Neurofibromatosis-1 heterozygosity increases microglia in a spatially and temporally restricted pattern relevant to mouse optic glioma formation and growth

Abstract

Whereas carcinogenesis requires the acquisition of driver mutations in progenitor cells, tumor growth and progression are heavily influenced by the local microenvironment. Previous studies from our laboratory have used Neurofibromatosis-1 (NF1) genetically engineered mice to characterize the role of stromal cells and signals to optic glioma formation and growth. Previously, we have shown that Nf1+/- microglia in the tumor microenvironment are critical cellular determinants of optic glioma proliferation. To define the role of microglia in tumor formation and maintenance further, we used CD11b-TK mice, in which resident brain microglia (CD11b+, CD68+, Iba1+, CD45low cells) can be ablated at specific times after ganciclovir administration. Ganciclovir-mediated microglia reduction reduced Nf1 optic glioma proliferation during both tumor maintenance and tumor development. We identified the developmental window during which microglia are increased in the Nf1+/- optic nerve and demonstrated that this accumulation reflected delayed microglia dispersion. The increase in microglia in the Nf1+/- optic nerve was associated with reduced expression of the chemokine receptor, CX3CR1, such that reduced Cx3cr1 expression in Cx3cr1-GFP heterozygous knockout mice led to a similar increase in optic nerve microglia. These results establish a critical role for microglia in the development and maintenance of Nf1 optic glioma.

Figure 1

Increased microglia in neurofibromatosis type 1 (NF1)-associated pilocytic astrocytomas. (A-C) Iba1 immunohistochemistry demonstrates increased percentages of microglia in anaplastic astrocytomas (AA) (p = 0.0236) and glioblastoma multiforme (GBM) tumors (p = 0.0377) vs. non-neoplastic brain (NB). (C) Sporadic pilocytic astrocytomas (SP-PA) have a greater percentage of Iba1+ cells compared to NB (p < 0.0001) (A, C), but the greatest percentage of microglia is seen in NF1-associated pilocytic astrocytomas (NF1-PA) (p < 0.0001 vs. NB) (p = 0.0008 vs. SP-PA).

Figure 2

Microglia in the mouse optic nerve at 6 weeks of age and in mouse optic gliomas at 3 months of age are mainly resident microglia. (A) Flow cytometry demonstrates that the majority of cells from 6-week-old wild type (WT) optic nerves (10 pooled optic nerves) are doubly positive for both CD11b and CD68. (B) The majority of these CD11b+ cells express low levels of CD45 (R1). (C) CD11b+ microglia in the 6-week-old optic nerve are also Iba1+. (D) Using double-labeling immunofluorescence, there are rare cells in the 6-week-old WT optic nerve that label with CD11b antibodies (green) alone (inset, arrowhead), whereas the vast majority (∼99%) of the microglia co-label with both CD11b and Iba1 antibodies (inset, arrows) (n = 5). (E) Iba1+ cells express low levels of CD45 (R1). (F) CD11b magnetic cell capture of microglia from 3-month-old Nf1^+/-GFAPCKO mouse optic gliomas (7 pooled optic nerves), followed by flow cytometry demonstrates that the CD11b+ cells are also almost exclusively CD45-low (R1).

Figure 2

Microglia in the mouse optic nerve at 6 weeks of age and in mouse optic gliomas at 3 months of age are mainly resident microglia. (A) Flow cytometry demonstrates that the majority of cells from 6-week-old wild type (WT) optic nerves (10 pooled optic nerves) are doubly positive for both CD11b and CD68. (B) The majority of these CD11b+ cells express low levels of CD45 (R1). (C) CD11b+ microglia in the 6-week-old optic nerve are also Iba1+. (D) Using double-labeling immunofluorescence, there are rare cells in the 6-week-old WT optic nerve that label with CD11b antibodies (green) alone (inset, arrowhead), whereas the vast majority (∼99%) of the microglia co-label with both CD11b and Iba1 antibodies (inset, arrows) (n = 5). (E) Iba1+ cells express low levels of CD45 (R1). (F) CD11b magnetic cell capture of microglia from 3-month-old Nf1^+/-GFAPCKO mouse optic gliomas (7 pooled optic nerves), followed by flow cytometry demonstrates that the CD11b+ cells are also almost exclusively CD45-low (R1).

Figure 2

Microglia in the mouse optic nerve at 6 weeks of age and in mouse optic gliomas at 3 months of age are mainly resident microglia. (A) Flow cytometry demonstrates that the majority of cells from 6-week-old wild type (WT) optic nerves (10 pooled optic nerves) are doubly positive for both CD11b and CD68. (B) The majority of these CD11b+ cells express low levels of CD45 (R1). (C) CD11b+ microglia in the 6-week-old optic nerve are also Iba1+. (D) Using double-labeling immunofluorescence, there are rare cells in the 6-week-old WT optic nerve that label with CD11b antibodies (green) alone (inset, arrowhead), whereas the vast majority (∼99%) of the microglia co-label with both CD11b and Iba1 antibodies (inset, arrows) (n = 5). (E) Iba1+ cells express low levels of CD45 (R1). (F) CD11b magnetic cell capture of microglia from 3-month-old Nf1^+/-GFAPCKO mouse optic gliomas (7 pooled optic nerves), followed by flow cytometry demonstrates that the CD11b+ cells are also almost exclusively CD45-low (R1).

Figure 3

Microglia ablation at 3 months of age reduces optic glioma proliferation. (A, B) Treatment of Nf1^+/-GFAPCKO-TK mice (n = 7) with ganciclovir (GCV) at 3 months of age reduces the number of Iba1-positive cells by 49% (p = 0.0280) and results in a 94% decrease in the number of Ki-67-positive cells in the optic nerve (p = 0.0118) vs. Nf1^+/-GFAPCKO-TK mice treated with vehicle (n = 7). (C) GCV treatment did not affect endothelial cell numbers, as assessed by CD34 immunohistochemistry (p = 0.2864).

Figure 3

Microglia ablation at 3 months of age reduces optic glioma proliferation. (A, B) Treatment of Nf1^+/-GFAPCKO-TK mice (n = 7) with ganciclovir (GCV) at 3 months of age reduces the number of Iba1-positive cells by 49% (p = 0.0280) and results in a 94% decrease in the number of Ki-67-positive cells in the optic nerve (p = 0.0118) vs. Nf1^+/-GFAPCKO-TK mice treated with vehicle (n = 7). (C) GCV treatment did not affect endothelial cell numbers, as assessed by CD34 immunohistochemistry (p = 0.2864).

Figure 4

Microglia ablation at 3 weeks of age reduces early optic glioma proliferation. (A, B) Nf1^+/-GFAPCKO-TK mice (n = 6) treated with ganciclovir (GCV) beginning at 3 weeks of age reduces the number of Iba1+ cells by 43% (p = 0.0012) and results in a 63% decrease in Ki-67-positive cells in the optic nerve vs. Nf1^+/-GFAPCKO-TK mice (n = 6) treated with vehicle (p = 0.0013). (C) GCV treatment did not affect endothelial cell numbers as assessed by CD34 immunohistochemistry (p = 0.5405).

Figure 4

Microglia ablation at 3 weeks of age reduces early optic glioma proliferation. (A, B) Nf1^+/-GFAPCKO-TK mice (n = 6) treated with ganciclovir (GCV) beginning at 3 weeks of age reduces the number of Iba1+ cells by 43% (p = 0.0012) and results in a 63% decrease in Ki-67-positive cells in the optic nerve vs. Nf1^+/-GFAPCKO-TK mice (n = 6) treated with vehicle (p = 0.0013). (C) GCV treatment did not affect endothelial cell numbers as assessed by CD34 immunohistochemistry (p = 0.5405).

Figure 5

Iba1+ cells are increased in Nf1^+/- optic nerve (ON) at 6 weeks of age, but not at 3 weeks or 3 months of age. (A, B) Equivalent numbers of Iba1-positive cells were found in the ON of Nf1^+/- (n = 5) (black bars) and wild type (WT) (gray bars) mice (n = 5) at 3 weeks of age (p = 0.9712). By 6 weeks of age, there was a 2-fold increase in the number of Iba1-positive microglia in the ONs of Nf1^+/- mice (n = 5) vs. WT littermates (n = 5) (p = 0.0002). By 3 months of age, Nf1^+/- (n = 4) and WT (n = 4) mice had equivalent numbers of Iba1-postive cells (p = 0.5155). (C, D) Nf1^+/- mice (n = 5) have similar numbers of Iba1-positive microglia in the brainstem (BS) and cortex (CTX) vs. WT (n = 5) at 3 weeks (BS, p = 0.7531) (CTX, p = 0.5248), 6 weeks (BS, p = 0.8260) (CTX, p = 0.0911) and 3 months of age (BS, p = 0.8138) (CTX, p = 0.6162).

Figure 5

Iba1+ cells are increased in Nf1^+/- optic nerve (ON) at 6 weeks of age, but not at 3 weeks or 3 months of age. (A, B) Equivalent numbers of Iba1-positive cells were found in the ON of Nf1^+/- (n = 5) (black bars) and wild type (WT) (gray bars) mice (n = 5) at 3 weeks of age (p = 0.9712). By 6 weeks of age, there was a 2-fold increase in the number of Iba1-positive microglia in the ONs of Nf1^+/- mice (n = 5) vs. WT littermates (n = 5) (p = 0.0002). By 3 months of age, Nf1^+/- (n = 4) and WT (n = 4) mice had equivalent numbers of Iba1-postive cells (p = 0.5155). (C, D) Nf1^+/- mice (n = 5) have similar numbers of Iba1-positive microglia in the brainstem (BS) and cortex (CTX) vs. WT (n = 5) at 3 weeks (BS, p = 0.7531) (CTX, p = 0.5248), 6 weeks (BS, p = 0.8260) (CTX, p = 0.0911) and 3 months of age (BS, p = 0.8138) (CTX, p = 0.6162).

Figure 6

Reduced CX3CR1 expression in the Nf1^+/- optic nerve (ON). (A) Real-time qPCR reveals no change in Cx3cl1 mRNA levels in ONs from Nf1^+/- (n = 5) (black bars) and wild type (WT) (n = 5) (gray bars) mice at 3 weeks (p = 0.1558), 6 weeks (p = 0.3478), or 3 months (p = 0.4788) of age. (B) qPCR reveals a >2-fold decrease in Cx3cr1 mRNA expression in the ONs of 6-week-old Nf1^+/- mice (n = 5) vs. WT controls (n = 5) (p = 0.0181). There is no difference in Cx3cr1 expression in the ONs from Nf1^+/- vs. WT mice at 3 weeks (p = 0.0855) or 3 months (p = 0.1413) of age. (C) There is a 2-fold increase in numbers of Iba1-positive microglia in ONs of NF^+/+Cx3cr1⁺/GFP mice (n = 6) vs. WT controls (n = 5) (p = 0.0042). Nf1^+/+-Cx3cr1+/GFP mice (n = 7) have greater numbers of microglia vs. WT (p < 0.0001) and Nf1^+/- mice (n = 5) (p < 0.0001).

Figure 6

Reduced CX3CR1 expression in the Nf1^+/- optic nerve (ON). (A) Real-time qPCR reveals no change in Cx3cl1 mRNA levels in ONs from Nf1^+/- (n = 5) (black bars) and wild type (WT) (n = 5) (gray bars) mice at 3 weeks (p = 0.1558), 6 weeks (p = 0.3478), or 3 months (p = 0.4788) of age. (B) qPCR reveals a >2-fold decrease in Cx3cr1 mRNA expression in the ONs of 6-week-old Nf1^+/- mice (n = 5) vs. WT controls (n = 5) (p = 0.0181). There is no difference in Cx3cr1 expression in the ONs from Nf1^+/- vs. WT mice at 3 weeks (p = 0.0855) or 3 months (p = 0.1413) of age. (C) There is a 2-fold increase in numbers of Iba1-positive microglia in ONs of NF^+/+Cx3cr1⁺/GFP mice (n = 6) vs. WT controls (n = 5) (p = 0.0042). Nf1^+/+-Cx3cr1+/GFP mice (n = 7) have greater numbers of microglia vs. WT (p < 0.0001) and Nf1^+/- mice (n = 5) (p < 0.0001).