Tumor samples
Resected tissue from 18 GBM patients (Additional file 1: Table S1) was collected from the neurosurgery department at Skane University Hospital in Lund, Sweden. Written consent was signed by each patient in accordance with the approved ethical permit from the regional Swedish ethics committee (Dnr 2018/37). Using a surgical navigation system (Medtronic, USA), peri-tumor tissue was resected according to the treatment plan and tumor tissue was obtained. Anatomical, pathological diagnosis and routine molecular analysis were ascertained in each case by Skane University Hospital pathology department according to WHO GBM criteria. The resected tissue was divided into two parts: one part was fixed with 4% Paraformaldehyde (PFA) for tissue staining and the other part was put into ice-cold artificial CSF [21] for primary cell culture.
Cell culture
Fresh surgical tissue in artificial CSF was immediately transferred to the lab and dissociated as a cell suspension. Dissociated cells were divided into two parts and grown in different conditions: serum-free condition (SFC), i. e. Neurobasal medium (Gibco) added with 1 mM sodium pyruvate (Gibco), 1 × B-27 supplement (Gibco), 1 × N-2 supplement (Gibco), 1 × non-essential amino acids (NEEA) (Gibco) or serum-condition (SC) DMEM/F12 (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco). Cells in SFC were supplemented with EGF (Thermo Fisher Scientific) and FGF (Thermo Fisher Scientific) 20 ng/ml and grown on the Poly-l-ornithine (Merck) and Laminin (Gibco) coated dishes.
The U87 cell line was maintained and expanded in DMEM medium (Gibco) supplemented with 1% NEAA and 10% FBS. The GL261 mouse glioma cell line was maintained in RPMI 1640 medium (Gibco) supplemented with 1 mM sodium pyruvate and 10% FBS. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) were collected from healthy donors as described previously [22]. BM-MSCs were maintained in StemMACS MSC Expansion Media (Miltenyi Biotec) added with 10% FBS and L-glutamine (Gibco). All the media were supplemented with 1% Penicillin–Streptomycin (Gibco) and mycoplasma detection was performed using MycoProbe Mycoplasma Detection Kit (R&D). Primary cells were expanded in culture up to passage five and then frozen at − 150 °C.
Flow cytometry
Primary cells were detached by incubating with Accutase (Gibco) at 37 °C for 4 min. After dissociation, cells were diluted with FACS buffer (eBioscience) and filtered through a 75 µm cell strainer (Fisher Scientific) to obtain a single cell suspension. Cells were counted and approximately 1 million cells were aliquoted into each sterile Eppendorf tube. Samples were stained with the following antibodies: PE Mouse anti-Human CD105 Clone 266 (RUO) (BD Biosciences), Human Endoglin/CD105 PE-conjugated Antibody (R&D), APC-R700 Mouse Anti-Human CD274 Clone MIH1 (RUO) (BD Biosciences), APC Mouse Anti-Human CD133 Clone W6B3C1 (BD Biosciences), APC-R700 Mouse IgG1, κ Isotype Control (BD Biosciences), PE Mouse IgG1, κ Isotype Control (BD Biosciences) and incubated on ice for 30 min. Samples were washed with FACS buffer for 3 times and 7-AAD (BD Biosciences) or Hoechst 33,342 (BD Biosciences) was added as cell viability markers. Cells were sorted on a FACSAria III cell sorter (BD Biosciences, USA). Data was analyzed by FLOWJO software (BD Biosciences, USA).
Immunohistochemistry
Frozen tissue was cut into 10 µm thickness using a cryostat (Leica, Germany). Cells was seeded on 8 well CultureSlides (Falcon, USA) and fixed by 4% PFA. Samples were washed with Phosphate Buffered Saline (PBS) (Gibco) and incubated for 1 h with PBS buffer containing 0.25% Triton X-100 and 5% Bovine Serum Albumin (BSA) (Merck) or 5% Donkey serum (Jackson ImmunoResearch) or 5% Goat serum (Jackson ImmunoResearch). The following primary antibodies was used for incubating at 37 °C for 1 h or 4 °C overnight: CD105 (1:100, AF1097, R&D), CD105 (1:200, AF1320, R&D), PD-L1(1:100, R&D), hFABP4 (1:100, R&D), hOsteocalcin (1:100, R&D) and hAggrecan (1:100, R&D), CD31 (1:100, Dako), NG2 (R&D, 1:200), Vimentin (1:500, Dako), Hu-Nu (1:250, Merck), SOX2 (1:200, Merck), CD73 (1:100, Merck), α-SMA (1:250, Merck), CD68 (1:100, Gene Tex), CD163 (1:100, Gene Tex), Nestin (1:200, Abcam), β III Tubulin (1:500, Abcam), Ki67 (1:250, Abcam), Iba1 (1:200, Abcam), CD11b (1:200, Abcam), NeuN (1:200, Abcam), GFAP ( 1:1000, Abcam), S100β (1:200, Abcam), CD34 (1:200, Abcam), vWF (1:100, Abcam), FAP (1:100, Invitrogen), CD90 (1:100, Santa Cruz), PDGF-β (1:200, Santa Cruz). Secondary antibodies conjugated to DyLight 488, 594 and 647 (1:200, Jackson ImmunoResearch) were applied for 1 h incubation and nuclei were stained with DAPI (1:1000, Invitrogen). Images were captured by an epifluorescence Olympus BX61 Microscope equipped with an Olympus DP80 Color Camera—9MP and CellSens acquisition software (Olympus Sverige AB, Solna, Sweden), or a Zeiss LSM 780 confocal microscope and Zeiss ZEN software (Carl Zeiss Microscopy GmbH, Germany).
Cell viability assay
Cell viability was detected by the PrestoBlue™ Cell Viability Reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol. Briefly, 1000 cells were seeded in 96 microplate wells (Greiner Bio-One) with 90 µl cell culture media in each well. 10 µl PrestoBlue regent was added for 10 min at 37 °C. Fluorescence units were read by a fluorescence microplate reader (Molecular Devices, USA) at 590 nm emission.
Mesenchymal stem cell assay
GBM CD105+ cells at low passage (P3-P5) were used for mesenchymal stem cell (MSC) differentiation assays using a human MSC identification kit (R&D) according to the protocol provided by the manufacturer. Briefly, GBM CD105+ cells were cultured in human/mouse StemXVivo Osteogenic/ Adipogenic Base Media (R&D) with Adipogenic Supplement(R&D) and Osteogenic Supplement(R&D), respectively, for up to 14 days for adipogenic differentiation or osteogenic differentiation. For chondrogenic differentiation, cells were culture in human StemXVivo Chondrogenic Media (R&D) supplemented with ITS Supplement (R&D) and pelleted in 5 ml tube for up to 20 days. Cells were fixed and detected by anti-hFABP4, anti-hOsteocalcin and anti-hAggrecan immunohistochemistry.
GBM CD105+ spheroid formation
Twelve-well dishes were precoated with 0.01% Poly-L-ornithine overnight and washed twice with PBS before coating with 10 µl/ml laminin for 4 h. 0.5 × 106 GBM CD105+ cells were seeded in DMEM/F12 medium supplemented with 10% FBS. b-FGF 20 ng/ml was added every 48 h and the medium was changed every 3 days. Cells were inspected daily using an inverted light microscope. Once spheres were formed, the culture was filtered through a 70 µm cell strainer and spheres transferred into a 5 ml tube with fresh medium. The tube was centrifuged at 200 × g to pellet the spheres. Spheres were then sectioned by the cryostat before staining.
DNA isolation and library preparation
Genomic DNA was isolated from the cells using the QIAamp DNA Micro kit (Qiagen) according to the manufacturer's protocol. The isolated DNA was dissolved in 25 μl of EB buffer (Qiagen) and the quality quantity was detected by the Nanodrop ND 1000 spectrophotometer (Thermo Fisher Scientific, USA), and stored at -20 °C. To construct the Library, DNA was diluted to 10 ng/μl in a total volume of 50 μl with 1 × EB buffer. Adaptor-ligated libraries was set by unique dual indices using KAPA-HyperPrep-Kit (Roche). Fragmentation of DNA was accomplished by sonication using the following parameters: 75 s Peak Power, 50.0 Duty Factor, 10.0 Cycles and burst 1000. After End repair and A-Tailing, adapter ligation was performed using 2 µl X Gen Duplex Adapters (15 µM) 30 min at 20 °C. The adaptor-ligated library was PCR amplified by 5 cycles according to the manufacturer's instructions with 8-bp unique dual index primer pairs. Hybridization of samples targeting predetermined regions of the genome provided by Twist Target Enrichment Protocol (Twist Bioscience) using Twist Hybridization and Wash kit (Twist Bioscience). The probes are labeled with biotin and streptavidin-conjugated magnetic beads (Lablife Nordic) purified DNA libraries. The adaptor libraries contain both primer sites used for post capture amplification by PCR methods. The quality and quantity of the exon-enriched capture library was measured by Agilent 2100 Bioanalyzer (Agilent, USA).
DNA sequencing and data analysis
The pooled libraries at 0.7 nM loading concentration and 1% of PhiX Control (Illumina) were sequenced in a NovaSeq 6000 System (Illumina, USA) using NovaSeq 6000 SP Reagent Kit (Illumina), following the manufacturer's instructions. The sequencing data was analyzed on Illumina DRAGEN Bio-IT Platform (Illumina, USA) with the following steps. Raw basecalls were converted to Fastq files. Adapters are trimmed if added to samplesheet. Fastqs were mapped and aligned to the reference genome (hg38) (http://www.ncbi.nlm.nih.gov). Variants (SNV and SV) were called in the target region. Clinical grade annotation of all variants was passed with basic filtering in the DRAGEN platform. The quality of metrics from raw sequencing reads was calculated by FastQC [23].
In vitro cytokine array
Cell culture medium from 1 × 106 cells was collected after 72 h and centrifuged at 1000 g for 5 min. The supernatant was aspirated and stored at − 80 °C. Cytokine array was performed using a Human Angiogenesis Array C1 kits (RayBiotech) according to the manufacturer's instructions. Culture medium with 10% FBS was used as control. The membrane was scanned by azure biosystems C600 (Azure Biosystems, USA), and protein levels were semi-quantified by measuring the gray level values using Image J software [24].
Temozolomide (TMZ) and bevacizumab resistant assays
1000 CD105+ cells per well were cultured in 96 Well plates (Greiner Bio-One). Cells were treated with different concentration of TMZ (0.1 µM to 50 µM) and bevacizumab (10–2000 ng/ml) and cocultured for up to 96 h. Cell viability was detected by PrestoBlue™ Cell Viability Reagent according to manufactures’ protocol at 24 h, 48 h and 96 h. Fresh drug and media were added after the detection. Data was analyzed by Soft Max Pro V6.4 (Molecular Devices, USA).
Small molecule screening and analysis
The 88 anticancer drugs were selected from Selleckchem (L3000) library. The cells were seeded in 96-well plates at 1000 cells per well. The chemical compounds were dissolved in DMSO with two concentrations (0.5 μM and 10 μM) and added to the previous 96-well plates. The same volumes of DMSO (0.005% and 0.1% in volume) were added to the control group. Cells were incubated in 5% CO2 at 37 °C for 96 h and the cell viability was detected at 48 h and 96 h. After the drug sequencing, the 4 most effective drugs were selected. A drug-gene interaction analysis of the effects of these compounds were predicted by the DGIdb database as previously described [25]. The relation between the 10 genes with the highest drug-gene interaction score and GBM patient survival (TCGA database) was analyzed on cBiopartia online platform (https://www.cbioportal.org).
Animal studies
All animal procedures were guided by the practices of the Swedish Board of Animal Research and approved by the Committee of Animal Ethics in Lund-Malmo, Sweden (permit 5372–20). The JAX®NSG® mouse model (Charles River) was a gift from Dr. Henrik Ahlenius. Female mice at 6 weeks were used for tumor cell injection. Mice were anesthetized with isoflurane and fixed in a stereotactic frame (David Kopf Instruments, CA). Cells were suspended in culture media at a concentration of 50,000 cells/µl and 5 µl was injected into the right striatal region using a Hamilton syringe (Hamilton, Switzerland). Immediately after the appearance of neurological symptoms, the brain was harvested following transcardial perfusion with 4% PFA. Tumor volumes were macroscopically assessed in coronal sections and calculated from the formula (length × width2)/2.
Statistical analysis
Statistical analyses were performed using GraphPad Prism (GraphPad Software Inc., CA). Results are presented as mean ± SD. Comparisons between groups were performed by two-tailed Student’s t test or by one-way ANOVA, followed by Tukey’s multiple comparisons test. Kaplan–Meier survival curves were compared using a log rank test. R Studio with R packages (http://www.rstudio.com) was used for statistical analysis of DNA sequencing and Drug sequencing. Survival curves were analyzed by the online platform: cBiopartial (https://www.cbioportal.org) and GEPIA2 (http://gepia2.cancer-pku.cn). P < 0.05 was considered statistically significant.