Finally, we inhibited the expression of DHFR, MSH3, ZFYVE16 and FAM151B at the HSR and ecDNA stages, respectively, and found that the relative IC50 changes of cells against MTX was significantly reduced, indicating that the amplification of these four genes played an important role in the resistance of tumor cells to MTX (Fig. 6). Here, DHFR is linked to neoplasm.