Interestingly, in the absence of MTX, the resistant cell lines had reduced copies of the DHFR gene in eccDNAs, which suggested that the dynamic regulation of eccDNAs determines the response toward chemotherapy.53,266,267 Furthermore, Meng et al. found that the inhibition of DNA-PKcs (a key NHEJ protein) inhibits the formation of DHFR-containing eccDNAs and therefore increased MTX sensitivity in colon cancer cells. This evidence concerns the gene DHFR and malignant colon neoplasm.