Fig 2: Suppression of OSMR improves glioblastoma response to therapy.a Schematic diagram of the experimental procedure in which OSMR CRISPR and control BTSC73 (3 × 105 cells per brain) were intracranially injected into randomized Fox Chase SCID mice and then treated with or without 4 Gy of IR. b Representative bioluminescence real-time images tracing BTSCs and tumor growth are shown. c Intensities of luciferase signal were quantified at different time points using Xenogen IVIS software. *pOSMR CRISPR vs. IR + OSMR CRISPR, day 26 = 0.0289; Unpaired two-tailed t-test, n = 4 mice. d Kaplan–Meier survival plot was graphed to evaluate mice lifespan in each group, mice were collected at end stage. *pCTL vs. IR = 0.0311, **pCTL vs. OSMR CRISPR = 0.0069, **pCTL vs. IR + OSMR CRISPR = 0.0069, **pOSMR CRISPR vs. IR + OSMR CRISPR = 0.0067; Two-sided log-rank test, n = 4 mice. e Coronal sections of mouse brains were stained with hematoxylin and eosin on day 22 after injection. Representative images of 3 different tumor sections are shown. Scale bar = 1 mm. Inset scale bar = 0.1 mm. f Tumor sections from OSMR CRISPR and control BTSC73 treated with or without IR (4 Gy) were stained with MitoSOX. The fluorescent intensity was quantified using ImageJ software. ***p < 0.0001 for each pairwise comparison except: *pCTL vs. OSMR CRISPR = 0.0215, *pCTL vs. IR = 0.0129; One-way ANOVA followed by Tukey’s test for multiple comparisons, n = 4 different tumor sections. Data are presented as the mean ± SEM.

Fig 3: OSM/OSMR confers resistance of BTSCs to IR.a, b LDA was performed following 4 Gy of IR in the absence or presence of OSM. BTSC73 (a): 200 cells (**pCTL vs. OSM = 0.0011, **pIR vs. IR + OSM = 0.0054, ***pCTL vs. IR = 0.0007), 100 cells (*pCTL vs. OSM = 0.0185, **pCTL vs. IR = 0.0078, **pIR vs. IR + OSM = 0.0093), 50 cells (*pCTL vs. OSM = 0.0246, *pCTL vs. IR = 0.0389, **pIR vs. IR + OSM = 0.0054); BTSC147 (b): 200 cells (**pCTL vs. OSM = 0.0012, **pIR vs. IR + OSM = 0.0088, ***pCTL vs. IR = 0.0009), 100 cells (*pIR vs. IR + OSM = 0.0236, **pCTL vs. OSM = 0.0026, **pCTL vs. IR = 0.0012), 50 cells: (*pCTL vs. OSM = 0.0301, **pCTL vs. IR = 0.0039, **pIR vs. IR + OSM = 0.0087), 25 cells (*pCTL vs. OSM = 0.0382, **pCTL vs. IR = 0.0094), 12 cells (*pIR vs. IR + OSM = 0.0207, **pCTL vs. OSM = 0.0044). c, d ELDA was performed following 4 Gy of IR in the absence or presence of OSM in either BTSC73 (c) or BTSC147 (d). e, f LDA were performed following 4 Gy of IR in OSMR KD and control BTSCs. ***p < 0.0001 except: BTSC73 (e): 200 cells (**pIR vs. IR + OSMR CRISPR = 0.0030, ***pCTL vs. OSMR CRISPR = 0.0004), 100 cells (*pIR vs. IR + OSMR CRISPR = 0.0286, **pCTL vs. IR = 0.0021, **pCTL vs. OSMR CRISPR = 0.0052), 50 cells (**pCTL vs. IR = 0.0061, ***pCTL vs. IR + OSMR CRISPR = 0.0004), 25 cells (**pCTL vs. IR = 0.0018, **pCTL vs. OSMR CRISPR + IR = 0.0016), 12 cells (*pCTL vs. IR + OSMR CRISPR = 0.0404); BTSC147 (f): 200 cells (*psiCTL vs. siOSMR = 0.0184), 100 cells (*pIR vs. IR + siOSMR = 0.0414, **psiCTL vs. siOSMR = 0.0035), 50 cells (**psiCTL vs. siOSMR = 0.0050), 25 cells (*psiCTL vs. siOSMR = 0.0264, ***pCTL vs. IR = 0.0001), 12 cells (*psiCTL vs. IR = 0.0194, **psiCTL vs. IR + siOSMR = 0.0070). g, h ELDA was performed following 4 Gy of IR in OSMR CRISPR vs. control BTSC73 (g) or in siOSMR vs. siCTL BTSC147 (h). i, j Cell viability was measured by alamarBlue assay following 4 Gy of IR in OSMR KD and control BTSCs. ***p < 0.0001 except: BTSC73 (i): *pIR vs. IR + OSMR CRISPR = 0.0490, **pCTL vs. OSMR CRISPR = 0.0044, ***pCTL vs. IR = 0.0003; BTSC147 (j): *pIR vs. IR + siOSMR = 0.0213. Data are presented as the mean ± SEM, n = 3 independent biological cell cultures, one-way ANOVA followed by Tukey’s test for multiple comparisons.

Fig 4: Presence of mitochondrial OSMR in human BTSCs.a–d Four different patient-derived BTSC lines were subjected to subcellular fractionation, and the lysates for each fraction were analyzed by immunoblotting using antibodies to OSMR. a-Tubulin, H3K4me3, BCL2/TOM20, Na+/K+ ATPase, and calnexin. WCL: Whole-cell lysates; Cyto: cytoplasmic; Mito: mitochondrial. The Western blots represent a minimum of three replicates from different passage numbers for each BTSC. e, f BTSC73 and BTSC147 were subjected to immunostaining using antibodies to OSMR (green) and the mitochondrial matrix protein ATP synthase inhibitor F1 (ATPIF1, red). Nuclei were stained with DAPI. White rectangles mark the inset to demonstrate the co-localization of OSMR with ATPIF1. g, h PLA of OSMR and ATPIF1 were performed in BTSC73 (g) and BTSC147 (h). Primary antibodies were omitted as controls. i Double labeling of the PLA signal (red) and the MitoTracker (green) in BTSC73 is shown. j A FRAP assay was performed on BTSC73 transduced with GFP-OSMR and stained with MitoTracker (red). Different regions of interest (ROIs) containing GFP-OSMR in the mitochondria were defined. ROI1 indicates a non-bleached area and ROI2, a photobleached area. The fluorescence recovery was monitored over time following photobleaching. Images were obtained on a laser scanning confocal microscope (ZEISS LSM 800). Scale bar = 10 µm; Inset scale bar = 1 µm. Representative images of three independent experiments are shown.

Fig 5: OSMR regulates mitochondrial OXPHOS and ROS generation.a–e Enzymatic activities of mitochondrial ETC were analyzed in OSMR CRISPR or control BTSC73. Complex I, **p = 0.0037 (a); Complex II, **p = 0.0027 (b); Complex III, *p = 0.0126 (c); Complex IV, *p = 0.0488 (d); ATP synthase, p = 0.2506 (e); Unpaired two-tailed t-test, n = 4. f ROS generation was measured by flow cytometry using H2DCFDA in OSMR CRISPR and control BTSC73. ***p < 0.0001; Unpaired two-tailed t-test, n = 4. g Mitochondrial superoxide abundance was assessed by flow cytometry using MitoSOX in OSMR CRISPR and control BTSC73. ***p < 0.0001; Unpaired two-tailed t-test, n = 4. h Tumor sections from OSMR CRISPR and control BTSC73 were subjected to staining using OxyIHC oxidative stress detection kit. Representative images of 4 different tumor sections are shown. Scale bar = 20 µm. i RFP OSMR or RFP control BTSC73 (in the absence and presence of OSM) were subjected to bioenergetic analysis using a Seahorse XFe96 Bioenergetic Flux Analyzer. Oxygen consumption rates (OCR) are plotted (top panel). Data is plotted to demonstrate the differences between basal, ATP-linked, proton leak, maximal, and non-mitochondrial (mito) respiration (middle panel). Spare respiratory capacity (SRC), which is maximal minus basal respiration, is plotted (bottom panel). ***p < 0.0001 for each pairwise comparison except: *pLeak (RFP CTL vs. RFP OSMR) = 0.0225, **pMaximal (RFP CTL vs. RFP OSMR) = 0.0021, ***pSRC (RFP CTL vs. RFP OSMR) = 0.0004; One-way ANOVA followed by Dunnett’s test, n = 5. j BTSC73 transfected with non-targeting (NT) gRNA control and two different gRNAs against OSMR were subjected to bioenergetic analysis as described in i. ***p < 0.0001; One-way ANOVA followed by Dunnett’s test, n = 5. k BTSC147 transduced with two different OSMR shRNA (shOSMR 1 and shOSMR 2) and scramble shRNA control (SCR CTL) were subjected to bioenergetic analysis as described in i. ***p < 0.0001 for each pairwise comparison except: ***pSRC (CTL vs. shOSMR 2) = 0.0003; One-way ANOVA followed by Dunnett’s test, n = 5. Data are presented as the mean ± SEM. n represents an independent biological sample.