Peer review of: OSMR controls glioma stem cell respiration and confers resistance of glioblastoma to (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
Sharanek and colleagues describe very convincingly that the cytokine receptor for Oncostatin M 
(OSMR) is localized in mitochondria of brain tumor stem cells (BTSCs) and regulates oxidative 
phosphorylation and thereby confers resistance to ionizing radiation. This was done by the help of 
western blot of subcellular fractions and by performing knockdown experiments. They identified 
interaction partners by IP-MS and were able to show that OSM regulates cellular respiration 
independent of EGFRvIII and that a knockdown of OSMR sensitizes BTSCs to irradiation in vitro 
and in vivo lowering sphere and tumor growth. Data are novel and impressing. However, in 
extended data fig 7 the authors describe a similar effect on primary CGN cells lowering the 
translational relevance of targeting OSMR. A few other criticisms are listed below: 
1.) Extended data fig. 6: Did authors also analyze other brain cells? Glial cells would have been of 
particular interest. How was the purity of CGNs confirmed? What was the purity? 
2.) Fig. e-f: Why are control values in f) higher as compared to e)? 
3.) Fig. 3: Not only sphere numbers but also sphere sizes should be assessed. Pictures in a) and d) 
should show a larger area and more representative spheres. 
4.) Page 8, last sentence: This is an overinterpretation. It has not been shown that especially 
slow-cycling BTSCs have been eliminated. 
5.) For some of the western blot pictures it appears as if the samples were not run on the same 
gel because especially for OSMR there is often a lot of background. Please comment. 
6.) Fig. 3f and g: these graphs should contain data with and without IR like in 3b, c and e). 
7.) Glioblastoma are not any more called glioblastoma multiforme but simply glioblastoma. Please 
correct. 
8.) Extended data, fig. 5a-b: Did the authors also test other time points and tissues? 
 
 
 
Reviewer #2: 
Remarks to the Author: 
The contribution by Sharanek and colleagues adds on a previous contribution (Nat. Neuroscience 
2016) that showed that the suppression of the receptor for oncostatin M (OSMR) prevents 
proliferation and tumor growth of glioblastoma cells and human xenografts in mice. OSMR is a co-
receptor of the plasma membrane EGFRvIII receptor. In this manuscript they put forward the 
puzzling idea that OSMR is localized in mitochondria of brain tumor stem cells (BTSC) and in 
mouse liver and neurons and is responsible for the activation of mitochondrial respiration by 
interaction with subunits of complex I of the respiratory chain. In addition, they show that 
knockdown of OSMR increases the response of brain xenografts to ionizing radiation (IR). My 
major concerns with this piece of work are the methodologies used to claim the mitochondrial 
localization of OSMR and details of the mechanism of action of OSMR in mitochondrial respiration 
as detailed in the following comments. Addressing these concerns is required before the 
manuscript could be considered for publication in Nat. Commun. 
Major points. 
1. Fig. 1a-f and Extended Fig. 5: It is claimed that OSMR is present in mitochondria of BTSCs, liver 
and brain. However, membrane fractions are known to co-purify with mitochondria. These blots 
should include biomarkers of plasma and ER membranes to exclude the contamination of 
plasma/ER membranes in the Mito-fraction. Plasma membrane as such needs to be included as a 
fraction of the separation and a blot of EGFRvIII could illuminate the findings. 
2. Fig. 1 g-h: The proteinase K/Triton X100 experiment supports that OSMR is protected in a 
membranous compartment but not necessarily mitochondria. They should address a specific 
import assay of OSMR in isolated mitochondria. 
3. Fig. 1 k-n, Fig.1 r-s and Supp. Table 1: Blots of IPs and the “interactome” study by IP-LC-
MS/MS are carried out from solubilized proteins from whole cellular lysates where the abundant 
mitochondrial proteins are highly represented. The IPs should be performed from solubilized 

proteins from isolated mitochondria. Moreover, the interactome data is really misleading they only 
show a set of mitochondrial proteins out of the more than thousand proteins that are obtained by 
this basic approach. This sort of studies need to be quantitatively carried out using SILAC. For 
instance, in their case, by labeling with heavy and light amino acids the OSRM and knockdown-
OSRM cells, respectively. For details in the way to procedure see the example in He et al., PNAS 
(2018) 115, 2988-2993. 
4. The mechanisms to explain the upregulation of mitochondrial respiration mediated by OSMR 
either alone or in response to the incubation of the cells with OSM (Figs. 2a,e and Extended Figs. 2 
and 3) are not explained and far from clear. The major downstream signaling pathways that are 
activated in response to OSM are JAK/STAT, Ras/Raf/MAPK and PI3K pathways which could be 
modulating the activity and phosphorylation status of oxidative phosphorylation complexes, these 
facts are not taken into consideration and, in contrast, they emphasize the potential interaction of 
OSMR with NDUFS1, a matrix protruding subunit of complex I, which is unconvincing (see point 3) 
and by itself does not explain the increase in respiration. They should determine the enzymatic 
activities of respiratory chain complexes and eventually the differential phosphorylation of 
immunocaptured respiratory complexes. 
Minor points: 
1.- The differential redox stress of the xenografts in response to IR should be documented.