Invadopodia formation assay: a promising experiment for targeting breast cancer invasion and metastasis

Abstract

Abstract Introduction/Objective Invadopodia has been found to invade the extracellular matrix (ECM) in multiple cancers, including breast, lung, brain, and prostate, which is highly associated with cancer metastasis. Invadopodia have been shown to form in various cancer cells, such as the MDA-MB-231 breast cancer cell line. Objectives In-vitro testing to validate the anti-cancer invasion drugs using an invadopodia formation assay. Methods/Case Report MDA-MB-231 triple-negative breast cancer cell line was used in this study due to its highly invasive characteristics obtained from American Type Culture Collection. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM), a high-glucose medium containing FBS 10% and 1% penicillin/streptomycin. Cells were maintained in a humidified atmosphere of 95% O2 and 5% CO2 incubator at 37 °C for normal conditions. Cells with low passage numbers were used in order to get accurate results. Then, the gelatin-coated coverslips were prepared for invadopodium assay, washed three times with ddH2O, and transferred to slides containing a drop of antifade, a mounting solution. For invadopodia quantification, invadopodia images on the F-actin, gelatin, and nucleus were taken using the same fluorescence microscope settings (Confocal Laser Leica fluorescent microscope, German). Cells were scored for the presence of underlying invadopodia. The mean percentage of cells with invadopodia formation was calculated for drug-validated tests. Results (if a Case Study enter NA) The study revealed how MDA-MB-231 cells penetrate the gelatin that mimics the ECM-like basement membrane in the human body; this is thought to be a key point during intravasation and extravasation steps in metastasis. The study showed actin-rich filament as red dots in the center of the MDA-MB-231 cells, representing invadopodia in a 3D image under normal conditions. In contrast, in a 2D image, invadopodia are represented as black dots on gelatin in a merged image. Conclusion Breast cancer is one of the leading causes of cancer-related death in the advanced stage. The study helps to promote using invadopodia assay to validate the effectiveness of the potential anti-cancer drugs and predict cancer metastasis in clinical studies. However, invadopodia formation assay can be especially useful for patients with early-stage cancers; it can help to predict which patients are more likely to benefit from aggressive treatment or further monitoring rather than immediate surgical intervention.