Peer review of: ARS2/MAGL signaling in glioblastoma stem cells promotes self-renewal and M2-like (2020)


 

Reviewers' Comments: 
 
Reviewer #2: 
Remarks to the Author: 
Overall Comments: In the revised manuscript, the authors attempt to clarify their report, in which 
the transcription factor ARS2, previously shown to control SOX2 expression and neural stem cell 
identity and self-renewal, is linked to glioblastoma progression through transcriptional activation of 
MAGL causing production of prostaglandin E2 causing M2 tumor associated macrophage 
polarization driving a cancer stem cell phenotype. There were significant problems precluding 
publication of the initial submission, and the authors have made insufficient progress in addressing 
these flaws. 
 
Specific Comments: 
1. During the last revision period, a paper entitled “Ars2 promotes cell proliferation and 
tumorigenicity in glioblastoma through regulating miR-6798-3p” (PMID: 30349053) was published. 
2. The story remains very convoluted, and the links between the steps, which are critical for the 
novelty of the paper, are still weak. For instance, the authors contend that the end product of 
ARS2 upregulation is signaling to cause M2 macrophage polarization, yet, the majority of the 
results in the paper take place in cell culture with no macrophages present, and the authors 
purport to show changes in stemness of glioma anyway. GSCs have been reported to cause M2 
polarization (PMID: 25580734) so I’m unclear why this inclusion is necessary. Is ARS2 necessary 
for stemness, or for stem cells to induce M2 polarization, or just for survival of cells in general? 
3. The authors have not addressed the concern of artificial amplification of signal at each step. 
Specifically: 
a. “Although ARS2 mRNA level was 1.5 times higher in glioma than normal tissue (Original Fig. 
1a), we thought protein expression might not be exactly correlated. To address this issue, we 
quantified protein expression from cohort from National Cancer Center Korea (NCC) in original Fig. 
1f and Supplementary Fig. 1. ARS2 protein was barely detectable in normal brains, but was widely 
and strongly expressed in patient tumor samples. These results support that our experimental 
overexpression of ARS2 is supposed to be within the clinically reproducible and reasonable 
alteration.” I’m confused by this point – the authors contend that the mRNA is not predictive of 
protein expression so figure 1A is either incorrect or misleading. Then why are the data still figure 
1A? Also, the new data are slightly suspect – the authors suggest ARS2 expression in brain is 
undetectable, yet ARS2 is a common essential gene, with ARS2 disruption almost universally lethal 
(https://depmap.org/portal/gene/SRRT?tab=overview), including in CNS samples. 
b. “Concentration of PGE2 treatment is also based on previous scientific references regulating gene 
expression on PGE2 administration in macrophages at the level of 10 μM1,2.” I don’t see the 
relevance of the references. PGE2 should be added at concentrations that the glioma cells are 
capable of producing – levels which are never directly measured in the manuscript. What if the 
positive staining in figures 5a/b reflect nanomolar concentrations of PGE2? 
4. The overreliance on immunofluorescence images is still unacceptable. The standard joke in 
science is that “representative” images means the best image you can find; without a more 
wholistic analysis the data are suspect. It is also unacceptable to force the reader to connect to a 
database with all of the higher visibility images – the manuscript should be a self-contained 
package that allows the reader to judge the work. Figures 6d-g are still problematic – even 
ignoring the doubts about the accuracy of the quantification raised by the previously incorrect 
results, the PGE2 staining in figure 6d is definitely more red than figure 6f, yet the bar chart in 6g 
has a higher percent positive than 6e - showing the limitation of representative images. 
5. The authors response to problems with the LDAs are insufficient. LDAs are not designed to 
measure proliferation, but proliferation is a confounding factor in an LDA – anything that affects 
proliferation will affect the results of an LDA. Figures 2c and 2f show that proliferation is greatly 
reduced by shARS2, so even if there is no effect on stemness, the LDA will show less sphere 
formation. This is where serial LDAs are critical – and a serial LDA involves taking the spheres 
formed by the initial LDA and replating them, not plating independent serial tissue culture 
passages for LDAs. Additionally, the time until reading should be consistent between experiments 

and 7 days is too short for adequate sphere formation. 
a. “As stemness grows stronger over time, we can easily expect that the stemness of the control 
X01 GSC in original fig. 4h harbors higher stemness.” This is an absolutely incorrect statement. 
Sphere formation increases with time, because of the effects of proliferation on the LDA, but those 
cells were always stem cells and the wells were false negatives until they were able to grow into a 
sphere. That’s why it’s critical to wait until saturation of sphere formation to get a readout of stem 
cell frequency independent of proliferation effects. 
6. The rebuttal states that β-catenin is highly expressed in the nucleus rather than the cytosol, yet 
the staining in figure 5a/b is clearly cytoplasmic. 
7. “Genes with statistical significance (p-value and q-value with less than 0.01) and fold-change 
(±1.5) were chosen as significant DEGs.” All of the samples on the table except AL593851.1 and 
MGLL have a q-value above 0.01? Also, was ARS2 itself explicitly excluded, or does it not show up 
as significantly altered? 
 
 
 
Reviewer #3: 
Remarks to the Author: 
The authors already did an effort to accommodate most of my concerns, but there are still a few 
important problems: 
 
1) The authors changed peritoneal macrophages into bone marrow-derived macrophages for their 
in vitro experiments, which is already some improvement, but BMDM are still quite distinct from 
real TAM. As already asked before, it would be much more relevant and informative to sort real 
TAM (CD11b+ F4/80+) from ARS2 or MAGL KD tumors and treat those cells with PGE2. 
2) The authors could use inhibitors that block PGE2 production in vivo, to test the invivo relevance 
of this pathway in their models. 
3) RNAseq to screen for macrophage genes was performed on cell preparations that contained less 
than 10% macrophages. This is unacceptable. How can the authors be sure that the regulated 
genes are intrinsic to macrophages? At the very least, genes whose expression were altered 
should be tested on purified TAM populations.