Six previously characterized GBM tissue samples (35) from 3 male and 3 female patients aged 42–81 years (mean, 64.2 years) were sourced from the Gillies McIndoe Research Institute Tissue Bank, for this study, which was approved by the Central Health and Disabilities Ethics Committee (ref. no. 15CEN28).

Four micrometer-thick formalin-fixed paraffin-embedded sections of GBM from six patients were used for hematoxylin and eosin (H&E) staining confirming the presence of GBM by an anatomical pathologist (HDB). Immunohistochemical (IHC) staining was performed on the Leica Bond Rx autostainer (Leica, Nussloch, Germany) as previously described (36). 3,3-Diaminobenzidine (DAB) IHC staining for SOX2 (1:500; cat# PA094, Thermo Fisher, Scientific, Scoresby, VIC, Australia), PRR (1:2000; cat# ab40790, Abcam, Cambridge, UK), ATIIR1 (1:30; cat# ab9391, Abcam), ATIIR2 (1:2000; cat# NBP1-77368, Novus Biologicals, LLC, Littleton, CO, USA), ACE (1:100; cat# MCA2054, AbD Serotec, Kidlington, UK) diluted with Bond™ primary antibody diluent (cat# AR9352, Leica) was done for all tissue samples. Immunofluorescent (IF) IHC staining was performed on two representative GBM tissue samples from the original cohort of patients used for DAB IHC staining, using identical primary antibodies and concentrations. Antibodies used for IF IHC detection of PRR and ATIIR2 combinations were Vecta fluor Excel anti-rabbit 594 (ready-to-use; cat# VEDK-1594, Vector Laboratories, CA, USA) and Alexa Fluor anti-mouse 488 (1:500; cat#A21202, Life Technologies, Carlsbad, CA, USA). Antibodies for IF IHC staining for ACE and ATIIR1 combinations were Vecta fluor Excel anti-mouse (ready-to-use; cat# VEDK2488, Vector Laboratories) and Alexa Fluor anti-rabbit 594 (1:500; cat# A21207, Life Technologies). All IF IHC-stained slides were mounted using Vectashield HardSet antifade mounting medium with DAPI (Vector Laboratories).

Appropriate positive control human tissues for the primary antibodies were placenta for PRR (37), liver for ATIIR1 (38) and ACE (39), kidney for ATIIR2 (38), and skin for SOX2 (35). A secondary and tertiary only negative control was performed on a GBM sample randomly selected from the original cohort of GBM samples used for DAB IHC staining.

All DAB IHC stained-slides were visualized with an Olympus BX53 light microscope (Tokyo, Japan) and images were captured with the CellSens 2.0 software (Olympus). IF IHC-stained slides were viewed, and images were captured using an Olympus FV1200 biological confocal laser scanning microscope (Olympus) with images processed using CellSens Dimension 1.11 2D deconvolution algorithm software (Olympus).

Five snap-frozen samples of GBM of the original cohort used for DAB IHC staining were washed in 1× PBS and homogenized in RIPA buffer (cat# R0278, Sigma-Aldrich, St Lewis, MA, USA) supplemented with Halt™ Protease and Phosphatase Inhibitor Cocktail (cat# 1861281, Thermo Scientific, Waltham, MA, USA) and dithiothreitol (DTT) (cat# DTT-RO, Sigma-Aldrich, St Lewis, MA, USA). Protein was precipitated using a Calbiochem^® ProteoExtract^® Protein Precipitation Kit (cat# 539180, EMD Millipore Corp., Billerice, MA, USA) for 1 h at −20°C, washed and re-suspended in 1× Laemmli sample buffer (cat# 161-0737, Bio-Rad, Hercules, CA, USA) with 1% DTT. Equal amounts of protein were heated at 85°C and separated on Bolt™ 4–12% Bis-Tris Plus gels (cat# NW04120BOX, Invitrogen, Carlsbad, CA, USA) via electrophoresis. Separated protein was transferred to a nitrocellulose membrane (cat# IB23001, Life Technologies, Carlsbad, CA, USA) and blocked in 1× TBST containing 2% skim milk powder for 90 min at 4°C. Primary antibody probing for each RAS marker was overnight in TBST at 4°C with the following primary antibodies at the given concentrations: PRR (ATP6IP2, 1:500, cat# ab40790, Abcam, Cambridge, UK), ATIIR1 (AT2R1, 1:500; cat# sc-1173, Santa Cruz, CA, USA), ATIIR2 (1:5000; cat# ab92445, Abcam), and ACE (1:200; cat# sc-12184, Santa Cruz). Secondary antibody probing was in 1× TBST for 1 h at 4°C with goat anti-rabbit HRP (1:10,000; cat# A16110, Thermo Fisher) or donkey anti-goat HRP (1:10,000; cat# ab97120; Abcam). ACE tertiary cascade used a rabbit anti-goat Superclonal™ biotin conjugated secondary antibody (1:20,000; cat# A27013, Thermo Fisher) followed by a Pierce™ Streptavidin Poly HRP (1:5000, cat# 21140, Thermo Fisher) at 4°C for 10 min. β-actin antibody probing was performed with the iBind™ Flex device (cat# SLF2000, Life Technologies) using primary mouse monoclonal anti-β-actin (1:2000 cat# ab8226, Abcam) and secondary donkey anti-mouse Alexa fluor 488 (1:2000; cat# A21202, Thermo Fisher). Clarity Western ECL (cat# 1705061, Bio-Rad) was used as the substrate for visualizing HRP detected protein bands, and the Chemi Doc MP Imaging System (Bio-Rad) and Image Lab 5.0 software (Bio-Rad) were used for both HRP and fluorescent band detection and analysis. Appropriate positive controls were human placenta for PRR (37) and ATIIR1 (40), PC3 cell lysate for ATIIR2 (41), and mouse lung for ACE (42). Negative controls were NTERA2 for ATIIR2, HeLa cell lysate for ACE, and no negative tissues or lysates could be found for either the PRR or ATIIR1.

Total RNA was extracted from ~20 mg of snap-frozen GBM tissue (n = 6) from the same cohort of patients included in DAB IHC staining using the MagJET RNA kit (cat# k2731, Thermo Scientific) and the Kingfisher Duo RNA extraction machine (Thermo Scientific). All samples were quantitated and quality controlled with the NanoDrop 2000 Spectrophotometer (Thermo Scientific) and the Qubit 2.0 Fluorimeter (Thermo Scientific). The samples with A260/A230 ≥ 1.5 and A260/A280 ~ 2 were used for further analyses. The integrity of the RNA was assessed by the New Zealand Genomics Ltd. (Dunedin, New Zealand) using Agilent 2100 BioAnalyzer (Agilent Technologies). The isolated RNA was then subjected to NanoString nCounter™ Gene Expression Assay (NanoString Technologies, Seattle, WA, USA) as completed by New Zealand Genomics Ltd (Dunedin, New Zealand), according to the manufacturer’s protocol. Probes for the genes encoding the PRR (NM_005765.2), ATIIR1 (NM_000685.3), ATIIR2 (NM_000686.3), ACE (NM_000789.2) and the housekeeping gene, and GAPDH (NM_002046.3) were designed and synthesized by NanoString Technologies. Raw data were analyzed with Microsoft Excel using standard settings and were normalized against the housekeeping genes.

3,3-Diaminobenzidine IHC staining for SOX2, PRR, ATIIR1, ATIIR2, and ACE was performed on six GBM samples with the diagnosis confirmed by H&E staining. SOX2 was widely expressed by cells within GBM (Figure 1A, brown). These SOX2+ CSCs, that we have previous identified (35), demonstrated cytoplasmic expression of PRR, which was also expressed on the endothelium of the microvessels (Figure 1B, brown). ACE was expressed on the endothelium of the microvessels only, with minimal staining seen on the CSCs (Figure 1C, brown). Nuclear and cytoplasmic expression of ATIIR1 (Figure 1D, brown) and ATIIR2 (Figure 1E, brown) was observed on the endothelium of the microvessels and the CSCs within GBM.

Expected staining patterns for SOX2 (Image S1A in Supplementary Material, brown), PRR (Image S1B in Supplementary Material, brown), ATIIR1 (Image S1C in Supplementary Material, brown), ATIIR2 (Image S1D in Supplementary Material, brown), and ACE (Image S1E in Supplementary Material, brown) were demonstrated in the respective positive controls. Staining with the omission of the primary antibodies in a GBM sample provided an appropriate negative control (Image S1F in Supplementary Material).

The presence of CSCs within GBM was demonstrated by the relatively abundant expression of the ESC marker SOX2 (Figure 2A, red) on the GFAP+ cells (Figure 2A, green) within GBM, as recently reported (35). We then investigated the expression of PRR (Figure 2B, red) in GBM, by performing IF IHC co-staining with GFAP (Figure 2B, green), which demonstrated that most of the GFAP+ CSCs within GBM expressed PRR. To determine the expression of ACE, we performed dual staining for ACE (Figure 2C, green) and SOX2 (Figure 2C, red) and showed mutually exclusive expression of these markers. Interestingly, ACE was expressed on the endothelial cells with erythrocytes evident within the lumina of the microvessels. We also showed the expression of ATIIR1 (Figure 2D, green) on the SOX2+ (Figure 2D, red) CSC population. ATIIR2 (Figure 2E, red) was expressed on the GFAP+ (Figure 2E, green) CSCs in GBM that were demonstrated to express SOX2 (35). Appropriate negative controls, consisting of omission of the primary antibodies did not reveal any staining (Figure 2F).

Western blotting was performed to examine the presence of components of the RAS in GBM samples of five patients included in DAB IHC staining. PRR (Figure 3A) and ATIIR1 (Figure 3B) were present in all five samples with bands of ~37 and 45 kDa, respectively. Bands of ~70 kDa represent PRR dimerization (Figure 3A). ATIIR2 was absent in all five samples (Figure 3C), while ACE was present, at low levels, in all five samples (Figure 3D).

NanoString analyses demonstrated that PRR and ACE were expressed in GBM samples of all six patients included in DAB IHC staining, while ATIIR1 was present in only two samples, and ATIIR2 was below detectable levels in all six samples examined (Figure 4).