Fig 1: Benign and malignant GCTs respond differently to the TME.A ELISA for SEMA4D on the supernatant of human monocytes and small/large osteoclasts after 9 days of differentiation; 3 osteoclast preparations. B Representative images of osteoclasts (TRAP-positive cells) used in (A). C Expression of SEMA4D and SCUBE3 by qPCR in GCTs (n = 32) and osteoblastic osteosarcoma (OS) samples with variable numbers of osteoclasts (n = 20). The GCT and OS samples represent a selection of those presented in Fig. 2D; levels of SCUBE3 and SEMA4D are inversely related reflecting that osteoclast-poor bone-forming OSs have high levels of SCUBE3 and low levels of SEMA4D whereas the opposite effect is observed in osteoclast-rich OSs. D Proliferation of hFOB transfectants in the presence of rSEMA4D, IgG control or mineralisation medium only (none) by Presto Blue assay after 7 days of proliferation at 34 °C; 2 experiments, 4 replicates each experiment. E Osteoimage assay of hFOB transfectants on day 6 of differentiation; 2 experiments, 2 replicates each experiment. F Proliferation of hTERT-hFOB transfectants by Presto Blue assay after 7 days of proliferation at 34 °C; 2 experiments, 4 replicates each experiment. G Osteoimage assay of hTERT-hFOBs on day 6 of differentiation; 2 experiments, 4 replicates each experiment. H Malignant GCT treated with denosumab: axial CT at T4-T5 vertebral level pre- and 3 months post-denosumab. In contrast to a conventional GCT, this tumour has grown (108–152 mm) (dotted outline) and mineralisation has not been induced by denosumab. Asterisk, vertebral body. I Proposed schema for GCT evolution. H3.3G34W-mutant osteoprogenitors express reduced levels of SCUBE3 resulting in increased formation of large osteoclasts, which secrete high levels of SEMA4D that block differentiation and promote proliferation of H3.3G34W-osteoprogenitors. The transition from benign to malignant GCT requires acquisition of at least one additional genomic alteration and malignant cells display cell-autonomous growth. Data are mean ± SD. A, D-G: 1-way ANOVA.