Preclinical evaluation of radiation and perifosine in a genetically and histologically accurate model of brainstem glioma

Abstract

Brainstem gliomas (BSG) are a rare group of central nervous system tumors that arise mostly in children and usually portend a particularly poor prognosis. We report the development of a genetically engineered mouse model of BSG using the RCAS/tv-a system and its implementation in preclinical trials. Using immunohistochemistry, we found that platelet-derived growth factor (PDGF) receptor alpha is overexpressed in 67% of pediatric BSGs. Based on this observation, we induced low-grade BSGs by overexpressing PDGF-B in the posterior fossa of neonatal nestin tv-a mice. To generate high-grade BSGs, we overexpressed PDGF-B in combination with Ink4a-ARF loss, given that this locus is commonly lost in high-grade pediatric BSGs. We show that the likely cells of origin for these mouse BSGs exist on the floor of the fourth ventricle and cerebral aqueduct. Irradiation of these high-grade BSGs shows that although single doses of 2, 6, and 10 Gy significantly increased the percent of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive nuclei, only 6 and 10 Gy significantly induce cell cycle arrest. Perifosine, an inhibitor of AKT signaling, significantly induced TUNEL-positive nuclei in this high-grade BSG model, but in combination with 10 Gy, it did not significantly increase the percent of TUNEL-positive nuclei relative to 10 Gy alone at 6, 24, and 72 hours. Survival analysis showed that a single dose of 10 Gy significantly prolonged survival by 27% (P = 0.0002) but perifosine did not (P = 0.92). Perifosine + 10 Gy did not result in a significantly increased survival relative to 10 Gy alone (P = 0.23). This PDGF-induced BSG model can serve as a preclinical tool for the testing of novel agents.

Figure 1. Generation of PDGF-induced Brainstem gliomas

A. Kaplan-Meier of 15 Ntv-a mice, and 71 Ntv-a; Ink4a-ARF−/− mice injected with RCAS-PDGFB. B. Low (left) and high (middle) magnification H&E of a PDGF-induced BSG in Ntv-a mouse (sacrificed at twelve-weeks of age). Right. T2 weighted image at twelve-weeks of age of a BSG induced by overexpression of PDGF in Ntv-a mouse. C. Low (left) and high (middle) magnification H&E of a PDGF-induced BSG in Ntv-a; Ink4a-ARF−/− mouse (sacrificed at five-weeks of age). Right T2 weighted image at four-weeks of age of a BSG induced by overexpression of PDGF in Ntv-a; Ink4a-Arf null mice.

Figure 2. PDGF-induced murine BSGs are histopathologically similar to human BSGs

A. H&E, Ki-67, PDGFRα, and Olig-2 of a pediatric high-grade BSG (human). B. H&E, Ki-67, PDGFRα, and Olig-2 of PDGF-induced BSG generated in an Ntv-a; Ink4a-ARF−/− mouse (high-grade BSG). C. H&E, Ki−67, PDGFRα, and Olig-2 of PDGF-induced BSG generated in an Ntv-a mouse (low-grade BSG). D. H&E, Ki-67, PDGFRα, and Olig-2 of normal brainstem of an Ntv-a; Ink4a-ARF−/− mouse.

Figure 3. Cell of Origin for murine Brainstem Gliomas

A. High magnification of a cross section from a four-week old Ntv-a mouse infected with RCAS-GFP. White arrows points to clusters of GFP labeled cells. B. Quantification of the number of GFP-labeled cells at particular locations of a schematic of the cross section of the 4^th ventricle. Y-axis denotes the number of GFP-positive cells and the Y-axis denotes to the location of the GFP-positive cells. This cartoon illustrates the cross-section of the 4^th ventricle as a triangle. Each side of the triangle (where the floor of the 4^th comprises two sides of the triangle) was divided into 4 equal areas and the number of GFP labeled cells were counted in each area, and tabulated. C. High magnification of a cross-section through the 4^th ventricle at P2 immunostained with nestin. Black arrows point to positive immunostaining. D. Examples of precursor lesions arising from the 4^th ventricle or aqueduct of Ntv-a mice infected with RCAS-PDGF (Bottom left is cross section of aqueduct (AQ)).

Figure 4. Dose Response Curve for RT of high-grade PDGF-induced BSGs

A. Graph of % pH3 quantification at 6 hours post RT at different doses: 2 Gy (n=6), 6 Gy (n=5), and 10 Gy (n=6) and unirradiated controls (n=7). Mann-Whitney analysis was significant at p=0.0025 between control group and 6 Gy group and was significant at p=0.0012 between control and 10 Gy. We used the following data more than once: control BSGs and 10Gy 6hrs in figure 4A, 5A, and supplemental Figure 4A. B. High magnification sections immunostained with pH3: top left: control, top right: 2 Gy, bottom left: 6 Gy and bottom right: 10 Gy. C. Graph of % TUNEL-positive nuclei of 2 Gy (n=5), 6 Gy (n=5), and 10 Gy (n=7) and unirradiated controls (n=5) at 24 hours post RT. Mann Whitney analysis was significant at p=0.008 between control and 2 Gy, p=0.008 between control and 6 Gy, and p=0.0243 between control and 10Gy. We used the following data more than once: control BSGs and 10Gy 24hrs in Figure 4C, 5B, and supplemental Figure 4B. D. High magnification TUNEL of 2, 6, 10 Gy 24 hours post RT, and unirradiated control.

Figure 5. Treatment of PDGF induced Ink4a-ARF null BSGs with Radiation and Perifosine

A. Graph of % pH3 quantification at 6 hours, 24 hours and 72 hours post RT with and without perifosine. Mice per group are denoted in parentheses: untreated (n=7), perifosine (n=7), RT 6hrs (n=6), RT 6hrs + perifosine (n=6), RT 24hrs (n=5), RT 24hrs + perifosine (n=8), RT 72hrs (n=6) and RT 72hrs+perifosine (n=6) B. Graph of % TUNEL-positive nuclei at 6 hours, 24 hours, and 72 hours post RT with and without perifosine. Mice per group are denoted in parentheses: untreated (n=5), perifosine (n=7), RT 6hrs (n=8), RT 6hrs + perifosine (n=7), RT 24hrs (n=7), RT 24hrs+ perifosine (n=5), RT 72hrs (n=6), RT 72hrs + perifosine (n=6) C. Low magnification H&E (left) and TUNEL(right) of high-grade BSG treated with perifosine+10Gy 6 hours post RT. D. Survival analysis of PDGF-induced Ink4a-ARF−/− BSG treated with single dose 2 Gy (10 mice), single dose 10 Gy (12 mice), perifosine (7 mice), perifosine + 10Gy (9 mice), untreated mice (9 mice).