Advanced MR imaging characterization of a novel in vivo xenograft model mimicking recurrent glioblastoma
Mona M Al-Gizawiy1, Robert T Wujek1, Melissa A Prah1, Hisham S Alhajala2, Ninh B Doan3, Jeffrey A Knipstein4,5, Jennifer M Connelly5,6, Shama P Mirza7, Christopher R Chitambar2, and Kathleen M Schmainda1,8

1Radiology, Medical College of Wisconsin, Milwaukee, WI, United States, 2Medicine, Medical College of Wisconsin, Milwaukee, WI, United States, 3Neurosurgery, Medical College of Wisconsin, Milwaukee, WI, United States, 4Pediatrics, Medical College of Wisconsin, Milwaukee, WI, United States, 5Neuro-Oncology, Medical College of Wisconsin, Milwaukee, WI, United States, 6Neurology, Medical College of Wisconsin, Milwaukee, WI, United States, 7Chemistry & Biochemistry, University of Wisconsin, Milwaukee, WI, United States, 8Biophysics, Medical College of Wisconsin, Milwaukee, WI, United States


We have developed a robust and reproducible rat xenograft model of recurrent GBM by irradiating adult and pediatric GBM cell lines in vitro prior to brain inoculation. Both advanced MR imaging and histological analyses highlight the amplified aggressiveness of the resultant tumor compared to the conventional U-87MG xenograft, as evidenced by profound vascularization and increased cell proliferation. Moreover, our recurrent GBM model exhibited invasive lesions with areas of infiltrating neutrophils and necrosis, all features that are not associated with conventional U-87MG xenograft tumors. Shortened survival of animals bearing irradiated U87-10Gy or SJGBM2-10Gy tumors further reinforces the aggressive nature of the model.


Recurrent or progressive glioblastoma (rGBM) frequently exhibits a vastly different biological behavior than de novo glioblastoma (GBM), thus making treatment for rGBM challenging, a factor that likely contributes to its poor prognosis.1 Therefore, clinically relevant preclinical models of rGBM are crucial for studying this devastating disease. Current approaches to model rGBM include in vitro drug testing,2,3 as well as in vivo irradiation,4 resection,5 or chemotherapy.6 However, the in vitro models frequently lack biological context, while the in vivo models primarily reflect treated de novo tumor, or are time-consuming and require highly specialized surgical skills. Here, we present an entirely new rat xenograft model of rGBM, which is grown from pretreated tumor cells and characterized with advanced MRI methods that distinguish tumor growth from treatment effect.


Animals: Care of the animals before and during the experimental procedures was conducted in accordance with the policies of the NIH Health Guide for the care and use of laboratory animals. All protocols were approved by the Institutional Animal Care and Use Committee at the Medical College of Wisconsin. Irradiated adult (U-87MG) and pediatric (SJ-GBM2) GBM cells were generated by exposure to five daily doses of 2Gy for a total radiation dose of 10Gy. The resultant irradiated U87-10Gy and SJGBM2-10Gy cells were tested for cell viability and allowed to reach confluence prior to stereotactic implantation into the right striatum of 7-week-old male athymic rats. Data Acquisition: In vivo advanced MR imaging was carried out over a three-week time course starting on day 14 after tumor implantation. All preclinical MR imaging was performed on a 9.4T Bruker BioSpec system fitted with a linear transmit and a 4-channel receive coil. Data collected included pre- and post-contrast T1W anatomical images, diffusion-weighted imaging (DWI), and dynamic susceptibility contrast imaging (DSC). The T1W (RARE) sequence was collected with TR=1000ms at minimum TE, flip-angle=90 degrees, 18 slices with slice thickness=0.5mm, inter-slice gap=0mm, acquisition-matrix=256x256, and FOV=3.5cm. The DWI was collected at 8 b-values (50, 100, 150, 200, 400, 800, 1000, 1500 s/mm2), with TR=4,500ms, TE=30-50ms, flip-angle=90 degrees, 9 slices with slice thickness=0.7-1mm, inter-slice gap=0-0.3mm, acquisition-matrix=128x128, and FOV=3.5cm. Following 0.2 mmol/kg gadodiamide (Omniscan™: GE Healthcare Inc., Princeton, NJ) contrast agent administration, a post-contrast T1W (RARE) anatomical scan was collected. To obtain dynamic relative cerebral blood volume (rCBV) measures, DSC gradient-echo echo-planar imaging (DSC GRE-EPI) was acquired with TR=1000 ms, TE=16/33ms, flip-angle=75-90 degrees, 9 slices with slice-thickness=0.7-1mm, inter-slice gap=0-0.3mm, acquisition-matrix=128x128, and FOV=3.5 cm. The DSC contrast injection (5 mg/kg MION; courtesy of Dr. Young Kim, Massachusetts General Hospital, Charlestown, MA) occurred exactly 60 seconds into the DSC scan at a rate of 10 ml/min using a power-injector. Image & Data Analysis: For this study, advanced MRI parameter maps, including the distributed diffusion coefficient (DDC),7 rCBV (determined from the dynamic MION injection scans), and T1 subtraction maps, were processed for enhancing tumor ROIs in OsiriX 8.5.1 (Lite) with Imaging Biometrics™ Software (Imaging Biometrics LLC, Elm Grove, WI). Fractional tumor burden (FTB) maps were constructed as previously described.8 Kaplan-Meier survival curves (Gehan-Breslow analysis) for the three U-87MG and five U87-10Gy rats were created in SigmaPlot 11.0. The level of significance was set at p=0.05. Immunohistochemical staining (H&E for anatomy and cellularity, KI67 for proliferation, and von Willebrand Factor for vasculature) was performed according to standard protocols by the CRI Histology Core at the Children’s Hospital of Wisconsin.


The study results are summarized in Figures 1 through 4.


We have developed an expedient, yet robust and reproducible, rat xenograft model of rGBM by irradiating GBM cell lines in vitro prior to brain inoculation. Both advanced MR imaging and histological analyses highlight the amplified aggressiveness of the resultant tumor compared to the conventional U-87MG xenograft. Our rGBM model exhibited invasive lesions with areas of infiltrating neutrophils and necrosis, all features that are not associated with conventional U-87MG tumors.9 Shortened survival of animals bearing U87-10Gy xenograft tumors further reinforces the aggressive nature of model. We were able to reproduce similar features using the pediatric SJ-GBM2 cell line. However, because those cells were obtained from a previously treated GBM, following in vitro irradiation we observed xenograft tumors exhibiting one of two phenotypes (invasive or angiogenic). This is a finding that warrants further study and characterization.


Our novel rGBM xenograft model represents a robust and reproducible in vivo model for studying recurrent GBM biology using in vitro irradiated cell lines.


Funding support was provided by NIH/NCI R01CA082500, the Daniel M. Soref Charitable Trust, and the Musella Foundation. Special thanks to the Children's Research Institute Histology Core at the Children's Hospital of Wisconsin, and to Matt Runquist of the MCW Center for Imaging Research for help with MR imaging.


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9. Candolfi M, Curtin JF, Nichols WS, et al. Intracranial glioblastoma models in preclinical neuro-oncology: neuropathological characterization and tumor progression. J Neurooncol. 2007;85: 133-148.


In vivo tumor growth mimics the clinical profile of recurrent tumors. Representative longitudinal post-contrast T1-weighted (T1+C) MR images of an animal with a U-87MG xenograft tumor (A, E, I) show a homogeneously enhancing slow-growing lesion. In contrast, longitudinal T1+C images of an animal bearing an U87-10Gy tumor (C, G, K) show a rapidly growing lesion with increasingly heterogeneous enhancement. The rCBV maps of the U87-10Gy tumor (D, H, L) highlight its aggressive biology from the onset, compared to the conventional U-87MG tumor (B, F, J).

Advanced MRI characterization of the novel rGBM and the conventional de novo U-87MG xenograft models. Heterogeneously enhancing U87-10Gy xenograft tumors were considerably larger on Day 28 (A) than homogeneously enhancing non-irradiated U-87MG control tumors (F). The T1 subtraction maps improve visualization of enhancing tumor (B, G). Relative cerebral blood volume (rCBV) maps revealed U87-10Gy as rim-enhancing lesions (C), while conventional U-87MG tumors showed more limited perfusion (H, yellow arrow). (D, I) Distributed diffusion coefficient maps (DDC) generated from diffusion-weighted imaging (DWI). (E, J) Fractional tumor burden (FTB) maps highlight the relative proportions of tumor (red, pink) and necrosis (white).

Irradiated GBM xenograft tumors exhibit features of increased aggressiveness compared to conventional GBM xenograft tumors. (A) Rats bearing non-irradiated U-87MG tumors lived significantly longer than those bearing U87-10Gy tumors. Unlike conventional untreated U-87MG xenograft tumors, U87-10Gy tumors exhibited features of increased aggressiveness, such as pronounced vessel engorgement and vascularization (B: H&E, vWF), increased proliferative index (B: KI67), necrosis characterized by infiltrating neutrophils and fibrin deposits (C), as well as invasion of surrounding brain tissue (D: blue arrows). H&E = Hematoxylin & Eosin; vWF = von Willebrand Factor

Advanced MRI and histological characterization of the pediatric rGBM model. Longitudinal post-contrast T1-weighted (T1+C) MR images of an animal with a SJGBM2-10Gy xenograft tumor (A, C, E) show a non-enhancing lesion on Day 14 (A) becoming fully enhancing by Day 28 (E). In contrast, regular SJ-GBM2 cells produced enhancing tumors from the beginning (G). The rCBV maps of the SJGBM2-10Gy tumor highlight the gradual evolution of tumor perfusion (B, D, F). The more aggressive nature of SJGBM2-10Gy tumors was evidenced by presence of necrosis (I), increased mitotic figures (K, arrows), and brain invasion (M), compared to SJGBM2 tumors (J, L).

Proc. Intl. Soc. Mag. Reson. Med. 26 (2018)