S1860

AsPC1 cells (a pancreatic ductal adenocarcinoma cell line) were purchased from American Type Culture Collection (CRL-1682) and grown in Roswell Park Memorial Institute medium (Thermo Fisher Scientific, 11875093) supplemented with 10% fetal bovine serum (FBS) (Biowest, S1860). HUVECs were purchased from PromoCell (C-12203) and grown in endothelial cell growth medium (PromoCell, C-22010). U2-OS osteosarcoma cells were purchased from the Leibniz Institute DSMZ, German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany; ACC 785) and grown in Dulbecco's modified Eagle medium (DMEM; Merck, D5671) supplemented with 10% FBS. MCF10DCIS.com Lifeact–RFP cells were generated previously (Jacquemet et al., 2017 (link)) and cultured in a 1:1 mix of DMEM and F12 (Merck, 51651C) supplemented with 5% horse serum (Gibco, 16050122), 20 ng/ml human epidermal growth factor (Merck, E9644), 0.5 mg/ml hydrocortisone (Merck, H0888-1G), 100 ng/ml cholera toxin (Merck, C8052-1MG), 10 mg/ml insulin (Merck, I9278-5ML) and 1% (vol/vol) penicillin/streptomycin (Merck, P0781-100ML). All cell lines tested negative for mycoplasma. AsPC1 cells were authenticated by DSMZ. All other cell lines were not authenticated.

U2-OS (human osteosarcoma) and MDA-MB-231 (triple-negative human breast adenocarcinoma) cells were grown in DMEM (Dulbecco’s Modified Eagle’s Medium with HEPES modification; Sigma, D1152) supplemented with 10% fetal bovine serum (FCS) (Biowest, S1860). CHO cells were cultured in alpha-MEM, supplemented with 5% FCS and L-glutamine.

Cells isolated by FACS were plated overnight on a 24-well glass bottom plate (P24G-0-10-F; MatTek) coated with Matrigel (BD Biosciences #354234) and placed in an incubator in pre-equilibrated medium (1:1 DMEM Glutamax:MCDB (Sigma-Aldrich), 20% fetal calf serum (FCS; Biowest S1860). The plate was then incubated at 37 °C, 5% CO₂ (Zeiss, Pecon). A Zeiss Observer.Z1 connected with a LCI PlnN × 10/0.8 W phase II objective and AxioCam camera piloted with AxioVision was used. Cells were filmed for up to 5 days, and images were taken every 30 min with bright-field and phase filters and MozaiX 3X3 (Zeiss). Raw data were transformed and presented as a video.