In the sensitivity and specificity assays, SK-MEL-28 and MCF7 cell lines were maintained in serum-free medium for 48 hours to generate conditioned culture medium. In the study of investigating drug effects on EV phenotypes, SK-MEL-28, LM-MEL-33, LM-MEL-35, and LM-MEL-64 cell lines were maintained in Medium 254 (Gibco) with Human Melanocyte Growth Supplement, and with/without 1 μM PLX4720 (Selleckchem), whose conditioned culture media were collected every 3 days. To remove detached cells and cellular debris, conditioned culture media were centrifuged at 800g for 10 min. The cell-free supernatant was then stored at −80°C for the following experiments.