Role of Ezrin in cell spheroids formation

NOELIA LUJEA; MARTIN MASNER; KUNDA, PATRICIA

Congreso; LV Annual SAIB Meeting and XIV PABMB Congress; 2019

ERM proteins (Ezrin, Radixin, and Moesin) link the actin cytoskeleton to the cell membrane forming the cell cortex. Thus, the actin cortex plays an important function in cell survival and proliferation. We suggest that the integrity of cortical actin and ezrin could play a role in spheroid formation in soft environments such as fluid. Understanding the mechanism of spheroids formation in fluids is crucial to understand tumor growth in ascitic fluid during ovarian cancer intraperitoneal metastasis. The epithelial ovarian cancer cell line SKOV3 and CRISPR-CAS9 modified cell line against ezrin (Ez6148) were used. Spheroids were generated by two methods: soft agar method, in which cells were cultured in 0.5% agar up to 10 DIV, and the aggregation method, in which cells were allowed to aggregate over an agar base for 24, 48, 72, and 96 h after seeding. Cytochalasin D (CD) was used to disrupt the actin cytoskeleton. Spheroid formation was analyzed by IF and confocal microscopy imaging. The experiments showed that, while SKOV3 in soft agar proliferated and formed spheroids from 3 DIV, Ez6148 did not proliferate at any time of the assay. Furthermore, when cells were allowed to aggregate over an agar base, Ez6148 failed to form a compact spheroid as the control cells did. Moreover, disruption of the actin cytoskeleton with CD did not alter the formation but changed the level of cell compaction at different times after cells seeding. These results show that the anchorage of the actin cytoskeleton to the plasma membrane is essential for the spheroid formation in soft environments, while actin dynamic and organization is not necessary. This reveals the importance of ezrin in favoring the formation of cell spheroids and could have an implication in the development of ovarian cancer intraperitoneal metastasis.