Contributions of inflammation and tumor microenvironment to neurofibroma tumorigenesis

Abstract

Neurofibromatosis type 1 associates with multiple neoplasms, and the Schwann cell tumor neurofibroma is the most prevalent. A hallmark feature of neurofibroma is mast cell infiltration, which is recruited by chemoattractant stem cell factor (SCF) and has been suggested to sustain neurofibroma tumorigenesis. In the present study, we use new, genetically engineered Scf mice to decipher the contributions of tumor-derived SCF and mast cells to neurofibroma development. We demonstrate that mast cell infiltration is dependent on SCF from tumor Schwann cells. However, removal of mast cells by depleting the main SCF source only slightly affects neurofibroma progression. Other inflammation signatures show that all neurofibromas are associated with high levels of macrophages regardless of Scf status. These findings suggest an active inflammation in neurofibromas and partly explain why mast cell removal alone is not sufficient to relieve tumor burden in this experimental neurofibroma model. Furthermore, we show that plexiform neurofibromas are highly associated with injury-prone spinal nerves that are close to flexible vertebras. In summary, our study details the role of inflammation in neurofibromagenesis. Our data indicate that prevention of inflammation and possibly also nerve injury at the observed tumor locations are therapeutic approaches for neurofibroma prophylaxis and that such treatment should be explored.

Keywords: Mouse models; Neurological disorders; Oncology; Tumor suppressors.

Figure 1. Scf expression in normal nervous tissues and neurofibroma tumors.

(A) Scf^gfp identified Scf expression in DRG. (B) Scf^gfp identified Scf expression in sciatic nerve. (C) Scf expression in S100β⁺ Schwann cells in DRG. (D) Scf expression in S100β⁺ Schwann cells in sciatic nerves. (E) Scf^gfp identified Scf expression in DRG with spontaneous plexiform neurofibroma. (F) GFP immunohistochemical staining identified Scf-expressing cells (by Scf^gfp) in plexiform neurofibroma tumor tissue. Scale bar, 500 μm in A, B, and E. Scale bar, 100 μm in C, D, and F.

Figure 2. Contributions of Nf1 heterozygosity and SCF to neurofibroma progression.

The speed of neurofibroma progression was determined by the survival of mice. (A) Survival comparison between neurofibroma-bearing (PlpCre^ERT2) and tumor-free (no Cre) mice. (B) Survival comparison between neurofibroma-bearing mice with germline homozygous Nf1 (Nf1^+/+) and heterozygous Nf1 (Nf1^+/–) status. (C) Survival comparison between neurofibroma-bearing mice with the following variable Scf status: (1) Scf^+/+, WT; (2) Scf^f/+, hemizygous deletion in Schwann cells; (3) Scf^+/gfp, hemizygous deletion in all cells; and (4) Scf^f/gfp, homozygous deletion in Schwann cells and hemizygous deletion in all other cells. The number of mice in each group ranged from 19–30 as labeled in each individual figure. The statistics were performed by Kaplan-Meier estimator with log-rank test. In this figure, the same survival curves from Nf1^f/– Scf ^f/gfp Plp-Cre^ERT2 and Nf1^f/f Scf ^f/gfp Plp-Cre^ERT2 double knockout mice were presented in A–C to compare the survival between different genotype cohorts.

Figure 3. Contributions of Nf1 heterozygosity and SCF to neurofibroma development.

(A) The structure of whole spinal cord from neurofibroma-nearing mice. X-gal staining marks Plp-lineage Schwann cells. Plexiform neurofibromas were highlighted as enlarged DRGs. A representative image in each group was selected from a 10- to 12-month-old mouse as shown in Supplemental Figures 4–11. (B) The histology of cervical plexiform neurofibromas. (C) S100β immunohistochemical staining for Schwann cells in the tumors and nontumor nerves. (D) Quantification of the severity of plexiform neurofibroma by a reference grading scale in the left panel. No statistically significant difference was identified by comparing any 2 groups. Cervical tumors in left panel are representative images for each level (0–5) selected from 116 whole spinal cords as shown in Supplemental Figures 4–11. For A–C, representative images were compared from mice at similar ages (10–12 months) in each group. Statistics were performed by 1-way ANOVA. Data are mean ± SEM. Scale bar, 1 cm in A. Scale bar, 100 μm in B and C.

Figure 4. Mast cells in the neurofibroma tumor microenvironment.

(A) Mast cells (arrows) in neurofibromas were identified by toluidine blue staining. Results were representative images compared with mice at similar ages (10–12 months) in each group. (B) Statistic analysis of mast cell density in neurofibroma. Statistics were performed by 1-way ANOVA. Significant differences were noted by asterisks: *P < 0.05, **P < 0.01, and ****P < 0.0001. Data are mean ± SEM. Scale bar, 100 μm.

Figure 5. Macrophages in the neurofibroma tumor microenvironment.

Iba1 (A) and iNOS (B) immunohistochemical staining for macrophages in the cervical plexiform neurofibroma tumor and nearby nontumor nerve regions. Note the density difference of positive cells in tumors and nerves. Representative images are shown from mice with similar ages (10–12 months) in each group (n = 3). Scale bars, 100 μm.

Figure 6. The prevalence of thoracic neurofibroma in different thoracic nerves.

The number and sizes of thoracic neurofibromas in different thoracic locations in different groups of neurofibroma-bearing mice. Figure represents the sizes and locations of each tumor with means. Note the high tumor prevalence in T5–T8 (n = 7–18 mice per group).

Figure 7. Contribution of tumor-derived SCF in SKP transplantation neurofibroma model.

(A) Scf^gfp reporter identified Scf expression in the SKP spheres. (B) Scf expression (determined by Scf^gfp) in the Nestin⁺ neural crestlike stem cells in the SKPs. (C) The strategy of generation of Nf1/Scf DKO SKPs and controls. (D) Illustration of SKP transplantation in sciatic nerves in nude mice for neurofibroma development. (E) The observation of sciatic nerves with LacZ staining (marked by dark blue color) after SKP transplantation. (F) The histology of sciatic nerves at low magnification (×40, upper panel) and high magnification (×400, lower panel). (G) Toluidine blue staining for mast cells. In E–G, results were representative from 5–9 tumors, as labeled. Scale bars, 100 μm in A, F, and G. Scale bar, 50 μm in B.

Figure 8. Hypothetical model of neurofibroma initiation and tumorigenesis.

Neurofibroma occurs more commonly at injury-prone nerves. Nerve injury induces inflammation as a mechanism of tissue repair. Nerve inflammation results in the infiltration of inflammatory cells and high concentrations of reactive oxygen species (ROS) and growth factors. NF1 mutant Schwann cells may interplay with the inflammatory microenvironment that contributes to neurofibroma tumorigenesis. Tumorous Schwann cells express chemokines and cytokines, such as SCF and CSF1, to recruit inflammatory cells, including mast cells and macrophages. This model may explain the presence of mast cells and macrophages in the neurofibroma tumor microenvironment, the high neurofibroma frequency in injury-prone nerves, and our observation that removal of mast cells alone is not sufficient to relieve tumor burden.