自噬在肿瘤发生中发挥着双重作用，既可以作为肿瘤抑制剂，又可以作为促进剂，取决于肿瘤发生的阶段。然而，目前尚不清楚自噬在肿瘤发生过程中的作用转变发生在何时。本研究旨在调查五种不同细胞转化阶段的基础水平和自噬作用的差异。我们发现，在较高的转化阶段，细胞对自噬抑制剂更为敏感，表明随着转化的进行，自噬的作用变得更加重要。我们的ptfLC3成像分析结果显示，与未转化细胞相比，转化细胞中的Atg5/LC3依赖型自噬通量增加。然而，Cyto-ID分析，用于测量Atg5依赖和非依赖自噬通量的方法，显示转化细胞中不仅存在高水平的自噬体形成，而且在初始细胞和Atg5 KO细胞系中也存在高水平的自噬体形成。这些结果表明，在初始和转化细胞系中，非Atg5依赖的自噬可能比在未转化细胞中更为关键。尤其是，我们观察到，在快速增殖条件下，转化细胞维持相对较高的基础自噬水平，但在高密度时表现出更低的基础自噬水平；然而，在未转化细胞中，即使在高密度时，自噬通量也没有显著减少。此外，在连续培养3周而不传代的情况下，衰老细胞对自噬抑制的敏感性明显降低，与其活跃增殖的对应物相比。这些结果表明，一旦细胞从增殖状态转变为衰老状态，抑制自噬只有最小的影响。综上所述，我们的结果表明，在应激条件下，自噬在不同阶段的肿瘤发生细胞中可能会发生差异调控。 © 2023 Wiley Periodicals LLC.

Autophagy plays a dual role in tumorigenesis by functioning as both a tumor suppressor and promoter, depending on the stage of tumorigenesis. However, it is still unclear at what stage the role of autophagy changes during tumorigenesis. Herein, we investigated the differences in the basal levels and roles of autophagy in five cell lines at different stages of cell transformation. We found that cell lines at higher transformation stages were more sensitive to the autophagy inhibitors, suggesting that autophagy plays a more important role as the transformation progresses. Our ptfLC3 imaging analysis to measure Atg5/LC3-dependent autophagy showed increased autophagic flux in transformed cells compared to untransformed cells. However, the Cyto-ID analysis, which measures Atg5-dependent and -independent autophagic flux, showed high levels of autophagosome formation not only in the transformed cells but also in the initiated cell and Atg5 KO cell line. These results indicate that Atg5-independent autophagy may be more critical in initiated and transformed cell lines than in untransformed cells. Specially, we observed that transformed cells maintained relatively high basal autophagy levels under rapidly proliferating conditions but exhibited much lower basal autophagy levels at high confluency; however, autophagic flux was not significantly reduced in untransformed cells, even at high confluency. In addition, when continuously cultured for 3 weeks without passage, senescent cells were significantly less sensitive to autophagy inhibition than their actively proliferating counterparts. These results imply that once a cell has switched from a proliferative state to a senescent state, the inhibition of autophagy has only a minimal effect. Taken together, our results suggest that autophagy can be differentially regulated in cells at different stages of tumorigenesis under stressful conditions.© 2023 Wiley Periodicals LLC.