Beta 1-integrins regulate the formation and adhesion of ovarian carcinoma multicellular spheroids

Abstract

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 beta 1-integrin-stimulating monoclonal antibody or exogenous fibronectin, but was inhibited by blocking monoclonal antibodies against the alpha 5- or beta 1-integrin subunits. By immunohistochemical staining, alpha 2-, alpha 3-, alpha 5-, alpha 6-, and beta 1-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 beta 1-integrin subunit completely inhibited adhesion of the spheroids to all three proteins. These results suggest that interactions between the alpha 5 beta 1-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.

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.

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.

Figure 3.

Formation of ovarian carcinoma spheroids is mediated by β1-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 β1-integrin subunits (b and e), or 10 μg/ml of a mAb that blocks β1-integrin subunits (c and f). Scale bar, 250 μm.

Figure 4.

Formation of ovarian carcinoma spheroids is mediated by the α5-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 α1-integrin subunit (b), α2-integrin subunit (c), α3-integrin subunit (d), α4-integrin subunit (e), α5-integrin subunit (f), α6-integrin subunit (g), integrin αvβ3 (h), or CD44 (i) for 16 hours. Scale bar, 500 μm.

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.

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 α1-integrin subunit (b), α2-integrin subunit (c), α3-integrin subunit (d), α4-integrin subunit (e), α5-integrin subunit (f), α6-integrin subunit (g), β1-integrin subunit (h), or CD44 (i). Scale bar, 1 mm.

Figure 7.

Localization of the α5- and β1-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 α5-integrin subunit (b), a mAb against the β1-integrin subunit (c), or a polyclonal antibody against fibronectin (d). The slides were then counterstained with methylene green. The α5- and β1-integrin subunits, as well as fibronectin, localized to the cell surface and were concentrated at points of cellular contact. Scale bar, 100 μm.

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.