HUVEC proliferation and viability assays.

CM from EBP and control Ewing sarcoma cells was generated by plating 5 × 10⁶ CHLA10 or 10 × 10⁶ A673 cells on 10-cm dishes. Once adherent, cells were serum starved for 24 hours, and serum-free CM was aspirated and centrifuged at 900 rcf for 5 minutes to remove cell debris. CM was added in a 50:50 concentration to HUVECs plated at a density of 7.5 × 10⁴ cells per well on 6-well plates, in M200 containing 0.25× LSGS. On days 1, 3, and 5, cells were dissociated with Accutase (Corning) and stained with trypan blue (Invitrogen, Thermo Fisher Scientific) before counting. Cell number and viability were determined using a Countess automated cell counter and confirmed manually on a hemocytometer.