Novel CSF1R-positive tenosynovial giant cell tumor cell lines and their pexidartinib (PLX3397) and sotuletinib (BLZ945)-induced apoptosis

Abstract

Tenosynovial giant cell tumor (TGCT) is a mesenchymal tumor derived from the synovium of the tendon sheath and joints, most frequently in the large joints. The standard of care for TGCTs is surgical resection. A new targeting approach for treating TGCTs has emerged from studies on the role of the CSF1/CSF1 receptor (CSF1R) in controlling cell survival and proliferation during the pathogenesis of TGCTs. We established four novel cell lines isolated from the primary tumor tissues of patients with TGCTs. The cell lines were designated Si-TGCT-1, Si-TGCT-2, Si-TGCT-3, and Si-TGCT-4, and the TGCT cells were characterized by CSF1R and CD68. These TGCT cells were then checked for cell proliferation using an MTT assay and three-dimensional spheroid. The responses to pexidartinib (PLX3397) and sotuletinib (BLZ945) were evaluated by two-dimensional MTT assays. All cells were positive for α‑smooth muscle actin (α‑SMA), fibroblast activation protein (FAP), CSF1R, and CD68. Except for Si-TGCT-4, all TGCT cells had high CSF1R expressions. The cells exhibited continuous growth as three-dimensional spheroids formed. Treatment with pexidartinib and sotuletinib inhibited TGCT cell growth and induced cell apoptosis correlated with the CSF1R level. Only Si-TGCT-4 cells demonstrated resistance to the drugs. In addition, the BAX/BCL-2 ratio increased in cells treated with pexidartinib and sotuletinib. With the four novel TGCT cell lines, we have an excellent model for further in vitro and in vivo studies.

Keywords: CSF1R; Pexidartinib; Pigmented villonodular synovitis; Sotuletinib; Tenosynovial giant cell tumor.

Fig. 1

Clinical imaging and pathological diagnoses of 4 patients with TGCT. A–D Magnetic resonance imaging revealed 2 localized-type TGCTs of the knee and ankle (A, B) and 2 diffused-type TGCTs of the hip and knee (C, D). Lesions exhibited areas of low T1-weighted signals with blooming artifacts on gradient echo. E–H Hematoxylin and eosin staining of TGCTs showing mononuclear stromal cells with stromal fibrosis, including the formation of a hyalinized collagen matrix and a small area of multinucleated osteoclast-like giant cells

Fig. 2

Morphology of the established TGCT cell lines: A Si-TGCT-1 (passage 32), B Si-TGCT-2 (passage 29), C Si-TGCT-3 (passage 23), and D Si-TGCT-4 (passage 18). Typical morphology of stable culture cells under a phase-contrast light microscopy (original magnification =  × 100; scale bars = 100 μm). The growth curves were analyzed using the MTS assay at 24, 48, 72, and 96 h normalized with time 0 h, quantified by measuring the absorbance at 490 nm. Statistical significances were set at *P < 0.05; **P < 0.01, compared with a 1% fetal bovine serum culture condition

Fig. 3

Biological marker detection in the in‑house TGCT cells (Si-TGCT-1, Si-TGCT-2, Si-TGCT-3, and Si-TGCT-4). A Immunofluorescence staining, consisting of PanCK (red fluorescence), CK-19 (green fluorescence), α-SMA (red fluorescence), FAP (green fluorescence), and CSF-1R (green fluorescence). Staining with Hoechst33342 (blue fluorescence) was conducted to visualize chromatin; images were captured at × 630 original magnification; scale bars = 50 μm. (Si-TGCT-1: passage 29, Si-TGCT-2: passage 25, Si-TGCT-3: passage 22 and Si-TGCT-4: passage 15). B CSF-1R expression by Western blot assay. β‑actin was used as the loading control. C Densitometry data of CSF-1R/β‑actin ratio from 3 separate experiments (expressed as mean ± standard deviation) is shown in the histograms. Statistical significances were set at *P < 0.05; ***P < 0.001, compared with negative-control cells, MDA-MB-231. (Si-TGCT-1: passage 12, Si-TGCT-2: passage 4, Si-TGCT-3: passage 16, and Si-TGCT-4: passage 11). D, E Expression of CD68 positive cells detected by flow cytometry. The percentages of positive cells for the anti-CD68 were compared with an isotype control and are represented in the graph as mean ± SD (isotype control, in black; specific antibodies, in red). *P < 0.05; **P < 0.01, compared with negative-control cells, monocyte isolated from whole blood. (Si-TGCT-1: passage 35, Si-TGCT-2: passage 32, Si-TGCT-3: passage 26, and Si-TGCT-4: passage 21)

Fig. 4

Proliferation ability is shown by 3‑D spheroids on days 2, 4, 6, 8, and 10. The images were captured under light microscopy at × 100 magnification; scale bars, 100 μm. Statistical significances were set at *P < 0.05; **P < 0.01, compared with day 2, normalized with time day 0 of culture time. (Si-TGCT-1: passage 29, Si-TGCT-2: passage 25, Si-TGCT-3: passage 22, and Si-TGCT-4: passage 15)

Fig. 5

The treatment of pexidartinib (PLX3397) induced cell death in TGCT cells. A Cytotoxicity analysis of pexidartinib treatment. Pexidartinib induced cell death in TGCT cells was performed by MTS assay at 0, 0.2, 2, 20, and 200 µM for 96 h. Quantitative results of MTS staining were performed in triplicate; data represented by mean ± SD. B–F TGCT cell pellets were subjected to check the expression of BAX and BCL‑2 in pexidartinib treatment at 0, 2, 20, and 200 µM for 96 h by Western blot analysis. β‑actin was used as the protein loading control. The ratio of BAX/BCL‑2 was analyzed and reported from the relative band intensity of the Western blotting. *P < 0.05; **P < 0.01 and ***P < 0.001 compared with the untreated control (0 µM). (Si-TGCT-1: passage 10, Si-TGCT-2: passage 26, Si-TGCT-3: passage 20, and Si-TGCT-4: passage 15)

Fig. 6

Sotuletinib (BLZ945) induced cell death in TGCT cells. A Cytotoxicity analysis of sotuletinib  treatment was performed by MTS assay at 0, 0.1, 1, 10, and 100 µM for 96 h. B–F TGCT cell pellets treated with sotuletinib at concentrations 0, 50, 250, and 500 µM for 96 h were subjected to check the expression of BAX and BCL‑2 by Western blot analysis. β‑actin was used as the protein loading control. The ratio of BAX/BCL‑2 was analyzed and reported from the relative band intensity of the Western blotting. *P < 0.05; **P < 0.01 and ***P < 0.001 compared with the untreated control (0 µM). Si-TGCT-1: passage 10, Si-TGCT-2: passage 26, Si-TGCT-3: passage 20, and Si-TGCT-4: passage 15)