PDGFRA mediates the PI3K/AKT signaling pathway to regulate the growth and phenotype of GCTB tumor cells

Clinical samples collection

We obtained 32 formalin-fixed paraffin-embedded (FFPE) GCTB tissue blocks from Huashan Hospital of Fudan University (Shanghai, China), along with one FFPE liver cancer sample from a female as a positive control for clonality analysis. For primary cell culture, we also obtained one pair of fresh GCTB surgical samples from a patient before and after denosumab treatment. The patient was a 30-year-old male with GCTB on distal right femur, who was treated at Huashan Hospital. All cases were initially diagnosed by an attending pathologist and reconfirmed by a consulting pathologist. Informed consent was obtained for tissue use in all cases. This study was approved by the Institutional Review Board of School of Basic Medicine, Fudan University (2023-008).

Histologic assessment

Histologic assessment was performed on hematoxylin and eosin (H&E)-stained and immunohistochemistry (IHC) of CD68. GCTB displayed diverse cell types, with multinucleated giant cells, mononuclear oval cells, and mononuclear spindle cells scattered in the focus. Among them, multinucleated giant cells and monocytes were accurately distinguished by H&E staining, while mononuclear spindle cells were identified as CD68-negative and fusiform in shape via IHC.

Immunohistochemistry

Immunohistochemistry for CD68 was performed using a primary antibody (Cat. No. ab201340; Abcam) at a dilution of 1:100. Briefly, the paraffin embedded tissue was sectioned at 5 µm thickness and placed on slides, dried at 65°C for over 3 hours. After deparaffinization in xylene and alcohol and blocking of endogenous peroxidase in 1% hydrogen peroxide, antigen retrieval was carried out in trypsin (Gibco, Thermo Fisher Scientific, USA) using a 37°C incubator for 1 hour. Sections were blocked with 5% goat serum for 30 minutes, incubated with primary antibody at 4°C overnight, washed with PBS, incubated with secondary antibody (Dako, Agilent) at room temperature for 1 hour, and washed with PBS. Detection was achieved using a standard horseradish peroxidase (HRP)-based detection system (Dako EnVision+ System-HRP, Agilent). Sections were counterstained with hematoxylin. Negative controls were performed by omitting the primary antibody.

Laser capture microdissection (LCM) and genomic DNA extraction

FFPE GCTB tissues, 5 μm thick, were affixed to slides (Applied Biosystems, Foster City, Germany), dried at 65°C for over 3 hours, dewaxed in xylene, and dehydrated with alcohol. Six sections of the same sample were H&E stained, and another six were subjected to CD68 IHC. Multinucleated giant cells, monocytes, and CD68-negative mononuclear spindle cells were accurately captured by laser capture microdissection (LCM) instrument. LCM was performed using an Arcturus XT instrument (Life Technologies). For each cell type, approximately 1,500-3,000 cells were captured per sample. The laser settings were as follows: spot size 7.5-12 µm, power 60-80 mW, duration 8.0-10.0 ms. Captured cells were collected onto CapSure Macro LCM caps (Life technologies).

The genomic DNA of captured cells on the cap was cleaved and extracted as per The PicroPure DNA Extraction Kit (Life Technologies) instructions. Whole tissue DNA was extracted using the QiAamp DNA Micro Kit (Qiagen, Hilden, Germany).

Restriction endonuclease HpaII digestion

The human androgen receptor gene (HUMARA) on the X chromosome contains variable CAG short tandem repeats (ranging from 11 to 30), making it a clonality marker. On the inactivated X chromosome, methylation of a CpG site upstream of these repeats prevents HpaII digestion [20], enabling HUMARA gene amplification. In heterozygous samples, both of the two homologous X chromosomes may be inactivated. Thus, two fragment sizes are amplified from the genomic DNA of polyclonal non-tumor cells after HpaII digestion. Whereas true tumor cells are monoclonal and exhibit only one fragment size, differing from non-tumor cells. This technique helps identify true tumors [21,22].

In each case, genomic DNA from LCM-captured multinucleated giant cells, monocytes, and CD68-negative spindle cells was individually assigned to enzyme-digested and undigested subgroups. For the enzyme-digested subgroups, genomic DNA was quantified to 100 ng and subjected to a 3-hour, 37°C digestion using HpaII (New England Biolabs, Beverly, MA). For detailed methods, please refer to our previous research [22].

PCR amplification and data analysis

After HpaII digestion, the reaction mixture was purified using the QIAquick PCR Purification Kit (Qiagen) to remove salts and enzymes. Then, 2 μL purified product was used as template for PCR amplification. HUMARA gene amplification was performed using nested PCR. The outer primers were: 5’-TCC AGA ATC TGT TCC AGA GCG TGC-3’ and 5’-GCT GTG AAG GTT GCT GTT CCT CAT-3’. The inner primers (fluorescently labeled with 6-FAM) were: 5’-GCA GCG CTC CGA GAA GTT CCT GTT C-3’ and 5’-GCT GGG AAC GGG GCT CCC CAG AAC-3’. The PCR reaction mixture (25 μL) contained 1× PCR buffer, 200 μM dNTPs, 0.2 μM of each primer, 1 U of Taq DNA polymerase (TaKaRa), and 2 μL of template DNA. Initial denaturation was performed at 95°C for 10 minutes, followed by 34 cycles of 30 seconds at 94°C, 30 seconds at 64°C, and 30 seconds at 72°C, with final extension at 72°C for 10 minutes.

The fluorescence intensity detected by capillary electrophoresis appears as a peak map. In theory, HpaII-undigested HUMARA DNA from heterozygote yields two peaks: Allele 1 and Allele 2. A set of tightly clustered multiple peaks was adjacent to the major product peak due to Taq polymerase slippage during amplification [12,22]. Obvious peaks were thus selected as main peaks for clonality analysis. To account for the preferential amplification of non-specific short fragments that could affect HUMARA gene amplification, we calculated the corrected cleavage ratio. It’s defined as:

Samples with cleavage ratio above 2.0 or below 0.5 were considered to be monoclonal tumor tissues [23], and the corresponding cells were identified as tumor cells.

Isolation of GCTB primary cells

The isolation of GCTB primary cells was carried out as previously described [24-26]. Briefly, fresh surgical samples of GCTB were cut into small tissue blocks of approximately 1 mm³ in size and washed with PBS. The tissue blocks were digested by 0.2% type II collagenase (Thermo Fisher Scientific, USA)-DMEM medium in a constant temperature incubator at 37°C overnight. The digest was filtered through a 70 μm cell screen and centrifuged at 1,500 rpm for 5 min. The supernatant was discarded, and the deposition was resuspended in 10% FBS-DMEM culture medium, and seeded into dishes.

CCK-8 assay

The cells were seeded at a density of 8,000 cells/well in a 96-well plate. For denosumab (Xgeva, Amgen, USA), cells were treated with concentrations ranging from 10 to 800 μg/mL for 48 h, and concentration of 800 μg/mL from 24 to 120 h. For Avapritinib (Selleckchem, USA) and Capivasertib (Selleckchem), cells were treated with concentrations ranging from 0.1 to 100 μM for 48 h. Control groups received an equal volume of vehicle (PBS for denosumab, DMSO for inhibitors, with final DMSO concentration <0.1%). After adding the CCK-8 reagent (Yeasen, Shanghai, China), the plate was incubated at 37°C for 2 h. The optical density (OD) value was measured at 450 nm wave length by Multiskan GO spectrophotometer (Thermo Fisher Scientific).

RT-qPCR

Total RNA was isolated with FreeZol Reagent (Vazyme, Nanjing, China) from cells. cDNA was reverse transcribed with HiScript IV RT SuperMix (Vazyme). The Real-time quantitative PCR reaction system (20 µL) contained 1× ChamQ Universal SYBR qPCR Master Mix (Vazyme), 0.4 µM each primer, and 1 µL of cDNA template. The expression level of PDGFRA, PDGFRB, PDGFD and RUNX2 RNA were normalized by GAPDH. The sequences of primers (Sangon Biotech, Shanghai, China) are shown in Table 1. The detection was carried out using a LightCycler 480 (Roche, Switzerland). The reaction conditions included an initial denaturation step at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 10 s and annealing/extension at 60°C for 30 s. The relative expression levels were calculated using the 2^-ΔΔCt method.

Statistical analysis

All data are presented as mean ± SEM. Statistical analysis was performed with unpaired two-tailed Student’s t test, or one-way or two-way analysis of variance (ANOVA) followed by Bonferroni’s posttest, in GraphPad Prism 5.0. p-value <0.05 was considered to be statistically significant.

Morphological characteristic of GCTB

Typical GCTB tissue was basically composed of three types of cells: multinucleated giant cells, mononuclear oval cells, and mononuclear spindle cells (Figure 1A). Multinucleated giant cells were scattered among mononuclear cells in large numbers, with many nuclei. IHC staining showed that multinucleated giant cells and part of the mononuclear oval cells were CD68-positive, while another part of mononuclear oval cells and mononuclear spindle cells were negative (Figure 1B). The CD68-negative mononuclear spindle cells, which were the focus of our clonality analysis, exhibited elongated, fusiform shapes with scant cytoplasm and oval nuclei, distinct from the rounder CD68-positive mononuclear cells.

Prior to clonality analysis, the polymorphism of the samples’ HUMARA gene must be confirmed. DNA was extracted from 32 GCTB cases. The HUMARA gene was amplified, and the gene polymorphism was detected (Figure 1C; Table 2). Eleven of 32 cases were HUMARA-heterozygous, which were suitable for further analysis.

The multinucleated giant cells in GCTB are polyclonal hyperplasia

As there are mainly three types of cells in GCTB, clonality analysis was performed on each type to confirm the genuine neoplastic cell population within this tumor.

Before analyzing GCTB, in order to ensure the methodological feasibility and result reliability, hepatoma cells were selected as a positive control. The hepatoma cells showed 1 peak through digestion with HpaII, indicating that the cells were monoclonal, while liver cancer was a neoplastic hyperplasia (Figure 2A).

We chose to analyze the clonality of the osteoclastic multinucleated giant cells first. The multinucleated giant cells were captured by LCM machine (Figure 2B). The DNA of captured multinucleated giant cells was extracted, digested and non-digested, and amplified by HUMARA primer with 6-FAM fluorescence. The PCR products were subjected to capillary electrophoresis, and the fluorescence intensity peak map was analyzed.

The results showed that the two representative cases (No.10 and 25) were both polyclonal (Figure 2C), which indicated that osteoclast-like multinucleated giant cells were polyclonal hyperplasia, and were not the neoplastic component of GCTB. Analysis of multinucleated giant cells from all 11 informative (heterozygous) cases revealed polyclonal patterns, consistent with reactive hyperplasia (Supplementary Table 1).

The mononuclear spindle cells in GCTB are the true tumor components of GCTB

We further accurately captured mononuclear cells around multinucleated giant cells to explore their clonality. The results showed that the mononuclear cells were polyclonal in most cases, while monoclonal in very few cases. Figure 3 demonstrated the results that the two cases (No. 10 and 25) were still polyclonal. Since mononuclear cells can be divided into two kinds: oval cells and spindle cells, we believed this needed further classification, as one of them could be the true neoplastic component.

Based on the established pathological concept that the neoplastic component in GCTB is the stromal-derived spindle cell, which are typically CD68-negative, we targeted CD68-negative mononuclear spindle cells for LCM and clonality analysis. We used LCM to capture CD68-negative mononuclear spindle cells and extract their DNA for clonality analysis (Figure 4A). In 2 representative cases, No. 10 and 25, there was only one peak after digestion with HpaII enzyme (Figure 4B). Analysis of CD68-negative spindle cells from all 11 informative (heterozygous) cases showed a monoclonal pattern (Supplementary Table 1). Monoclonality, as determined by X-inactivation analysis, is a well-established hallmark of neoplastic proliferation, indicating origin from a single progenitor cell. The results showed that CD68-negative mononuclear spindle cells were monoclonal and were true neoplastic cells of GCTB, which further demonstrated that GCTB was a true tumor.

Isolation and identification of GCTB primary tumor cells

Primary cell isolation was performed on fresh tumor tissue from a patient with GCTB before and after denosumab treatment. One day after isolation and culture, three main types of cells in GCTB were observed: mononuclear spindle cells, mononuclear round cells, and multinucleated giant cells (Figure 5A). After 2-3 passages, the only remaining primary cells were mononuclear spindle-shaped cells, while multinucleated giant cells and other cells disappeared (Figure 5B). The passaged primary cells were used for further study.

To identify the isolated primary cells, cellular immunofluorescence staining was performed. Immunofluorescence staining showed that mutant histone H3.3 (G34W) was strongly positive in the nucleus in approximately 90% of the isolated adherent spindle cells, while CD68 was negative. (Figure 5C). Negative controls, performed by omitting the primary antibody, showed no signal. A known GCTB tissue section served as the positive control for the antibody. For further confirmation, DNA was extracted from primary cells and PCR amplification and Sanger sequencing were performed on the fragment near H3-3A G34W. Sequencing revealed a mutation at H3-3A G34W site in primary cells (Figure 5D). These results proved that the isolated primary cells were the tumor cells of giant cell tumor of bone.

Denosumab has no direct effect on the growth of GCTB tumor cells but indirectly changes the phenotype of GCTB tumor cells

Denosumab has a certain clinical effect on GCTB that makes it difficult to resect or it can cause serious complications after resection. However, the optimal administration time and dosing of denosumab are still not completely unified [16].

A high concentration (800 μg/mL) was included based on some in vitro studies [27,28], although it exceeds typical serum levels. The primary tumor cells were treated with denosumab from 10 to 800 μg/mL for 48 h. Compared with the control group, different concentrations of denosumab had no direct effect on cell growth (Figure 5E). The tumor cells were treated with 800 μg/mL denosumab continuously. The growth of cells at 24 h, 48 h, 72 h, 96 h and 120 h did not change compared with the control group (Figure 5F).

We inquired whether denosumab could change the phenotype of GCTB tumor cells. RUNX family transcription factor 2 (RUNX2) is essential for osteoblastic differentiation and skeletal morphogenesis and acts as a scaffold for nucleic acids and regulatory factors involved in skeletal gene expression [29]. qPCR showed that RUNX2 RNA increased after denosumab treatment in vitro (P<0.05), but decreased after treatment in vivo (P<0.01, Figure 5G). These results indicate that denosumab, at the tested concentrations, has no direct anti-proliferative effect on cultured GCTB tumor cells in vitro. The opposite changes in RUNX2 RNA expression observed in vitro versus in vivo samples suggest that the drug’s effects on tumor cell phenotype may be context-dependent, potentially involving the bone microenvironment.

PDGFRAi has a direct effect on the growth of GCTB tumor cells and changes the phenotype of GCTB tumor cells

Platelet derived growth factor receptor alpha (PDGFRA), a member of receptor tyrosine kinase family, plays an important role in embryonic development, cell proliferation, survival and chemotaxis [30]. Avapritinib is a potent and selective inhibitor of PDGFRA (PDGFRAi) and KIT kinases. In addition to inhibiting the activation loop of wild-type PDGFRA and KIT kinases, it also has potent inhibition of PDGFRA D824V and KIT D816V mutants. High-throughput drug screening showed that Avapritinib at 3 µM concentration had no significant inhibitory effect on other kinases [31].

Primary tumor cells were treated with Avapritinib (0.1-100 uM) for 48 h. Avapritinib inhibited tumor cell growth in a dose-dependent manner, as the cell viability decreased to 50.54% with the concentration of 100 µM (Figure 6A).

Subsequently, we treated primary tumor cells with 10 µM Avapritinib for 24 h and detected the expression of related genes. Avapritinib up-regulated the mRNA expression of PDGFRA (P<0.001), PDGFRB (P<0.05), PDGFD (P<0.01) and RUNX2 (P<0.05) in primary tumor cells (Figure 6B). These findings indicate that PDGFRAi could inhibit the growth of primary tumor cells and alter the phenotype of primary tumor cells.

PDGFRAi regulates the growth and phenotype of GCTB tumor cells through the PI3K/AKT pathway

The PI3K/AKT/mTOR pathway plays a key role in the occurrence and development of many kinds of tumors, regulating cell proliferation, apoptosis and metabolism. There are three isoforms of PI3K: I, II and III. Type I PI3K is a heterodimeric structure composed of a catalytic subunit and a regulatory subunit, which can be further subdivided into types IA and IB. The former is activated by receptor tyrosine kinases (RTKs) and the latter by G protein-coupled receptors (GPCRs). PDGFRA is a member of the RTK family. When the ligand specifically binds to it, signal transduction adaptor proteins are phosphorylated, thereby activating type IA PI3K and activating downstream pathways [32].

Capivasertib is an orally active, broad-spectrum AKT inhibitor with inhibitory activity against AKT1, AKT2, and AKT3. Capivasertib has anti-tumor activity and can be used to treat breast cancer, and it is currently in phase I clinical trials.

Primary tumor cells were treated with Capivasertib (0.1-100 µM) for 48 h. Capivasertib inhibited the growth of tumor cells in a dose-dependent manner, and the IC50 of Capivasertib on tumor cell growth was 21.53 µM (Figure 6C).

Subsequently, we treated the primary tumor cells with 10 µM Capivasertib for 24 h and detected the expression of related genes. Capivasertib up-regulated the mRNA level of PDGFRA (P<0.05), PDGFRB (P<0.05), PDGFD (P<0.01) and RUNX2 (P<0.05) in tumor cells (Figure 6D). AKT inhibitor can inhibit the growth of GCTB cells and change the cell phenotype, which is consistent with the effect of PDGFRAi. Moreover, Avapritinib modulated the expression of AKT-related genes in tumor cells, up-regulating the mRNA level of B-cell lymphoma 2 apoptosis regulator (BCL2, P<0.05) and RB transcriptional corepressor like 2 (RBL2, P<0.001), and down-regulating the mRNA level of Myeloid cell leukemia 1 apoptosis regulator (MCL1, P<0.05, Figure 6E).

Western blot analysis showed that treatment with 5 μM Avapritinib for 24 h decreased the levels of phosphorylated AKT (Ser473, p-AKT, P<0.01) and its downstream target phosphorylated GSK3β (Ser9, pGSK3β, P<0.01) in primary tumor cells. Co-treatment with the AKT agonist SC79 (10 μM) partially restored the levels of pAKT and pGSK3β (Figure 6F). These finding indicate that PDGFRAi regulates the growth and phenotype of GCTB tumor cells through the PI3K/AKT pathway.