Spheroid-based Angiogenesis Assay in SCID Mice

Female CB17 SCID mice (Charles River, Wilmington, MA, USA) were used for the spheroid-based angiogenesis assay. Spheroids consisting of HUVECs and human microvascular PCs, pre-transfected with adenoviruses in a 1:1 ratio, were generated following established protocols [20 (link)]. Spheroids were formed by culturing cells in hanging drops of ECM containing 0.24% methylcellulose (HY-125,861, MCE, China). Spheroids of HUVECs pre-transfected with adenoviruses Ad-NC, Ad-P4HA1, a combination of Ad-P4HA1 and Ad-FBP1, or a combination of Ad-P4HA1 and Ad-TET2 were generated using the same procedure. The spheroids were then harvested, washed, and centrifuged for collection. A plug mixture, consisting of Matrigel matrix (354234, BD Biosciences, USA), spheroids, fibrinogen (2 mg/mL, 341576, Sigma, USA), methylcellulose, EC basal medium, and thrombin (10602400001, Sigma, USA) was injected into the groin of the mice (500 µL total volume) following a previously reported method [21 (link)]. After 21 days, mice were euthanized, and the plugs were harvested, embedded in paraffin, and sectioned for immunofluorescent staining of CD31.