Multicellular Tumor Spheroid Migration Assay

Cell spheroids were formed in round-bottomed 96-well tissue culture plates. Briefly, a mixture of 1 × 10⁴ cells, 80–90% unlabeled cells and 10–20% hypoxia fate-mapping cells derived from orthotopic tumors, were plated per well in spheroid formation media 1:1 DMEM (Sigma-Aldrich) and Methocult H4100 (STEMCELL Technologies). Plates were centrifuged at 1200 rpm for 7 min, twice. After 72 hr of incubation, each spheroid was transferred to a Petri dish, where they were individually isolated with the collagen solution (2 mg/mL) and quickly transferred to the center of a semi-cross-linked collagen gel in a 96-well plate at 37°C. After complete cross-linking, warm media was added. Following 4 days in culture, spheroids were imaged in an environmentally controlled microscope every 15 min for 16 hr using an Olympus (UPLFLN 4×) objective in Cytation 5 (BioTek Instruments). Cell trajectories were tracked using MetaMorph software to obtain x,y coordinates at each time. More details are available in the studies by Ju et al. (2017) (link) and Valencia et al. (2015) (link).