Immune co-stimulatory blockade permits human glioblastoma xenografting in immunocompetent mice: model validation with MRI and bioluminescence imaging
Samantha Lynn Semenkow1, Shen Li2, Eric Raabe1,3, Jiadi Xu2,4, Miroslaw Janowski2,5, Byoung Chol Oh6, Gerald Brandacher6, Jeff W. Bulte2,4, Charles Eberhart1,3,7, and Piotr Walczak2

1Department of Pathology, Johns Hopkins Medical Institue, Baltimore, MD, United States, 2Department of Radiology and Radiological Science, Johns Hopkins Medical Institue, Baltimore, MD, United States, 3Department of Oncology, Johns Hopkins Medical Institue, Baltimore, MD, United States, 4F. M. Kirby Center for Functional Brain Imaging Kennedy Krieger Institute, Johns Hopkins Medical Institue, Baltimore, MD, United States, 5NeuroRepair Department, Mossakowski Medical Research Centre, Warsaw, Poland, 6Department of Plastic and Reconstructive Surgery, Vascularized Composite Allotransplantation (VCA) Laboratory, Johns Hopkins Medical Institue, Baltimore, MD, United States, 7Department of Opthalmology, Johns Hopkins Medical Institue, Baltimore, MD, United States

Synopsis

Immunodeficient mice are currently used for modeling human brain tumor xenografts; however, immunodeficiency is a serious limitation precluding studies based on immunotherapy or inducing tumors in a variety of transgenic animal models. We therefore investigated whether disruption of co-stimulatory signaling using blocking antibodies induces tolerance to intracerebrally transplanted human glioblastoma xenografts in immunocompetent mice. With longitudinal MRI and bioluminescence we established that the growth rate of xenografts is comparable between immunodeficient and tolerance-induced immunocompetent mice. Quantitative MRI including T2/T1 relaxation time, MTR, diffusion parameters and perfusion were not significantly different, validating this new approach as a reliable brain tumor model.

Purpose and Background

1) To establish a human brain tumor xenograft model in immunocompetent mice based on induction of tolerance via co-stimulation blockade.

2) To use multimodal, longitudinal imaging (MR imaging and bioluminescence imaging) for validation of this new approach as a reliable human brain tumor model.

Modeling human brain tumors in immunocompetent animals is highly desirable, because it would represent a more relevant system allowing studies on interactions between tumor and the microenvironment, facilitate immunotherapy treatments, as well as permitting the study of human xenografted tumors in transgenic animals 1-8. Immune checkpoint co-stimulation blockade with CTLA4-Ig and anti-CD154mAb is effective in preventing rejection of allografted organs, but to date it has not been used for inducing tolerance to brain tumor xenografts 9, 10.

Methods

Cell Culture: The HSR-GBM-1 neurosphere cell line was established from a human primary glioblastoma (GBM) and transduced with a lentivirus to induce constitutive expression of a firefly luciferase gene (luc). HSR-GBM-1 cells were maintained and cultured as neurospheres at 37°C and 5% CO2 as previously described 11-13.

Intracranial Xenografts: HSR-GBM-1-Luc cells were dissociated into single cells and suspended in PBS at the final concentration of 1x105/μL. Mice were positioned in a stereotaxic frame and 2x105 cells were injected into the right striatum (AP=0.0; ML=2.3mm; DV=2mm) using 10μl Hamilton syringe with attached 31-gauge needle. Hamster anti-mouse-CD154mAb (MR1, BioXcell, Lebanon, NH) and CTLA4-Ig (Bristol-Myers Squibb, Princeton, NJ) were administered to the experimental group of C57/Bl/6 mice (n=10) (25 mg/kg I.P.) injection daily between days 0-7, and on day 14. Control groups of C57/Bl/6 mice (n=10) and athymic (nu/nu) (n=13) mice received no treatment. Mice were monitored daily for neurological symptoms and weighed weekly (Figure 1).

Multimodal BLI and MR Imaging: For bioluminescence imaging (BLI) of tumor growth rate mice were injected I.P. with 250 μL luciferin (15 mg/mL) and images were acquired 5-15 minutes after injection. BLI was quantified (photon flux (p/s)) by drawing regions of interest (ROIs) over the signal hot spot using LivingImage software (Perkin Elmer, Hopkinton, MA) 14. MR imaging was performed 4 and 8 weeks after tumor implantation. Animals were anesthetized by isoflurane, positioned in a custom built animal holder and scanned using an 11.7 T Bruker Biospec system (Bruker, Ettlingen, Germany). A 72 mm volume resonator and a 2×2 phased array coil were used for transmission and image acquisition. Quantitative magnetization transfer (qMT) MRI was recorded using on-resonance version of the variable delay multi-pulse (VDMP) approach 15. Images were acquired using a RARE sequence with TR/TE= 8 s/ 4 ms, RARE factor= 8. Four binomial pulses (B1=93.6 μT; 2 ms pulse width) with 10 mixing times ranging from 0-150 ms were used for the quantification of macromolecule fraction (f). The T1 relaxation times were measured using variable TR (TR=0.5, 1, 1.5, 2, 3.5, 5, 8 s) method. The T2 relaxation times were measured using a MSME sequence with 10 echo times from 10-110 ms. Perfusion maps were acquired using Steady-state Pulsed Imaging and Labeling (SPIL) scheme 16. A fast spin-echo (FSE) sequence with TR= 5 s; RARE factor = 16; slice thickness= 1 mm were used in SPIL and number of averages= 20. A DTI-EPI sequence was used for ADC map. 30 diffusion directions were recorded with TR= 9 s and TE= 25 ms. 100μL of Gadolinium was injected I.P. For contrast enhanced T1 scans and T1+Gadolinium images were acquired at 5, 10 and 15 minutes post injection.

Results

Bioluminescence Imaging: Weekly imaging of mice showed that the xenograft growth for immunodeficient mice and tolerance-induced immunocompetent C57/B6 was similar over the course of the experiment for ~50days (Figure 2a, b, d). At that time the mice deteriorated and were euthanized. Conversely, immunocompetent control mice began rejecting the tumor between days 7 and 10, with complete rejection occurring by day 14 (Figure 2c, d). Mice in this group did not show any neurological deficits.

MRI: Longitudinal MRI demonstrated rapid progression of tumor growth between weeks 4 and weeks 8 after implantation, and appearance of the tumor on the anatomical T2-weighted images was similar for both immunodeficient and tolerance-induced immunocompetent mice (Figure 3). Multiparametric quantitative MRI (T1, T2, diffusion, perfusion, qMT) did not detect significant differences between the groups (Figure 3). Imaging at 8 weeks revealed a striking difference in blood brain barrier permeability with tolerance-induced immunocompetent mice showing a more heterogeneous pattern that more accurately resembles clinical scenarios (Figure 4).

Conclusions

Co-stimulation blockade is an effective strategy for promoting growth of human brain tumor xenografts in immunocompetent mice and accurately models human disease.

Acknowledgements

The Eberhart and Walczak labs would like to thank Irina Shats, Antionette Price, and Kazi Dilruba Akhter for their technical assistance.

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Figures

A schematic of the experimental design. CTLA4-Ig and anti-CD154mAb were given to immunocompetent C57/B6 mice to induce tolerance to the brain tumor xenograft. Control groups of immunocompetent C57/B6 mice and athymic (nu/nu) mice did not receive any treatment. Bioluminescence imaging and MRI were performed on the indicated days.

Immunodeficient mice (a), immunocompetent, tolerance induced mice (b) and immunocompetent control mice (c) were monitored by bioluminescent imaging (BLI) after xenograft inoculation. BLI averages for immunodeficient and tolerance-induced mice showed similar growth patterns, while the xenografts in the control mice were rejected by day 14.

MRI of immunodeficient control mice (a-f) and tolerance induced mice (g-l) demonstrate that the size and characteristics of tumors in these two models is very similar. Quantitative MRI (m-q) confirmed similarity of the two models with greatest difference recorded for tumor perfusion parameter with better perfusion in tolerance-induced tumors.

MR images of T1+Gadolinium acquired approximately 10 minutes after the injection of contrast show a difference in the contrast enhancement for immunodeficient mice (a, b) and tolerance-induced immunocompetent mice (c, d). The enhancement for the tolerance-induced mice more closely represents human GBM presentation seen in the clinic.



Proc. Intl. Soc. Mag. Reson. Med. 24 (2016)
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