Supplementary Figure S3. Inhibition of myosin IIa activity increases the kinetics of cell aggregation. Down-regulation of myosin IIa by transfection of MYH9 siRNA increased the kinetics of cell aggregation in HCT116 cells (see Fig. 5). A, western blotting (left) and immunofluorescent staining with anti-myosin IIA antibodies (right) demonstrates that MYH9 siRNA transfection inhibits myosin IIA (Myo IIA) expression in HCT116 cells after 72 hours. B, the selective myosin II inhibitor blebbistatin had a similar effect on aggregation as had MYH9 siRNA, as shown in time-lapse images (left) and measurements of area variation (right).
ARTICLE ABSTRACT
Cell aggregation is frequently impaired during the growth of primary tumors and the formation of metastatic lesions. Cell aggregation depends on cell–cell adhesion; however, no rigorous approach exists to monitor and quantify it accurately in the absence of the confounding factors of cell–substrate adhesion and the resulting cell motility on the substrate. We report here a highly reproducible, automated, microscopy-based quantification of tumor-cell spheroid formation in the absence of cell–substrate adhesion and use it to characterize cell aggregation dynamics in the early steps of this process. This method is based on fluorescence and bright-field microscopy and on a custom MATLAB program to quantify automatically the cells' aggregation kinetics. We demonstrate that the cell–cell adhesion protein E-cadherin and the desmosome proteins DSG2 and DSC2 are important for aggregation. Furthermore, we show that inhibition or silencing of myosin IIa enhances aggregation, suggesting that cytoskeleton tension inhibits tumor cell aggregation. This work opens new avenues to study the principles that govern multicellular aggregation, to characterize the aggregation properties of various tumor cell types, as well as to screen for drugs that inhibit or promote aggregation. Cancer Res; 75(12); 2426–33. ©2015 AACR.
