Development of tdTomato-transfected C6 glioma cells. Rat C6 glioma cells (Cell number, JCRB9096; Lot number, 09112000) were purchased from Japanese Collection of Research Bioresources Cell Bank (National Institutes of Biomedical Innovation, Health and Nutrition). pBIKS(-)loxPPGKneopA (pPGKneo) conferring neomycin resistance, was kindly provided by Dr. J. Miyazaki at Osaka University. pCX-tdTomato containing tdTomato inserted into the pCAGGS expression vector was kindly provided by Dr. R. Kaneko at Osaka University.
C6 glioma cells were cultured in Dulbecco’s modified Eagle’s medium (D-MEM; FUJIFILM Wako Pure Chemical Corporation, Japan) supplemented with 10% fetal bovine serum (FBS; GIBCO, Thermo Fisher Scientific K.K., Japan), and 1% penicillin-streptomycin (FUJIFILM Wako Pure Chemical Corporation, Japan), and maintained under tissue culture conditions at 37°C and 95% air, 5% CO₂, and 100% humidity. Two micrograms of pPGKneo DNA and 3 µg of pCX-tdTomato DNA were mixed with Lipofectamine 3000 Reagent (Life Technologies, Thermo Fisher Scientific K.K., Japan) in Opti-MEM (Life Technologies) and applied to C6 glioma cells in a 60-mm culture dish according to the manufacturer’s protocol. Drug selection began 2 days later with the addition of 0.6 mg/mL G418 sulfate solution (FUJIFILM Wako) to the medium and was maintained for 13 days. Clonal G418-resistant cells were isolated by using cloning rings and expanded into 60-mm dishes. Then, we confirmed that the G418-resistant cells expressed the tdTomato protein by fluorescence microscopy (BZ-X810, Keyence Co., Japan) and selected five clones, which demonstrated bright red fluorescence. Subsequently, we compared the morphology and viability of the five clones with those of parent C6 glioma cells and selected one clone demonstrating similar morphology and viability to the parent cells. We refer to this clone as C6-tdTomato.
Cell viability was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT; Tokyo Chemical Industry Co. Ltd., Tokyo, Japan) assay. In brief, the cells were seeded (1 × 103 cells/well) in quintuplicate in four 96-well plates and cultured for 24, 48, 72 and 96 hours in each plate. Then, the cells were treated with MTT solution (final concentration; 0.25 mg/mL) and further incubated under tissue culture conditions for 3 hours. Afterward, the cells were lysed by 5% sodium decasulfonate (SDS; FUJIFILM Wako) in N,N-dimethylformamide (DMF; Nacalai Tesque, Inc., Kyoto, Japan) solution and the optical density at 570 nm (OD570) was measured by the microplate reader xMark (Bio-Rad Laboratories, Inc.) as the index of cell viability. The MTT assay was repeated four times, and the averaged values of OD570 with standard errors were calculated.
Animals. All experiments were performed in accordance with the guidelines for the Animal Experimentation at Gunma University Graduate School of Medicine. All experimental protocols were evaluated and approved by the Gunma University Ethics Committee (permit number: 14-006, 19-009). This study complied with ARRIVE guidelines (https://arriveguidelines.org).
Male Wistar rats aged 8-9 weeks and male VGAT-Venus rats13 aged 9-13 weeks were used for the experiments. Wistar rats were purchased from CLEA Japan, Inc. (Tokyo, Japan) and acclimated to the laboratory environment several weeks before the experiments. VGAT-Venus rats were previously developed by us and have been housed in our laboratory. The animal room was maintained at 22 ± 3 ℃ with a 12-h light-dark cycle (lights on at 6:00, lights off at 18:00). The animals were housed with 2-3 rats per cage and had free access to food and water.
Implantation of C6 glioma cells into rat brains. C6-tdTomato cells were implanted into the rat brains according to the implantation method38 with minor modifications. The somatosensory cortex (1.3 mm posterior and 4.5 mm lateral right to bregma, 3.5 mm depth) was targeted for implantation referenced to the rat brain atlas 39.
Cultured C6-tdTomato cells were harvested, and then the cell suspension (1.0 × 106 cells in 1 µL PBS) was prepared. Rats were anesthetized with continuous inhalation of isoflurane and placed in a stereotactic frame. After an incision was made in the scalp, a small hole was drilled into the skull. A 33-gauge needle (33G. Super Short, Dentsply Sirona, Japan) was inserted at the target area and the cell suspension (1 µL) was injected at a constant flow rate (1.0 µL/min). Two minutes after the injection, the needle was removed, and the incision was sutured. The glioma-implanted rats were then returned to the home cage.
Magnetic resonance imaging (MRI). Small animal MRI was carried out over time on days 4-14 after glioma implantation by a 1-T benchtop MR scanner (Icon; Bruker Biospin GmbH, Ettlingen, Germany) according to a previous report9. Anesthesia was induced by the inhalation of 5% isoflurane and maintained by the inhalation of 3% isoflurane in room air. The respiration rate was monitored throughout the procedure, and body temperature was maintained at 37 ℃. The T2-RARE (Rapid Acquisition with Relaxation Enhancement) sequence was used to determine the colonization and growth of tumors. The measurement parameters were follows: rapid-acquisition relaxation enhancement factor 5, repetition time 2,500 ms, echo time 60 ms with in-plane resolution of 30×30 mm², thickness 1,000 µm, and 5 slices.
Immunofluorescence analysis. Animals were deeply anesthetized with continuous inhalation of isoflurane, and then fixed by perfusion with 4% paraformaldehyde (PFA) in 0.1 M PBS (pH 7.4) through the left ventricle. Thereafter, the brain was removed and postfixed overnight in 4% PFA at 4 ℃.
Coronal sections (20 µm in thickness) were made by a vibrating blade tissue slicer (Neo-LinearSlicer MT, Dosaka EM Co.,Ltd., Kyoto, Japan). After preincubation with 0.3% Triton X-100 and 2% BSA in PBS, the sections were incubated with primary antibodies in PBS containing 0.3% Triton X-100 and 2% BSA overnight at room temperature. These sections were rinsed in Tris-buffered saline with 0.1% Tween 20 and repeated three times. Thereafter, sections were incubated with secondary antibodies and DAPI (1:500, D523, Dojindo Laboratories, Japan) in PBS containing 0.3% Triton X-100 and 2% BSA for 30 minutes at room temperature. After rinsing, the stained sections were mounted on MAS-coated glass slides (Matsunami Glass Ind., Ltd., Osaka, Japan) with Fluoromount (K024, Diagnostic BioSystems, USA). Fluorescence images were captured with the fluorescence microscopy (BZ-X810, Keyence, Osaka, Japan).
The primary antibodies used in this study were mouse anti-neuronal nuclei (NeuN) (clone A60, 1:500, MAB377, Millipore Co.), rabbit anti-glial fibrillary acidic protein (GFAP) (1:100, GFAP-Rb-Af800, Frontier Institute Co. Ltd.) and rabbit anti-Iba1 (1:500, 019-19741, FUJIFILM Wako Pure Chemical Co.). In addition, the primary antibody of rabbit anti-green fluorescent protein (GFP) (1:1,000, GFP-Rb-Af2020, Frontier Institute Co. Ltd., Hokkaido, Japan) was used to enhance the fluorescent signal of Venus protein. The secondary antibodies used in this study were donkey anti-rabbit IgG conjugated with Alexa Fluor 488 (1:300, A-21206, Invitrogen) and donkey anti-mouse IgG conjugated with Alexa Fluor 647 (1:300, A-31571, Invitrogen).
Fluorescence images were captured with a fluorescence digital microscope (BZ-X810, Keyence, Osaka, Japan). The number of NeuN-positive cells and Venus-positive cells in the peritumoral area, and the contralateral healthy area was measured by ImageJ (v1.51) software (National Institutes of Health). The peritumoral area was up to 400 µm from the tumor limb. The ratio of Venus-positive cells per NeuN-positive cell in each area was calculated. The data were obtained from five sections from five rats (total 25 sections).
Statistical analyses were conducted using BellCurve for Excel ver. 3.20 (Social Survey Research Information Co., Ltd., Tokyo, Japan). Significant differences between regions were assessed by Student’s t-test. Data are expressed as the mean with standard error (SE). Statistical significance was defined as a p-value less than 0.05.
Microdissection. For laser capture microdissection (LCM), the PFA-fixed brains of C6-tdTomato-implanted VGAT-Venus rats (n = 6) were prepared in accordance with the protocol described above. The brains were cut into 20 µm-thick coronal sections by a vibrating blade tissue slicer and mounted on 1 mm polyethylene naphthalate (PEN) membrane-coated slides (MembraneSlide NF 1.0 PEN, Order No. 415190-9081-000, Carl Zeiss Microscopy GmbH, Göttingen, Germany). The peritumoral tissues were microdissected by laser pressure catapulting using a Palm Zeiss UV laser capture microdissection system (PALM MicroBeam Ⅳ, ZEISS Version 4.6) equipped with a fluorescence microscope (ZEISS Axio Observer D1). The samples were collected into PALM AdhesiveCap 500 (Order No.415401-4400-255, Zeiss) according to the Carl Zeiss MicroImaging PALM protocol for RNA handling. Before and after microdissection, fluorescent images of brain slices were captured by the fluorescence microscopy (BZ-X810, Keyence, Osaka, Japan). The tissues of the tumor and the contralateral healthy area were manually dissected and collected.
RNA extraction from dissected tissues. Total RNA was extracted from the dissected tissues by the High Pure FFPE RNA Micro Kit (Roche Diagnostics GmbH, Germany) according to the manufacturer’s instructions without performing the deparaffinization step. The RNA quantity and quality in the eluted samples were assessed using an Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) as recommended.
RNA-seq. One-hundred fifty nanograms of total RNA was subjected to rRNA removal using the FastSelect-rRNA HMR Kit (Cat No.: 334386, QIAGEN Inc., Hilden, Germany), according to the manufacturer’s protocol. Library preparation was performed using the KAPA RNA HyperPrep Kit (Cat No.: KK8540, Nippon Genetics Inc., Tokyo, Japan) from rRNA removed RNA, according to the manufacturer’s protocol. The resulting libraries were subjected to paired-end sequencing using a NextSeq500 High Output v2.5 Kit (Cat No.: 2002490, Illumina Inc. San Diego, CA, USA) and the Illumina NextSeq 500 system (75-base paired-end reads; Illumina Inc.). Data processing and analyses were performed using STAR v2.5.2b40, samtools-1.241 and HTSeq-0.6.1p142. Briefly, reads were aligned against the UCSC Rattus norvegicus (Rat) reference genome 6 (rn6) from the iGenome webpage (https://support.illumina.com/sequencing/sequencing_software/igenome.html; Illumina Inc.) using a STAR pipeline with the default setting. Sorted BAM files by coordinate using samtools were subjected to generate a count matrix table by HTSeq with the -f bam -r pos -t exon option. Normalization and differentially expressed genes (DEGs) were detected with the TCC43 package of R version 3.6.2 (R Foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org/). Genes with a false discovery rate (FDR)-adjusted p-values less than 0.05 were defined as significantly DEGs.
Ingenuity Pathway Analysis (IPA). To examine the biological alterations in the peritumoral area, IPA (QIAGEN Redwood City; Content version 52912811, Release Date 2020-06-01) 1) molecular network functional enrichment analysis and 2) core analysis were performed as followings. 1) Nineteen peri-T specific DEGs were uploaded to IPA and connected to the direct interaction of these molecules. The overlay function of the “Diseases and BioFunctions” was performed to identify the “Epilepsy or neurodevelopmental disorder” related molecules. Log2 fold change values of peri-T vs. HC were overlaid with the corresponding molecules. 2) The respective DEG (HC vs. Peri-T, T vs. Peri-T, HC vs. T) lists were uploaded as the input dataset, and then canonical pathway analyses were performed with default settings. As the next step, the comparison analysis was performed by using the results of the canonical pathway analyses. To determine predictive biological alterations specific to the peritumoral area, canonical pathways with activation z-scores between HC and Peri-T and between T and Peri-T greater than 2 (or less than -2) were included, but annotation terms with activation z-scores between HC and T greater than 2 (or less than -2) were excluded.