β1-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.

Spheroids Formation on Non‐Adhesive Surfaces by Liquid Overlay Technique: Considerations and Practical Approaches

“…[42,60] Agar and agarose were the first biomaterials applied for the growth of spheroids by LOT. [15,37,38,[61][62][63][64][65][66][67][68][69][70] Nowadays, these two materials are extensively used due to their low cost and easy manipulation, which reduces the time spent in the plate-coating procedure ( Table 3). For the preparation of agar or agarose coated plates, first the biomaterials are fully dissolved in water or serum-free medium and then they are sterilized by autoclave.…”

Revisiting the Complexity of the Ovarian Cancer Microenvironment—Clinical Implications for Treatment Strategies

“…Integrin-related mechanisms have been shown to be essential at different stages of EOC progression. For example, multicellular aggregated formation is mediated via b1-integrins and their subsequent attachment to the mesothelium is partially dependent on a5b1-integrins (63,64). Efforts to antagonize a5b1-integrin interactions were implemented in phase II clinical trials, using a chimeric antibody, Volociximab (Table 1), in patients with platinum-resistant advanced EOC, although clinical efficacy was not accomplished (65).…”

Cancer cell spheroids as a model to evaluate chemotherapy protocols

“…7,8 Spheroids, three dimensional architectures of cancer cells, are found in cancer patients with sizes of 250-750 μm. 9 Spheroids formed with cancer cell lines mimic both architecture and share the limited drug penetration properties since drugs are largely confined to the outer cell layers. 5 In other ways, spheroids represent a better model for drug resistance compared with monolayer cultures.…”