A recombinant lentiviral PDGF-driven mouse model of proneural glioblastoma

Abstract

Background: Mouse models of glioblastoma (GBM), the most aggressive primary brain tumor, are critical for understanding GBM pathology and can contribute to the preclinical evaluation of therapeutic agents. Platelet-derived growth factor (PDGF) signaling has been implicated in the development and pathogenesis of GBM, specifically the proneural subtype. Although multiple mouse models of PDGF-driven glioma have been described, they require transgenic mice engineered to activate PDGF signaling and/or impair tumor suppressor genes and typically represent lower-grade glioma.

Methods: We designed recombinant lentiviruses expressing both PDGFB and a short hairpin RNA targeting Cdkn2a to induce gliomagenesis following stereotactic injection into the dentate gyrus of adult immunocompetent mice. We engineered these viruses to coexpress CreERT2 with PDGFB, allowing for deletion of floxed genes specifically in transduced cells, and designed another version of this recombinant lentivirus in which enhanced green fluorescent protein was coexpressed with PDGFB and CreERT2 to visualize transduced cells.

Results: The dentate gyrus of injected mice showed hypercellularity one week post-injection and subsequently developed bona fide tumors with the pathologic hallmarks of GBM leading to a median survival of 77 days post-injection. Transcriptomic analysis of these tumors revealed a proneural gene expression signature.

Conclusion: Informed by the genetic alterations observed in human GBM, we engineered a novel mouse model of proneural GBM. While reflecting many of the advantages of transgenic mice, this model allows for the facile in vivo testing of gene function in tumor cells and makes possible the rapid production of large numbers of immunocompetent tumor-bearing mice for preclinical testing of therapeutics.

Fig. 1

Development and characterization of a gliomagenic recombinant lentiviral vector encoding shCdkn2a-PDGFB-T2A-CreERT2. (A) Vector design and (B) expression of PDGFB and CreERT2 from the vector in 293FT cells. (C) Quantitative real-time PCR analysis of Cdkn2a expression in primary mouse fibroblasts infected with the shCdkn2a virus compared with a control shRNA (2 multiplicity of infection). Mean (C) ± 1 SD of 3 independent experiments are shown. **P < 0.01.

Fig. 2

Stereotactic injection of lentiviruses encoding shCdkn2a-PDGFB-T2A-CreERT2 into the dentate gyrus gives rise to GBM. (A) A schematic diagram of the injection site (dentate gyrus marked in red) on a coronal section depiction of an adult mouse brain. (B) Kaplan–Meier survival curve showing overall survival of mice injected with shCdkn2a-PDGFB-T2A-CreERT2 lentiviruses. (C) Gross appearance of injected brains during terminal moribund status of mice. Hematoxylin and eosin (H&E) stained tumor sections from mice injected with the shCdkn2a-PDGFB-T2A-CreERT2 lentivirus after (D) 45 days from initial injection and (E) during terminal moribund status. The right panel in (E) displays the sharp contrast in cellularity between tumor and normal tissue (indicated in the image by T and N, respectively, separated by dashed line). H&E stained tumor sections from PDGF-driven tumors showing (F) increased cellularity, (G) pseudopalisading necrosis, (H) invasion of normal stroma, and (I) hemorrhage. (C) and (E–I) contain 2 panels showing 2 representative examples of what we observed in our study. White scale bar: 100 µm. Yellow scale bar: 10 µm.

Fig. 3

Immunohistochemical staining of shCdkn2a-PDGFB-T2A-CreERT2 GBM tumor sections with antibodies against GBM markers. Stains for OLIG2, GFAP, Ki-67, PDGFRA, and CD31 on tumor sections obtained from mice injected with the gliomagenic lentivirus under low (left panels, 4X magnification) and high (10X) magnification (right panels). Sections were counterstained with hematoxylin. We show 2 representative images for each of the GBM markers under both 4X and 10X magnifications. Scale bar: 100 µm.

Fig. 4

Fluorescent tracking of shCdkn2a-PDGFB-T2A-eGFP-E2A-CreERT2 transduced cells reveals early stages of tumorigenesis. (A) Vector design of shCdkn2a-PDGFB-T2A-eGFP-E2A-CreERT2 which expresses eGFP in the same coding locus as PDGFB and CreERT2. Infected cells in the dentate gyrus of an adult mouse (B) 1 week post lentiviral injection under 10X and 20X magnification. GFP (from the shCdkn2a-PDGFB-T2A-eGFP-E2A-CreERT2 lentivirus infected cells) and Hoechst (nuclei) channels are shown. Scale bar: 100 µm.

Fig. 5

GFP staining of shCdkn2a-PDGFB-T2A-eGFP-E2A-CreERT2 GBM tumor sections shows that tumor cells are predominantly infected with the lentiviral vector. Low and high magnification (top and bottom panels, respectively) of shCdkn2a-PDGFB-T2A-eGFP-E2A-CreERT2 GBM tumor sections stained with a GFP antibody. Sections were counterstained with hematoxylin. Scale bar: 100 µm.

Fig. 6

Transcriptomic profiling classifies shCdkn2a-PDGFB-T2A-CreERT2 induced tumors as proneural GBM. (A) Heatmap showing the subtype signature classification by TCGA (classification bar on top) and validation correlation scores of primary human GBM tumors using Spearman correlation. (B) Heatmap of 8 independent tumor samples from shCdkn2a-PDGFB-T2A-CreERT2 GBM shows an enrichment of a proneural signature using the same method as (A).