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

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. Supplementary Information The online version contains supplementary material available at 10.1007/s13577-022-00823-0.


Introduction
Tenosynovial giant cell tumors (TGCTs), formerly termed pigmented villonodular synovitis, are neoplasms that develop in the synovium of joints, tendon sheaths, and bursae. TGCTs are present in two types. The predominant form is diffuse and encompasses the entire synovium, whereas the minor type is localized, involving only a portion of the synovium [1]. Typically, TGCTs are found in 20-50-year-old patients, with an approximately equal distribution between men and women [2]. However, the diffused form is more common among young women [3]. TGCT tissue comprises many cell types. They include fibroblast-like synovial cells, sideroblasts, foam cells, histiocyte-like cells, hemosiderinladen macrophages, and multinucleated giant cells [1,4]. In addition, a small proportion of TGCT cells make up a neoplastic clone that expresses colony-stimulating factor 1 (CSF1). Approximately one-third of TGCT cases have a t (1;2) translocation linking the COL6A3 gene on chromosome 2q35 and the CSF1 gene on chromosome 1p13 [5,6]. High levels of CSF1 expression in TGCTs result from this COL6A3-CSF1 fusion [6,7]. Consequently, the underlying cause of TGCTs can be targeted by inhibiting signaling between CSF1 and the CSF1 receptor (CSF1R) [8,9]. Research has shown that the monocyte-macrophage lineage marker CD68 stains synovial-lining cells; double staining revealed that CD68 is also expressed by TGCT cells that express CSF1 [10]. Other work found high CD68 expression levels in several tumor types, particularly TGCTs, compared with normal tissue samples [11].
The current standard treatment for TGCTs includes arthroscopic or open synovectomy. However, the diffused TGCT is more difficult to resect. It also has a 20% to 55% chance of local recurrence, resulting in joint destruction requiring joint replacement or amputation [3,12]. Besides surgery, the CSF1R inhibitor pexidartinib and the monoclonal antibody emactuzumab are used to treat TGCTs [13,14].
Pexidartinib (PLX3397) is a small-molecule tyrosine kinase inhibitor that targets CSF1R, KIT (KIT protooncogene, receptor tyrosine kinase), and FLT3 (FMS-like tyrosine kinase 3) harboring an internal tandem duplication (ITD) mutation [15,16]. Overexpression of the CSF1R ligand promotes cell proliferation and accumulation in the synovium [17]. In vitro studies revealed that the growth of osteosarcoma cell lines that depended on CSF1R and the receptor's ligand-induced autophosphorylation property was inhibited by pexidartinib [18]. Pexidartinib is the first drug approved by the United States Food and Drug Administration for treating adult patients with TGCTs who have severe morbidity or functional limitations that are not amenable to surgery [15]. A study showed that in 62% of patients taking pexidartinib for 38 month median follow-up, TGCTs shrank by 30% or more, resulting in pain relief and less stiffness [16,19]. However, a subset of patients did not respond to pexidartinib. Side effects including lightening of hair color, fatigue, and reversible hepatotoxicity meant the drug is not appropriate for all patients with TGCTs [20]. There is a need for effective and less harmful treatment for TGCTs. Sotuletinib (BLZ945) is a small-molecule inhibitor that inhibits CSF1R and is being tested in a clinical phase II trial (NCT04066244).
Having TGCT cell lines would facilitate studies of the pathological interactions between the cell components of TGCTs, leading to alternative treatment approaches. In the present study, we established and characterized novel TGCT cell lines, designated Si-TGCT-1-4, from surgically resected tumor tissues. To demonstrate the usefulness of these cells, we studied their proliferation and spheroid formation characteristics and examined their responses to two CSF1R inhibitors: pexidartinib and sotuletinib. The results revealed that all Si-TGCT-1-4 cells can be used in preclinical research and the last one, Si-TGCT-4, can be used especially on TGCT drug resistance.

Patient background
The research protocol was evaluated and approved by the Siriraj Institutional Review Board (Si894/2020). Written informed consent was obtained from participants before their enrollment between November 2020 and November 2021. The patients' demographic data is detailed in Table 1.

Cancer cell isolation and culture
TGCT cancer tissues were obtained from 4 patients who underwent surgery at Siriraj Hospital, Bangkok, Thailand. The samples were designated Si-TGCT-1, Si-TGCT-2, Si-TGCT-3, and Si-TGCT-4. A fresh TGCT tissue sample (1 × 1 × 1 cm 3 ) was isolated from TGCT tissue and surgically resected according to our previous guidelines [21]. Briefly, TGCT tissues were incubated in a 10X antibiotic mixture (1 U/ml penicillin G sodium and 1 mg/ml streptomycin; Thermo Fisher Scientific Inc.), diluted in DMEM/ F12. The obtained tissue was minced into 0.1 × 0.1 × 0.1 cm 3 sections and incubated with an enzyme cocktail mix (Miltenyi Biotec GmbH) for 1 h at 37 °C. The digested cells were passed through a 70 μm nylon filter (SPL Life Sciences) and cultured in DMEM/F-12 media (Gibco BRL) supplemented with 10 ng/ml of epidermal growth factor (EGF, PeproTech

Western blot analysis
Cell pellets were resuspended in a cell lysis buffer (Cell Signaling Technology Inc.). After centrifugation, the protein content of the supernatants was determined using a Bradford Protein Assay Kit (Bio-Rad Laboratories Srl.). Then, 60 mg of protein was prepared in a sample buffer containing 10% SDS, 1.0 M Tris-HCl pH 6.8, 8% glycerol, and 0.05% (w/v) bromophenol blue. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. The membranes were incubated overnight with the following primary antibodies: CSF1R (ab205921; Abcam); BAX (mouse anti-human BAX antibody; 610,983; Becton Dickinson Holdings Pte. Ltd.); BCL-2 (rabbit anti-human BCL-2 antibody; ab196495; Abcam); and β-actin (mouse anti-β-actin antibody; sc-47,778; Santa Cruz Biotechnology Inc.). Further incubation was undertaken with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h. The signals were visualized by ECL (Thermo Fisher Scientific Inc.) under Gel Document Syngene (Syngene), and the bands were quantified by ImageJ (version 1.48v; National Institutes of Health, Bethesda, MD, USA). The densitometric values of all protein bands were normalized to that of β-actin and quantified using ImageJ (version 1.52a).

CD68 detection by flow cytometry
TGCT cells (5 × 10 5 ) were harvested and blocked in 5% FBS in PBS 1X before incubation for 30 min with PEconjugated monoclonal antibody Y1/82A human CD68 (21,270,684; ImmunoTools GmbH) or the isotype control (PE-conjugated mouse IgG1 isotype control; 21,815,014; ImmunoTools GmbH) for 30 min at 4 °C. After 3 washes in 1X PBS buffer, the cells were detected by a CytoFLEX flow cytometer (Beckman Coulter Inc.) and analyzed using CytExpert software (version 2.1; Beckman Coulter Inc.). Monocytes isolated from peripheral blood mononuclear cells and MDA-MB-231 cells were used as positive control cells.

Cell proliferation
was used to assay cell viability, following the manufacturer's instructions. Si-TGCT-1-4 cells were plated in triplicate in 96-well plates at 5000 cells/200 μl. Twenty μl of MTS reagent was added to each well at 24, 48, 72, and 96 h. The wells were incubated in a humidified, 5%-CO 2 atmosphere for a minimum of 2 h. Absorbance at 490 nm was recorded.

Three-dimensional spheroid formation
In vitro spheroids were obtained. TGCT single-cell suspensions were generated from trypsinized monolayers, with 1000 cells supplemented with 2.5% cold Matrigel (BD Biosciences) in 200 μl of complete DMEM F/12 medium and seeded into pre-cooled, 96-well, ultra-low attachment plates (CLS7007; Costar/Corning Inc.). Centrifugation at 4 °C at 300 × g for 3 min was performed, and the cells were maintained at 37 °C in a humidified 5% CO 2 atmosphere. The spheroid proliferation rate and size were monitored for up to 10 days under an inverted-light microscope Olympus IX71 using Olympus CellSens standard software.

Statistical analysis
The values are represented as the mean ± standard deviation (SD) from 3 independent assays. All statistical calculations were performed using GraphPad Prism, version 7.04 (GraphPad Software Inc., La Jolla, CA, USA). The data from 2 groups were analyzed by paired Student's t-tests, and multiple groups were assessed by 1-way repeated-measure analysis of variance (ANOVA). The level of statistical significance was set at P < 0.05.

Cell authentication by STR profiling
DNA fingerprint was performed by fluorescent-based PCR technique using capillary electrophoresis at Human Genetic Laboratory, Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand. Twenty short tandem repeat (STR) loci plus the gender determining locus, Amelogenin, were amplified by six multiplex PCR and separated on ABI 3730XL Genetic Analyzer. The signals were then analyzed by the software GeneMapper [22].

Patient backgrounds
The Si-TGCT-1, -2, -3, and -4 cells were retrieved from 4 patients aged between 38 and 70 who had been treated surgically at Siriraj Hospital, Mahidol University (Table 1). Two cases involved diffuse-type TGCTs. The localized type of TGCT was Si-TGCT-1 in the posterior knee and Si-TGCT-2 in the ankle. All patients were treated by synovectomy alone. No adjuvant such as CSF1R inhibitor or radiation was administered. Magnetic resonance imaging revealed joint effusion, hemosiderin deposition, expansion of the synovium, and marginal bony erosion in all four patients (Fig. 1A-D). TGCT tissue showed low signal intensity on T1W and T2W with blooming artifacts on gradient-echo due to iron in hemosiderin. Gross pathology was observed in the operative field and showed proliferative villi extending from the synovium. Hematoxylin and eosin staining (Fig. 1E-H) and a low-power field revealed mononuclear stromal cells infiltrating the synovium. High vascularization of the villi line within plump synovium was evident in hemosiderin-stained multinucleated giant cells, with pigmented foam cells or lipid-laden histiocytes and high mitotic figures.

Characterization of TGCT cell lines
The Si-TGCT-1, -2, -3, and -4 cell lines were established from the primary tumor tissues of patients with TGCTs. Three months after the adherent cells were initiated, the cells were maintained in culture through over 30 passages over the following 2 years. Mycoplasma contamination was negative, as no mycoplasma-specific DNA was detected in the cellconditioned medium via quantitative real-time polymerase  (Table 2; Fig. 3D-E). Interestingly, CD68 was positive in all TGCT cells. However, Si-TGCT-4 cells had a significantly higher percentage of CD68 than monocyte negative control cells.
Si-TGCT-1-4 cells' ability to form spheroids was observed on low-attachment substrates (Fig. 4). The spheroid outline was less regular and appeared round. To calculate the sphere size, the diameters of at least 10 spheres were measured every second day; the average dimensions of the TGCT spheres are detailed in Table 2. On day 10, all TGCT cells formed larger spheres whose diameters varied from 0.61 × 10 7 to 1.88 × 10 7 μm 3 . However, colonospheres from all lines displayed similar morphology. No invasive behavior was observed. Si-TGCT-1-4 cells were immortalized and showed constant growth at their latest passage of 36, 33, 27, and 22, respectively. They were capable of invasion and spheroid formation from the early to the later passage. Tumorigenesis in nude or SCID mice had not been done in this study. Additional in vivo experiments using TGCT cell lines are needed for further study.

Cell authentication by STR profiling
Fingerprint confirmed novelty of all 4 cell lines compared to STR data from ATCC, DSMZ, JCRB, ECACC, GNE and RIKEN databases (Supplementary data 1). The TGCT fingerprint was identical to that of the white blood cells of the patient whose tissue was used to establish four lines.

Discussion
While complete synovectomy is a standard treatment for TGCTs, it is often challenging because of the high rate of local recurrence [3,12,23]. The United States Food and Drug Administration approved pexidartinib for use with patients with advanced diseases for whom surgical treatment was not feasible [24]. The effectiveness of pexidartinib, a CSF1R inhibitor, has been proven in TGCTs. However, the response rate evaluated by RECIST criteria or tumor volume score has ranged between 39 and 60% [24,25]. Therefore, a novel treatment is needed. Patient-derived cell lines facilitate discoveries in cancer biology and translational research. Only 2 TGCT cell lines are available from the public cell bank [26,27]. Considering the genetic diversity of TGCTs, the number of TGCT cell lines remains inadequate. We established four novel TGCT cell lines-Si-TGCT-1, -2, -3, and -4-derived from the primary tumors of patients with TGCTs. Regarding the backgrounds of the patients from whom the Si-TGCT-1-4 cell lines were sourced, two lines were derived from localized-type TGCTs (Si-TGCT-1 and -2) and two were sourced from diffused TGCTs (Si-TGCT-3, and -4). The original tumors were found in typical locations of TGCT: the knee, ankle, and hip joints. Given the patients' mean age of 48 (range, 38-70) years and their variety of TGCT types and localizations, the Si-TGCT-1-4 cell lines were derived from patients with clinical features dissimilar to those of patients used to source previously established TGCT cell lines.
The morphology of the Si-TGCT-1-4 cells was mainly spindle-and polygonal-shaped under culture conditions in both the two-dimensional and spheroid forms. The Si-TGCT-1-4 cells showed constant but slow growth. Neoplastic TGCT cells with CSF1 translocation are most likely to recruit CSF1R-expressing macrophages, which may induce the formation of multinucleated giant cells [28]. Regarding giant cell appearance during cultivation, we found that the morphology of Si-TGCT-1-4 cells was mainly spindleshaped. Osteoclast-like multinucleated giant cells were not found in the plate or spheroid of Si-TGCT-1-4, and the major component of the spheroid was the spindle. Since HE-stained tumor tissues of TGCT contain multinucleated cells, the Si-TGCT-1-4 cell lines are clonal cell lines of the spindle cell population. The Si-TGCT-1-4 cells also formed spheroids on a low attachment substrate. Thus, they can be used to examine the effects of complex architecture on drug sensitivity. Although, one cell (Si-TGCT-1), the localized type, showed slower two-dimensional-growth, the rest of TGCT cells, both localized and diffused type showed no differences in their two-dimensional-growth or spheroidgrowth abilities.
Translocations involving chromosome 1p13 are present in a majority of cases of TGCTs. However, only approximately 30% of cases, CSF1 is fused to COL6A3 (2q35) [29,30]. Overexpression of CSF1 occurs only in a minority of the TGCT cells, whereas most cells express CSF1R, not CSF1. [5,6]. Similar results were obtained in the Si-TGCT-1-4 cells in that CSF1R was highly expressed, whereas CSF1 was rarely found on immunofluorescence staining and Western blotting (Table 2; Fig. 3). All four cell lines did not show CSF1-COL6A3 fusion on the short tandem assay (STR), which may represent most of the TGCTs.
In our drug testing, pexidartinib reduced the proliferation of Si-TGCT-1-4 markedly better than sotuletinib for all cell lines, with sotuletinib's IC50 values being 5-33 times higher than those produced with pexidartinib. Notably, Si-TGCT-1, and -3 showed the highest sensitivity to pexidartinib by their CSF1R affinity. The BAX/BCL-2 ratio increased with pexidartinib and sotuletinib treatment. As a tyrosine kinase inhibitor, pexidartinib targets CSF1R, KIT, FLT3, and platelet-derived growth factor receptor-β. These are receptor tyrosine kinases that are involved in regulating critical processes within cells. Sotuletinib is a highly effective, selective, and brain-penetrating inhibitor of CSF-1R (c-Fms). Clinical trials of two tyrosine kinase inhibitors with activity against CSF1R, imatinib [31] and nilotinib [32], have shown fair response rates, as have the CSF1R monoclonal antibody emactuzumab [14,33]. Along with clinical studies, there is a need to identify more effective and less toxic drugs or drug combinations added on to pexidartinib to treat TGCTs based on in vitro studies.
In conclusion, we established novel TGCT cell lines, Si-TGCT-1-4, which exhibited continuous proliferation and spheroid formation. We identified CSF1R in each cell line, with different expression levels among cells. We also added the antitumor effects of pexidartinib and sotuletinib on TGCT cell lines, which worked according to how much CSF1R they had. The results show that Si-TGCT-1-4 cells have the potential to facilitate numerous advances in preclinical and basic research on TGCTs.
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