Cell cycle dynamics analysis of drug-tolerant persister cells in non-small cell lung cancer

Molecular-targeted therapy remains effective in cancer treatment; however, drug resistance inevitably arises after prolonged administration for several years. Drug-tolerant persister cells (DTPs), which are subpopulations of cells derived from drug-sensitive cells that develop tolerance after short-term exposure (lasting days or weeks) to drugs, have gained substantial attention. However, the specific characteristics of DTPs remain unclear as they encompass a diverse cell population, although they generally exhibit low proliferative potential. Therefore, analysis of cell cycle fluctuations during and after the formation of DTPs is expected to elucidate DTPs characteristics and contribute to future therapies. Here, we introduced the Fucci probe into the PC9 cell line to create PC9-Fucci to observe dynamic cell cycle variations during and after the DTPs formation process. The results showed that during the formation of DTPs, the majority of cells showed red fluorescence and were arrested in the G1 phase, whereas a small number of S-phase cells showed green fluorescence. Furthermore, some cells proliferated after the DTPs phase under drug-free medium and drug-containing medium culture conditions. These results indicate cell cycle diversity, with the majority of cells exhibiting arrested cell cycle during the formation of DTPs, whereas others existed in the S phase. In addition, cell cycle diversity occurs not only in the process of DTPs formation but also in subsequent processes.