Optic Nerve Glioma in Mice Requires Astrocyte Nf1 Gene Inactivation and Nf1 Brain Heterozygosity

[CANCER RESEARCH 63, 8573–8577, December 15, 2003] Advances in Brief

M. Livia Bajenaru,1 M. Rosario Hernandez,2 Arie Perry,3 Yuan Zhu,6 Luis F. Parada,6 Joel R. Garbow,4,5 and David H. Gutmann1 Departments of 1Neurology, 2Ophthalmology, 3Pathology, 4Radiology, and 5Chemistry, Washington University School of Medicine, St. Louis, Missouri, and 6Center for Developmental Biology and Kent Waldrep Foundation Center for Basic Research on Nerve Growth and Regeneration, University of Texas Southwestern Medical Center, Dallas, Texas

Abstract bryonic day 14 using Cre/LoxP technology (11). Glial fibrillary acidic protein (GFAP) Cre; Nf1flox/flox mice are viable and fertile and exhibit Whereas biallelic neurofibromatosis 1 (NF1) inactivation is observed in increased numbers of brain and optic nerve astrocytes, but they do not NF1-associated gliomas, astrocyte-restricted Nf1 conditional knockout develop gliomas. The absence of glioma formation in these mice, even mice do not develop gliomas. These observations suggest that NF1 glioma formation requires additional cellular or genetic conditions. To determine after 20 months of age, suggests that additional cellular or genetic the effect of an Nf1 heterozygous brain environment on NF1 glioma events are necessary for glioma tumorigenesis in the setting of NF1. -formation, we generated Nf1؉/؊ mice lacking Nf1 expression in astro- Because patients with NF1 are heterozygous for a germ-line inacti cytes. In contrast to astrocyte-restricted Nf1 conditional knockout mice, vating NF1 mutation, and Nf1 mutant mice develop neurofibroma, Nf1؉/؊ mice lacking Nf1 in astrocytes develop optic nerve gliomas. This another NF1-associated nervous system tumor, only in the setting of mouse model demonstrates that Nf1؉/؊ cells contribute to the pathogen- constitutional Nf1 heterozygosity (12), we generated Nf1ϩ/Ϫ mice esis of gliomas in NF1 and provides a tool for the preclinical evaluation of lacking Nf1 expression in astrocytes. In this report, we describe this potential therapeutic interventions for these tumors. unique model of NF1-associated optic nerve glioma, in which Nf1ϩ/Ϫ mice with conditional Nf1 inactivation in astrocytes develop Introduction low-grade optic nerve and chiasm astrocytomas. Low-grade astrocytomas (gliomas) are common central nervous system tumors affecting children (1). Children with the inherited Materials and Methods tumor predisposition syndrome neurofibromatosis 1 (NF1) are prone Transgenic Mice. Nf1ϩ/Ϫ mice (10) were bred with Nf1flox/flox mice (13) to the development of WHO grade I pilocytic astrocytomas (PAs), to produce Nf1flox/mut mice, which were subsequently crossed with GFAPCre; which predominantly involve the optic nerve and chiasm (2). Al- Nf1flox/flox mice (11) to generate GFAPCre; Nf1flox/mut mice. Optic nerves and though these tumors are low-grade neoplasms, they can demonstrate brains from 8–12-month-old GFAPCre; Nf1flox/mut mice (n ϭ 12) and 8–18- aggressive biological features, diffusely infiltrate the optic pathway month-old GFAPCre; Nf1flox/flox mice (n ϭ 20) were analyzed. We used and neighboring hypothalamus, and culminate in visual loss and control mice of genotypes Nf1flox/flox, Nf1flox/mut, and Nf1ϩ/Ϫ at 8–12 months precocious puberty (2). Previous studies have shown that NF1-asso- of age (n ϭ 10). ciated optic pathway gliomas involve biallelic inactivation of the NF1 Histopathology and Immunohistochemistry. Control and mutant mice gene, whereas histologically similar sporadic PAs do not exhibit NF1 were perfused transcardially with 4% paraformaldehyde in 0.1 M sodium loss (3–6). In addition, PAs do not harbor genetic changes typical of phosphate buffer (pH 7.4). Eyes, optic nerves, optic chiasms, and brains were diffuse fibrillary astrocytomas, such as inactivation of CDKN2A, dissected and postfixed in 4% paraformaldehyde overnight at 4°C before TP53, or PTEN/MMAC1 or amplification of CDK4 or the epidermal examination for gross anatomical changes and photography with a digital camera (Optronics) attached to a dissection microscope (Nikon). All specimens growth factor receptor gene (7). were then processed for paraffin embedding and sectioning in the Pharmacol- Current animal models of astrocytoma have focused on oncogenic ogy Histology Core or Ophthalmology Histology Core at Washington Univer- events (e.g., constitutive RAS activation) or on combinatorial inacti- sity School of Medicine. vating events involving p53 (reviewed in Ref. 8). These mice develop Serial 4-␮m paraffin sections of the eyes and optic nerve heads, optic nerves high-grade gliomas, which are histologically and biologically distinct and chiasms, and brains were stained with H&E and examined under the from those observed in NF1 patients. Using an alternate approach, microscope for the presence of tumors or abnormal collections of cells by an heterozygous Nf1 mutant mice are cancer prone but do not develop experienced neuropathologist (A. P.). gliomas (9). Because mice lacking Nf1 expression do not survive Immunohistochemistry was performed on adjacent paraffin sections with rat embryonic development (9, 10), we developed a conditional knockout anti-GFAP (1:100; Zymed) and rabbit anti-Ki67 (1:1000; Novocastra) anti- mouse in which the Nf1 gene was inactivated in astrocytes by em- bodies. We used microwave antigen retrieval and detected the primary antibodies with biotinylated secondary antibodies, followed by amplification with peroxidase-conjugated avidin (Vectastain Elite ABC Kit; Vector Laboratories) Received 8/31/03; revised 10/10/03; accepted 10/17/03. and treatment with 3,3Ј-diaminobenzidine or Vector VIP substrate kits (Vector Grant support: NIH Grants NS36996 (to D. H. G.), F32-NS42428 (to M. L. B.), and 1 R24 CA83060; a grant from the Small Animal Imaging Resource Program (to J. R. G.); Laboratories), followed by counterstaining with hematoxylin or methyl green a grant from National Institutes of Neurological Disorders and Stroke (to L. F. P.); The (Vector Laboratories). Representative sections were photographed with a dig- United States Army Medical Research and Materiel Command’s Office of Congression- ital camera (Optronics) attached to an inverted microscope (Nikon). ally Directed Medical Research Programs Grant DAMD17-03-1-0215 (to D. H. G.), and a grant from The National Neurofibromatosis Foundation (to Y. Z.). Microglia cells were identified in optic nerve and chiasm sections by lectin The costs of publication of this article were defrayed in part by the payment of page histochemistry (14) using FITC-conjugated Bandeiraea (Griffonia) simplici- charges. This article must therefore be hereby marked advertisement in accordance with folia agglutinin isolectin-B4 (BSI-B4; Sigma). Briefly, optic nerve and chiasm 18 U.S.C. Section 1734 solely to indicate this fact. sections were deparaffinized and washed in PBS, followed by overnight Requests for reprints: David H. Gutmann, Department of Neurology, Box 8111, 660 ␮ South Euclid Avenue, St. Louis, Missouri 63110. Phone: (314) 362-7379; Fax: (314) 362- incubation at 4°C with FITC-conjugated BSI-B4 (10 g/ml) in PBS containing 2388; E-mail: [email protected]. 0.5% Triton X-100. After subsequent washes in PBS, fluorescence images 8573

were recorded by digital photomicrography (Spot Advanced; Diagnostic In- ities. Pathological examination of their brains failed to demonstrate struments, Sterling Heights, MI). astrocytoma (data not shown). These results suggest that Nf1 inacti- Proliferative indices were determined by counting the number of Ki67- vation, either alone or in combination with Nf1 heterozygosity, does labeled nuclei per 100 cell nuclei within the prechiasmatic optic nerves (areas not result in parenchymal brain tumor formation. ϫ 1 and 2) and the optic chiasm (area 3), using a 10-mm ocular grid at 400 Because gliomas in NF1 most typically involve the optic nerve, magnification. chiasm, and hypothalamus, we examined the optic nerves and chiasm Magnetic Resonance Imaging. Images were collected in an Oxford In- flox/mut ϭ struments 4.7-Tesla magnet (33 cm, clear bore) equipped with 15-cm inner in GFAPCre; Nf1 mice. All mice examined (n 12) demon- diameter, actively shielded gradient coils (maximum gradient, 18 G/cm; rise strated areas of gross optic nerve and/or chiasm enlargement, which flox/flox time, 100 ␮s). The magnet/gradients are interfaced with a Varian (Palo Alto, were not seen in either Nf1ϩ/Ϫ or GFAPCre; Nf1 mice. These CA) INOVA console, and data were collected using a 1.5-cm outer diameter gross abnormalities were seen in mice as early as 8 months of age, surface coil (receive) and a 9-cm inner diameter Helmholtz coil (transmit). although younger mice have not been systematically examined to Before the imaging experiments, the mice were anesthetized with isoflu- date. We observed multiple patterns of gross pathology, including rane/O2 [4% (v/v)], and they were maintained on isoflurane/O2 [1.5% (v/v)] unilateral as well as bilateral optic nerve enlargement, with and throughout the experiments. Diffusion tensor imaging data were acquired using without chiasmal involvement (Fig. 1). These patterns are highly a conventional spin-echo imaging sequence, modified by the addition of a reminiscent of those found in children with NF1. Stejskal-Tanner diffusion-sensitizing gradient pair. Six images with different 2 Microscopic examination of these optic nerve masses demonstrated gradient directions were acquired with a b value of 785 s/mm , together with Ϫ a reference spin-echo (b ϭ 0) image. Other experimental parameters were 16 increased cellularity (Fig. 2, top panel, A G) with collections of Ϫ signal averages, time between scans (TR) ϭ 0.75 s, echo time (TE) ϭ 0.05 s, GFAP-immunoreactive astrocytes (Fig. 2, top panel, I K). In addi- slice thickness ϭ 0.5 mm, field of view (FOV) ϭ 1.5 cm. tion to exhibiting elevated Ki67 proliferative indices (Fig. 3A, aϪc), The diffusion tensor matrix was generated from a series of diffusion- these proliferating individual or clustered cells exhibited enlarged, weighted images, in which the primary diffusion parameters (three eigenvalues mildly to moderately atypical, hyperchromatic nuclei with small nu- ␭ ␭ ␭ or diffusivities, 1, 2, and 3) were calculated by matrix diagonalization. cleoli, similar to their human counterparts (Ref. 1; Fig. 2, top panel, These primary parameters were combined into two secondary parameters D, H, and L). As is typical for low-grade gliomas, there was no useful for describing water diffusion (15): (a) apparent diffusion constant (the evidence of endothelial hyperplasia or necrosis. However, unlike mean of the three directional diffusivities); and (b) relative anisotropy (the human optic pathway PAs, these mouse gliomas lacked microcystic normalized standard deviation of the three diffusivities). These parameter spaces, Rosenthal fibers, or eosinophilic granular bodies and shared maps, together with conventional T2-weighted spin-echo images, were used to visualize the mouse optic nerves. some histological features with WHO grade II fibrillary astrocytomas. It should be noted that even within human NF1-associated PAs, Results and Discussion portions of the tumor may be infiltrative, lack Rosenthal fibers, eosinophilic granular bodies, and protein droplets; and can be con- Nf1 Inactivation in Astrocytes Alone Is Insufficient for Astro- fused with low-grade WHO II fibrillary astrocytoma (18). In this cytoma Formation. Whereas both Nf1ϩ/Ϫ and GFAPCre; respect, human pilocytic optic nerve gliomas most often appear as Nf1flox/flox mice exhibit increased numbers of brain astrocytes (11, 16, glial tumors with alternating dense-spindled and loose microcystic 17), neither strain develops astrocytoma in vivo. Similarly, Nf1Ϫ/Ϫ astrocytes have an incremental increase in cell-autonomous growth in vitro compared with Nf1ϩ/Ϫ astrocytes, but they lack tumorigenic properties (11). Because our GFAPCre mice (both lines 1 and 8) exhibit robust Cre expression in astrocytes throughout the optic nerve and chiasm (data not shown), in addition to the brain and spinal cord (11), we sought to determine whether Nf1 inactivation in optic nerve astrocytes was sufficient for optic nerve/chiasm tumor formation. Compared with Nf1ϩ/Ϫ mice, GFAPCre; Nf1flox/flox mice demonstrate complete loss of neurofibromin expression in astrocytes (11), increased astrocyte numbers in the optic nerve, and minor increases in astrocyte proliferation. However, examination of the optic nerves from both Nf1ϩ/Ϫ and GFAPCre; Nf1flox/flox mice failed to demonstrate any gross or microscopic evidence for optic nerve glioma. These results demonstrate that Nf1 inactivation in astrocytes, beginning on embryonic day 14, confers a growth advantage in vitro and in vivo that is not sufficient for astrocytoma formation in the optic nerve and raise the possibility that constitutional Nf1 heterozygosity in the supporting nonneoplastic cells is required for tumorigenesis. Nf1 Inactivation in Astrocytes Results in Optic Nerve/Chiasm Glioma Formation in the Context of Nf1 Heterozygosity. In an effort to model the human condition, where patients are heterozygous for an inactivating germ-line NF1 mutation, we first injected Nf1-null postnatal day 2 neocortical astrocytes, generated by Cre adenovirus- mediated Nf1flox inactivation, into the brains of syngeneic Nf1ϩ/Ϫ mice. None of the 22 mice that received the injection exhibited any evidence of glioma formation after 1 year (data not shown). Second, we generated Nf1ϩ/Ϫ mice lacking Nf1 expression in Fig. 1. GFAPCre; Nf1flox/mut mice manifest swollen optic nerves and chiasms. The flox/mut arrows denote two representative examples of enlarged optic nerves and chiasms in astrocytes (GFAPCre; Nf1 mice). These mice are viable and GFAPCre; Nf1flox/mut mice (A and C), not seen in GFAPCre; Nf1flox/flox mice (B)or healthy at 12 months of age, without obvious neurological abnormal- Nf1ϩ/Ϫ and Nf1flox/flox mice (D and E). Scale bar, 1 mm. 8574

Fig. 2. Histological analysis of optic pathway glioma in the GFAPCre; Nf1flox/mut mice (top panels). H&E staining (H&E) demonstrates enlarged optic nerves with areas of increased celullarity (C and G)in GFAPCre; Nf1flox/mut mice, compared with Nf1flox/flox (A and E)orGFAPCre; Nf1flox/flox mice (B and F). The areas of increased cellularity in the optic nerves of GFAPCre; Nf1flox/mut mice are highly enriched in GFAP-immunoreactive astrocytes (K), compared with control mice (I). GFAPCre; Nf1flox/flox mice exhibit moderate increases in astrocyte numbers in the chiasm and optic nerve (J). Many atypical nuclei were found clustered within the hypercellular optic nerve masses of GFAPCre; Nf1flox/mut mice. These atypical nuclei (arrows) are significantly larger than nonneoplastic cell nuclei (arrowheads), have an irregular contour, and are hyperchromatic (D and H). Mildly atypical nuclei (arrows) appear rarely in association with normal cell nuclei (arrowheads) in optic nerves from GFAPCre; Nf1flox/flox mice (L), and may represent reactive or hyperplastic astrocytes. Scale bar, 100 ␮m. Bottom panels: human pilocytic optic nerve gliomas typically appear as astrocytic tumors with alternating dense spindled and loose microcystic areas (A) with scattered brightly eosinophilic rod-shaped Rosenthal fibers and mulberry-shaped eosinophilic granular bodies (B), but may exhibit mild hypercellularity and nuclear pleiomorphism (C) with scattered individual and clustered atypical tumor nuclei (D) at the infiltrative edge of the same tumor, reminiscent of the Nf1 mouse model. Magnification, ϫ200 (A) and ϫ400 (BϪD).

areas (Fig. 2, bottom panel, A) with Rosenthal fibers and granular Therefore, it is difficult to determine with certainty whether the optic bodies (Fig. 2, bottom panel, B), but they may exhibit nuclear plei- gliomas in our mouse model represent low-grade fibrillary astrocyto- omorphism (Fig. 2, bottom panel, C) with clustered atypical tumor mas, an otherwise rare subtype in NF1 patients, or an earlier stage of nuclei (Fig. 2, bottom panel, D), reminiscent of the Nf1 mouse model. PA, lacking some of the more characteristic findings seen in human

Fig. 3. Optic gliomas in GFAPCre; Nf1flox/mut mice contain proliferating astrocytes. A, Ki67 immunoreac- tivity was detected in clusters of cells corresponding to areas of increased cellularity, in prechiasmatic regions as well as along the optic nerves and in the chiasm of GFAPCre; Nf1flox/mut mice. The arrow points to a representative mitotic figure (b). The Ki67-labeled cells are GFAP-immunoreactive astrocytes as demonstrated by Ki67/GFAP double immunohistochemistry (c). No Ki67-labeled nuclei were identified in the Nf1flox/flox control mice (a). Scale bar, 400 ␮m. B, the Ki67 proliferative indices are tabulated for each optic nerve and chiasm from GFAPCre; Nf1flox/mut, GFAPCre; Nf1flox/flox, Nf1ϩ/Ϫ, and Nf1flox/flox mice. C, Ki67 proliferative indices were determined in the prechiasmatic optic nerves (areas 1 and 2) and in the chiasm (area 3), denoted by the blue squares. D, microglia cells infiltrate the low-grade gliomas in GFAPCre; Nf1flox/mut mice. B. simplicifolia isolectin (BSI-B4) histochemistry identifies microglial cells (arrowheads) in the optic nerve areas (primarily optic nerve and chiasm), corresponding to the gliomas in GFAPCre; Nf1flox/mut mice. In contrast, there were few, if any, microglia in the optic nerves of control mice: Nf1flox/flox and GFAPCre; Nf1flox/flox. V, vessel. Scale bar, 100 ␮m.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2003 American Association for Cancer Research. OPTIC NERVE GLIOMA IN Nf1 MUTANT MICE examples that had grown for several years before resection. Never- theless, we have classified these collections of GFAP-immunoreactive astrocytes as neoplastic, based on their formation of grossly and radiologically (see below) recognizable tumor masses, the presence of nuclear clustering/atypia, and demonstrable cellular proliferation. Because these tumors grow asymmetrically within the optic nerves in these mice, we determined the proliferative index for the prechiasmatic optic nerves (areas 1 and 2) and chiasm (area 3; Fig. 3C) for each of eight tumors. As shown in Fig. 3B, there was a spectrum of increased proliferative indices in GFAPCre; Nf1flox/mut mouse optic pathway tumors, ranging from 1% to 10%, using Ki67 as a proliferation marker, as has been reported for human low-grade astrocytomas (18). The highest proliferation rates were consistently observed in older GFAPCre; Nf1flox/mut mice (Ͼ10 months old). Lower proliferative indices were observed in GFAPCre; Nf1flox/flox and Nf1ϩ/Ϫ mice (1–2%), even after 18 months of age. No Ki67 labeling was observed in the optic nerve or chiasm of control Nf1flox/flox mice. Previous studies on another nervous system tumor in NF1, the peripheral nerve-associated neurofibroma, demonstrated that Nf1ϩ/Ϫ mast cells infiltrate tumors composed of neoplastic Nf1Ϫ/Ϫ Schwann cells and likely contribute to the genesis of these benign tumors (12). In human optic nerve glioma, microglia are often observed at the periphery and within the tumor (19). To determine whether infiltrating Nf1ϩ/Ϫ microglia were found in GFAPCre; Nf1flox/mut mouse optic nerve tumors, we performed lectin histochemistry using B. (Griffonia) simplicifolia agglutinin isolectin-B4 (BSI-B4) to identify microglia. BSI-B4 labels D-galactose residues that are expressed on both resting and activated microglia cells, cells of the monocyte-macrophage lin- Fig. 4. Magnetic resonance images of transaxial slices of mouse brains. A, T2-weighted spin-echo image of one representative control C57Bl/6 mouse; the yellow box shows the eage, and vascular endothelial cells. We observed increased numbers expanded region displayed in all subsequent images; (B) relative anisotropy (RA) and (C) of microglia in the GFAPCre; Nf1flox/mut mouse optic nerve tumors apparent diffusion constant (ADC) maps of this same control mouse. Optic nerves, regions of high diffusion anisotropy that appear as bright spots in the RA map, are circled in red. compared with nonneoplastic optic nerve areas in these same mice or The remaining images in this figure are T2-weighted spin-echo images collected from one flox/flox flox/flox GFAPCre; Nf1 , Nf1ϩ/Ϫ,orNf1 mouse optic nerves. As representative C57Bl/6 control (D), one representative GFAPCre; Nf1flox/flox mouse (E), reported for human low-grade gliomas (20), the microglia infiltration and two different GFAPCre; Nf1flox/mut mice (F and G). In each of these images, the optic nerves, as determined from RA and ADC maps are shown as red circles. The regions was variable from one optic nerve glioma to another in GFAPCre; between the red circles in the bottom panels, highlighted by blue arrows, demonstrate the Nf1flox/mut mice (Fig. 3D). Whereas microglia are associated with development of optic nerve gliomas only in GFAPCre; Nf1flox/mut mice, evidenced by the these mouse optic nerve gliomas, it remains to be determined whether separation of the optic nerves by abnormal growth of tissue. Nf1ϩ/Ϫ microglia, oligodendrocytes, or neurons directly contribute GFAPCre; Nf1flox/mut mice, and we demonstrated the presence of to the pathogenesis of Nf1-null astrocyte transformation. abnormal tissue between the optic nerves only in GFAPCre; Relevance of Nf1 Heterozygosity to NF1 Tumorigenesis. Recent Nf1flox/mut mice (four of four mice imaged; age, 10–12 months; Fig. studies by Zhu et al. (12) have demonstrated a role for Nf1ϩ/Ϫ cells 4). Studies are in progress to determine the earliest time point when in the molecular pathogenesis of a related NF1-associated tumor, the these tumors can be detected by magnetic resonance imaging. plexiform neurofibroma. In this model, Nf1ϩ/Ϫ mast cells are hy- In this report, we describe a mouse model of optic nerve glioma, the pothesized to elaborate factors that promote Nf1Ϫ/Ϫ Schwann cell most common brain tumor seen in individuals affected with NF1. transformation and neurofibroma formation. Studies on Nf1ϩ/Ϫ mast Whereas astrocyte Nf1 inactivation is insufficient by itself for glioma cells (21), oligodendrocytes (22), and astrocytes (16, 17) have dem- formation, in the context of an Nf1 heterozygous environment, optic onstrated that Nf1 heterozygosity results in abnormalities in cell nerve gliomas develop. These observations extend previous results on migration, proliferation, and survival. These observations strongly Nf1 mouse modeling of nervous system tumors and strongly implicate support the emerging concept that Nf1ϩ/Ϫ cells have unique proper- Nf1ϩ/Ϫ cells in the cellular pathogenesis of NF1 brain tumor forma- ties, which might be important in the cellular pathogenesis of NF1- tion. Future studies aimed at defining the contributing Nf1ϩ/Ϫ or associated tumors. Nf1Ϫ/Ϫ cells to glioma formation as well as characterizing the factors Development of a Preclinical Model for NF1-Associated Optic that promote Nf1Ϫ/Ϫ astrocyte transformation will lead to an im- Nerve Glioma. All mice examined to date have gross and histopatho- proved understanding of the cellular and molecular events involved in logical evidence of optic nerve glioma by 8 months of age, suggesting the development of this benign tumor in patients with NF1. that this mouse might represent a preclinical model for NF1-associated optic pathway tumors. Because these tumors are routinely de- Acknowledgments tected and followed by magnetic resonance imaging in children with NF1, we sought to determine whether small animal magnetic reso- We thank Drs. Mercedes Salvador-Silva, Shu-Wei Sun, and David Louis for nance imaging could detect abnormal optic nerve pathology in the valuable technical advice and helpful scientific discussions. We thank Belinda living mouse. 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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2003 American Association for Cancer Research. Optic Nerve Glioma in Mice Requires Astrocyte Nf1 Gene Inactivation and Nf1 Brain Heterozygosity

M. Livia Bajenaru, M. Rosario Hernandez, Arie Perry, et al.

Cancer Res 2003;63:8573-8577.

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