We appreciate this question. Autophagy is a process that is elevated in order to cope with cellular stress, but as stress level elevate, autophagy can no longer provide the protection the cell needs, and the cell will undergo apoptosis. This kind of kinetics means that the levels of autophagy markers will depend on the time point the cells are examined. This may be appreciated from figure 2 panel c and d, with the different kinetics displayed by the two cell lines. While for Huh-7D12 autophagy levels increase from 24 to 48 hours of treatment, in the HepG2 cells autophagy levels at 48 hours are lower than at 24 hours, indicating these cells are already after the autophagy peak and on the way to apoptosis. Indeed, figure 1d shows higher levels of apoptosis for HepG2 cells. To clarify we have added a few sentences to the text. In results sub-section 3.2: “However, autophagy kinetics seems to be faster in the HepG2 cells, in which LC3 markers are lower at 48 versus 24 hours, whereas elevation is seen from 24 to 48 hours for the Huh-7D12 cells.” In the discussion “While autophagy serves as a survival strategy of cells, when stress levels continue raising it may be over-activated and mediate cell death [9]. The faster autophagy kinetics seen for the HepG2 relative to Huh-7D12 cells following application of TTFields is in agreement with the higher apoptosis levels displayed by this cell line, and may serve as an additional rational for the higher efficacy of TTFields against it. Examination of the reasons for faster autophagy in HepG2 relative to Huh-7D12 cells is out of the scope of this work.” For more clarity we have also added a more in-depth kinetic study, including additional relevant markers and additional time points, examinations that were performed for the combined treatment as compared to TTFields and sorafenib alone. The higher changes in expression levels and faster kinetics when TTFields and sorafenib were applied together rather than alone indicate higher stress levels imposed on the cells in the former case” Figure 3. In the animal study we may only measure one time point. The lower autophagy seen at this time for the combined treatment together with the higher apoptosis indicate that we are further along the kinetic timeline of autophagy relative to the monotherapies, suggesting higher stress in animals receiving the combined treatment. We have now added IHC examination of beclin-1, an additional autophagy marker, and of GRP78, a marker for ER stress, as described in results sub-section 3.4.
Is there any explanation why the combination of TTFields and sorafenib did not induce a significant level of autophagy as compared to untreated animals which invalid the initial hypothesis that “concomitant application of sorafenib and TTFields may increase stress levels enough to tilt autophagy towards the cell death pathway”.