The rat F98 glioma cell line was obtained from ATCC (Manassas, VA) and grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and penicillin at 37°C in an atmosphere of 5% CO2. These materials for the culture medium were purchased from Gibco Invitrogen (Grand Island, NY, USA).
Animal experiment A: X-ray irradiation to the rat brain
All of the experimental procedures used in this study were approved by the Animal Review Board and Ethical Committee of Osaka Medical College (Approval No.: 27058). This study was conducted in accordance with the 'Ethics Declaration' for the ARRIVE guidelines' (https://arriveguidelines.org). Eight-week-old, male Fischer 344 rats weighting between 270-320 g (Japan SLC, Inc. Shizuoka, Japan) were anesthetized with an intraperitoneal injection of a mixture of medetomidine 0.15 mg/kg, midazolam 2 mg/kg, and butorphanol 2.5 mg/kg.
During X-ray irradiation, the 5 mm-thick lead shield with a 1 cm square window were used to protect the whole body of the rats. For rats in the irradiation groups (IR: n=8), 100-kV X-ray was irradiated to the right cerebral hemisphere through the 1 cm square window, with a single dose of 65 Gy using a linear X-rat accelerator (SOFTEX M-150WE; Kanagawa, Japan). The irradiation area was determined with the bregma as reference points. The sham-irradiation was performed for rats in the control group (Sham-IR: n=7).
RNA and protein extraction from the irradiated rat brain
Three months after the X-ray irradiation, the rats were euthanized to obtain their brain tissue. After the brains were divided along the midline, the tissue protein and whole RNA were separately extracted from the right (IR/Ipsi-brain: n=8) and left cerebral hemispheres (IR/Contra-brain: n=8) of the animals irradiated on the right brain as mentioned in Figure1A. As a control, we prepared proteins and total RNA extracted from the right cerebral hemisphere of the sham-irradiated animals (Sham-IR/Brain: n=7). The RNA was extracted using miRNeasy Mini Kit (Qiagen, Hilden, Germany), and cDNA library preparation was performed using a SuperScript VLIO cDNA Synthesis kit (Thermo Fisher Scientific, Cleveland, USA) according to the manufacturer’s instructions. The brain tissue protein was extracted with the 3-min total protein extraction kit for animal tissues (101 Bio, Palo Alto, CA). The concentrations of the extracted protein were determined by the PierceTM BCA Protein Assay Kit (Thermo Scientific-Life Technologies, Waltham, MA).
In vitro cell proliferation assay using F98 glioma cells
The proliferation ability of the tumor cells was analyzed by a WST-8 assay. F98 cells were seeded in 96-well plates at a density of 10,000 cells/well in 100 μl of culture serum-free DMEM. At 24 hours after this pre-incubation, 100 μl of medium containing the extracted brain protein with 1.5, 3.0 or 6.0 μg/ml was added to the wells. 72 hours after the incubation, 10 μl of WST-8 labeling reagent (Cell Counting Kit; Dojindo Laboratories, Kumamoto, Japan) were added to each well and the absorbance at 450 nm was measured with the corona grating microplate reader (Hitachi High-Tech Corporation, Japan).
In vitro tube-formation assay using HUVEC
For the in vitro tube-formation assays, 96-well plates were coated with growth-reduced factor Matrigel (BD Biosciences, catalog number: 354230) on ice and incubated at 37°C for 2 hours until the Matrigel became solid. Human umbilical vein endothelial cells (HUVECs) (Life Technologies, Invitrogen, catalog number: C-003-5C) were seeded in 96-well plates at a density of 20,000 cells/well and cultured in 150 μl of medium containing 3.0 or 6.0 μg/ml of the cerebral proteins extracted the above. After a 3-hour (incubation in 5% CO2 at 37°C, images were taken with at a magnification of 100× (10 ocular×10 objective), and the lengths of the tubes in four random fields were quantified by Image J software version1.52. (U.S. National Institutes of health, Maryland, USA)
Animal experiment B: glioma cell transplantation into the irradiated rats
Animals groups and study design
Thirty-two male Fischer 344 rats (8-week-old, weighting between 270-320 g) were used.
65-Gy of X-ray was irradiated to the right cerebral hemisphere of 20 rats as described above, and sham-irradiation was performed for 12 rats to prepare a control group.
Three months after the irradiation, the 20 rats were randomly divided into two groups (IR/Ipsi-tumor or IR/Contra-tumor) as mentioned in Figure1B. In “IR/Ipsi-tumor” group (n=12), the F98 glioma cells were implanted to the right cerebral hemisphere of the irradiated animals. On the other hand, in the “IR/Contra-tumor” group (n=8), F98 cells were implanted to the left hemisphere of the irradiated animals. All the sham-irradiated animals (n=12) were implanted with F98 cells into right hemisphere (Sham-IR/Tumor group). The rats were observed daily for body weight and neurological deficits (n=5). They were euthanized by inhalation of Sevoflurane one days before of death, and the brains were removed for the following analyses.
At the chronic phase (3 months) after the X-ray irradiation, F98 glioma cells were implanted into the cerebral hemisphere of rats in the above three groups. Under anesthesia, the rat’s head were fixed with a stereotactic frame (Model 900; David Kopf Instruments, Tujunga, CA, USA), and a small burr hole was made on the skull with an electric drill at 1 mm anterior and 3 mm lateral of the bregma. 1×104 of F98 cells were suspended in 10 μl of DMEM and injected at a rate of 1 μl/min into the brain at a depth of 5 mm from the skull burr hole, using a 25-μl Hamilton microsyringe with a 26-gauge needle (model 1700 RN, Hamilton Bonaduz AG, Bonaduz, Switzerland) and a micro-injector (WPI model UMP3, Sarasota, FL, USA). After the injection was complete, the burr hole was sealed with bone wax.
Immunohistochemical analyses for the tumor
Cell proliferation index: The primary antibody, Ki-67 (clone MIB-1; dilution of 1:100) (Abcam, Cambridge, MA, USA), and a universal secondary antibody (dilution of 1:300) (Roche, Basel, Swizerland) were used. The cell proliferation index was calculated by taking five random pictures of each slide at a magnification of 200× (10 ocular×20 objective). The index was calculated as the number of MIB-1-stained cells divided by the total number of cells. HematoxylinⅡwas used for the counterstaining for cell nuclei.
Apoptotic index: To detect the cell apoptosis, the terminal deoxynucleotidyl transferase (TdT) -mediated dUTP nick end labeling (TUNEL) assay was performed using an ApopTag Peroxidase In Situ Apoptosis Detection Kit (Cosmo Bio, Tokyo, Japan). The apoptotic index was evaluated by counting the number of TUNEL-positive cells over the total number of cells at a magnification of 200× (10 ocular×20 objective) in five random fields.
Microvascular density (MVD) index: The primary antibody, CD34 (dilution of 1:2500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), was used with a universal secondary antibody (dilution of 1:300) (Roche, Basel, Swizerland). The MVD index was calculated by taking five random pictures of each slide at a magnification of 200× (10 ocular×20 objective). The index was calculated as the number of CD34-stained cells divided by the total number of cells.
Evaluation of glioma invasiveness
F98 rat glioma model shows tumor cell invasion into normal brain parenchyma at the tumor periphery. Implanted brain tumors were removed from the three groups; IR/Ipsi-tumor, IR/Contra-tumor, and Sham-IR/Tumor groups, fixed with formalin and embedded in paraffin. The FFPE blocks were sliced and stained with hematoxylin and eosin. The invasion index was evaluated by taking pictures of each slide at a magnification of 50× (10 ocular × 5 objective). The index was calculated as the total area of glioma crossing tumor rim divided by the field of tumor regions in serial sections using Image J software . The invasive tumor ratio of the Sham-IR/Tumor group was defined as 1.0.
Total RNA was extracted and prepared cDNA from the F-98 cell implanted tumors in the three groups; IR/Ipsi-tumor, IR/Contra-tumor, and Sham-IR/Tumor as described above.
Whole transcriptome sequencing was applied to the RNA samples of brain tissues in triplicate assay (n=3 for each group), with use of on an Illumina HiSeq 2500 platform in a 75-base single-end mode. Illumina Casava ver.1.8.2 software was used for base calling. Sequenced reads were mapped to the rat reference genome sequences (rn6) using TopHat ver. 2.0.13 in combination with Bowtie2 ver. 2.2.3 and SAMtools ver. 0.1.19. The number of fragments per kilobase of exon per million mapped fragments (FPKMs) was calculated using Cuffnorm ver. 2.2.1. The raw data have been deposited in the Gene Expression Omnibus database of the U.S. National Center for Biotechnology Information (NCBI).
As compared to Sham-IR/Brain, the differentially expressed genes (DEGs) in IR/Ipsi-brain and IR/Contra-brain were obtained using a threshold of 0.05 for statistical significance (p-value) and a log fold change of expression with absolute value of at least 1.5. To identify significantly altered pathways, we used an analysis software, “iPathwayGuide” (Advita Corporation, Plymouth, MI, USA; [email protected]). These DEGs were analyzed with the impact analysis method [54-56], in the context of pathways obtained from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (Release 81.0+/01-20, Jan 17) [57,58], and gene ontologies from the Gene Ontology Consortium database (2016-Sep26) [59,60]. (See Supporting information: Methods for pathway analysis)
Real-time quantitative PCR (qPCR)
Total RNAs were reverse-transcribed into cDNA with a SuperScript VLIO cDNA Synthesis kit (Thermo Fisher Scientific, Cleveland, USA). The validation gene for the quantitative polymerase chain reaction (qPCR) was selected based on the results of RNA-sequencing. A quantitative real-time PCR was performed with TaqMan probes and primers using the LightCyclerⅡ (Roche Applied Science, Penzberg, Germany). The PCR conditions were as follows: a single denaturation cycle at 95°C for 10 min, followed by 45 amplification cycles of 95°C for 10 seconds and 60°C for 25 seconds. The relative expression ratio was calculated after normalization with reference to the expression of the housekeeping gene 18S RNA. The PCR primers were designed by the Roche Universal Probe Library. The expression of cytokines such as tumor necrosis factor alpha (TNFα), tumor growth factor β1 (TGF-β1), vascular endothelial growth factor-A (VEGF-A), C-X-C motif chemokine 12 (CXCL12), C-X-C chemokine receptor type 4 (CXCR4), epidermal growth factor receptor (EGFR), fibroblast growth factor-2 (FGF-2), and extracellular signal-regulated kinase2 (ERK2) was assessed by qPCR.
Data are expressed as the mean ± standard deviation. Statistical analysis of the groups' MST values was performed using the Wilcoxon log-rank test, and other results were analyzed using Student's t-test. Values of p<0.05 were considered statistically significant.
All data generated during the current study are included in this article and are available from the corresponding author on reasonable request.