subunits, but not {alpha}1-integrin subunits, were detected on NIH:OVCAR5 spheroids by immunohistochemistry. Taken together, these data suggest that condensation into multicellular aggregates resulted in decreased expression of the {alpha}1-integrin subunit, which in turn may result in a similarly decreased ability to adhere to a substratum. The condensation of ovarian carcinoma cells into spheroids or multicellular aggregates may induce other alterations in adhesion molecule expression or ECM composition, with concurrent effects on their adhesive abilities.

An earlier study of single-cell suspensions of NIH:OVCAR5 cells reported >90% adhesion of these cells to fibronectin, laminin, and type IV collagen within 30 minutes15 However, in this study we found that NIH:OVCAR5 spheroids required 4 hours to achieve maximum adhesion. Factors that may contribute to the longer time period required for spheroid adhesion to various substrates include: decreased expression of receptors, decreased avidity of receptors, competing cell-cell and cell-matrix interactions within the spheroids, physical constraints that limit the cells’ ability to spread onto the anchoring surfaces, and the effects of mechanical forces on a structure with a greater surface area/volume ratio. Any of these factors may contribute to the presence of free-floating multicellular aggregates found in ovarian carcinoma patient ascites.2

In this study, we report that the adhesion of ovarian carcinoma spheroids to ECM components is a ß1-integrin-mediated event. The nearly complete inhibition of adhesion by a blocking mAb against the ß1-integrin subunit, coupled with partial inhibition in the presence of blocking mAbs against {alpha}-integrin subunits, suggest that multiple integrin-ECM interactions are involved in the process. Our results suggest that the interactions of the {alpha}5ß1-integrin with fibronectin, the {alpha}6ß1-integrin with laminin, and the {alpha}2ß1-integrin with type IV collagen mediate ovarian carcinoma spheroid adhesion. Recently, Kawano and colleagues,41 reported that the {alpha}2ß1-, {alpha}6ß1-, {alpha}3ß1-integrins mediated the adhesion of squamous epithelial multicellular aggregates to type I collagen, laminin 1, and laminin 5, respectively. Taken together, these data suggest that spheroid adhesion is an integrin-dependent event, although the precise integrin-ligand interactions involved may be tissue-specific. However, additional cell-matrix and cell-cell interactions, which have been reported in spheroids generated from other cell types,36-40 may mediate the initial formation and continued maintenance of spheroid morphology as they progress with time.

The examination of multicellular aggregates in ovarian carcinoma may be of clinical importance. This study demonstrates that ovarian carcinoma cells cultured as spheroids exhibit decreased proliferative and adhesive properties compared to ovarian carcinoma cells cultured as monolayers. Ovarian carcinoma spheroids are less sensitive than monolayers to cancer drugs and ionizing radiation,4,5,9,42 which may be partly because of the low proliferative rates we report in this study. Also, the kinetics of drug absorption are altered in ovarian carcinoma spheroids, compared to ovarian carcinoma monolayers.43 Taken together, these data suggest that spheroids may represent a tenacious, long-term source of secondary tumor growth in ovarian carcinoma that is not addressed by current therapeutic strategies, which target highly proliferative cancer cells. The biological properties of ovarian carcinoma spheroids require further study to better understand their significance in secondary tumor growth and to effectively eradicate them during the treatment of the disease. The model of ovarian carcinoma spheroid formation and adhesion presented in this study will also lay the groundwork for future studies in which ovarian carcinoma spheroids isolated from patient ascites fluid can now be monitored for their cell surface receptors and adhesive properties.


   Acknowledgements
 
We thank Dr. James McCarthy for providing fibronectin, Dr. Leo Furcht for providing laminin and the mAb P5D2 against the ß1-integrin subunit, Dr. Robert C. Bast, Jr. for providing the SKOV3 cell line, Drs. Thomas Hamilton and Judah Folkman for providing the NIH:OVCAR5 cell line; and the Tissue Procurement Facility of the University of Minnesota (a Comprehensive Cancer Center designated by the National Cancer Institute, supported by a Cancer Center Support grant from NIH/NCI, grant P30CA77598) for providing the patient ascites samples.


   Footnotes
 
Address reprint requests to Dr. Amy P. N. Skubitz, Department of Laboratory Medicine and Pathology, MMC 609, 420 Delaware St. SE, University of Minnesota, Minneapolis, MN 55455. E-mail: skubi002{at}tc.umn.edu

Supported by a Cancer Biology Training grant from the National Institutes of Health/National Cancer Institute (CA09138-25 to R. C. C.), a grant from the Department of the Army (DA/DAMD17-99-1-9564), a grant from the Minnesota Medical Foundation (SMF-2078-99), and a Grant-in-Aid of Research from the Office of the Vice President for Research and Dean of the Graduate School of the University of Minnesota (no. 18118).

The content of the information presented in this manuscript does not necessarily reflect the position of the United States Government.

Accepted for publication August 17, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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