Protocol for generating dormant human brain metastatic breast cancer spheroids in vitro.

Here, we present a protocol to generate dormant brain metastatic breast cancer (BMBC) spheroids utilizing hyaluronic acid (HA) hydrogels. We describe the steps for construction of spheroids from human BMBC cell lines MDA-MB-231Br and BT474Br3, HA hydrogel preparation, and spheroid plating on HA hydrogels and in suspension culture. We then detail the impact of HA hydrogel on the dormant phenotype of spheroids by measuring spheroid cross-sectional area, cell numbers, and EdU staining. For complete details on the use and execution of this protocol, please refer to Kondapaneni et al..