Nerve growth factor promote VCAM-1-dependent monocyte adhesion and M2 polarization in osteosarcoma microenvironment: Implications for larotrectinib therapy

Abstract

Osteosarcoma is the most prevalent form of primary malignant bone tumor, primarily affecting children and adolescents. The nerve growth factors (NGF) referred to as neurotrophins have been associated with cancer-induced bone pain; however, the role of NGF in osteosarcoma has yet to be elucidated. In osteosarcoma samples from the Genomic Data Commons data portal, we detected higher levels of NGF and M2 macrophage markers, but not M1 macrophage markers. In cellular experiments, NGF-stimulated osteosarcoma conditional medium was shown to facilitate macrophage polarization from the M0 to the M2 phenotype. NGF also enhanced VCAM-1-dependent monocyte adhesion within the osteosarcoma microenvironment by down-regulating miR-513c-5p levels through the FAK and c-Src cascades. In in vivo xenograft models, the overexpression of NGF was shown to enhance tumor growth, while the oral administration of the TrK inhibitor larotrectinib markedly antagonized NGF-promoted M2 macrophage expression and tumor progression. These results suggest that larotrectinib could potentially be used as a therapeutic agent aimed at mitigating NGF-mediated osteosarcoma progression.

Keywords: M2 macrophage; NGF; larotrectinib; miR-513c-5p; osteosarcoma.

Figure 1

NGF facilitates the polarization of macrophages to the M2 phenotype in the osteosarcoma microenvironment. (A&B) mRNA expression of macrophage and neurotrophin factors in osteosarcoma tissue based on analysis of samples from the GDC data portal; (C) Schematic of Macrophage Differentiation and Polarization; (D) qPCR analysis of THP-1 cells, following incubation with PMA for 24 h; (E) qPCR analysis showing mRNA expression after treating osteosarcoma cells with NGF for 24 h and then applying conditioned medium to M0 macrophage; (F&G) Fluorescence microscope images of THP-1 cells adhered to osteosarcoma cells, following incubation with NGF for 24 h. All experiments were repeated at least three times. * p < 0.05 compared with the control group.

Figure 2

NGF enhances VCAM-1 generation and monocyte adhesion to osteosarcoma. (A) qPCR results indicating mRNA expression after treating osteosarcoma cells with NGF for 24 h; (B-D) Western blot and qPCR results indicating VCAM-1 expression after treating osteosarcoma cells with NGF for 24 h; (E&F) Western blot analysis indicating VCAM-1 expression following transfection of osteosarcoma cells with VCAM-1 siRNA; (G&H) THP-1 adhesion in osteosarcomas transfected with VCAM-1 siRNA and then stimulated with NGF for 24 h. All experiments were repeated at least three times. * p < 0.05 compared with the control group; # p < 0.05 compared with the NGF-treated group.

Figure 3

NGF increases VCAM-1 production and monocyte adhesion to osteosarcomas via FAK signaling. (A&B) Western blot analysis showing FAK phosphorylation in osteosarcomas stimulated with NGF; (C-E) THP-1 adhesion and VCAM-1 expression in osteosarcomas incubated with FAK inhibitor or transfected with FAK siRNA and then stimulated with NGF for 24 h; (F) The supernatant collected from osteosarcoma cells to THP-1 cells affected the expression of M2 macrophage marker CD206 mRNA expression; (G&H) Western blot analysis showing FAK expression following transfection of osteosarcoma cells with FAK siRNA. All experiments were repeated at least three times. * p < 0.05 compared with the control group; # p < 0.05 compared with the NGF-treated group.

Figure 4

NGF increases VCAM-1 production and monocyte adhesion to osteosarcomas via c-Src signaling. (A&B) Western blot analysis indicating c-Src phosphorylation after stimulating osteosarcoma cells with NGF; (C-E) THP-1 adhesion and VCAM-1 expression in osteosarcoma cells incubated with PP2 or transfected with c-Src siRNA and then stimulated with NGF for 24 h; (F) The supernatant collected from osteosarcoma cells to THP-1 cells affected the expression of M2 macrophage marker CD206 mRNA expression; (G&H) Western blot analysis showing c-Src expression following transfection of osteosarcoma cells with c-Src siRNA; (I&J) Western blot analysis showing c-Src phosphorylation after incubating osteosarcoma cells with FAK inhibitor followed by stimulation with NGF. All experiments were repeated at least three times. * p < 0.05 compared with the control group; # p < 0.05 compared with the NGF-treated group.

Figure 5

NGF promotes VCAM-1 production and monocyte adhesion through the down-regulation of miR-513c-5p expression. (A) miRNAs predicted to bind with VCAM-1 3'-UTR based on analysis of two miRNA prediction databases; (B-D) qPCR results showing miRNA expression after treating osteosarcoma cells with NGF; (E) miR-513c-5p binding site in VCAM-1 3'-UTR; (F) Osteosarcoma cells were transfected with the VCAM-1 3'-UTR wild-type or mutant plasmid for 24 h, then stimulated with NGF for 24 h, and relative luciferase activity was measured; (G-I) THP-1 adhesion and VCAM-1 expression in osteosarcomas transfected with miR-513c-5p mimic and then stimulated with NGF for 24 h; (J) The supernatant collected from osteosarcoma cells to THP-1 cells affected the expression of M2 macrophage marker CD206 mRNA expression; (K&L) miRNA expression in osteosarcoma cells incubated with FAK inhibitor and PP2 or transfected with FAK and c-Src siRNA and then stimulated with NGF for 24 h. All experiments were repeated at least three times. * p < 0.05 compared with the control group; # p < 0.05 compared with the NGF-treated group.

Figure 6

Larotrectinib blocks NGF-induced monocyte adhesion to osteosarcomas. (A-C) Western blot and qPCR results showing NGF and VCAM-1 expression in 143B and NGF-overexpressing (143B/NGF) cells; (D&E) Adhesion of THP-1 cells to 143B and 143B/NGF cells; (F) MTT assay results indicating viability of 143B/NGF cells incubated with larotrectinib for 24 or 48 h; (G&H) THP-1 adhesion in osteosarcoma cells incubated with larotrectinib and then stimulated with NGF for 24 h. All experiments were repeated at least three times. * p < 0.05 compared with the control group; # p < 0.05 compared with the NGF-treated group.

Figure 7

Larotrectinib antagonizes NGF-induced osteosarcoma growth in vivo. (A-C) Osteosarcoma cells were subcutaneously injected into the right flanks of mice. The 143B/NGF+Larotrectinib group was administered larotrectinib (50 mg/kg) orally three times a week. The tumor size and weight were measured after sacrificing the mice at four weeks; (D-G) IHC analysis indicating protein expression levels of CD163, CD206, and VCAM-1 in tumors; (H&I) Correlation between CD163 or CD206 versus VCAM-1 expression levels. All experiments were repeated at least three times. * p < 0.05 compared with the 143B group; # p < 0.05 compared with the 143B/NGF group.

Figure 8

Schematic diagram illustrating the mechanism underlying the effects of NGF in an osteosarcoma microenvironment. NGF facilitates VCAM-1-dependent monocyte invasion into the osteosarcoma microenvironment and then enhances M2 macrophage polarization by inhibiting miR-513c-5p levels through the FAK and c-Src signaling cascades. Larotrectinib effectively suppresses NGF-induced cell growth.