Gliomagenesis in NF1 occurs with remarkable spatial and temporal specificity, predominantly affecting the optic nerves and chiasm of young children (19
). This pattern of glioma formation indicates that a necessary interaction occurs between glial NF1
gene inactivation and signals present in the optic pathway microenvironment during NF1-associated gliomagenesis.
In prior studies involving human specimens and a genetically-engineered mouse model of NF1-associated optic glioma (“OPG” mice), we identified CXCL12 as a candidate stromal signal capable of stimulating Nf1
-deficient glial cell growth. (9
). Multiple brain-derived cells within optic pathway glioma express CXCL12 including endothelial cells (), entrapped axons and infiltrating microglia. The presence of a functional paracrine relationship between CXCL12-expressing stromal elements and tumor cells is demonstrated by the frequent presence of the CXCL12 receptor, CXCR4, in a CXCL12-induced phosphorylated form within bipolar pilocytic cells (, inset
). The potential importance of stromal activation of CXCR4 lies in its ability to suppress intracellular levels of cAMP, which we found was necessary for the stimulation of Nf1
-/- astroglial cell growth (9
). Moreover, we found region-specific differences in cAMP levels in the mouse brain that correlated with the pattern of glioma formation in NF1. The optic pathway had the lowest, while the cortex had the highest cAMP levels. Based on these observations, we hypothesized that differences in cAMP levels underlie the distinct pattern of gliomagenesis in NF1.
Optic nerve gliomas in humans and mice
To evaluate this hypothesis in vivo
, we sought to alter the pattern of brain region-specific cAMP levels to determine whether this would coordinately change the pattern of tumor formation in an established genetically-engineered mouse (GEM) model of NF1 OPG. Astrocytomas in Nf1
GEM models share many, but not all, of the features of human OPG. Gliomas within the optic nerves of OPG mice () contain bipolar GFAP positive astrocytoma cells () (20
) that also express the transcription factor Olig2 (Supplemental Figure 1A
). Misexpression of Olig2 was recently shown to be highly correlated with pilocytic astrocytoma (22
). In addition, murine OPG exhibit microglial infiltration reminiscent of human tumors (Supplemental Figure 1B
). In contrast to human pilocytic astrocytomas, murine tumors do not contain Rosenthal fibers or eosinophilic granular bodies.
To generate foci of decreased cAMP, we ectopically expressed lentiviral-encoded cAMP specific phosphodiesterase-4A1 (PDE4A1) (27
) via stereotactic injection into the cortex of OPG mice. PDE4A1 was chosen for these experiments based on our prior demonstration that increased PDE4A1 expression decreased intracellular cAMP levels and enhanced tumor growth in a xenograft model of malignant glioma in vivo
). The cortex was selected as the site for PDE4A1 injection based on the following considerations that suggest it is usually not a permissive microenvironment for glioma formation in NF1: (1) Patients with NF1 rarely develop cortical tumors, (2) glioma formation in Nf1
mouse models does not occur in the cortex, and (3) the cortex exhibits the highest levels of cAMP (9
Catalytically-active and inactive (PDE4A1-H229Q (15
)) forms of PDE4A1 were cloned into a lentiviral packaging vector also encoding for mCherry fluorescent protein (11
). In control studies, Daoy medulloblastoma cells were infected with lentivirus encoding PDE4A1 or PDE4A1-H229Q. While both forms of PDE4A1 were equally expressed (Supplemental Figure 2A
) and localized to the Golgi apparatus (Supplemental Figure 2B
), only catalytically-active PDE4A1 decreased intracellular cAMP levels (Supplemental Figure 2C
). To mimic the low levels of cAMP normally present within the optic pathway, we co-injected PDE4A1/mCherry lentivirus with a second lentivirus encoding firefly luciferase (14
) into the cortex of wild-type, Tumor Progenitor, and OPG mice. Bioluminescence imaging (BLI) of firefly luciferase expression was used to monitor transgene expression and served as a potential measure of neoplastic growth. In control studies, the expression of PDE4A1 or PDE4A1-H229Q in Daoy cells, or treatment with the pan-PDE4 inhibitor Rolipram had no effect on luciferase activity (Supplemental Figure 2D, E
Since wild-type mice do not develop cortical gliomas, tumor induction following ectopic PDE4A1 expression in wild type mice (Nf1flox/flox)
would indicate that cAMP suppression is sufficient for glioma initiation and promotion. Although Tumor Progenitor mice (Nf1flox/flox;
GFAP-Cre) possess occasional foci of hyperproliferating astrocytes, they do not develop gliomas (3
). PDE4A1-induced tumor formation in these mice would occur only if suppression of cAMP alone induced the combined tumor-promoting properties of an Nf1
+/- microenvironment and the optic pathway. Finally, cortical glioma formation in OPG mice (Nf1flox/mut;
GFAP-Cre) would suggest that cAMP suppression is sufficient, within the context of an Nf1
+/- microenvironment to bring about the neoplastic growth of Nf1
-/- tumor progenitors. PDE4A1-H229Q injections serve as controls for non-catalytic effects of lentiviral injection and PDE4A1 overexpression.
In 51 of 58 injected mice, we detected a bioluminescent signal, indicating successful viral transduction and transgene expression. Increased BLI on serial studies demonstrated replication of virally infected cells. The brain of each animal was subjected to an extensive blinded evaluation for the presence of neoplasia according to the algorithm outlined in Supplemental Figure 3
Hematoxylin and eosin-stained sections from micro-injected cortices were examined for common features of neoplasia, including hypercellularity, cellular clustering, and nuclear atypia, as well as the specific features of low-grade glioma seen in the Nf1
OPG GEM model (, Supplemental Figure 1
). Twelve of 13 OPG mice injected with catalytically-active PDE4A1 lentivirus demonstrated increased BLI and expression of mCherry fluorescent protein/PDE4A at the injection site (). The diagnosis of an induced glioma was made when hypercellular lesions expressing mCherry fluorescent protein and PDE4 also contained GFAP-positive cells with the bipolar morphology characteristic of tumor cells (piloid cells) commonly found in the low-grade gliomas of NF1 patients (). Further indication of glioma was evident in increased Olig2 expression (). In each case, induced tumor sites exhibited greater numbers of Olig2 positive nuclei per high-powered field than did the same location in contralateral cortex. As a group, the induced tumors demonstrated significantly increased Olig2 expression compared to the uninvolved contralateral cortex (). Comparable histological findings were not observed in the contralateral cortex, nor are they observed in normal human or mouse cortex and optic nerve (Supplemental Figure 4
Induced tumors occur at PDE4A injection sites
Induced tumors exhibit increased Olig2 expression and evidence for decreased cAMP levels
Importantly, bipolar piloid cells expressed PDE4 in a peri-nuclear distribution characteristic of PDE4A1 localization ( inset), suggesting a role for PDE4A1 expression in gliomagenesis. The mechanistic significance of this finding was further underscored by our finding that induced tumors had lower levels of cAMP. Since the induced tumors are small and preclude direct cAMP measurements, cAMP levels were inferred from an immunohistochemical analysis using a surrogate marker, the intensity of Protein Kinase A (PKA) substrate phosphorylation. Tumor sites exhibited decreased labeling with phosphoPKA (pPKA) substrate antibody, indicating decreased PKA activity () and suggestive of decreased levels of cAMP. These findings are consistent with a relationship between lower levels of cAMP and gliomagenesis in NF1.
In total, 41 of the 51 mice with increased BLI had injection sites with evidence for exogenous PDE4A1 or PDE4A1-H229Q expression and were deemed evaluable for tumor formation. Eight of the 13 OPG mice with increased BLI following PDE4A1 injection were considered to have induced cortical gliomas (). This was a statistically significant rate of induced tumor formation, as determined by Fisher's Exact Test (P=0.02). In contrast, only 1 of 7 Tumor Progenitor mice and 1 of 6 wild-type mice had evidence of induced glioma. None of the 15 control injections of PDE4A1-H229Q resulted in a tumor. The frequency of tumor formation in control mice was not significantly significant.
While PDE4A (not shown) and mCherry positive cells () were observed at injection sites in mice without tumors, these differed from induced tumors by the absence of abnormal astrocyte morphologies () and their lack of increased Olig2 expression (). Disruption in tissue architecture at these injection sites was limited to inflammation, as evidenced by the presence of LCA-positive cells (). LCA-positive inflammatory cells were only a minor component of the cellular constituents in the induced tumors (data not shown). Together with our prior studies, the pathological features of PDE4A1-induced cortical tumors in OPG mice demonstrate that low cAMP levels are sufficient to promote glioma formation when Nf1-/- glioma progenitors arise within the context of an Nf1+/- microenvironment.
Histological features of non-tumor reactions
To determine whether restoration of normal cAMP levels would attenuate optic glioma growth in vivo
, we treated OPG mice for one or four weeks with Rolipram or vehicle following the detection of an optic glioma by manganese-enhanced MRI (). We previously showed that PDE4 inhibition with Rolipram blocked in vivo
malignant glioma growth through elevation of cAMP levels (11
). Using this preclinical paradigm, four weeks of Rolipram resulted in tumor regression as measured by a restoration of optic nerve volumes to near normal values (). Moreover, Rolipram treatment suppressed tumor cell proliferation (), but had no effect on tumor cell apoptosis as measured by TUNEL staining (data not shown). These observations demonstrate that low levels of cAMP are necessary for the maintenance of optic glioma proliferation in vivo
and further support the critical role of cAMP signaling in gliomagenesis and continued tumor growth.
Targeted inhibition of PDE4 attenuates tumor growth in OPG mice