Fabrication of Tumor Spheroid-Laden Hydrogels

Tumorspheres were fabricated using Aggrewell Microwell Plates (Stemcell Technologies). Briefly, 1.2×10⁵ cells were seeded into a single well of the Aggrewell plate to form spheroids consisting of 100 cells. After a 48-hour incubation, spheroids were resuspended in phenol red-free serum-free Dulbecco’s Modified Eagle’s Medium (DMEM, Thermo Fisher Sci, 21-063-029) at a density of 1.5 spheroid/μL and used as solvent for HA hydrogel crosslinking. To form hydrogels, 6 wt. % Me-HA was crosslinked in phenol red-free serum-free Dulbecco’s Modified Eagle’s Medium (DMEM, Thermo Fisher Sci, 21-063-029) with a protease-cleavable peptide (KKCG-GPQGIWGQ-GCKK, Genscript). HA-RGD gels were crosslinked with peptide crosslinkers at varying ratios to yield hydrogels with a shear modulus ~300 Pa and a final 1.5 wt. % Me-HA (Supp. Fig. 1). For all experiments, unless otherwise mentioned, a concentration of 3.405 mM peptide crosslinker was selected to yield a shear modulus of 300 Pa. After 1 hour crosslinking in a humidified 37°C chamber, complete cell culture media was added to the hydrogels. For all experiments unless otherwise noted, cell culture media was replenished every 2 days.