Lentiviral Vector Induced Modeling of High-Grade Spinal Cord Glioma in Minipigs

Abstract

Background: Prior studies have applied driver mutations targeting the RTK/RAS/PI3K and p53 pathways to induce the formation of high-grade gliomas in rodent models. In the present study, we report the production of a high-grade spinal cord glioma model in pigs using lentiviral gene transfer. Methods: Six Gottingen Minipigs received thoracolumbar (T14-L1) lateral white matter injections of a combination of lentiviral vectors, expressing platelet-derived growth factor beta (PDGF-B), constitutive HRAS, and shRNA-p53 respectively. All animals received injection of control vectors into the contralateral cord. Animals underwent baseline and endpoint magnetic resonance imaging (MRI) and were evaluated daily for clinical deficits. Hematoxylin and eosin (H&E) and immunohistochemical analysis was conducted. Data are presented using descriptive statistics including relative frequencies, mean, standard deviation, and range. Results: 100% of animals (n = 6/6) developed clinical motor deficits ipsilateral to the oncogenic lentiviral injections by a three-week endpoint. MRI scans at endpoint demonstrated contrast enhancing mass lesions at the site of oncogenic lentiviral injection and not at the site of control injections. Immunohistochemistry demonstrated positive staining for GFAP, Olig2, and a high Ki-67 proliferative index. Histopathologic features demonstrate consistent and reproducible growth of a high-grade glioma in all animals. Conclusions: Lentiviral gene transfer represents a feasible pathway to glioma modeling in higher order species. The present model is the first lentiviral vector induced pig model of high-grade spinal cord glioma and may potentially be used in preclinical therapeutic development programs. High-grade glioma has a clinical picture of untenable morbidity and mortality 1,2. Given this clinical need, the U.S. National Library of Medicine has over 350 completed phase II-III clinical trials registered as of this writing. Unfortunately, there have been limited changes to the clinical outcomes in patients with high-grade glioma over the past years. In part, this represents the malignant nature of a disease that is refractory to a variety of treatment approaches. On the other hand, this raises the question of the translational value of existing pre-clinical animal models as a pathway to the clinic 3,4. Given numerous strategies with measured preclinical optimism including immunotherapy, oncolytic vectors, and targeted drug delivery, more appropriate large animal model systems are necessary to bridge the translational gap 5-10. In patients, high-grade gliomas present with marked invasion along white matter tracts and surrounding parenchyma, an immunosuppressive microenvironment, and significant inter and intra-tumoral heterogeneity 11. Existing models of high-grade gliomas are variable in recapitulation of these features. The most common are xenograft and syngeneic models which have drawbacks including non-invasive growth (9L, U87, U251), immunogenicity (U251, U87, C6, 9L), and restriction to murine (CT-2A, ...

Large Animal Models of Glioma: Current Status and Future Prospects

“…MRI of the spinal cord mass demonstrated radiological characteristics similar to those of xenografted glioma models (47,50). Lesions confirmed as highgrade glioma on histopathological analysis demonstrated invasion of white and gray matter, high cellularity, elevated Ki-67 expression, astrocytic morphology, and immunohistochemical evidence of GFAP and oligodendrocyte transcription factor 2 (OLIG2) expression (50). Lentiviral induction of a glioma model with multiple genetic abnormalities can be used to model the genotypic heterogeneity of glioma, allowing for the study of several common driver mutations (63).…”

“…In rodents, retroviral expression of platelet-derived growth factor-β, HRAS-G12V, shRNA-p53 resulted in glioma formation (60)(61)(62). Employing a similar strategy, Göttingen minipigs were injected with lentiviral vectors expressing platelet-derived growth factor-β, constitutively active HRAS, and shRNA-p53 (50). MRI of the spinal cord mass demonstrated radiological characteristics similar to those of xenografted glioma models (47,50).…”

“…While the BBB provides partial immunological isolation from the rest of the organism, a detailed characterization of the pig immune system and its impact on xenograft models have not been achieved (57,58). Further, the loss of intratumoral heterogeneity arising from serial cell culture leads to cell lines that lack the genotypic and phenotypic heterogeneity of (50). In rodents, retroviral expression of platelet-derived growth factor-β, HRAS-G12V, shRNA-p53 resulted in glioma formation (60)(61)(62).…”

“…Employing a similar strategy, Göttingen minipigs were injected with lentiviral vectors expressing platelet-derived growth factor-β, constitutively active HRAS, and shRNA-p53 (50). MRI of the spinal cord mass demonstrated radiological characteristics similar to those of xenografted glioma models (47,50). Lesions confirmed as highgrade glioma on histopathological analysis demonstrated invasion of white and gray matter, high cellularity, elevated Ki-67 expression, astrocytic morphology, and immunohistochemical evidence of GFAP and oligodendrocyte transcription factor 2 (OLIG2) expression (50).…”

Lentiviral-Induced Spinal Cord Gliomas in Rat Model

“…Concerns about the ability of existing models to faithfully replicate human gliomas led us to produce the first lentiviral-vector-induced models of high-grade spinal cord glioma in rats and pigs [ 12 , 21 ]. Recently, we reported that the virus cocktail (PDGF-B, HRAS-G12V, and shRNA-p53) induces glioma in the minipig spinal cord [ 21 ].…”