STAT3 inhibition reduces macrophage number and tumor growth in neurofibroma

Jonathan S Fletcher, Mitchell G Springer, Kwangmin Choi, Edwin Jousma, Tilat A Rizvi, Eva Dombi, Mi-Ok Kim, Jianqiang Wu, Nancy Ratner, Jonathan S Fletcher, Mitchell G Springer, Kwangmin Choi, Edwin Jousma, Tilat A Rizvi, Eva Dombi, Mi-Ok Kim, Jianqiang Wu, Nancy Ratner

Abstract

Plexiform neurofibroma, a benign peripheral nerve tumor, is associated with the biallelic loss of function of the NF1 tumor suppressor in Schwann cells. Here, we show that FLLL32, a small molecule inhibitor of JAK2/STAT3 signaling, reduces neurofibroma growth in mice with conditional, biallelic deletion of Nf1 in the Schwann cell lineage. FLLL32 treatment or Stat3 deletion in tumor cells reduced inflammatory cytokine expression and tumor macrophage numbers in neurofibroma. Although STAT3 inhibition downregulated the chemokines CCL2 and CCL12, which can signal through CCR2 to recruit macrophages to peripheral nerves, deletion of Ccr2 did not improve survival or reduce macrophage numbers in neurofibroma-bearing mice. Interestingly, Iba1+; F4/80+;CD11b+ macrophages accounted for ~20-40% of proliferating cells in untreated tumors. FLLL32 suppressed macrophage proliferation, implicating STAT3-dependent, local proliferation in neurofibroma macrophage accumulation, and decreased Schwann cell proliferation and increased Schwann cell death. The functions of STAT3 signaling in neurofibroma Schwann cells and macrophages, and its relevance as a therapeutic target in neurofibroma, merit further investigation.

Conflict of interest statement

Competing Interests

The authors have no relevant competing interests to disclose.

Figures

Figure 1.
Figure 1.
JAK2/STAT3 inhibition in Dhh-Cre;Nf1fl/fl mice treated with FLLL32. (a) Western blotting demonstrates reduced (P-Y705, Cell Signaling #9145) STAT3 activation in neurofibroma lysates from mice treated for 5 days with 200 mg/kg/day FLLL32 (n = 4 per group). (b) Global tumor burden as measured by volumetric MRI was reduced in neurofibroma-bearing mice after 60 day FLLL32 treatment relative to in-group (grey bars) and historic (white bars) vehicle controls (p < 0.05, random effects model analysis on log transformed tumor volume data). (c) Activated (P-Y705, Cell Signaling #9145) STAT3 signal was present in neurofibroma tissue sections from Dhh-Cre;Nf1fl/fl vehicle control mice, but absent in FLLL32-responders and weak in FLLL32-non-responders treated for 60 days with FLLL32. (d) Proliferation, measured as Ki-67+ (Cell Signaling, 12202) cells per high-powered field (HPF), was reduced (** p <0.01, unpaired t-test, n = 4 control, n = 6 FLLL32 responders, n = 8 FLLL32 non-responders) in neurofibroma tissue from FLLL32 responders relative to vehicle controls. (e) Tumor macrophages, measured as Iba-1+ (019-19741, Wako) cells per HPF, were reduced in both FLLL32 responder and non-responder neurofibroma tissue relative to vehicle controls (*** p <0.001, unpaired t-test, n = 3 control, n = 5 FLLL32 responders, n = 7 FLLL32 non-responders). (f) Differences in the number of tumor mast cells, identified by toluidine blue staining, were not observed between vehicle and FLLL32-treated neurofibroma tissue (n = 4 control, n = 6 FLLL32 responders, n = 10 FLLL32 non-responders).
Figure 2.
Figure 2.
STAT3 signaling stimulates the production of macrophage attractants by neurofibroma Schwann cells. (a) Single-end RNA-sequencing was performed on vehicle (n = 11) and FLLL32-treated (n = 10) neurofibromas from Dhh-Cre;Nf1fl/fl mice and Dhh-Cre;Nf1fl/fl;Stat3fl/fl (n = 4) neurofibromas. Reads were trimmed and aligned to UCSC mm10 genome using TopHat. Gene-level read counts were computed using FeatureCounts. Gene expression was normalized by the trimmed mean of M-values method implemented in edgeR. (a) Signaling pathway impact analysis identified a single pathway (#04060; cytokine-cytokine receptor interaction) satisfying a global pathway significance p-value filter (pNDE=6.850337e-05, pPERT=0.191, and pGFDR=0.017) decreased by FLLL32 treatment. (b) Expression of the CCR2 ligand Ccl12 was decreased (p < 0.05, ANOVA with Tukey’s HSD) in FLLL32-treated and Dhh-Cre;Nf1fl/fl;Stat3fl/fl neurofibromas relative to vehicle, a trend towards decreased Ccl2 was also observed. (c) Vehicle samples were subgrouped by their rate-of-growth (Fast-growth:>35mm3 over 60 days, Slow-growth:<5mm3 over 60 days). FLLL32 treated samples were segregated by response to therapy (responders: tumor shrinkage during 60 day FLLL32 treatment, non-responders: continued growth during the treatment period). Both Ccl2 and Ccl12 were increased in fast-growing neurofibromas relative to slow-growing, and were decreased in responders and Dhh-Cre;Nf1fl/fl;Stat3fl/fl relative to vehicle treatment and non-responders (p < 0.05, ANOVA with Tukey’s HSD). (d) Analysis of CCL2 and CCL13 (Ccl12 human orthology) in a previously published human neurofibroma microarray dataset. CCL2 expression is significantly increased in human plexiform neurofibroma (pNF) and plexiform neurofibroma SCs (pNFSCs) relative to normal human SCs (NHSCs) and nerve (p < 0.05, ANOVA with Tukey’s HSD). Relative CCL13 expression was low in NHSCs and pNFSCs, and similar in pNF and nerve (p < 0.05, ANOVA with Tukey’s HSD).
Figure 3.
Figure 3.
Effects of Ccr2 deletion on the survival of Dhh-Cre;Nf1fl/fl neurofibroma-bearing mice and the accumulation of neurofibroma macrophages. (a) Proposed interaction between Schwann cells and hematopoietic monocytes/macrophages. FLLL32 reduces tumor macrophages by decreasing the pSTAT3 dependent expression of macrophage recruiting chemokines, especially Ccl2. (b) Loss of Ccr2 did not alter survival in neurofibroma-bearing mice (log-rank test, n= 11 Dhh-Cre;Nf1fl/fl, n = 10 Dhh-Cre;Nf1fl/fl;Ccr2−/−, n = 5 Dhh-Cre;Nf1fl/fl;Ccr2+/−). (c) Iba-1+ macrophages were quantified in frozen neurofibroma sections from Dhh-Cre;Nf1fl/fl and Dhh-Cre;Nf1flox/flox;Ccr2−/− survival mice,. Nuclei were counterstained with DAPI. Neurofibroma macrophage accumulation was not affected by loss of Ccr2 (t-test, n = 4 each group).
Figure 4.
Figure 4.
(a) Colorimetric double labeling for pSTAT3 (DAB, brown) demonstrates pSTAT3 expression in (VIP, purple) macrophages (Iba-1+) and Schwann cells (Sox10+). (b) Quantification of pSTAT3 expression in macrophages and Schwann cells. (c) Co-staining of EdU stained neurofibroma sections for macrophages (F4/80, BioRad, MCA497) and Schwann cells (Sox10, Santa Cruz, sc-17342) demonstrates that these cell populations proliferate in neurofibroma. FLLL32 decreased both macrophage and non-macrophage proliferation in neurofibroma relative to vehicle (**p <0.01, two-way ANOVA with post-hoc Sidak’s multiple comparisons test, n = 5 each group). (d) Representative images of Sox10, F4/80, EdU triple staining with DAPI counterstain in FLLL32 and vehicle-treated neurofibromas. The rightward pointing white arrowheads indicate a pair of proliferating macrophages and the leftward pointing arrowhead a proliferating Schwann cell in a vehicle-treated neurofibroma. A rare F4/80−;Sox10− proliferating is shown in an FLLL32-treated neurofibroma. (e) Frozen sections from 10-day FLLL32-treated and vehicle neurofibroma were stained for macrophage (F4/80) and apoptotic cell (cleaved caspase 3, Abcam, ab49822) markers and counterstained with DAPI. Cleaved caspase 3+ cells were rare in both vehicle and FLLL32 treated neurofibromas. These cells frequently had extensive contact with neighboring macrophages. FLLL32 treatment modestly increased apoptosis in non-macrophage cells in neurofibroma (*p <0.05, two-way ANOVA with post-hoc Sidak’s MCT, n = 5 each group). (f) Diagram representing possible mechanisms by which STAT3 signaling regulates proliferation and macrophage accumulation in neurofibromas: by regulating the SC expression of chemokines that recruit hematopoietic macrophages, and by directly or indirectly (through autocrine and paracrine secreted factors) that drive proliferation in neurofibroma SCs and resident macrophages.

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