Gliomagenesis requires the combination of susceptible preneoplastic cells coupled with spatially- and temporally-restricted signals emanating from the tumor microenvironment. This cellular collaboration may account for the unique pattern of glioma formation within the CNS in children with NF1. With few exceptions, gliomas are predominantly located along the optic pathway and less frequently in the brainstem in young children. Based on this unique pattern of gliomagenesis, we previously showed that optic nerve and brainstem, but not neocortex, astrocytes increase their proliferation in response to Nf1
gene inactivation in vitro and in vivo (48
). This region-specific susceptibility to the effects of neurofibromin loss provides a receptive preneoplastic cell type, which in cooperation with specific signals from the local environment facilitates gliomagenesis.
The requirement for key stromal signals is illustrated by studies of Nf1
GEM strains. In both peripheral and CNS tumors, Nf1
loss in Schwann cell or glial cell precursors, respectively, is not sufficient for tumor formation (5
). Neurofibromas or gliomas only form when Nf1
inactivation occurs in Schwann or glial cell precursors in Nf1+/-
). The obligate role of Nf1+/-
cells in the process of tumor formation and continued growth is further underscored by studies that identify mast cells (3
) and microglia (20
) as important stromal cell types in neurofibroma and optic glioma development and maintenance, respectively. In the current study, we show that NF1-associated human pilocytic astrocytomas have increased numbers of microglia and use several Nf1
GEM strains to establish a pivotal role for resident microglia in both optic glioma development and maintenance.
First, we demonstrated that astrocytic tumors of all malignancy grades harbor increased percentages of Iba1-positive microglia vs. non-neoplastic brain. Previous studies have employed CD68 or Iba1 to identify microglia and found correlations between the malignancy grade and CD68/Iba1 immunopositivity (51
), but others have reported differences in microglia morphology (12
) or proliferation (40
) in gliomas of varying histologic grade. We observed a statistically insignificant trend towards increased CD68-positive cells in gliomas vs. non-neoplastic brain. We suspect that the difference between CD68 and Iba1 as microglia markers reflects the fact that CD68 recognizes other monocyte-like cells in addition to microglia and may also stain non-monocyte/macrophage lineage cells, especially under pathologic conditions (33
). Indeed, we demonstrated that macrophages in an ischemic region in the human brain were strongly CD68-positive, whereas few cells in that pathological focus coincidentally labeled with Iba1. These data strongly suggest that Iba1 is a more selective marker for brain microglia (29
), but also highlight the need to identify additional specific markers for resident microglia.
We found that GCV-mediated reductions in microglia numbers were similar during tumor evolution (5-6 weeks of age) and in the established glioma (3 months of age). Using this method to attenuate microglia function, we provide definitive evidence for the role of microglia in glioma proliferation in established tumors and show for the first time their role in glioma proliferation during tumor formation. While this method of microglia ablation should target only CNS microglia, several independent experiments confirmed the identity of the CD11b+ cells. Using flow cytometry, we found that the normal optic nerve macrophage/monocyte population is primarily composed of CD11b+ CD45low
cells, consistent with the profile of resident brain microglia (34
). Moreover, these microglia were CD68-positive (by flow cytometry) and Iba1-positive (by immunofluorescence and flow cytometry). Similar to our previous studies in human NF1-asspciated optic gliomas (20
), the CD11b+ population in Nf1+/-GFAPCKO
mouse optic gliomas was CD45low
resident brain microglia. Together, these results support the conclusion that genetic elimination of resident microglia reduces optic glioma proliferation both during tumor formation and tumor maintenance.
Because of the limited reagents currently available to characterize brain microglia, it is possible that there are different populations of microglia in the optic nerve at 5-6 weeks of age and 3 months of age. In an analogous fashion, macrophages have been proposed to have varying functions relevant to tumorigenesis as a function of tumor evolution (54
). Early during tumor development, macrophages are active participants in eliminating tumor cells, but as the stroma and cancer cells co-evolve an “equilibrium” phase emerges in which the neoplastic cells acquire resistance to immune editing and elimination (56
). Finally, these “adapted” tumor cells expand and escape from immunologic pruning and create a new condition where macrophages may facilitate tumor growth. It is not clear whether these phases exist for microglia-glioma cell interactions, but if so it will be important to identify specific subpopulations of microglia with unique properties germane to a given period of glioma development and maintenance.
We also showed that Nf1
heterozygosity creates spatially- and temporally-restricted differences in microglia abundance relevant to optic glioma evolution. The fact that the numbers of Iba1-positive microglia increase in Nf1+/-
mice in the optic nerve during a defined window of development suggests that this specific microglial population might be uniquely susceptible to the effects of reduced Nf1
expression during this period of optic nerve maturation. We hypothesize that the accumulation of Nf1+/-
microglia in the optic nerve at 5 to 6 weeks of age reflects a defect in microglia homing in response to chemokines that instruct microglia dispersal. It is unlikely that Nf1+/-
optic nerve microglia have impaired migratory abilities as we have previously shown that Nf1+/-
microglia have increased motility in Boyden chamber assays in vitro (20
). Rather, we propose that Nf1+/-
microglia are defective in directed migration.
One of the major determinants controlling microglia homing is CX3CR1 (58
), which was reduced in the Nf1+/-
optic nerve. Here, we showed that reduced Cx3cr1
expression in CX3CR1-GFP heterozygous knockout mice resulted in a similar increase in optic nerve microglia at 6 weeks of age. These results are consistent with previous reports demonstrating that Cx3cr1
-deficient mice exhibit microglia accumulation in the retina (47
). Together, these findings suggest a model in which microglia accumulation reflects the impact of Nf1
heterozygosity on the developmental pattern of microglia migration in the optic nerve, thus delaying microglia dispersal and resulting in the presence of microglia and microglia-produced signals during a time when Nf1-/-
glial progenitors are most susceptible to expansion by stromal elements.
Previous studies have demonstrated the presence of CD11b-, Iba1-, CX3CR1-positive microglia in human gliomas, leading to the hypothesis that this cell population might be important for glioma growth (61
). Moreover, polymorphisms in the human CX3CR1
gene are linked to increased survival of patients with high-grade glioma, and patients with the common “improved survival” V249I polymorphism also had reduced numbers of tumor-associated microglia (62
). These findings raise the intriguing possibility that neurofibromin regulation of CX3CR1 expression might control microglia function in a specific fashion to modulate neoplastic glial cell growth in both the evolving and established optic glioma. Collectively, our new observations suggest a mechanistic model of stroma-tumor cell interaction relevant to optic gliomagenesis and maintenance that envisions Nf1
heterozygosity as an essential event that alters microglia abundance during optic nerve development. Future studies aimed at defining the function of microglia during gliomagenesis and glioma maintenance will be required to begin to develop treatments that target the relevant microenvironmental cell types in this common pediatric brain tumor.