Intravital imaging reveals synergistic effect of CAR T-cells and radiation therapy in a preclinical immunocompetent glioblastoma model.

Cell lines

GL26 and SB28 murine glioma cell lines were acquired as gifts from Dr. Gerald Grant (Stanford University, Stanford, CA) and Dr. Hideho Okada (University of California San Francisco, San Francisco, CA), respectively. Culture media consisted of DMEM supplemented with 10% heat inactivated fetal bovine serum (FBS), and antibiotic-antimyocotic (ThermoFisher, Waltham, MA). Cells were maintained in a humidified, 5% CO2 incubator at 37°C. GL26-luc2/GFP cell line was generated by transfection with Lipofectamine 3000 (ThermoFisher) and three rounds of sorting for the highest 5% of GFP expressors. SB28-luc2/GFP was generated by lentiviral transduction followed by puromycin selection (125 ng/mL) and one round of sorting for the highest 5% of GFP expressors. SB28 luc2-GFP and GL26 luc2-GFP cell lines stably overexpressing GD2 were produced by with genes coding for the GD2 and GD3 synthases as described. 26 A stable cell line was derived through three rounds of sorting of the bulk population stained using the anti-GD2 antibody (14G2a, BioLegend, San Diego, CA) and sorted for the highest 2% of GD2 expressors. Cells were regularly tested as negative for mycoplasma by PCR and not maintained in culture for longer than 6 months. Production of retroviral supernatant Retroviral supernatant for the GD2 CAR was produced by transient transfection of 293GP cells with GD2.28z CAR plasmid (MSGV-14g2a-28z) and the pCL-Eco envelope plasmid. Design of the MSGV-14g2a-28z has been previously reported. 25 Briefly, 293GP cells were transfected via Lipofectamine 2000 (Life Technologies, Carlsbad, CA) with the plasmids encoding the CARs and the RD114 envelope protein. Supernatants were collected 48 and 72 hours after transfection.

T-cell transduction

Primary murine T-cells were isolated from spleens of healthy 6–8 week old C57Bl/6 mice (The Jackson Laboratory, Bar Harbor, ME) or transgenic mT/mG (007676, Jackson Laboratory) with cell membrane-localized tdTomato fluorescence using the EasySep TM Mouse T-cell Isolation Kit (STEMCELL Technologies, Vancouver, Canada) following manufacturer instructions. Cells were activated for 24 hours in RPMI supplemented with 10% FBS, 1% antibacterial/antimycotic solution, 50 μM 2-mercaptoethanol (ThermoFisher), 100 U/ml IL-2 (Peptrotech, Rocky Hill, NJ), 10 ng/mL IL-7 (Peprotech), and CD3/CD28 Mouse T-cell Activation DynabeadsTM (ThermoFisher) at a bead to cell ratio of 1:1. Retrovirus encoding the GD2 CAR with CD3ζ and CD28 co-stimulatory domains was centrifuged for 3 hours at 3200 RPM on non-adherent 6-well plates which had been coated overnight at 4ºC with 24 μg of RetroNectin (Takara Bio, Kusatsu, Shiga Prefecture, Japan) in 2 mL PBS per well. Viral supernatant was then removed and 1 × 10 6 naïve T-cells were added in 4 mL of media per well. Mock T-cells were maintained in identical activation conditions but were not transduced with the CAR vector. After 48 hours of transduction, CD3/CD28 activation beads were removed and both mock and transduced CAR T-cells were transferred to fresh medium supplemented with 100 U/ml IL-2 and 10 ng/mL IL-7. Transduction efficiencies were routinely 60-80% as measured by 1A7 staining 24 after removal of activation beads and cells were used thereafter. T-cell functional assays 1 x 10 4 GL26 luc2-GFP/GD2 or SB28 luc2-GFP/GD2 tumor cells were plated per well in black 96-well plates. The following day, GD2 CAR T-cells or mock activated T-cells were added in each well at a specific effector to target ratios, ranging from 1:1 to 1:32 effector to target (312 to 1 × 10 4 T-cells). For cell killing assays, imaging of luciferase was performed after 24 hours of T-cell and tumor cell co-incubation on an IVIS-50 system (PerkinElmer, Waltham, MA) after addition of 56 μg D-luciferin (PerkinElmer) to each well. Images were taken with an exposure time of 30 s, f/stop of 1, and medium binning. For cytokine release assays, the supernatant was removed from individual wells and analyzed for IFN-γ, TNF-α, and IL-2 via an ELISA assay per the manufacturer instructions (Invitrogen, Carlsbad, CA).

Flow cytometry GD2

CARs were detected with the 14g2a anti-idiotype antibody 1A7 (National Cancer Institute BRB Repository). Additional antibodies for flow cytometry include: anti-CD4 (GK1.5, BioLegend), anti-CD8a (53–6.7, BioLegend), anti-CD62 L (IM7, BioLegend), anti-CD44 (MEL-14, BioLegend), anti-GD2 (14G2a, BioLegend). Cells were washed with PBS containing 2% Bovine Serum Albumin before the addition of the antibody at a concentration of 0.2 µg per 1 × 10 6 cells in 100 µL volume. After 30 minutes of incubation with antibodies at 4°C in the dark, cells were washed once with PBS before being resuspended in PBS with 2% Bovine Serum Albumin (BSA). Quantitative flow cytometry was conducted using the QIFIKIT (Agilent, Santa Clara, CA). The GD2 primary antibody (14G2a, BioLegend) was added to 1 × 10 5 cells at a saturating dose (1 µg) and incubated at 4°C for 45 minutes. This was followed by washing and the addition of 0.5 µg of the secondary stain with APC-F(ab’)2-Goat anti-Mouse IgG (H + L) secondary antibody (Thermofisher) to samples and calibration beads. After a 30 minute incubation at 4°C, samples were washed and DAPI was added. All samples were analyzed with an LSR Fortessa or FACSAria II (BD Bioscience, San Jose, CA) and data were analyzed using FlowJo. In vitro time-lapse imaging Time-lapse videos were acquired using a Leica DMi8 inverted microscope with a 20x/0.40 dry objective. 1 × 10 5 GL26 luc2-GFP/GD2 or GL26 luc2-GFP were plated within a 6-well plate. The following day, 4 × 10 5 GD2 CAR T-cells were added in each well and immediately imaged at 30 second intervals. Videos were processed with ImageJ. In vivo therapy studies All animal experiments were performed under a protocol approved by the Stanford University Administrative Panels on Laboratory Animal Care (APLAC) and conducted in accordance with ethical guidelines prescribed therein. 6–8 week old female C57Bl/6 were implanted intracranially into the right hemisphere (0.5 mm anterior and 2 mm lateral to the lambda, 3 mm below the cranial surface) with either 1 × 10 4 SB28 luc2-GFP/GD2 or 1 × 10 5 GL26 luc2-GFP/GD2 cells in 3 µL HBSS using a Kopf stereotactic frame (David Kopf Instruments, Tujunga, CA). The cells were injected at a depth of 3 mm over a period of 7 min using an AS blunt-ended Hamilton syringe. Mice were anesthetized with 2% isofluorane over the duration of the tumor implantation and imaged with 3 mg D-luciferin 7–10 days of tumor implantation to confirm stable tumor growth. Ten days following tumor implantation, mice were randomly assigned to experimental groups based on tumor burden. Radiation treated mice were given either 5 Gy whole body irradiation (WBI) or focal RT and administered 1 × 10 7 CAR or mock T-cells in 100 µL of HBSS intravenously via the lateral tail vein. Tumor response was monitored by bioluminescence imaging via the IVIS 200 system (PerkinElmer). Twenty-five sequences at 30 second exposures were acquired to obtain the peak average radiance. Animals were humanely euthanized upon displaying symptoms of morbidity. Sample sizes were determined based on power calculations for one-way independent ANOVA (3 groups, power = 0.8, p < .05). Radiation therapy For WBI, mice were treated with 5 Gy using an x-ray cabinet (Kimtron Polaris SC-500). For focal RT, mice were treated with 5 Gy using a conformal small-animal irradiation system (X-RAD SmaRT, PXi, North Branford, CT) outfitted with a 5 mm diameter collimator. Radiation treatment planning was performed using RTImage. Cone-beam CT images were acquired prior to RT to set the radiation isocenter on the center of the tumor. In vivo intravital imaging through a cranial window Six to eight week old female C57Bl/6 mice were anesthetized with 2-3% isofluorine positioned on a Kopf stereotactic frame. A circular craniotomy (diameter of 3–4 mm) was performed on the right parietal bone. Tumor cells were injected as described above. Following tumor implantation, a round glass coverslip (diameter of 5 mm; Warner Instruments, Hamden, CT) was glued on the surrounding bone and further fixed to the skull by dental cement (Parkell, Edgewood, NY). To perform intravital microscropy, mice were anesthetized with 1.5-2% isoflurane injected intravenously with 40 ug of rat anti-mouse CD31 (clone 390, BioLegend) prior to imaging. Mice were positioned on a custom-designed stereotactic frame and imaged using a Nikon confocal microscope (A1 MP+) and imaged with lasers set at 488. 560, and 633 nm. 20x water objective was selected based on the field of view and resolution considerations. Images were typically acquired over a depth of 200 µm and time-lapse videos were taken over a one-hour period.

Statistical analysis

Statistical analysis was performed using ordinary two-way ANOVA or two-tailed unpaired t tests. Statistical differences in survival curves were determined using Log-rank tests. All statistical analysis was performed in GraphPad Prism Version 8.0.2 (San Diego, CA).

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