Neurofibroma-associated macrophages play roles in tumor growth and response to pharmacological inhibition

Abstract

Neurofibromatosis type 1 (NF1) is a common genetic disease that predisposes 30-50 % of affected individuals to develop plexiform neurofibromas. We found that macrophage infiltration of both mouse and human neurofibromas correlates with disease progression. Macrophages accounted for almost half of neurofibroma cells, leading us to hypothesize that nerve macrophages are inflammatory effectors in neurofibroma development and/or growth. We tested the effects of PLX3397, a dual kit/fms kinase inhibitor that blocks macrophage infiltration, in the Dhh-Cre; Nf1(flox/flox) mouse model of GEM grade I neurofibroma. In mice aged 1-4 months, prior to development of nerve pathology and neurofibroma formation, PLX3397 did not impair tumor initiation and increased tumor volume compared to controls. However, in mice aged 7-9 months, after tumor establishment, a subset of mice demonstrating the largest reductions in macrophages after PLX3397 exhibited cell death and tumor volume regression. Macrophages are likely to provide an initial line of defense against developing tumors. Once tumors are established, they become tumor permissive. Macrophage depletion may result in impaired tumor maintenance and represent a therapeutic strategy for neurofibroma therapy.

Fig. 1

Iba1+ macrophages in nerve and neurofibroma. a Staining of paraffin sections with anti-Iba1 (brown) to mark macrophages. GEM genetically engineered mouse model. b Quantification of Iba1+ macrophages per high-powered field (hfp) (*p < 0.001). Averages from individual mice are shown as individual dots. c Staining of Dhh-Cre; Nf1^flox/flox sciatic nerve sections with anti-Iba1 to mark macrophages (red) and with anti-GFP to mark Schwann cells (green). d M2 macrophages are rare in Nf1− deficient nerves and tumors. Iba1 (brown) and CD163 (purple) in paraffin sections. Green arrowheads show rare double-labeled cells. In all other cases, cells are single labeled. e Staining of human plexiform neurofibroma (PNF) and malignant peripheral nerve sheath tumor (MPNST) paraffin sections with anti-Iba1 (brown) to mark macrophages. f Average numbers of Iba1+ cells in human neurofibroma and MPNST. g FACS shows populations of CD11b+ cells (macrophages) isolated from human neurofibromas after in vitro culture; table presents quantification. Scale bar in Fig. 1a 50 m

Fig. 2

Pharmacological intervention prior to neurofibroma formation. a Plasma concentration of PLX3397 in Dhh-Cre; Nf1^flox/flox mice over time. b Experimental design. c Fur pigmentation loss in mice receiving PLX3397 (lower panel) and not in control mice (upper panel). d MRI shows increase in volume of neurofibromas treated with PLX3397. Images are from representative tumor-bearing mice given vehicle control (left) and PLX3397 (right). e Volumetric measurement. f PLX3397 treatment induced mast cell and macrophage depletion in neurofibromas. Toluidine blue staining (red arrows) indicates detection of mast cells and brown staining indicates detection of Iba1+ cells in paraffin sections from control and PLX3397-treated Dhh-Cre; Nf1^flox/flox mouse neurofibromas. g Remak bundle disruption was not prevented by treatment with PLX3397. Electron microscope photographs (×12,000) from sciatic nerve. h Graphs show average cell counts for Iba1+ cells (*p < 0.001), and toluidine blue cells (*p < 0.01) per high-powered field in control (n = 6) and PLX3397 (n = 6) treated neurofibromas

Fig. 3

PLX3397 effect on neurofibroma growth. a Experimental design, b Waterfall plots of tumor volume for control and PLX3397-treated Dhh-Cre; Nf1^flox/flox mice showed a subset of with decrease neurofibroma volume (negative values). Each bar shows data from a single mouse. The y axis shows neurofibroma tumor volume change from 7 to 9 months of age quantified by measurements of MRI scans, so that bars below the x axis indicate that tumors regressed over the treatment period. c Western blots showed that PLX3397 inhibited p-Kit and total Kit protein expression in Dhh-Cre; Nf1^flox/flox mouse neurofibromas at the end of treatment. β-actin serves as a loading control. d Quantification of numbers of toluidine blue-positive mast cells per high-powered field (hfp) (*p = 0.011). At least 10 fields were counted per section; averages from individual mice are shown as individual dots. e Tumor pharmacokinetics. Responding (Resp) and non-responding (NR) tumors contained >30 ng/PLX3397/g tissue. Dotted line shows efficacious concentration

Fig. 4

Macrophage activation and response to therapy. a Brown staining indicates Iba1 or F4/80 immunoreactivity in paraffin sections from Dhh-Cre; Nf1^flox/flox neurofibromas treated with vehicle of control or responder (Resp) and non-responder (NR) PLX3397-treated neurofibromas. b Assessment of macrophage content in neurofibromas after treatment with PLX3397 for 60 days showed a significant (*p < 0.001) reduction in Iba1+ cells in the Responder PLX3397-treated Dhh-Cre; Nf1^flox/flox mice. c Assessment of cell death in neurofibromas by quantification of Tunel+ cells. d Tunel staining (green) of neurofibroma cryostat sections in Dhh-Cre; Nf1^flox/flox treated mice. Blue are DAPI-stained nuclei. e Cell death only in neurofibromas taken from Dhh-Cre; Nf1^flox/flox mice with response to therapy (Resp) versus non-responders (NR). f Assessment of proliferation in neurofibromas by quantification of Ki67+ cells. g PLX3397 induces apoptosis in Nf1− deficient Schwann cells. Double staining of CNPase (purple) and cleaved caspase-3 (brown) in tissue sections from Dhh-Cre; Nf1^flox/flox treated mice. h Assessment of Iba1+ macrophage content in neurofibromas after treatment with minocycline. i Cell death was quantified by Tunel+ staining in Dhh-Cre; Nf1^flox/flox neurofibroma after exposure to minocycline