Tumor-promoting function of apoptotic caspases by an amplification loop involving ROS, macrophages and JNK in Drosophila

Essential revisions:

The authors must quantify the data in the manuscript (as detailed below), as well as provide additional background information, and soften some conclusions or provide more support for these statements.

As requested, we have quantified all data in the manuscript. We have provided additional background information in the Introduction and have softened controversial statements.

1) Several points are vague and often lacks needed quantification. For example when examining DHE staining of scrib-/- ras^V12 clones, they comment that some clones have diffuse cytosolic staining, others stain intensely, while others have web-like staining. No attempt is made to define these differences. The DHE staining must be quantified. Other readouts of ROS could be useful. Similarly, when examining ROS and JNK, in scrib-/ ras^V12 clones the authors state that "many clones" that were depleted of ROS or had inhibited caspase had less JNK. How many and what percent of clones lose JNK? How many (and what percent) of ey>MARCM scrib-/-; ras^v12 adult animals live when ROS is inhibited ?

Quantification of hemocyte numbers/area they cover relative to the total area of the clones is also needed for Figure 4. The number of hemocytes seems very different from Figures 4C-J to Figures 4K-Q

We agree with the reviewers that quantification is needed to support our conclusions. Therefore, we have spent a considerable amount of time to quantify all the data. This includes the DHE, pJNK, MMP1, hemocytes, cleaved caspase-3 and TUNEL labelings. We have also added and quantified H₂DCF-DA labelings as additional ROS readout in Figure 1—figure supplement 1. The H₂DCF-DA data are very consistent with the DHE labelings. We have also significantly expanded on the counts of pupal and adult survival and have now much more reliable numbers for all genotypes (Figures 1G and 2N). Overall, the quantified data are very consistent with our conclusions.

Figures 4K-Q are not concerned with the number of hemocytes, but rather with the morphology of hemocytes on scrib^-/- Ras^V12 discs and discs that have reduced caspase activity and reduced ROS. We have clarified this point in the text.

2) The authors observe Cc3 and TUNEL staining inside and outside of scrib-/- ras^v12 clones. The authors suggest that this death is essential to the tumor, and that the clones are supercompetitors. If they want to support this claim, they should inhibit death only in the surrounding wildtype cells and see if this is essential. Alternatively they can modify this in the Discussion.

In the long term, it is certainly a very important question to address if the apoptotic cell death outside the clones (non-autonomously) is essential for tumor growth. However, experimentally, it is not possible to address the allotted time of the revision. We have therefore removed these statements and any reference to supercompetitors.

3) Introduction: At present, the link to mammalian settings is insufficient, and the manuscript would benefit from more clearly spelling out previous observations hinting at a pro-tumorigenic role of caspases. Given the numerous studies that demonstrate tumor promoting roles of caspases, it might be appropriate to better reflect the current state of knowledge on this issue.

We agree with the reviewers and have expanded and clarified the different forms of AiP and the role of caspases for tumor growth in mammals. We also have cited all relevant original papers on this topic.

4)The authors suggest that cell death of non-Scrib/Ras^V12 cells contribute to the growth of Scrib/Ras^V12 tumor cells. It would be interesting to test this hypothesis, and evaluate the contribution of JNK activation and cell death of neighbouring wild-type cells (inactivating caspases only in wild-type cells). Although this would be interesting to address, I realize that by-stander cell death is not exactly to the issue of this manuscript. Hence, this could be an added benefit, but is non-essential for the current study.

This point is similar to point #2. To inactivate caspases only in wild-type cells in a scrib^-/- Ras^V12 mosaic background is experimentally quite challenging and not possible in the allocated time of the revision.

5) Figure 3 would profit from a side-by-side comparison with Scrib and Scrib/Ras^V12/drICE RNAi. While the results from the TUNEL staining are clear and convincing, it is somewhat difficult to evaluate the extent of caspase activation in Figure 3. Clearly, inhibition of caspase activation suppresses ROS production, hemocyte recruitment and tumour overgrowth. The phenotype is clear. But it is difficult to evaluate the extent of caspase activation in the Scrib/Ras^V12 clones.

We have added the scrib^-/- Ras^V12 /drICE RNAi analysis to Figure 3. We have omitted a scrib analysis, because scrib clones are very small. In order to address the extent of caspase activation in scrib^-/- Ras^V12 clones, we have quantified the CC3 data inside and outside the clones (autonomously and non-autonomously) in control (wild-type mosaics), scrib^-/- Ras^V12 and scrib^-/- Ras^V12 /drICE RNAi mosaics. We found that the caspase activity outside scrib^-/- Ras^V12 clones is about 2.5 times higher than inside the clones. These data are presented in Figure 3D.

It is important to show double staining of CC3 (cleaved caspase3) and TUNEL in the scrib Ras^V12 clones. CC3 staining outside the clones, as shown in Figure 3B, looks different to the TUNEL staining in Figure 3C. Furthermore, staining with CC3 and TUNEL would be the way to show which cells are indeed activating caspases without undergoing cell death. CC3 staining should be performed in scrib Ras^V12 Duoxi, hCatS, SOD1, SOD2, Catalase… to provide a stronger evidence for the loop. It would also be nice to have CC3 staining in p35, dronci and dricei as controls.

We performed a large amount of CC3/TUNEL double labelings, but the CC3 signal in these double labelings was too weak to give meaningful and satisfying results. However, the TUNEL labelling in these experiments went very well which we were able to quantify. We found that almost 90% of all TUNEL-positive cells are outside (non-autonomous) of the scrib^-/- Ras^V12 clones. The remaining TUNEL-positive cells are located inside these clones. Thus, although our CC3/TUNEL double labelings were inconclusive, these statistical data strongly confirm that most apoptotic cells are located outside of scrib^-/- Ras^V12 cells.

The CC3 and TUNEL labelings outside the clones look different, because one stain is cytosolic (CC3), while the other one is nuclear (TUNEL). We have also added CC3 labelings of scrib^-/- Ras^V12 with Duox RNAi, hCatS, Catalase and SOD2 in the supplement to Figure 3. In all cases, the CC3 signal is strongly reduced, further strengthening the positive feedback amplification loop.

6) A very interesting observation is the fact that JNK is activated in the periphery of the scrib Ras^V12 clones when ROS or caspases are inhibited. Unfortunately, only few not very clear examples are provided, and instead most of the times staining with MMP1 is shown, where this phenomenon can't be observed. pJNK staining (which would be a better readout for JNK staining) or alternatively puc-lacZ, should be shown for the rest of the genotypes, including the scrib Ras^V12 alone, which is not shown in the paper. Again, it would also be nice to have this staining in the JNKDN experiment as a control.

We have replaced the MMP1 labelings in Figure 5 with pJNK stainings and moved the MMP1 labelings as supporting information into the supplement. As requested, we have also added the JNKDN experiment as control. In addition, all data are quantified.