We thank the reviewer for raising this important issue. Using a xerograph model is indeed a good suggestion, however it would require working in immunosuppressed mice. It is currently not feasible for us to perform studies in which we apply TTFields to immunosuppressed animals, as these animals are of smaller weight thus the burden of wearing the electrodes may induce too much stress. Furthermore, these animals are more prone to contract infections during the process of electrodes placement and replacement on the animals. On the other hand, using the rat cells for in vitro experiments in the inovitro dishes turned out to be very technically challenging, as the cells were non-adherent, and so we could not pursue this important avenue. Nevertheless, we agree with the reviewer that there is a possibility that the optimal frequency may differ in the N1S1 relative to the human cell lines. Therefore, we have addressed this issue by including results of an additional frequency scan experiments done with N1S1 cells. We now mention this in the text, results sub-section 3.4: “The efficacy of combining TTFields with sorafenib relative to each modality alone was examined in the N1S1 HCC rat orthotopic model (timeline in Figure 4a). In vitro experiments confirmed that the 150 kHz TTFields frequency found optimal for treatment of the human cell lines was also optimal for treatment of the murine N1S1 cells used for the in vivo study (Figure S2).” We provide the relevant frequency scan figure in the supplementary material. Per the good question by the reviewer regarding the p53 status of the N1S1 cells we added: “It is also worth mentioning that the N1S1 murine cells used for this study, like the HepG2 cells, are p53 wild type.” Figure S2.
HepG2 cells were treated for 6, 24, or 48 hours with 150 kHz TTFields, 3 µM sorafenib, or the two treatments combined, followed by Western blot examination of the autophagy markers beclin-1 and LC3 (d), the ER stress marker GRP78 (e), and the apoptosis marker cleaved PARP (f).