Patient Samples
All samples were continuously obtained from the Department of Neurosurgery, Huashan Hospital, Fudan University. The use of these samples was approved by the Medical Ethical Committee of the Huashan Hospital. GAMs used for transcriptome sequencing were obtained between 2016–2019. Glioma samples used for immunohistochemistry and their GAMs used for western blotting and real-time quantitative reverse transcription PCR (qRT-PCR) were continuously obtained between 2020–2021 (Ethics Number: KY2020-009) (Table. S1). The tissue microarray (TMA) section was obtained as described in our previous study23.
GAMs Isolation
As shown in flowchart (Figure. S1A), glioma tissues were minced with scissors, digested with acctuase (SCR005, Sigma), and filtered into a single-cell suspension in a 40-µm cell strainer (352340, Falcon). After the removal of red blood cells by red blood cell lysis buffer (B541001, Sangon Biotech), cell suspension was diluted in magnetic affinity cell sorting (MACS) buffer (130-091-221, Miltenyi Biotec) and incubated with CD11b microbeads (130-093-634, Miltenyi Biotec) for 15 min at 4 ℃. The cell suspension was applied onto the MS column (130-042-201, Miltenyi Biotec). CD11b-positive cells were then labelled by a washing column. After pushing the plunger firmly into the column, positive cells were eluted and collected24. Flow cytometry was used to verify the cell purity (Figure. S1 B, C).
Cell Culture and Transfection
GL261 glioma cells transfected with firefly luciferase expression vector, GL261-luc cells, were kindly donated by Sichuan University. BV-2 microglia cells were donated by Sun Yat-sen University. To stably express STAP1, BV-2 cells were transfected with lentivirus with pHBLV-EF1-STAP1-Flag-CMV-puro vector (Hanheng Biotechnology), named BV-2-STAP1 and cells transfected with empty vectors were named BV-2-CON (Figure. S4 A, B). After 48 h of transfection, BV-2-STAP1 and BV-2-CON cells were treated with puromycin (5µg/mL; Ant-pr, Invivogen) for 5 d and resistant cells are selected. Cells were grown in Dulbecco’s modified Eagle’s medium (SH30243.01, Hyclone) supplemented with 10% foetal calf serum (10099141C, Gibco), penicillin, and streptomycin (15140-122, Gibco). The cells were cultured in a 37 ℃ incubator with a 5% CO2 atmosphere.
mRNA Isolation and qRT-PCR
mRNA was isolated using the Total RNA Extraction Kit (R1200, Solarbio) and reverse-transcribed into cDNA by qRT-PCR Kit (DBI-2000, DBI Bioscience). Gene amplification was performed using Sybrgreen qPCR mix (DBI-2043, DBI Bioscience) under the following PCR conditions: 95 ℃ for 10 min, 95 ℃ for 15 s, 59 ℃ for 1 min, and 72 ℃ for 30 s for 40 cycles, 45 ℃ for 1 min. We measured transcripts of STAP1 in human GAMs. For BV-2, BV-2-CON and BV-2-STAP1 cells, we measured the transcripts levels of Stap1 and Arg1. All transcripts were normalised to GAPDH. The primer sequences used are listed in Table. S2.
RNA-Sequencing (RNA-seq)
RNA-seq of GAMs was performed by Gemple Biotechnology (Shanghai, China). RNA-seq of BV-2-STAP1 and BV-2-CON was carried out by Mingma Technologies (Shanghai, China) using a second-generation sequencing technique (Illumina Novaseq 6000 platform). Raw reads were quality-controlled using FastQC (v0.11.9) (Andrews, 2010). Gene expression was quantified using RSEM (v1.2.29). Differential analysis was performed using the edgeR (v3.28.1).
Western Blotting
Cells were lysed in RIPA lysis buffer (P0013B, Beyotime) supplemented with protease inhibitors (BL507A, Biosharp) and phosphatase inhibitors (BL615A, Biosharp). Cell lysates were then added to the loading buffer (P1040, Solarbio) and boiled for 10 min at 100°C. Samples were loaded onto sodium dodecyl sulfate-polyacrylamide gel, electrophoresed, transferred onto nitrocellulose membranes, incubated with appropriate antibodies and finally exposed by Western Horseradish Peroxidase (HRP) Substrate (WBKLS0500, Sigma).
Immunohistochemistry and Immunofluorescence Analyses
For immunohistochemistry, the samples were fixed in formalin, embedded in paraffin, and sectioned. The sections were de-paraffinised and blocked with 3% H2O2 (16B22C, Boster) for 20 min. To repair antigens, sections were boiled in sodium citrate buffer (C1032, Solarbio) for 20min and cooled at room temperature. After blocking in 5% bovine serum albumin (BSA) blocking solution (17E23C, Boster) for 30 min, the sections were incubated with primary antibodies overnight at 4 ℃ and secondary antibodies at 37 ℃ for 30min. The colour was developed using a diaminobenzidine (DAB) substrate kit (SK-4100, Vector). Sections were counterstained with haematoxylin solution (G1150, Solarbio) to stain nuclei. For immunofluorescence, the sections were de-paraffinised and boiled in citrate buffer before blocking with 3% BSA (A8010, Servicebio). The sections were then incubated with anti-STAP1 (1:200; HPA038529, Sigma 1:200) or anti-ARG1 (1:100; 93668, Cell Signaling Technology) overnight at 4 ℃ and then incubated with secondary antibodies at room temperature for 50 min. The above steps wre repeated to stain CD11b (1:500, ab8878, Abcam) and CD45 (1:500; GB113885 for human, GB113886 for mouse, Solarbio). After incubation with 4,6-diamidino-2-phenylindole (DAPI) solution at room temperature for 10 min, the sections were sealed with an antifade mounting medium (S2100, Servicebio). For cell immunofluorescence, cells were cultured on glass slides. Then cells were stained with the following steps: fixed with 4% paraformaldehyde (BL539A, Biosharp) at 4 ℃ for 20 min, permeabilised with 0.5% Triton X-100 (9002-93-1, Sangon Biotech) at room temperature for 20 min and blocked with 1% BSA (A8010, Servicebio) at room temperature for 30 min. Sections were incubated with anti-ARG1 (1:200; 93668, Cell Signalling Technology) overnight at 4 ℃, fluorescent secondary antibody (SA00013, Proteintech) at 37 ℃ for 60 min, and DAPI solution (C0065, Servicebio) at room temperature for 10 min the next day. Finally, the sections were sealed with an antifade mounting medium (S2100, Servicebio). For immunohistochemistry, the histological score (H-score) was calculated as the proportion score × intensity score. For immunofluorescence, images were analysed using the HighPlex FL algorithm in HALO software (Indica Labs).
Orthotopic and Subcutaneous Glioma Mice Models
Adult female C57BL/6 mice (6 weeks old, 18–22 g) were purchased from the Jiesijie Experimental Animal Company (Shanghai, China). For orthotopic glioma models, mice were anaesthetised with 1.25% avertin (150µL/10 g; M2920, Nanjing AIBI Bio-Technology) and placed on a stereotaxic apparatus (Ruiwode Shenzhen). To develop the orthotopic glioma model, 5 × 104 GL261-luc cells mixed with 1.25 × 104 BV-2-CON cells or BV-2-STAP1 cells were injected into the right striatum. For subcutaneous glioma models, 1 × 106 GL261-luc cells mixed with 2.5 × 105 BV-2-CON cells or BV-2-STAP1 cells were injected into the right dorsum subcutaneously of the mice. Animal experiments were approved by the Department of Laboratory Animal Science of Fudan University (Ethics Number: 201906007s).
Public Data Access
RNA-sequencing data, clinical information and molecular data were obtained from The Cancer Genome Atlas (TCGA) (https://tcga-data.nci.nih.gov/tcga/) and Chinese Glioma Genome Atlas (CGGA) (http://www.cgga.org.cn). The inclusion criteria and exclusion criteria are shown in Figure. S3.
Statistical Analysis
Data were obtained from at least three independent experiments. Data with a normal distribution were analyzed by T-test. Otherwise, Mann–Whitney U-test would be used. Survival curves are plotted by the Kaplan–Meier method and compared by log-rank tests. Significance levels are as follows: NS (no significance); * (P < 0.05); **(P < 0.01); *** (P < 0 .001); **** (P < 0 .0001).