Images of the spheroids were captured during 72 h and analyzed using ImageJ software. 2D Migration and Invasion Assays Two-dimensional migration was investigated using 8 m FluoroBlok? (Corning? Fluoroblok? Cell Culture Inserts, BD biosciences). cell line and on human CD16-transfected Jurkat cells (Physique S1). Consistent with our previous data (21, 22), MesobsFab exhibited a high apparent affinity for CD16 with a low KDapp value (1.8 0.8 nM), compared to that of conventional human IgG Fc fragment (>100 nM) (Table S1). The KDapp values Schisantherin A on HCC1806 cells (4.7 0.9 nM) were in good agreement with previous data obtained with the anti-MSLN sdAb A1 on HELA cells (23), indicating that the specific binding activity of this sdAb was retained in the bispecific format. MesobsFab specificity was also confirmed by competition assays (Physique S1). MesobsFab Decreases the Invasive Properties of TNBC Cell Lines migration/invasion properties of MDA MB 231 and HCC1807 cells. Schisantherin A MesobsFab displayed a reproducible tendency to slightly decrease the migration of HCC1806 and MDA MB 231 cells without reaching significance (Physique 1A). By contrast, a significant decrease of both MDA MB 231 and HCC1806 invasiveness was observed in the presence of MesobsFab (Physique 1B). Open in a separate window Physique 1 MesobsFab binding to mesothelin reduces Schisantherin A HCC1806 invasiveness. Effect of MesobsFab or control bsFab (50 nM) around the migration and invasion of MDA MB 231 and HCC1806 cells. (A) CFSE-stained tumor cells were allowed to migrate toward culture medium supplemented with 5% FBS for 6 or 24 h at 37C. The fluorescence signals measured in the Fluoroblok bottom chambers correspond to migrating cells. A control of low migration was performed by omitting the FBS in the culture medium. (B) CFSE-stained MDA MB 231 and HCC1806 cells were seeded onto a matrigel coated fluoroblok inserts and allowed to migrate in response to serum gradient. 100% inhibition corresponds to the absence of CFSE-stained tumor cells in the bottom chamber. In all panels, data represent the mean SEM from 3 impartial experiments. Statistical significance was determined by Schisantherin A two-tailed Student’s < 0.05, **< 0.001, MesobsFab vs. control bsFab. Formation of Homotypic Multicellular Tumor Spheroids Derived From TNBC Cells Homotypic spheroids were generated from the two TNBC cell lines using the static liquid overlay method (3, 25). Growth of spheroids and changes in morphology were monitored by phase contrast light microscopy. TNBC cells formed cell clusters within 24 h after seeding and reached a characteristic 3D business after 2C4 days as Schisantherin A shown by the formation of more or less compact and round-shaped spheroids and the disappearance of cells in suspension in the growth medium. The mean radius of 4-days spheroids (CV > 10%) was comparable for MDA MB 231 and HCC1806 spheroids (222.9 16.8 vs. 224.1 17.9 m, respectively). HCC1806 spheroids displayed a rather rounded and compact morphology while MDA MB 231 spheroids were less regular and less compact likely due to weaker cell-cell contacts (Physique 2A). As described in the literature, the spheroid periphery consisted of viable cells while necrotic cells were located in the core as evidenced by Hoechst 3342 and Propidium Iodide (PI) staining (Physique S2A). Evolution of cell death during spheroid growth was monitored by PI staining at different time points, revealing a discrete area of necrosis HSPC150 already at day 4 which increased over time (Figures S2B,C). The necrosis process was more pronounced in the HCC1806-spheroids than in the MDA MB 231-spheroids and was accompanied by a visible cellular migration phenomenon. Epithelial/mesenchymal phenotypes of TNBC spheroids were investigated by immunochemistry on 7-day spheroids through the expression of epithelial (E-cadherin) or mesenchymal (Vimentin) markers. MDA MB 231 spheroids presented a high vimentin staining (Physique 2B) and a low E-cadherin expression (Physique 2C) characterizing a mesenchymal-like phenotype while HCC1806 spheroids displayed a strong E-cadherin staining and a lack of vimentin expression suggesting an epithelial phenotype. Open in a separate window Physique 2 Characterization and phenotypic properties of TNBC spheroids. (A) Representative bright field images of MDA MB 231.