GL261 cells were a generous gift from Dr. Michael R. Olin (University of Minnesota), and were tested and found free of adventitious agents, before being injected into mice. GL261 cells (H-2b haplotype) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen), added with 10 % of fetal bovine serum (FBS, Atlanta Biologicals). We also tested the resistance of these cells in medium containing various concentrations of the neomycin analog Geneticin (G418), and found that a medium containing 200 µg/mL of G418 could kill GL261 within 2 weeks. Our stock of GL 261 cells also expresses a transfected firefly luciferase gene, which allow us to monitor tumor engraftment by bioluminescence imaging technology (see below). RAG cells were purchased from the American Type Culture Collection (ATCC), tested and found free of adventitious agents, before being injected into mice. RAG cells were cultured in DMEM added with 10 % FBS. RAG cells are deficient in the X-linked hypoxanthine-guanine phosphoribosyl transferase gene (HGPRT−). They are a non-reverting, 8-azaguanine-resistant clone of the Renal-2a cell line, originally derived from a kidney adenocarcinoma of BALB/c origin (H-2d haplotype); therefore, they are killed in culture media containing a supplement of hypoxanthine, aminopterin, and thymidine (HAT, Invitrogen). We have previously confirmed that RAG cells are non-reverting, 8-azaguanine-resistant, and HAT-sensitive [5, 6]. Neomycin-resistant RAG cells were generated by DNA-mediated gene transfer of the pRSV-neo plasmid (RAG-neo), as previously described . RAG-neo cells can grow in medium containing >400 µg/mL of G418. To generate semi-allogeneic somatic cell hybrids, single-cell suspensions of GL261 and RAG-neo cells were mixed at a 3:1 ratio in serum-free DMEM containing 50 μM sodium dodecyl sulfate (SDS) and centrifuged at 300×g for 5 min (min) at room temperature. The mixed cell pellet was then slowly suspended in 1 mL 50 % polyethylene glycol (PEG)-1450 (cell-culture grade from the ATCC and diluted in serum-free DMEM) over a 1 min period while gently stirring. The cell suspension was then slowly diluted over a 2 min period with DMEM supplemented with 10 % FBS. After centrifuging at 300xg for 5 min, fused cells were plated in selective medium (DMEM + 10 % FBS, containing 400 µg/mL G418 and HAT supplement). Under these culture conditions only RAG x GL261 semi-allogeneic somatic cell hybrids can survive, since RAG-neo cells are killed by aminopterin and GL261 cells are killed by G418.
Fluorescence-activated cell sorting (FACS)
Drug-resistant cultures were expanded and tested by FACS for co-expression of MHC surface antigens using phycoerythrin (PE)-labeled, H-2Kb-specific monoclonal antibody (MAb) AF6-88.5 (BD Bioscience cat.# 553570), and allophycocyanin (APC)-labeled, H-2Kd-specific MAb SF1.1 (eBioscience cat.# 17-5957-82).
Mouse studies were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) and were conducted in accordance with all applicable national and institutional guidelines for the care and use of animals. Ten to twelve week-old C57BL/6 male mice (Jackson Laboratories) were anesthetized by i.p. injection of Ketamine (JHP Pharmaceuticals—90 mg/per kg) and Xylazine (Lloyd Laboratories—10 mg/kg), and positioned in a Kopf stereotaxic frame for intracranial injection into the striatum (2.2 mm medio-lateral, 0.2 mm anterior-posterior, and 3 mm dorso-ventral). Injection into the right cerebral hemisphere of GL261 cells suspended in PBS (2 × 104/mouse) was performed using a 5 μL Hamilton micro-syringe under mechanical control to avoid brain injuries during the procedure. Three days after tumor inoculation, treated mice were injected s.c. with lethally irradiated [30 Gray (Gy)] GL261xRAG-neo hybrids (106 cells per mouse) in 0.5 mL PBS, and control mice were injected s.c. with 0.5 mL PBS alone (mock-vaccination). Mice were checked daily until the end of the experiment. Mice were euthanized as soon as they looked sick. Length of animal survival was measured by means of Kaplan–Meier graphs. Data from the animal experiments were analyzed using GraphPad Prism software program (GraphPad Software Inc., La Jolla, CA, USA), with p < 0.05 indicating statistical significance. Approximately 120 mice were tested in these experiments, including mice used in preliminary studies aimed at optimizing the surgical procedures and determining the timing of vaccine administration. Less than 5 % of mice receiving intracranial inoculation of GL261 cells died within 48 h after surgery; these mice were excluded from the survival analyses.
Bioluminescence Imaging (BLI)
For BLI studies, luciferase activity was monitored using an Ivis 200 Spectrum Instrument (Perkin Elmer, Waltham, MA). Two and four days post-implantation of GL261 cells in the brain, mice were anesthetized in a chamber by inhalation of isoflurane (Florane, Baxter) at 2–3 % in oxygen for the duration of the imaging session, and injected i.p. with 150 μL (150 mg/kg) of the bioluminescent substrate ReadyJect D-Luciferin (PerkinElmer). Up to three mice were placed within the BLI chamber and imaged until measurable luciferase activity could be recorded. Serial images were obtained at 1, 4, 7, and 10 min after i.p. injection of luciferin. Mice were imaged using the following parameters: field of view C, exposure time 60 s, medium binning and F/Stop:1. Luciferase activity was calculated using Living Image Software (Perkin Elmer).
Pathology and immunohistochemistry
At time of sacrifice, mouse brains were removed and fixed for 24 h in 4 % paraformaldehyde in PBS. After fixation, brains were immersed in 30 % sucrose for cryosectioning; sections were 5–30 μm-thick depending on the study. For morphological analysis, sections were stained with Hematoxylin and Eosin (H&E). Tumor volumes (in mm3) were estimated using image analysis (NIH Image software ImageJ 1.37v).
For immunohistochemical studies, tissue sections were placed into wells containing 0.5 mL of Tris-buffered saline (TBS) and incubated in blocking buffer (TBS + 5 % FBS) for 1 h at room temperature. Primary antibodies diluted in blocking buffer were applied for 24 h at 4 °C. Primary antibodies used were: microglia-specific anti-Iba1 (1:500; Wako cat.# 019-19741); anti-Ki-67 (1:100 abcam cat.# ab16667); anti-CD3 (1:100 abcam cat.# ab5690); anti-CD4 (1:100 abcam cat.# ab25475); anti-CD8 (1:100 abcam cat.# 22378). After the first incubation, sections were washed in TBS three times for 5 min and incubated for 60 min with secondary antibody (VECTSTAIN ABC Kit), followed by development using 3,3′-diaminobenzidine (DAB) horseradish peroxidase (HRP) substrate (Vector Laboratories). Control sections were processed in identical fashion, except that the primary antibodies were omitted.