Human specimens
We examined 28 surgical specimens from patients with LGG (WHO grade 2, n = 13, Supplementary Table 1) or HGG (WHO grades 3 or 4, n = 15, Supplementary Table 2). The diagnosis of glioma was confirmed by a neuropathological examination [5]. Normal-appearing cortex specimens obtained from patients with traumatic injuries were used for comparison (control, n = 5, Supplementary Table 3). These patients did not have a history of neurological diseases. All specimens were immediately frozen with liquid nitrogen after surgical resection and then stored in an ultralow temperature freezer (Sanyo, Japan) at −80 ℃ until subsequent use. Data on the patients were obtained from the databases compiled by the Neurosurgery Department of the General Hospital of Western Theater Command of PLA (China). All procedures and experiments were conducted according to the guidelines approved by the ethics committee of this hospital. All human specimens were used in a manner compliant with the Declaration of Helsinki.
Western blot analysis
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was selected as the loading control. Tissue was homogenized in protein extraction reagent (Keygen, China). The obtained homogenate was centrifuged at 12,000 rpm for 30 min at 4 °C. The protein samples collected from the supernatant were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) at 8% concentration and transferred to 0.45-μm polyvinylidene difluoride membranes, which were subsequently blocked with TBST buffer for 2 h at 37 °C. The membranes were probed with primary rabbit anti-TSP1 (1:1000, MA5-13,398, Thermo Fisher Scientific, USA), rabbit anti-TSP2 (1:600, PA5-97,117, Thermo Fisher Scientific, USA), rabbit anti-TSP4 (1:1000, ab156258, Abcam, USA), and rabbit anti-GAPDH (1:2000, BA2913, Boster, USA) antibodies for 12 h at 4 °C. Next, the membranes were incubated with a horseradish peroxidase (HRP)-conjugated secondary goat anti-rabbit antibody (1:10,000, BA1054, Boster, USA) for 1 h at 37 °C. The immunoreactive bands were visualized with a chemiluminescent substrate (Thermo Fisher Scientific, USA). The optical densities (ODs) of the bands on the Western blots were quantified using Fiji software (USA). The relative expression levels of specific proteins were normalized and calculated as the optical density of the specific protein band/OD of the GAPDH band.
Immunohistochemical (IHC) and immunofluorescence (IF) analyses
Tissues from surgical specimens, including tumor center tissues from patients with glioma and cerebral cortex tissues from patients with surgical injuries, were fixed with a 4% paraformaldehyde solution for 24 h at 4 °C. After dehydration and paraffin embedding, 7-μm-thick tissue sections were subjected to IHC staining using the avidin–biotin-peroxidase method. A primary rabbit anti-TSP2 antibody (1:200, GTX51797, GeneTex, USA) was diluted in 5% bovine serum albumin (BSA) in 0.01 M phosphate-buffered saline (PBS). Immunostaining was performed without primary antibodies as a negative control for each case.
IHC staining was performed in a double-blinded manner to assess the average optical density values. The TSP2 signal was quantified in randomly selected brain slices. The optical density was quantified in regions of interest in the tumor center of samples from patients with glioma and the cerebral cortex of patients with traumatic injuries. Three fields of view were quantified for each site. Semiautomated routines in Fiji were used to calculate the pixel area.
For IF staining, a Microm HM 525 Cryostat (Thermo Fisher Scientific, USA) was used to slice the fixed surgical specimen tissue into 35-μm sections. Primary antibodies, including a rabbit anti-TSP2 antibody (1:600, GTX51797, GeneTex, USA), rabbit anti-Iba1 antibody (1:1000, GTX100042, GeneTex, USA), mouse anti-GFAP antibody (1:600, ab10062, Abcam, UK), and mouse anti-NeuN antibody (1:100, GTX30773, GeneTex, USA), were diluted in 0.01 M PBS. FITC-conjugated goat anti-mouse (1:1000, bs-0296G-FITC, Bioss, China) and Cy3-conjugated goat anti-rabbit (1:300, GB21303, Servicebio, China) secondary antibodies were diluted in 0.01 M PBS. A Nikon A1R confocal microscope (Nikon, Japan) was used for image acquisition with consistent laser intensities and imaging settings. All compared images were acquired with the same exposure time.
Cell culture
Human U251 glioma cells (U251) and human U87MG glioblastoma cells (U87MG) were obtained from Procell Life Science & Technology Co., Ltd (China). Rat C6 glioma cells (C6) were obtained from Cyagen Biosciences Inc. (China). All cells were cultured in high-glucose DMEM (HyClone, USA) supplemented with 10% fetal bovine serum (Gibco, USA) and a 1% penicillin–streptomycin solution (HyClone, USA) at 37 °C in a 5% CO2 atmosphere. All cells were used between passages 2 and 8 for experiments.
Wound healing assay
Cells in logarithmic growth phase were detached with 0.25% trypsin–EDTA (Gibco, USA) to prepare cell suspensions. A total of 4 × 105 cells were inoculated in 6-well plates and cultured at 37 °C in a 5% CO2 atmosphere until they adhered to the plate wall. A 10-μl pipette tip was used to create a vertical scratch in the cell layer after the confluence reached approximately 90%, and the medium was replaced with fetal bovine serum-free high-glucose DMEM. At the same time, the human TSP2 polypeptide (ab96113, Abcam, UK) was added. An optical microscope was used to acquire images at the marked points immediately and at different time points after the wound was created. The unhealed area was measured and analyzed with Fiji software to assess migration.
Transwell (migration) assay
For the preparation of cell suspensions, cells in logarithmic growth phase were detached with 0.25% trypsin–EDTA after culture in serum-free medium for 24 h. Five-thousand cells were inoculated into the upper compartment of the transwell chamber, medium containing 10% fetal bovine serum was added to the lower compartment of the chamber, and the cells were incubated at 37 °C in a 5% CO2 atmosphere for 24 h. The human TSP2 polypeptide was added to the upper compartment of the transwell chamber. The cells on the upper surface of the transwell membrane were removed, and those on the lower surface were fixed with 4% paraformaldehyde for 10 min. After staining with crystal violet at room temperature for 15 min and rinsing with running water, the cells were counted under a microscope.
Cell Counting Kit-8 (CCK-8) assay
Cells (50 cells/μl, total volume of 100 μl) harvested during logarithmic growth phase were seeded in 96-well plates at 37 °C in a 5% CO2 atmosphere. After the cells adhered to the plate wall, the TSP2 protein was added to the experimental group. The medium was replaced with fresh medium containing 10% CCK-8 reagent after 24, 48, or 72 h. The OD value at 450 nm was measured using a microplate reader (Thermo Fisher Scientific, USA) after 60 min of incubation at 37 °C in a 5% CO2 atmosphere.
TSP2 overexpression
A lentivirus expressing TSP2 (LV-EF1a > rat Thbs2-CMV > eGFP/T2A/Puro) was obtained from Cyagen Biosciences Inc. (China). The lentivirus (multiplicity of infection (MOI) = 10) was added to glioma cells, and stable cells were selected with 1.6 μg/ml puromycin (4–5 passages). TSP2 overexpression in the stable cells was verified using qPCR [36] (data not shown).
TSP2 knockout
A total of 2 × 105 glioma cells were seeded into six-well plates. The lenti-CRISPR/Cas9-eGFP-puro-TSP2 knockout construct (gRNA-A1: ACCTTTGACCTTGCCGCACGTGG, gRNA-A2: AGCTCTGCGTCATATAGCTTAGG) obtained from Cyagen Biosciences Inc. (China) was transduced into C6 cells. Stable TSP2 knockout was verified in C6 cells by DNA sequencing [36] (data not shown).
Stereotactic implantation
Twenty-one-day-old healthy male Wistar rats (Chengdu Dossy Experimental Animals Co., Ltd., China) were selected for glioma cell implantation [36,37,38,39]. Rats were anesthetized by administering an intraperitoneal injection of 10% chloral hydrate at 0.3 ml/100 g, and the head was then fixed in a stereotactic frame (RWD, China). The skull was fully exposed, and a 1-mm-diameter hole (3 mm anterior to the bregma, 3 mm lateral to the sagittal suture) was drilled on the left side. C6 cells (4 × 106 cells/ml, total volume of 10 μl) were injected into the cortex with a stereotactic device at a rate of 1 μl/min. The vertical injection depth was 3.0 mm. The needle was retained in place for 10 min and was then slowly pulled out vertically. After the hole was sealed with bone wax, the scalp was sutured, and the skin was disinfected. One week after the C6 cells were implanted, the rat glioma transplantation model was used for subsequent experiments. This study was approved by the Ethics Committee of the General Hospital of Western Theater Command of PLA and was conducted in accordance with the international animal care guidelines established by the Declaration of Helsinki. Every effort was made to relieve the pain and discomfort of the animals.
MRI analysis
After rats were anesthetized and fixed in a 7-cm small animal coil (MR750 3.0 T, GE, USA), coronal and sagittal MRI scans were performed. The scanning parameters were set as follows: layer thickness, 2 mm; layer spacing, 2 mm; and FOV, 120 mm × 120 mm. For T1WI, a 0Ax T1-FSE sequence was used, where the TR/TE was 6.072 ms/597 ms; for T2WI, a 0Ax T2-FSE sequence was used, where the TR/TE was 123.9 ms/5000 ms. The MRI analysis and tumor volume calculations were performed using syngo fastView software (Siemens, Germany). The image of the largest level of the tumor was selected to measure the volume. The following formula (mm3) was used for the calculation: length × width × height × (π/6).
Statistical analysis
All data are presented as the mean ± SEM values. SPSS 26.0 (IBM, Armonk, NY, USA) and GraphPad Prism 8.0 software (GraphPad Software Inc., La Jolla, CA, USA) were used for statistical analyses. ImageJ software was used to analyze the gray values of the Western blots, optical density values of IHC images, gray values of IF images, and areas of cell wounds. An unpaired t-test was used for comparisons between two groups, and ANOVA followed by the Bonferroni post hoc test was used for comparisons among three or more groups. P < 0.05 was considered statistically significant.