Collagenase type 3

For in vitro analysis, cells were washed with phosphate-buffered saline and dissociated from the plate with accutase (Gibco) for 5–10 min at 37 °C to generate single cell suspensions. For in vivo studies, tumors were excised post-mortem, mechanically digested using gentle MACS tubes (Miltenyi) and enzymatically digested using a mixture of 0.5 mg/ml collagenase type III (Sigma-Aldrich), 0.01 mg/mL dispase, and 0.125 mg/ml DNAase (Sigma-Aldrich) with antibiotic, 30-min at 37 °C. Tissue dissociates were passed through a 70-µm filter to collect a single cell suspension. Single cell suspensions were washed once in flow staining buffer and incubated with respective flow antibodies at 4 °C for 30 min in the dark. DAPI was used to discriminate viable and dead cells. Tumor cells were gated on EpCAM-positive cells. Flow cytometry was performed using the following antibodies: HLA-A,B,C/Alexa Fluor488 (Biolegend, clone W6/32, 1:200), HLA-DR/PE-Cy7 (Biolegend, clone L243, 1:200), mouse MHC-I (H-2Kd/H-2Dd)/PE (Biolegend, clone 34-1-2S, 1:200), mouse MHC-II (I-A/I-E)/Alexa Fluor488 (Biolegend, clone M5/114.15.2, 1:200), mouse CD274(PD-L1)/APC (Biolegend, clone 10F.9G2, 1:200), and mouse EpCAM/PE-Cy7 (Biolegend, clone G8.8, 1:350). Samples were analyzed on an Attune NxT system (Life Technologies).

Primary meningioma were obtained from consented individuals following the ethical guidelines included in the ‘Investigation into the expression of signalling molecules in human brain tumour samples (Research Ethics Committee (REC) number 6/Q2103/123)’ and the ‘Identifying and validating molecular targets in low grade brain tumours (REC number 14/SW/0119) study’. Primary tumours were mechanically disrupted after overnight incubation with a digestion medium. Meningioma medium consisted of DMEM, 10% FBS, 100 U/mL penicillin/streptomycin and 20 U/mL collagenase type III (Sigma-Aldrich, Dorset, UK). The digested sample was collected and centrifuged, the cells were then cultured in meningioma medium [DMEM, 10% FBS, 100 U/mL penicillin/streptomycin, 2 mM L-glutamine (Gibco) and 1% v/v D(+) glucose solution (Sigma)]. Merlin protein expression was determined by Western blot and only samples with no expression were used in subsequent Merlin-deficient experiments (Figure S1A). Human meningeal cells (HMC) (Sciencell, Carlsbad, CA, USA) and 293T cells were cultured as directed by the manufacturer.

Ten pinch biopsies taken from each site were placed in RPMI medium (Life Technologies) containing 10% fetal calf serum (FCS) (Bovogen, VIC, Australia), as well as 100 U of penicillin/streptomycin (Life Technologies; R10/P/S) to minimize bacterial contamination from the GALT samples (37 (link)). Biopsies were washed three times with HBSS (Life Technologies) containing 1% PenStrep, 0.1M DTT (Life Technologies) (pH 8.0), and 0.15% EDTA (Sigma-Aldrich, MO, USA) (0.5M) (38 (link)) on a shaking incubator at 130 rpm at 37°C for 20 min. Supernatants collected after each wash step were retained for each site. Biopsies were then minced using sterile scissors and enzymatically digested with 200 U/ml collagenase type III (Sigma-Aldrich) (37 (link)–39 (link)) and 200 mg/ml DNase (Sigma-Aldrich) in R10/P/S for 1 h at 37°C in a shaking incubator at 110 rpm. A single-cell suspension was prepared by pushing digested tissue through a 70-μm cell strainer for flow cytometry.

Tumor tissues were disaggregated with an enzyme cocktail containing collagenase type III (Sigma), hyaluronidase (Sigma), and collagenase type IV (Sigma), washed several times, and resuspended in phosphate-buffered saline (PBS) to produce a single cell suspension. Fluorescence was measured using a FACScalibur instrument and FACSDiva 6.0 software (BD Bioscience). Tumor cells were gated according to DsRed expression and side scatter (SSC). The hypoxic subpopulations were further gated or isolated based on the analysis of Hoechst 3342 and GFP fluorescence in dot plots. The control cells are derived from disaggregated the orthotopic GBM8401 or U87 xenografts, which are both Hoechst 3342 and GFP-negative, and were set in the lower left quadrant of the plot. Same setting conditions were used thereafter, cell populations located outside of this quadrant of the plot were defined as either Hoechst 3342⁻ and GFP⁺ cells (chronic hypoxic cells), Hoechst 3342⁺ and GFP⁺ cells (cycling hypoxic cells) or Hoechst 3342⁺ and GFP⁻ cells (Normoxic cells).