Tumor spheroid invasion assay

Tumor spheroids were formed using the hanging drop technique^34–36. A cell suspension of 2.5 × 10⁴ cells/ml was prepared and hanging drops were made using 20 μl from this suspension. Thus, hanging drops containing 500 cells each were placed on the inside of the cover of a culture dish and incubated at 37 ^oC²². Formed spheroids were transferred into wells containing 1 mg/ml 3D collagen I gel (Collagen I high concentration solution, Corning Cat.# 354249) using sterile glass Pasteur pipettes and left to grow for the designated time (5 h for MDA-MB-231-LM2 cells and 18 h for MCF-7 cells). The transfer was performed three days later (for SshRNA or RSU-1L shRNA stable cells) (Fig. 2) or the next day (for cells transiently expressing NSC, or RSU-1-X1 siRNA) (Fig. 5). The diameter of the spheroids was monitored by a Nikon Eclipse optical microscope equipped with a digital camera. Pictures were taken immediately (time zero) and at the end of the designated time period. Cell invasion through the surrounding collagen was measured using the ImageJ software and the final spheroid size (average of the major and minor axis length) was compared to the initial size at time zero^22,37. In experiments where cells were subjected to RSU-1-X1 siRNA-mediated silencing, siRNA transfection was performed 24 h prior to formation of hanging drops and invasion was monitored up until 72 h post-transfection. At least 8 spheroids were analyzed per condition and at least two independent experiments were performed²².