Cell culture and preparation of conditioned medium
Human umbilical vein endothelial cells (HUVECs), the human glioblastoma cell lines U87 and U251, and human microglial cell line HMC3 were acquired from the cell library of the Chinese Academy of Sciences. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin reagent (Gibco), and incubated at 37◦C in a humidified atmosphere with 5% CO2. The medium was refreshed every two days. Controlled cell numbers of U87, U251, and HMC3 cells were cultured in serum-free medium for 24 h. Then, the cell supernatant was collected as conditioned medium for use in the human cytokine antibody array.
Human cytokine antibody array
A human cytokine antibody array (RayBio® C-Series Human Cytokine Antibody Array C5, Ray Biotech, USA) including 80 different cytokines was used to measure the levels of several cytokines in the conditioned medium of U87, U251 and HMC3 cells, following the manufacturer’s instructions.
Clinical specimens
Serum specimens were collected from normal human volunteers (n = 20), pre-operative GBM patients (n = 20) and post-operative GBM patients within 72 h (n = 20), and stored at -80◦C. Patients (n = 20) received surgical treatment at Fudan University Shanghai Cancer Center between January 2021 and June 2021. Informed consents were collected from all patients and volunteers. Ethical approval was obtained from the ethics committee of the Fudan University Shanghai Cancer Center.
Enzyme-linked immunosorbent assay (ELISA)
ELISA was performed to assess the SPP1 levels in serum samples using a commercial kit according to the manufacturer’s instructions (ELH-OPN-1; R&D company, USA). Serum samples were diluted 25-fold, and then directly added to the specific 96-well plate previously coated with a human SPP1 antibody contained in the kit. The absorbance value was assessed at 450 nm using a microtest plate spectrophotometer. The SPP1 levels were quantified based on a standard SPP1 curve.
Bioinformatic analysis
The clinical analysis of SPP1 and HIF1α, including expression levels, the Kaplan-Meier curves of overall survival and the receiver operator characteristics (ROC) curve, was performed using The Cancer Genome Atlas (TCGA) database datasets. The correlations of targeted genes were assessed using GEPIA (http://gepia.cancer-pku.cn/). The open-access transcription factor database JASPAR (http://jaspar.genereg.net) was used to explore potential transcription factors binding to PSMA promotor.
RNA isolation and quantitative real-time PCR
Total RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The RNA quality and quantity were evaluated as previously described [12]. Quantitative real-time PCR was also performed as previously described [12]. The expression levels of GAPDH, used as control, and targeted genes were assessed. All reactions were run in triplicate. Primer sequences were as follows:
h-GAPDH-F, 5-ACAACTTTGGTATCGTGGAAGG-3,
h-GAPDH-R, 5-GCCATCACGCCACAGTTTC-3;
h-PSMA-F, 5-ACACAGATACCACATTTAGCAGG-3,
h-PSMA-R, 5-TTTGGGTAGGACAACAGGACA-3;
h-HIF1α-F, 5-ATCCATGTGACCATGAGGAAATG-3,
h-HIF1α-R, 5-TCGGCTAGTTAGGGTACACTTC-3.
Protein extraction and western blot analysis
Cell pellets were washed twice in cold PBS (Gibco), and then lysed in RIPA buffer supplemented with 1% PMSF and 1% phosphatase inhibitor on ice. The protein concentration was determined using a BCA protein assay kit (Beyotime Biotechnology).
Protein samples (10 μg) were handled and transferred to membranes as previously described, then incubated with PSMA (1:1,000), HIF1α (1:1,000), and GAPDH (1:2,000) antibodies at 4◦C overnight [12]. Proteins were washed there times with TBST, and incubated with the corresponding secondary antibodies (1:5,000) at room temperature for 2 h. The membranes were washed again, and then incubated with enhanced chemiluminescence (Thermo Fisher Scientific) for 1 min. Finally, the protein band images were captured and analyzed.
Tube formation assay
HUVECs with HIF1α knocked down or not were cultured under different treatment conditions, adding recombinant protein SPP1 (1 µg/ml) or PBS, in a 24-well plate pre-coated with Matrigel (50 µl/well, corning) for 24 h. Capillary-like tube formation was photographed under an inverted microscope. Tube length and branching points were calculated using the NIH ImageJ software.
Scratch assay
HUVECs were seeded and cultured under different treatment conditions (+/- HIF1α knockdown, +/- SPP1 or PBS, respectively) in a 6-well plate and grew to 100% confluency. Pipette tips of 200 µl were used to scratch the cell monolayer. Then, the plate was gently washed with PBS to clear detached cells. Images were captured at 0 h and 48 h under an inverted microscope.
Promoter activity assessment using a dual-luciferase assay
The Dual-Luciferase® Reporter Assay kit (Promega) was used to evaluate promoter activity following the manufacturer’s instructions. The luciferase reporter construct PSMA-pGL3-Promoter-Luc was transiently co-transfected into HUVECs grown in 96-well plates using LipofectamineTM 3000 (Invitrogen). HUVECs were previously treated and grouped correspondingly (+/- HIF1α knockdown, +/- SPP1 or PBS, respectively). Both firefly and Renilla luciferase activities were analyzed at 72 h after infection using a dual-luciferase system on GloMax® Discover (Promega, Madison, USA).
Chromatin immunoprecipitation (ChIP) assay
For ChIP analysis, HUVECs were treated with 1 µg/ml SPP1 or PBS and harvested separately. Cell samples in each dish with 8 ml medium were fixed using 210 µl 37% formaldehyde for 10 min, and then the fixation was stopped by adding 400 µl 2.5 M glycine. The mix was slowly shaken for 2 min until the liquid turned yellow and then washed three times with precooled PBS. Next, 1 ml PBS was added to each dish, the cells were scraped down into the EP tube, and centrifuged at 9000 rpm for 30 s. Finally, the cells were collected and supernatants discarded. The precipitate was lysed with 400 µl 1% SDS lysate, mixed and incubated on ice for 10 min, followed by sonication on ice with 22% power and centrifugation at 13200 rpm for 10 min at 4◦C. Finally, 300 µl supernatant was collected. Electrophoresis was conducted to ensure the majority of the DNA fragments were between 300–700 bp and the rest of the supernatant was stored at -80◦C. A total of 300 µl supernatant was diluted with 0.6 mL dilution buffer containing PMSF (sigma). Agarose A or G was washed three times with TE. Each EP tube was prepared with 300 µl chromatin, 1.2 ml dilution buffer and 80 µl beads (50% turbidness), and then rotated for 1 h at 4◦C. After centrifugation at 4,000 rpm for 2 min, 50 µl supernatant was obtained as input. The supernatants were divided into two portions (475 µl each), one of which was incubated with rotation overnight in a cold chamber with the PMSA antibody (2 µg) and the other with the same amount of normal IgG. The precipitated DNA was recovered using a PCR purification kit (TransGen Biotech) and analyzed using qRT-PCR with a SYBR Green Real-Time PCR Master Mix (Fermentas). ChIP values were normalized to their respective input values, and the fold changes in concentration were assessed based on the relative enrichment in anti-PSMA compared with anti-IgG immunoprecipitates.
Statistical analysis
Data are presented as the mean ± SD. The data were analyzed using Graphpad Prism 9.0 software, and independent Student's tests (two-tailed) and one-way ANOVA tests were used to analyze the differences between groups. Pearson and Spearman correlation analysis was used for correlation analysis. P value < 0.05 was considered statistically significant.