To determine if C-Circles were secreted by cancer cells inside the ECV, we performed the CCA on each differential centrifugation fraction of 24 h-old culture medium from the ALT+ cancer cell lines SaOS-2 and SK-N-FI and the ALT- cancer cell line HT1080, and only saw the CCA signal in the fractions from the ALT+ cells lines (Figure 2a,b). This evidence concerns the gene GPT and cancer.