Spheroid-Induced Mesothelial Clearance Assay

Spheroids were generated as in [34 (link)]. OVCAR-3 and SKOV3 cell spheroids were formed by incubating 1 × 10³ cells per well in a 96-well U-bottom-shaped culture dish with a cell-repelling surface (Cat# F202003, faCellitate) at 37 °C for 96 hours in the presence of MS-275 (1 𝜇M for OVCAR3, 2.5 𝜇M for SKOV3 cells). The MC monolayer was prepared by plating 30 × 10³ MeT5A cells per well 48-well microplate. Cells were treated with TGFβ1 and IL-1β for 48 hours and with MS-275 for 72 hours. Spheroids were transferred to the dish with the MC monolayer and the images of two cell populations were taken. Spheroid-induced mesothelial clearance was monitored by time-lapse microscopy using an epifluorescence inverted microscope Celldiscoverer 7 (Carl Zeiss AG, Oberkochen, Baden-Württemberg, Germany) equipped with a cage incubator for temperature and CO₂ control. Fluorescence and phase-contrast images (5x objective) were collected for each experimental condition for 24 hours at 4 hours intervals. At 24 hours, the non-fluorescent area in the MC monolayer underneath the spheroid was measured by Celldiscoverer 7 (Carl Zeiss) and normalized to the initial spheroid area. Experiments were conducted at least in triplicate.