RESUMEN
Three-dimensional (3D) ex vivo cultures allow the study of cancer progression and drug resistance mechanisms. Here, we present a protocol for measuring on-target drug sensitivity in a scaffold-free 3D culture system through quantification of apoptotic tumor cells. We provide detailed steps for sample processing, immunofluorescence staining, semi-high-throughput confocal imaging, and imaged-based quantification of 3D cultures. This protocol is versatile and can be applied in principle to any patient-derived material.
Asunto(s)
Neoplasias Ováricas , Humanos , Neoplasias Ováricas/patología , Neoplasias Ováricas/tratamiento farmacológico , Femenino , Antineoplásicos/farmacología , Técnicas de Cultivo Tridimensional de Células/métodos , Resistencia a Antineoplásicos , Apoptosis/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Células Tumorales Cultivadas , Línea Celular TumoralRESUMEN
The extracellular matrix (ECM) plays critical roles in tumor progression and metastasis. However, the contribution of ECM proteins to early metastatic onset in the peritoneal cavity remains unexplored. Here, we suggest a new route of metastasis through the interaction of integrin alpha 2 (ITGA2) with collagens enriched in the tumor coinciding with poor outcome in patients with ovarian cancer. Using multiple gene-edited cell lines and patient-derived samples, we demonstrate that ITGA2 triggers cancer cell adhesion to collagen, promotes cell migration, anoikis resistance, mesothelial clearance, and peritoneal metastasis in vitro and in vivo. Mechanistically, phosphoproteomics identify an ITGA2-dependent phosphorylation of focal adhesion kinase and mitogen-activated protein kinase pathway leading to enhanced oncogenic properties. Consequently, specific inhibition of ITGA2-mediated cancer cell-collagen interaction or targeting focal adhesion signaling may present an opportunity for therapeutic intervention of metastatic spread in ovarian cancer.