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Neurotherapeutics

, Volume 11, Issue 3, pp 623–635 | Cite as

Lentiviral-Induced High-Grade Gliomas in Rats: The Effects of PDGFB, HRAS-G12V, AKT, and IDH1-R132H

  • John Lynes
  • Mia Wibowo
  • Carl Koschmann
  • Gregory J. Baker
  • Vandana Saxena
  • A. K. M. G. Muhammad
  • Niyati Bondale
  • Julia Klein
  • Hikmat Assi
  • Andrew P. Lieberman
  • Maria G. Castro
  • Pedro R. LowensteinEmail author
Original Article

Abstract

In human gliomas, the RTK/RAS/PI(3)K signaling pathway is nearly always altered. We present a model of experimental gliomagenesis that elucidates the contributions of genes involved in this pathway (PDGF-B ligand, HRAS-G12V, and AKT). We also examine the effect on gliomagenesis by the potential modifier gene, IDH1-R132H. Injections of lentiviral-encoded oncogenes induce de novo gliomas of varying penetrance, tumor progression, and histological grade depending on the specific oncogenes used. Our model mimics hallmark histological structures of high-grade glioma, such as pseudopalisades, glomeruloid microvascular proliferation, and diffuse tumor invasion. We use our model of gliomagenesis to test the efficacy of an experimental brain tumor gene therapy. Our model allowed us to test the contributions of oncogenes in the RTK/RAS/PI(3)K pathway, and their potential modification by over-expression of mutated IDH1, in glioma development and progression in rats. Our model constitutes a clinically relevant system to study gliomagenesis, the effects of modifier genes, and the efficacy of experimental therapeutics.

Key Words

Gene therapy HSV1-TK adenoviral vectors brain tumors 

Notes

Acknowledgments

This work was supported by National Institutes of Health/National Institute of Neurological Disorders & Stroke (NIH/NINDS) grants 1RO1-NS 054193, 1RO1-NS 061107, and 1RO1-NS082311 to P.R.L.; and grants 1UO1-NS052465, 1RO1-NS 057711, and 1RO1-NS074387 to M.G.C. We thank Drs. I. Verma, E.C. Holland, and P. Canoll for providing valuable expression vectors utilized in this work. We thank Mr. Philip Jenkins and the Department of Neurosurgery at the University of Michigan, School of Medicine for their support of our work. We also thank Dr. Karin Murasko for her academic leadership, and D. Tomford and S. Napolitan for superb administrative support. The schematic of tumor infiltration (Fig. 4G) was created by Alan Traxler (alan.traxler@gmail.com). Full conflict of interest disclosure is available in the electronic supplementary material for this article.

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Disclosure forms provided by the authors are available with the online version of this article.

Supplementary material

13311_2014_269_MOESM1_ESM.pdf (419 kb)
Supplementary Fig. 1 Lentivirus characterization/biological activity of IDH1–R132H. (A) A schematic of the lentiviral vectors encoding platelet-derived growth factor (PDGF), H-RAS-G12V, and AKT under the transcriptional control of the major immediate-early human cytomegalovirus (MIEhCMV) promoter. (B) Confirmation of expression of transgene with transfection of 293T cells with lentiviral vector expressing PDGF using an enzyme-linked immunosorbent assay. (C) Immunostaining of tumors induced in rats injected with PDFG/HRAS-G12V/ATK to confirm transgene expression, with FLAG (HRAS-G12V) and HA (AKT). (D) Validation of IDH1 wildtype and IDH1-R132H expression in vitro. 293T cells were transfected with LV–IDH1 (wildtype) and LV-IDH1-R132H vectors. Confirmed with Western blot. (E) Confirmation of IDH1-R132H activity as hypermethylated histone phenotype, via immunocytochemistry of 293T cells. Cells transfected with IDH1-R132H lentivirus (citrine-positive), costained with the triple-methylated version of histone variant H3.3K27 (H3.3K27triMe; shown in red). There was no increase in the immunolabeling of control cells not transfected with IDH1-R132H. LTR = long terminal repeat; RRE = Rev Response Element; hCMV = human cytomegalovirus; PGK = phosphoglycerate kinase (PGK) promoter; PURO = puromycin resistance cassette; WPRE = woodchuck hepatitis post-transcriptional regulatory element (enhancer); GFAP = glial fibrillary acidic protein; DAPI = 4’,6-diamidino-2-phenylindole; HA = human influenza hemagglutinin (used as a tag, marker) (PDF 418 kb)
13311_2014_269_MOESM2_ESM.pdf (328 kb)
Supplementary Fig. 2 Lentivirus infects astrocytes, oligodendrocytes, endothelial cells, and microglia. Colocalization of Flag+ (HRAS-G12V) and different cell markers show the versatility of lentiviral introduction of genes of interest to a variety of cell types. Signals colocalized for astrocytes [glial fibrillary acidic protein (GFAP)], oligodendrocyte precursor cells (Olig2), endothelial cells [Von Willebrand factor (vWF)], and microglia (ED1). DAPI = 4’,6-diamidino-2-phenylindole (PDF 328 kb)
13311_2014_269_MOESM3_ESM.pdf (448 kb)
Supplementary Fig. 3 Detailed neuropathology of high-grade tumors. High-grade tumors in an animal injected with HRAS/AKT/IDH1–R132H display typical characteristics of human glioblastoma multiforme: (A) glomeruloid vascular proliferation (dotted lines), (A) thrombosis (arrow), (B) pseudopalisade necrotic core (*), and (C) perivascular invasion of tumor cells in otherwise-healthy parenchyma (high power, shown in boxes; lower power, arrows). RBCs = red blood cells (PDF 447 kb)
13311_2014_269_MOESM4_ESM.pdf (141 kb)
Supplementary Table 1 Antibodies used in all experiments (PDF 140 kb)
13311_2014_269_MOESM5_ESM.pdf (311 kb)
Supplementary Table 2 Experiments involving injection of viruses expressing IDH1–R132H demonstrated no positive or inhibitory effect on tumor formation or progression (PDF 310 kb)
13311_2014_269_MOESM6_ESM.pdf (20.9 mb)
ESM (PDF 21390 kb)

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Copyright information

© The American Society for Experimental NeuroTherapeutics, Inc. 2014

Authors and Affiliations

  • John Lynes
    • 1
    • 2
  • Mia Wibowo
    • 1
    • 2
  • Carl Koschmann
    • 1
    • 2
  • Gregory J. Baker
    • 1
    • 2
  • Vandana Saxena
    • 1
    • 2
  • A. K. M. G. Muhammad
    • 1
    • 2
  • Niyati Bondale
    • 1
    • 2
  • Julia Klein
    • 1
    • 2
  • Hikmat Assi
    • 1
    • 2
  • Andrew P. Lieberman
    • 3
  • Maria G. Castro
    • 1
    • 2
  • Pedro R. Lowenstein
    • 1
    • 2
    Email author
  1. 1.Department of NeurosurgeryUniversity of Michigan, School of MedicineAnn ArborUSA
  2. 2.Department of Cell and Developmental BiologyUniversity of Michigan, School of MedicineAnn ArborUSA
  3. 3.Department of PathologyUniversity of Michigan, School of MedicineAnn ArborUSA

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