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. We thank the reviewer for this comment, as these additions add much clarity to the mechanism of action of TTFields in combination with sorafenib and provide a more coherent explanation for the in vivo results. These additions may be seen in Figure 3 and are described in results sub-section 3.4, Autophagy-apoptosis Interplay For Treatment with Concomitant TTFields and Sorafenib: “In order to investigate the mechanism of action of TTFields-sorafenib co-application, HepG2 and Huh-7D12 cells were treated for 6, 24, or 48 hours with TTFields, sorafenib (3µM), or the two modalities together, and then examined for expression levels of various proteins. For HepG2 cells, the autophagy marker beclin-1 demonstrated elevation after 6 hours of treatment, which was later replaced with diminished expression levels (Figure 3d). This type of behavior was seen in all treatment groups, but was most pronounced for TTFields-sorafenib co-application. The autophagy marker LC3 also displayed such bi-phasic characteristics, but with a somewhat slower kinetics, showing some elevation at 6 hours of treatment, but higher elevation at the 24 hours time point (Figure 3d). As in the case of beclin-1, the magnitude of the effect was higher for co-treatment of TTFields and sorafenib relative to the monotherapies. GRP78, a marker of ER stress, remained low in all treatment groups for 6 and 24 hours of treatment, but demonstrated elevated levels at the later, 48-hours time point (Figure 3e). The apoptosis marker cleaved PARP displayed increased expression in the combined group already after 24 hours, elevating even further after 48 hours of treatment. For the monotherapies, cleaved PARP increase was only evident at 48 hours of treatment, and to a lower extent than that in the co-treatment group (Figure 3f). The slower kinetics of the autophagy-apoptosis path in the Huh-7D12 cells, as seen from the elevation of LC3 after as much as 48 hours (Figure 2c and d), prevented from detecting such changes in the levels of these markers in this cell line (Figure S1).” And in the discussion part “Kinetic examination in the HepG2 cells revealed elevation in autophagy levels as early as 6 hours of TTFields or sorafenib treatment, which diminished and were replaced with ER stress and apoptosis for 48 hours of treatment. These results are in line with a previous study that focused on the effects of sorafenib on such markers in HepG2 cells [32]. 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.
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”.