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 submitted manuscript, the authors set out to link the transcription 
factor ARS2, previously shown to control SOX2 expression and neural stem cell identity and self-
renewal, to glioblastoma progression. The proposed link involves transcriptional activation of MAGL 
causing production of prostaglandin E2 causing M2 tumor associated macrophage polarization to 
drive a cancer stem cell phenotype. However, the path from ARS2 to macrophage polarization is 
very convoluted and many of the links are insufficiently established. Without the links between 
steps, the novelty of the manuscript is greatly diminished, as many independent parts (e.g. PGE2 
causing macrophage polarization, macrophage polarization affecting GSCs) have been previously 
published. Additionally, much of the data shown in the manuscript is presented as difficult to 
interpret immunofluorescence images rather than more quantitative assays. 
 
Specific Comments: 
Major Comments: 
1. The manuscript lacks a cohesive narrative. The authors show that ARS2 is slightly elevated in 
glioma compared to non-tumor (1.5 fold according to figure 1A). They then show that dramatically 
altering ARS2 expression via overexpression constructs or shRNA has minor effects on MAGL 
expression. Next, dramatic changes in MAGL expression (overexpression or shRNA) are purported 
to show changes in PGE2 production, via an immunofluorescence image of less than ten cells. 
Then the authors switch to directly adding high concentrations of PGE2 to cells. By artificially 
amplifying the magnitude of changes at each step, the authors are artificially inflating the 
observed effects – given the actual magnitudes shown, it seems highly unlikely that a 1.5 fold 
increase in ARS2 expression following tumorigenesis will drive any significant downstream effects 
that the authors present. This is especially important given that many of the separate findings lack 
novelty without the connection to the previous findings. 
2. A large proportion of the data in the paper is presented as single immunofluorescence images 
depicting very few cells, and any presented quantification is questionable. Notably, related to 
figure 6d, the authors state that shARS2 completely eliminates PGE2 and the bargraph in 6e shows 
0% PGE2 positive cells, however there is very clearly visible red signal in 6d. The authors should 
include a quantitative experimental approach for each analysis that currently relies on IF. This is 
especially problematic given that when quantitative data is shown, the results are quite modest 
(e.g. protein data in figure 3, the effects of PGE2 in figure 6a). 
3. The ARS2 ChIP showing binding at the MGLL promoter is a critical link for ARS2 to the rest of 
the manuscript, but there are a lot of problems with these data. First and foremost - the labeling is 
unclear, but it appears that Supplementary Figure 3 presents negative data from a ChIP 
experiment. It is not explained in the text, but assuming that H3 represents a ChIP for histone H3, 
then there is no enrichment for any MGLL promoter sequence by ARS2 ChIP versus either input 
control or the histone H3 ChIP. These are the more accurate controls versus an IgG, which should 
pull down no DNA. The differences in figure S3 appear to be restricted to primer efficiency – 
primer 4 seems to amplify the most efficiently based on input and H3. Additionally, the Genetex 
ARS2 antibody mentioned in the methods is not listed as approved for ChIP, and there are no 
validation experiments presented by the authors. 
4. The authors do not actually test stemness in glioblastoma cells. The limiting dilution sphere 
formation assay performed in figures 2 and 4 measures a combination of proliferation/survival and 
self-renewal. Based on the other data presented (figs 2c, 2f especially), it appears as though the 
knockdowns are simply causing either cell cycle arrest or cell death. In order to actually address 
stemness, the authors would need to perform serial LDAs to show effects on sustained sphere 
formation. An analysis of non-stem glioma cells could also be potentially useful. 
5. Additional problems with the LDAs – in figure 4h, the control X01 GSC line has a higher stem 
cell fraction than the MAGL overexpression construct in the same cell line in figure 4j. In figure 4j, 
the control has a much lower stem cell frequency than the 4h control. Why are the controls so 
different? Also, the ELDA program used by the authors presents both the graph and an estimate of 

the stem cell frequency, these numbers should be presented. Finally, the timescale between 
plating and counting spheres is not mentioned in the methods. 
6. In figure 8a, the authors present an analysis of RNAseq data to argue for an overlap between 
JZL184 genes and M2 signature genes. The text is slightly unclear, but it seems the RNAseq data 
is generated from macrophages isolated from subcutaneous xenograft models and then aligned to 
the human genome (from methods - hg19 specifically). Why wasn’t this data aligned to a mouse 
genome (e.g. mm9)? The infiltrating macrophages should be from the mouse host, rather than 
GSC-derived? The methods section for the RNAseq is missing essential information, specifically 
read length and alignment parameters such as mismatch permission, which would affect the ability 
of mouse RNA to align to the human genome, but this data is suspect and should be re-aligned 
and reanalyzed. 
 
Other comments: 
1. Figure 6b is difficult to interpret, and given that it is an analysis of surface markers on 
macrophages, should probably be performed using flow cytometry rather than imaging. 
2. In figure 6d/f there should be some kind of stain to distinguish human cells from mouse cells 
when looking at cancer or macrophage markers. The images are too low resolution to distinguish 
the characteristic nuclear distinctions between species. 
3. For the mouse tumor formation studies, median survival times should be presented. 
4. Figure 1f lacks a loading control. 
5. The methods section is lacking many critical details on experiments. 
6. For the genome-wide analysis of differentially expressed genes, a P-value of 0.05 is far too 
high. Some form of multiple testing correction is essential for such a large analysis. 
 
 
 
Reviewer #3: 
Remarks to the Author: 
Yin et al uncover a tumor-promoting signalling axis, whereby ARS2 upregulates MAGL in 
glioblastoma stem cells, which leads to an increased production of prostaglandin E2, cancer 
stemness and M2-like TAM orientation. To my knowledge, this is a novel tumor-promoting pathway 
with potentially clinical relevance. Important issues remain: 
 
- Fig 3. One siRNA knocks down ARS2 completely but not MAGL, suggesting that part of the MAGL 
expression is ARS2-independent. Conversely, the other siRNA only knocks down ARS2 partially, 
but completely abolishes the expression of MAGL. How to explain? 
- Fig 5c. beta-catenin as a whole does not seem to be affected. Sometimes there is even an 
increase in beta-catenin. This contradicts the statement that MAGL knockdown decreased b-
catenin accumulation in GSCs. 
- The authors treated WT cells with PGE2 to show that b-catenin expression is increased (Fig 5e) 
and the sphere-forming ability was increased (Sup Fig 5d). These experiments should be done in 
MAGL KD cells, not in WT cells, to firmly establish a reversal of the KD phenotype by PGE2 
administration. 
- P11, line 230-231. The authors suggest a link between a decreased Iba1 staining and 
tumorigenicity of GSCs. There is no clear link provided between these two phenomena. 
- All the in vitro work with PGE2 on peritoneal macrophages is of little relevance for TAM. 
Moreover, the effect of PGE2 has been reported before and conclusions are too simplistic, only 
based on a selection of a very limited number of genes. One can prove anything if a very limited 
selection of genes is shown. The in vitro effect of PGE2 on bona fide TAM from ARS2 or MAGL KD 
tumors would be much more instructive and needs to be checked. 
- The authors also need to design experiments to prove that PGE2 is really responsible for 
instructing the TAM phenotype in vivo. Do TAM really enhance GSC stemness via PGE2 in vivo? 
- Line 248. Another disturbing point is that IL-4-polarized peritoneal macrophages are all of a 
sudden equal to M2-like TAM. I am convinced there are huge differences. In my opinion, there is 
no relation between both types of macrophages, which are derived from different tissues and have 

seen different triggers. 
- How does the JZL-148 inhibitor work? Inhibitors usually do not cause the breakdown of its target 
protein, merely inhibit its function. Why is there a breakdown here? 
- Why is the inhibitor tested on subcutaneous and not orthotopic models? Should work as well 
- For RNAseq, the authors used CD11b+ cells from tumors. This is absolutely not state-of-the-art! 
CD11b+ cells include all myeloid cells, which in a tumor include monocytes, neutrophils, 
eosinophils, mast cells, macrophages, DC. The authors should prove that macrophages are the 
cells being affected, not the other ones.