Cell culture and reagents
American Type Culture Collection (ATCC, Manassas, VA, USA) was the source of glioblastoma cell lines: U87, U251, H4, A172, U118, LN229, SHG-44 and GL261. Cell culture was performed in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin (HyClone, Thermo, USA) at 37°C and 5% CO2. NHAs (Normal human astrocytes) were procured from Lonza (Switzerland) and cultured as per supplied instructions. Rapamycin (Selleckchem) and MK-2206 (Selleckchem) were diluted with DMSO (Sigma).
Human glioblastoma samples
Glioblastoma tissues (n = 40) and neighboring healthy tissues (n = 40) were surgically excised at The People’s Hospital of China Medical University and the First Hospital of China Medical University. Table 1 displays the clinicopathological features of the patients. Regular follow-up was performed for patients along with informed consent. Other data included relapse-free survival and overall survival (OS). This work received approval from the ethics committee of The People’s Hospital of China Medical University and the First Hospital of China Medical University.
Immunohistochemistry (IHC)
In accordance with previous works[29, 30], staining of tissues was performed with a SETDB1 antibody (Sigma-Aldrich, USA). The scoring of cells was performed in accordance with the following guide: 0 (no positive staining); 1 (1 to 25% positive cells); 2 (26 to 50% positive cells); 3 (51 to 75% positive cells); and 4 (> 75% positive cells). The scoring for staining intensity was performed as follows: 0, negative; 1, weak; 2, moderate; and 3, high. The formula for SETDB1-positive cells was as follows: staining intensity score × percentage. This score involved both the nucleus and cytoplasm and was analyzed by two pathologists.
Cell invasion assay
The migration assay used Falcon cell culture inserts from BD (Franklin, USA). The invasion assay utilized a BioCoat™ Matrigel™ Invasion Chamber (BD) in accordance with the instructions of the manufacturer. A phase contrast microscope was utilized to count cells; the average of 5 various fields/well was considered.
Wound-healing assay
Indicated ells were cultured in 6-well plates in triplicate for each cell line until approximately 90% confluent. Wounds were made in each confluent monolayer of cells with a sterile 200-μl pipette tip, and fresh growth medium was replaced. Microscopic pictures were taken of the same field at 24 hours.
Macrophage migration assay
The 24-well transwell plates (Corning Inc) were used to examine the macrophages migration induced by CM from U87 cells with indicated treatment. Macrophages were collected and added into the top chamber of 24-well transwell plates. Simultaneously, CM were added into the bottom of transwell chamber. After 24 hours, the cells that crossed the inserts were stained with crystal violet and counted under phase-contrast microscopy.
CCK-8 assay
The indicated cells (5,000 per well) were plated in 96-well plates and grown in normal culture conditions. Cell proliferation was determined every 24 hours for 4 days using a CCK-8 assay.
Clone formation assay
Six-well plates were used to plate single-cell suspensions at a density of 1,000 cells per plate. Every 3 days, the culture medium was replaced. After allowing 2 weeks for clone formation, fixation and staining of the clones was performed using 0.1% crystal violet/40% methanol. Microscopy was performed to count colonies with >50 cells/colony.
RNA extraction and real-time PCR
Real-time PCR was performed as previously described[31, 32]. Briefly, TRIzol (Invitrogen, USA) was utilized for the extraction of total RNA. cDNA was synthesized with the PrimeScript™ RT reagent kit (Takara, Dalian) in accordance with the instructions of the manufacturer. SYBR Premix ExTaq II (Takara, Dalian) was utilized for real-time PCR of this cDNA on an ABI PRISM 7300 (Applied Biosystems, USA) to analyze the chemokines of the immune system. GAPDH was used to normalize mRNA levels. The primers used are listed in Table 2.
Transfection and Knockdown
Transfections with targeted siRNA against AKT were performed using lipofectamine 3000 according to the manufacturer's instructions. Stable SETDB1 knockdown cells were generated by transducing U87 or U251 cells with the pLKO.1-puro lentiviral vector (Addgene) expressing shRNA. Lentiviral particles were generated by co-transfecting 293T cells with the lentiviral vector, pMD2.G (VSVG), pMDLg/pRRE, and pRSV-REV (Addgene). Following lentiviral transduction, cells were plated in 96-well plates in the presence of puromycin (2 μg/ml; EMD/Millipore). SETDB1 expression of the puromycin-resistant clones was then analyzed by Western blotting. The sequences are listed in Table 3.
Western blotting
Western blotting was performed as previously described[33, 34]. Briefly, cell lysis was performed with the RIPA buffer protein extraction reagent (Pierce, Rockford, IL, USA) containing a protease inhibitor cocktail (Roche, USA). The proteins were resolved by 10% SDS-PAGE followed by transfer to polyvinylidene fluoride (PVDF) membranes (Sigma-Aldrich). Next, the membranes were blocked using 5% bovine serum albumin (BSA) and incubated with primary antibodies at 4°C overnight. Appropriate secondary antibodies were later added and then visualized by using an ECL chemiluminescence kit. The primary antibodies used are listed as follows: SETDB1 (HPA018142, Sigma-Aldrich), cleaved caspase 3 (9661, Cell signaling technology), cleaved caspase 8 (9748, Cell signaling technology), slug (9585, Cell signaling technology), vimentin (5741, Cell signaling technology), E-cadherin (14472, Cell signaling technology), mTOR (2983, Cell signaling technology), p-mTOR (5536, Cell signaling technology), AKT (4685, Cell signaling technology), p-AKT (4060, Cell signaling technology), CSF-1 (3155, Cell signaling technology), β-actin (3700, Cell signaling technology).
Macrophage cells isolation and differentiation
The preparation of human monocytes from buffy coats of healthy volunteers was performed using Ficoll-Hypaque (Pharmacia Corporation) for 50 min at 400 g. Twenty-four-well plates were seeded with 2×106 cells/mL in RPMI 1640 medium containing 10% heat inactivated human AB serum, 50 U of penicillin/mL, 50 U of streptomycin/mL, 2 mM L-glutamine, and 100 ng/mL human M-CSF (which allows differentiation into macrophages). Warm medium was used to gently wash away non-adherent cells 6 days post-culture. CD14+ macrophages were found to account for greater than 95% of the adherent cells. The activation of these monocytes to macrophages in vitro involved the treatment of 2×106 cells/L with 25 μg/mL lipopolysaccharide (LPS, Sigma-Aldrich) to produce M1-polarized macrophages and 45 ng/mL recombinant human interleukin-4 (IL-4; R&D) to produce M2-polarized macrophages. Flow cytometry was employed to detect the formation of macrophages. For the following in vitro assays, cells were cultured for 24 hours with RPMI media minus supplements and meticulously washed with PBS prior to the experiments.
Animal experiments
For the xenograft model, 5 × 105 U251-EV, U251-SETDB1, U87-shCon or shSETDB1 cells in 100 μL of PBS was performed followed by subcutaneous injection in the flanks of nude mice. The mice were sacrificed 15 days and tumor weights were assessed. The mice were kept at the Mouse Experimentation Core premises of the China Medical University.
The syngeneic glioblastoma mouse model was generated in accordance with previous reports[35, 36]. Briefly, 2% isoflurane in O2 was used to sedate C57BL/6J mice (4-6 weeks old). The addition of 5 × 105 GL261-EV and GL261-SETDB1 cells in 100 μL of PBS was performed followed by subcutaneous injection in the flanks of C57BL/6J mice. The mice were sacrificed after 19 days and tumor weights were assessed.
Statistical analysis
The mean ± standard deviation (SD) was used to represent the data of triplicate assays. Student’s t-test was applied to assess significant differences between groups. Repeated measures analysis of variance was performed to assess variations between tumor parameters (growth rate and cell growth) of the animals.