Monoclonal rabbit anti-H3G34W antibody (clone RM263) was purchased from RevMab Biosciences (San Francisco, CA, USA). Monoclonal rabbit anti-β-catenin antibody (#32572), anti-alkaline phosphatase, tissue non-specific antibody (#126820), goat anti-rabbit IgG H&L preadsorbed (Alexa Fluor® 488, #150081), and goat anti-mouse IgG H&L preadsorbed (Alexa Fluor® 594, #150120) were purchased from Abcam (Cambridge, UK). Monoclonal mouse anti-actin clone C4 (MAB1501) was purchased from Merck Millipore (Burlington, MA, USA). A selective inhibitor of geranylgeranyltransferase1, GGTI-286 (#22756), was purchased from Cayman Chemical (Ann Arbor, MI, USA), and a selective Rac1 (a Rho-family small GTPase) inhibitor, NSC23766, was purchased from Merck Millipore.
Establishment of primary culture and treatment
Some fresh GCTB tumor samples were obtained from surgeries and washed with warmed PBS. The samples were minced in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (HyClone Laboratories, Logan, UT, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin. Together with small pieces of chipped tissues, the cell suspension was transferred to culture dishes and cultured at 37°C in a humidified atmosphere of 5% CO2 and 95% air. GCTB-OCs were present only in the first passage, whereas primary GCTB-SCs (pGCTB-SCs) were further amplified. Upon reaching confluence, pGCTB-SCs were sub-cultured, and the third through sixth passages were used for subsequent experiments. To induce osteogenic differentiation, confluent cells were treated with osteogenic medium (OGM) containing Minimum Essential Medium (Thermo Fisher Scientific) supplemented with 10% FBS, 100 nM dexamethasone (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 100 μM ascorbic acid (FUJIFILM Wako), and 10 mM β-glycerophosphoric acid (NACALAI TESQUE, INC., Kyoto, Japan) for various periods.
Preparing cell brocks and immunocytochemistry of GCTSCs
To determine the purity of primary cultures, we prepared cell blocks and performed immunostaining with anti-H3G34W. Confluent suspensions of pGCTB-SCs were harvested, and cell pellets were prepared by centrifugation for 5 minutes at 1,500 rpm. The pellets were incubated overnight at 37°C in DMEM, and then fixed for 3 hours at room temperature (RT) in 10% Formalin Neutral Buffer Solution (FUJIFILM Wako). After fixation, the supernatant was aspirated, and 1% sodium alginate (FUJIFILM Wako) was added to the pellets. Gelatinous cell blocks were immediately obtained by addition of 100 μl of 1 M CaCl2 (FUJIFILM Wako), and the blocks were embedded in paraffin.
Immunostaining was performed as described previously 43. Briefly, antigen retrieval of deparaffinized sections was performed with 10 mM citric acid pH 6.0 (FUJIFILM Wako), and then the samples were incubated with anti-H3G34W monoclonal Abs (1:200) at 4°C overnight 44. Specimens were then incubated with Dako EnVision Dual Link System-HRP (Agilent, Santa Clara, CA, USA), visualized using the diaminobenzidine substrate system (FUJIFILM Wako), and counterstained with hematoxylin 44. Section images were obtained on a Keyence BZ-X800 microscope (Keyence Corporation, Osaka, Japan).
Alkaline phosphate staining
ALP activity is widely used to assess the early osteogenic ability of osteoblast-like cells. We seeded pGCTB-SCs into a 24-well plate at a density of 5 × 104 cells per well. After 48 hours of incubation, the culture medium was exchanged and further incubated for the indicated periods. The cells were washed with PBS, fixed in 10% formalin, and stained with premixed ALP substrate solutions (FUJIFILM Wako).
pGCTB-SCs were seeded into 96-well plates at a density of 5 × 103 cells per well and incubated for 48 hours. After the cells reached confluence, the medium was exchanged, and the samples were incubated further. ALP assays were performed using the TRACP & ALP assay kit (Takara Bio, Kusatsu, Shiga, Japan). Briefly, treated cells were lysed in extraction solution (saline with 1% NP-40), mixed with freshly prepared p-nitrophenyl phosphate substrate (12.5 mM), and incubated at 37°C for 30 minutes. The optical density of p-nitrophenol at 405 nm was determined using iMark™ Microplate Absorbance Reader (Bio-Rad, Hercules, CA, USA). Finally, ALP activity was normalized with a standard curve derived from Escherichia coli C75 (Takara Bio).
Quantitative real-time PCR (qRT-PCR)
Total RNA of treated pGCTB-SCs was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and reverse-transcribed with PrimeScript RT Reagent Kit (Takara Bio). qRT-PCR was conducted with a LightCycler 1.5 (Perfect Real Time, Takara Bio, Kusatsu, Shiga, Japan) as previously described 45. Primers are listed in Supplementary Table 2. Data were standardized against the housekeeping gene GAPDH. At least four separate experiments were conducted.
pGCTB-SCs were seeded on poly-L-lysine (Fujifilm Wako)–coated cover glass at a density of 10 × 104 cells and cultured for 48 hours. After the cells were treated with each reagent for the indicated periods, they were fixed with 4% paraformaldehyde (FUJIFILM Wako) for 10 minutes at RT, permeabilized with 0.2% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 15 minutes, and blocked with 10% goat serum (FUJIFILM Wako) for 30 minutes. Subsequently, the cells were incubated at 4°C overnight with a mixture of primary antibodies diluted in 1:200 in Can Get Signal Immunostain Solution A (TOYOBO, Osaka, Japan). Samples were then washed three times with PBS and incubated with Alexa Fluor® 488 and 594 diluted in 1:200 for 1 hour at RT. SlowFade Diamond antifade mountant with DAPI (Invitrogen) was used as a mounting solution. Immunostaining was visualized using fluorescence microscopy (BZ-X800; Keyence).
Nuclear protein extraction and Western blot analysis
pGCTB-SCs were seeded in 6-well dishes at a density of 1.2 × 106 cells/well and incubated overnight. The following day, the culture media were replaced for each reagent, and the cells were incubated for an additional 12 hours. After incubation, the cells were washed twice with ice-cold PBS, scraped, and centrifuged. Cytoplasmic and nuclear proteins were isolated using nuclear and cytoplasmic extraction reagents (Thermo Fisher Scientific) to which Cell Lytic M (Sigma-Aldrich) with protease inhibitor cocktail (cOMplete™ Mini: Sigma-Aldrich) were added.
Western blotting was performed as previously described 46 with the following primary antibodies: β-catenin (1:1000) and actin clone C4 (1:5000), with or without rabbit polyclonal Lamin A/C antibody (1:3000, sc-20681; Santa Cruz Biotechnology, Dallas, TX, USA). Relative intensity was calculated using the ratio of each target protein's signal intensity to internal controls' intensity, using ImageJ ver1.52p (NIH, Bethesda, MD, USA).
Ethics and Guidelines
Our all mothods were conducted in accordance with the Declaration of Helsinki, and written informed consent was obtained from all human subjects.
Patients and quantitative CT image analysis
To evaluate nuclear β-catenin translocation in naïve GCTB clinical samples, we performed a retrospective analysis using samples of GCTB registered in the files of the Department of Anatomic and Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan 44. A total of 91 clinical samples of GCTB from 88 patients were prepared for immunohistochemistry. These tumor specimens had been acquired from biopsy or surgeries, and the existence of the H3G34W mutation had been immunohistochemically confirmed. Samples collected after denosumab treatment were excluded from the study.
Immunohistochemical staining and assessment of the nuclear β-catenin labeling index (NBLI) were performed as previously described 47,48. Histogram analysis was conducted to calculate the cut-off value for the NBLI. The presence of intra-tumoral ossification of GCTB was also assessed using H&E-stained sections.
Twenty-one patients were diagnosed with GCTB or received treatment for this cancer at our hospital between July 2011 and November 2020. Of those, 18 patients had received denosumab treatment (primary, n = 12; recurrent, n = 4; both, n = 2), and 16 had also undergone non-contrast CT or PET-CT evaluation before and after denosumab treatment. CT DICOM (Digital Imaging and Communications in Medicine) image datasets from identified patients were analyzed using SYNAPSE VINCENT ver6.1 (VINCENT, FUJIFILM Medical Co., Ltd.). A single musculoskeletal radiologist with eight years of experience manually delineated the regions of interest (ROI) in the axial CT images of whole slices, and three-dimensional CT images were semi-automatically acquired. Slice thickness was set at 2 or 5 mm. We identified the calcified tissue volumes (≥ 130 Hounsfield Units, HU) using VINCENT histogram analysis and quantified tumor calcification using the previously described Agatston scoring system 36,49. In addition, we assessed the association between nuclear β-catenin translocation and intra-tumoral ossification of GCTB.
All experiments were repeated at least three times. Data are presented as means ± SD. Student’s t-test or Wilcoxon's rank-sum test was used for two-group comparisons. Multiple comparisons were assessed using one-way ANOVA with the Tukey–Kramer post hoc test. Fisher’s exact test was used to examine the significance of the association between the categorical data. All data analyses were performed using the JMP 13 statistical software (SAS Institute, Cary, NC, USA). P < 0.05 was considered statistically significant.
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.