Giant Cell Tumor Of Bone Cell Research Articles

Effect of Thermoablation with pH Change on Giant Cell Tumor of Bone: An In Vitro Study

Objective: To evaluate the effect of pH on the apoptosis and necrosis rate of giant cell tumor of bone (GCTB) cells during thermoablation. Materials and Methods: GCTB tissues were collected from 15 patients. Cells were incubated at 25 ºC , 37 ºC, 45 ºC, and 50 ºC, with the variation of the pH at 4.7, 5.6, 6.5, 7.4, 8.3, and 9.2 for 20 minutes (in triplicate for each condition). The effect of thermoablation and pH variation on GCTB cells death was evaluated by staining with Annexin V-FITC and propidium iodide solution after 3 days of incubation. The fluorescence intensity was evaluated by flow cytometry to evaluate the percentage of tumor cells death. Results: Thermoablation alone increased the percentage of tumor cells death. However, when combined with an increase in pH, the percentage of GCTB cells death increased more. Conversely, lowering the pH did not increase the tumor cells death compared with thermoablation treatment alone, while changing the pH alone had only a low effect on increasing the percentage of GCTB cells death. Conclusion: Thermoablation the temperature between 37 ºC and 45 ºC plus a pH level slightly higher than physiologic pH (between 7.4 and 8.3) for 20 minutes increased GCTB cell death. However, determining the optimum condition to kill tumor cells while causing minimal harm to normal cells requires more study.

H3.3-G34W in giant cell tumor of bone functionally aligns with the exon choice repressor hnRNPA1L2.

RNA processing is an essential post-transcriptional phenomenon that provides the necessary complexity of transcript diversity prior to translation. Aberrations in this process could contribute to tumourigenesis, and we have previously reported increased splicing alterations in giant cell tumor of bone (GCTB), which carries mutations in the histone variant H3.3 encoding glycine 34 substituted for tryptophan (H3.3-G34W). G34W interacts with several splicing factors, most notably the trans-acting splicing factor hnRNPA1L2. To gain a deeper understanding of RNA processing in GCTB and isogenic HeLa cells with H3.3-G34W, we generated RNA-immunoprecipitation sequencing data from hnRNPA1L2 and H3.3-G34W associated RNAs, which showed that 80% overlapped across genic regions and were frequently annotated as E2F transcription factor binding sites. Splicing aberrations in both GCTB and HeLa cells with H3.3-G34W were significantly enriched for known hnRNPA1L2 binding motifs (p value < 0.01). This splicing aberration differed from hnRNPA1L2 knockouts, which showed alterations independent of H3.3-G34W. Of functional significance, hnRNPA1L2 was redistributed to closely match the H3.3 pattern, likely driven by G34W, and to loci not occupied in normal parental cells. Taken together, our data reveal a functional overlap between hnRNPA1L2 and H3.3-G34W with likely significant consequences for RNA processing during GCTB pathogenesis. This provides novel opportunities for therapeutic intervention in future modus operandi.

Establishment and characterization of two novel patient-derived cell lines from giant cell tumor of bone: NCC-GCTB8-C1 and NCC-GCTB9-C1.

Giant cell tumor of bone (GCTB) is a rare osteolytic bone tumor consisting of mononuclear stromal cells, macrophages, and osteoclast-like giant cells. Although GCTB predominantly exhibits benign behavior, the tumor carries a significant risk of high local recurrence. Furthermore, GCTB can occasionally undergo malignant transformation and distal metastasis, making it potentially fatal. The standard treatment is complete surgical resection; nonetheless, an optimal treatment strategy for advanced GCTB remains unestablished, necessitating expanded preclinical research to identify appropriate therapeutic options. However, only one GCTB cell line is publicly available from a cell bank for research use worldwide. The present study reports the establishment of two novel cell lines, NCC-GCTB8-C1 and NCC-GCTB9-C1, derived from the primary tumor tissues of two patients with GCTB. Both cell lines maintained the hallmark mutation in the H3-3A gene, which is associated with tumor formation and development in GCTB. Characterization of these cell lines revealed their steady growth, spheroid-formation capability, and invasive traits. Potential therapeutic agents were identified via extensive drug screening of the two cell lines and seven previously established GCTB cell lines. Among the 214 antitumor agents tested, romidepsin, a histone deacetylase inhibitor, and mitoxantrone, a topoisomerase inhibitor, were identified as potential therapeutic agents against GCTB. Conclusively, the establishment of NCC-GCTB8-C1 and NCC-GCTB9-C1 provides novel and crucial resources that are expected to advance GCTB research and potentially revolutionize treatment strategies.

Establishment and characterization of two novel patient-derived cell lines from giant cell tumor of bone.

Giant cell tumor of bone (GCTB) is a rare bone tumor with osteolytic features, composed of stromal cells with a monotonous appearance, macrophages, and osteoclast-like giant cells. GCTB is commonly associated with a pathogenic mutation in the H3-3A gene. While complete surgical resection is the standard cure for GCTB, it often results in local recurrence and, rarely, metastasis. Thus, an effective multidisciplinary treatment approach is necessary. Although patient-derived cell lines is an essential tool for investigating novel treatment strategies, there are only four GCTB cell lines available in public cell banks. Therefore, this study aimed to establish novel GCTB cell lines and successfully created NCC-GCTB6-C1 and NCC-GCTB7-C1 cell lines from two patients' surgically removed tumor tissues. These cell lines exhibited H3-3A gene mutations, consistent proliferation, and invasive properties. After characterizing their behaviors, we performed high-throughput screening of 214 anti-cancer drugs for NCC-GCTB6-C1 and NCC-GCTB7-C1 and integrated their screening data with those of NCC-GCTB1-C1, NCC-GCTB2-C1, NCC-GCTB3-C1, NCC-GCTB4-C1, and NCC-GCTB5-C1 that we previously established. We identified histone deacetylase inhibitor romidepsin as a possible treatment for GCTB. These findings suggest that NCC-GCTB6-C1 and NCC-GCTB7-C1 could be valuable tools for preclinical and basic research on GCTB.

Histone Deacetylase Inhibitors as a Therapeutic Strategy to Eliminate Neoplastic "Stromal" Cells from Giant Cell Tumors of Bone.

Simple SummaryGiant cell tumor of bone (GCTB) is an intermediate bone neoplasm which consists of several cell populations, including the neoplastic “stromal” cells. These cells harbor a mutation in one of the histone H3.3 genes (H3F3A), and are therefore considered as the driving component of GCTB. This mutation causes changes in the epigenetic landscape, leading to aberrant gene expression patterns that may drive tumor growth. Surgery is currently the only curative treatment option because contemporary systemic therapies cannot remove the neoplastic cells from GCTB lesions, leading to re-outgrowth of the tumor when the treatment is discontinued. Therefore, the aim of this study was to explore whether therapeutic targeting of the epigenome can eliminate the neoplastic cells from GCTB lesions. The findings from this study indicate that histone deacetylase (HDAC) inhibitors may represent such a treatment strategy, which could improve the quality of life of GCTB patients who currently require life-long treatment.The neoplastic “stromal” cells in giant cell tumor of bone (GCTB) harbor a mutation in the H3F3A gene, which causes alterations in the epigenome. Current systemic targeted therapies, such as denosumab, do not affect the neoplastic cells, resulting in relapse upon treatment discontinuation. Therefore, this study examined whether targeting the epigenome could eliminate the neoplastic cells from GCTB. We established four novel cell lines of neoplastic “stromal” cells that expressed the H3F3A p.G34W mutation. These cell lines were used to perform an epigenetics compound screen (n = 128), which identified histone deacetylase (HDAC) inhibitors as key epigenetic regulators in the neoplastic cells. Transcriptome analysis revealed that the neoplastic cells expressed all HDAC isoforms, except for HDAC4. Therefore, five HDAC inhibitors targeting different HDAC subtypes were selected for further studies. All GCTB cell lines were very sensitive to HDAC inhibition in both 2D and 3D in vitro models, and inductions in histone acetylation, as well as apoptosis, were observed. Thus, HDAC inhibition may represent a promising therapeutic strategy to eliminate the neoplastic cells from GCTB lesions, which remains the paramount objective for GCTB patients who require life-long treatment with denosumab.

Single-cell transcriptome analysis reveals T-cell exhaustion in denosumab-treated giant cell tumor of bone.

Denosumab (DMAB), a human monoclonal antibody against the receptor activator of the nuclear factor-kappa B ligand, is used for the treatment for unresectable giant cell tumor of bone (GCTB). However, little is known about the molecular and functional characteristics of GCTB-infiltrating lymphocytes after DMAB treatment. Here, we performed single-cell RNA sequencing and immunostaining assays to delineate the immune landscape of GCTB in the presence and absence of DMAB. We found that exhausted CD8+ T cells were preferentially enriched in DMAB-treated GCTB. A distinct M2-skewed type of tumor-associated macrophages (TAMs) comprises the majority of GCTB TAMs. We identified cytokines, including interleukin-10, and inhibitory receptors of M2 TAMs as important mediators of CD8+ T cell exhaustion. We further revealed that DMAB treatment notably increased the expression levels of periostin (POSTN) in GCTB cells. Furthermore, POSTN expression was transcriptionally regulated by c-FOS signaling and correlated with GCTB recurrence in patients after DMAB treatment. Collectively, our findings reveal that CD8+ T-cells undergo unappreciated exhaustion during DMAB therapy and that GCTB cell-derived POSTN educates TAMs and establishes a microenvironmental niche that facilitates GCTB recurrence.

Interactions in CSF1-Driven Tenosynovial Giant Cell Tumors.

A major component of cells in tenosynovial giant cell tumor (TGCT) consists of bystander macrophages responding to CSF1 that is overproduced by a small number of neoplastic cells with a chromosomal translocation involving the CSF1 gene. An autocrine loop was postulated where the neoplastic cells would be stimulated through CSF1R expressed on their surface. Here, we use single-cell RNA sequencing (scRNA-seq) to investigate cellular interactions in TGCT. A total of 18,788 single cells from three TGCT and two giant cell tumor of bone (GCTB) samples underwent scRNA-seq. The three TGCTs were additionally analyzed using long-read RNA sequencing. Immunofluorescence and IHC for a range of markers were used to validate and extend the scRNA-seq findings. Two recurrent neoplastic cell populations were identified in TGCT that are highly similar to nonneoplastic synoviocytes. We identified GFPT2 as a marker that highlights the neoplastic cells in TCGT. We show that the neoplastic cells themselves do not express CSF1R. We identified overlapping MAB features between the giant cells in TGCT and GCTB. The neoplastic cells in TGCT are highly similar to nonneoplastic synoviocytes. The lack of CSF1R on the neoplastic cells indicates they may be unaffected by current therapies. High expression of GFPT2 in the neoplastic cells is associated with activation of the YAP1/TAZ pathway. In addition, we identified expression of the platelet-derived growth factor receptor in the neoplastic cells. These findings suggest two additional pathways to target in this tumor.

Establishment and characterization of NCC-GCTB5-C1: a novel cell line of giant cell tumor of bone

Giant cell tumor of bone (GCTB), is a rare intermediate malignant bone tumor with high local infiltrative ability, and is genetically characterized by mutation in the H3-3A gene. Standard treatment is curative surgical tumor resection. GCTB demonstrates both local recurrence and pulmonary metastasis after surgical treatment, and effective systematic chemotherapy is yet to be established. Therefore, development of novel chemotherapies for GCTB is necessary. Although patient-derived tumor cell lines are potent tools for preclinical research, 15 GCTB cell lines have been reported to date, and only four are publicly available. Thus, this study aimed to establish and characterize a novel GCTB cell line for preclinical studies on GCTB. Herein, we described the establishment of a cell line, NCC-GCTB5-C1, from the primary tumor tissue of a patient with GCTB. NCC-GCTB5-C1 was shown to harbor a mutation in the H3-3A gene, which is typical of GCTB; thus, it has useful properties for in vitro studies. We conducted the largest integrated screening analysis of 214 antitumor agents using NCC-GCTB5-C1 along with four GCTB cell lines. Romidepsin (a histone deacetylase inhibitor), camptothecin, and actinomycin D (topoisomerase inhibitors) demonstrated remarkable antitumor effects, suggesting that these antitumor agents are potential therapeutic candidates for GCTB treatment. Therefore, the NCC-GCTB5-C1 cell line could potentially contribute to the elucidation of GCTB pathogenesis and the development of novel GCTB treatments.

Establishment and characterization of the NCC-GCTB4-C1 cell line: a novel patient-derived cell line from giant cell tumor of bone.

Giant cell tumor of bone (GCTB) is a rare osteolytic intermediate bone tumor that harbors a pathogenic H3F3A gene mutation and exhibits characteristic histology. The standard curative treatment for GCTB is complete surgical resection, but it frequently results in local recurrence and, more rarely, metastasis. Therefore, effective multidisciplinary treatment is needed. Although patient-derived tumor cell lines are promising tools for preclinical and basic research, there are only four available cell lines for GCTB in public cell banks. Thus, the aim of this study was to establish a novel GCTB cell line. Using surgically resected tumor tissues from a patient with GCTB, we established a cell line named NCC-GCTB4-C1. The cells harbored the typical H3F3A gene mutation and exhibited constant proliferation and invasive capabilities. After characterizing NCC-GCTB4-C1 cell behaviors, we conducted high-throughput screening of 214 anti-tumor drugs and identified seven effective drugs. Comparing the results of high-throughput screening using NCC-GCTB4-C1 cell line with the results using NCC-GCTB1-C1, NCC-GCTB2-C1, and NCC-GCTB3-C1 cell lines that we previously established, four drugs were in common effective. This study showed potential drugs for the treatment of GCTB. These data indicate that NCC-GCTB4-C1 has the potential to be a powerful tool in preclinical and basic research on GCTB.

Ultrastructural changes in giant cell tumor of bone cultured cells exposed to quercetin

ABSTRACT Giant cell tumor of bone (GCTB) is a primary bone tumor that affects skeletally mature people and whose main treatment is surgical. Because there are few pharmacological alternatives for the treatment of this tumor to find other molecules or compounds that could be potential therapeutic agents is desirable. Quercetin is a flavonoid with described antitumoral effect in different types of cancer cell lines that could be a possible option in GCTB treatment. However, there is no literature about the effect of quercetin on GCTB. In the present paper, we reported the ultrastructural changes in GCTB cells exposed to quercetin and also determined the expression of RIP1K, Caspase 3 and Caspase 8 on the exposed cells. For this purpose, GCTB sample was obtained from one patient and cultured. Quercetin affected all the histological components of the GCTB. The ultrastructural changes consisted mainly in necroptosis, autophagocytosis and secondary necrosis. This is the first report about quercetin effects on giant cell tumor of bone cultured cells. Further studies in other models could be done to support the use of quercetin as a complementary treatment in giant cell tumor of bone. Abbreviations: Giant cell tumor of bone (GCTB); transmission electron microscopy (TEM); reverse transcription – polymerase chain reaction (RT-PCR); receptor interacting protein kinase 1 (RIP1K); Dulbecco’s Modified Eagle’s Medium (DMEM).

Combination of Melatonin and Zoledronic Acid Suppressed the Giant Cell Tumor of Bone in vitro and in vivo.

Melatonin (Mlt) confers potential antitumor effects in various types of cancer. However, to the best of our knowledge, the role of Mlt in the giant cell tumor of bone (GCTB) remains unknown. Moreover, further research is required to assess whether Mlt can enhance the therapeutic effect of zoledronic acid (Zol), a commonly used anti-GCTB drug. In this research, we investigated the effects of Mlt, Zol, and the combination of these two drugs on GCTB cells’ characteristics, including cell proliferation, apoptosis, osteogenic differentiation, migration, and invasion. The cell counting kit-8 (CCK-8) assay, colony formation assay, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay (TUNEL), alkaline phosphatase (ALP) staining, alizarin red staining (ARS), scratch wound healing assay, and transwell experiment were performed, respectively. Our results showed that Mlt could effectively inhibit the proliferation, migration, and invasion of GCTB cells, as well as promote the apoptosis and osteogenic differentiation of tumor cells. Of note, a stronger antitumor effect was observed when Mlt was combined with Zol treatment. This therapeutic effect might be achieved by inhibiting the activation of both the Hippo and NF-κB pathways. In conclusion, our study suggests that Mlt can be a new treatment for GCTB, which could further enhance the antitumor effect of Zol.

A novel model to culture cells from giant cell tumor of bone using three-dimensional (3D) polycaprolactone scaffold.

Two‐dimensional (2D) culture of cells from giant cell tumor of bone (GCTB) is affected by loss of the multinucleated giant cells in subsequent passages. Therefore, there is limited time to study GCTB with all its histological components in 2D culture. Here, we explored the possibility of culturing GCTB cells on a polycaprolactone (PCL)‐printed scaffold. We also evaluated the viability of the cultured cells and their adherence to the PCL scaffold at day 14 days using immunofluorescence analysis with calcein, vinculin, and phalloidin. Using the histological technique with hematoxylin and eosin staining, we observed all the histological components of GCTB in this 3D model. Immunohistochemical assays with cathepsin K, p63, and receptor activator of nuclear factor (NF)‐κB ligand (RANKL) yielded positive results in this construct, which allowed us to confirm that the seeded cells maintained the expression of GCTB markers. Based on these findings, we concluded that the PCL scaffold is an efficient model to culture GCTB cells, and the cell viability and adherence to the scaffold can be preserved for up to 14 days. Moreover, this model can also be used in subsequent studies to assess in vitro cell–cell interactions and antineoplastic efficacy of certain agents to establish a treatment against GCTB.

Extended Curettage for Giant Cell Tumors of Bone: A Surgeon's View.

The surgical procedure involves 4 essential components.(1) Surgical exposure and isolation of the soft tissues. The technique begins with a complete exposure that allows visualization of the entire tumor cavity. The initial part of the exposure involves getting to the bone and to the soft-tissue mass outside the bone, when present. The muscles are separated from the bone and retracted away from the soft-tissue component of the tumor. Soft tissue is adequately retracted to allow complete visualization of the tumor cavity. The tissues around the cavity are protected by placing hydrogen peroxide-soaked mops around the tumor cavity. The aim is to isolate the bone opening and avoid any contamination of soft tissue by the tumor, as hydrogen peroxide kills GCTB cells on contact1. The soft-tissue mass of the tumor is removed en bloc with a cover of normal tissue in order to prevent spillage into uninvolved tissues. Ward and Li advise the use of cautery for this part of the exposure in order to minimize contamination because high-temperature cautery kills the tumor2. The initial opening into the osseous tumor cavity is made smaller in order to control the tumor spillage, and then enlarged in order to gain complete visualization.(2) Curetting and burring for tumor clearance. The walls are curetted, using the sharp edges of the curet. Good visualization is the key to meticulous and complete curetting. We recommend the use of a surgical loupe and headlight for adequate visualization1. Tumor cavities often have overhanging osseous ridges with tumor hidden behind them. A curet may not be useful for breaking these hard osseous ridges; a burr is best utilized for this task. In addition to breaking the ridges, a high-speed burr helps to extend the curettage for a few millimeters beyond the grossly visible tumor margin3,4.(3) Use of adjuvants to achieve extended curettage. Various physical and chemical agents have been utilized to control the microscopic disease remaining in the walls following a thorough curettage. Liquid nitrogen, phenol, hydrogen peroxide, alcohol, electrocautery, bone cement, and argon plasma cautery have been utilized as adjuvants. Balke et al. showed that the rate of recurrence decreases with use of more adjuvants, with high-speed burring having the greatest effect on the rate of recurrence, likely as a result of the larger resection and the thermal effect of the burring5. We suggest a case-by-case use of specific adjuvants, but in our experience, a high-speed burr is always utilized, hydrogen peroxide is utilized commonly when safe, and argon-plasma cautery is utilized judiciously.(4) Reconstruction of the cavity. The defect may be filled with bone, cement, or a combination of both. We suggest an individual, case-by-case approach to the reconstruction. In cases in which >25% of the articular surface is undermined2, subchondral bone grafting is recommended prior to cementing (i.e., a sandwich procedure). GCTB requires tumor clearance for local control. One alternative to intralesional surgical treatment performed around the knee is resection of the tumor-affected segment of bone and reconstruction with an endoprosthesis. This is a joint-sacrificing alternative and, in our experience, should be reserved for patients with joint involvement, multiply recurrent disease, or insufficient remaining wall to curet. When adequate tumor clearance is possible, joint-salvaging intralesional surgical treatment remains the superior option to achieve physiological joint function6. Indications for this procedure include maintained or restorable joint congruity and construct stability allowing early mobilization. The rate of local recurrence following intralesional surgical treatment ranges from 16% to 25%1,7. The use of a high-speed burr is an essential part of the curettage5, and hydrogen peroxide is a safe choice for an adjuvant, if desired and when suitable, to provide greater local control8. Reconstruction with bone, cement, or both is acceptable, but we recommend building up a few millimeters of subchondral bone with bone graft before cement filling1,9. Illumination and magnification within the tumor cavity provide better visualization.Isolation of the soft tissue around the tumor with use of hydrogen peroxide-soaked mops can prevent seeding and contamination.Meticulous tumor clearance is more important to minimize recurrence than the use of adjuvants.Use of a C-arm helps to better guide extension of the curettage and avoid inadvertent joint penetration.Maintaining joint congruity is essential.

Establishment and characterization of NCC-GCTB1-C1: a novel patient-derived cancer cell line of giant cell tumor of bone.

Giant cell tumor of bone (GCTB) is a rare osteolytic bone tumor, accounting for approximately 5% of all primary bone tumors. GCTB is characterized by unique giant cells. It is also characterized by recurrent mutations in the histone tail of the histone variant H3.3, H3F3A, on chromosome 1, therapeutic implications of which have not been established yet. There are few effective standardized treatments for GCTB, and a novel therapy has long been required. Patient-derived cancer cells have facilitated the understanding of mechanisms underlying the etiology and progression of multiple cancers. Thus far, only 10 GCTB cell lines have been reported, and none of them are publicly available. The aim of this study was to develop an accessible patient-derived cell line of GCTB, which could be used as a screening tool for drug development. Here, we describe the establishment of a cell line, designated NCC-GCTB1-C1, from the primary tumor tissue of a male patient with GCTB on the right distal radius. NCC-GCTB1-C1 cells were maintained as a monolayer culture for over 23 passages for 7months. These cells exhibited continuous growth, as well as spheroid formation and invasive ability. Using an oncology agent screen, we tested the effect of anticancer drugs on the proliferation of NCC-GCTB1-C1 cells. The cells displayed a remarkable response to romidepsin and vincristine. Thus, we established a novel GCTB cell line, NCC-GCTB1-C1, which could be a useful tool for studying GCTB tumorigenesis and the efficacy of anticancer drugs.

Functions of Exogenous RUNX2 in Giant Cell Tumor of Bone In Vitro.

ObjectivesThis research aimed to investigate the relative level of Runt‐related transcription factor 2 (RUNX2) in giant cell tumor of bone (GCTB). Through the histopathological similarities between osteoporosis and GCTB, the biological functions of exogenous RUNXS were demonstrated in GCTB cell lines. This generated awareness of the molecular mechanism of the biogenesis and metastasis of GCTB, as well as showing the pathways and processes involved in this study. This research also expected to provide hints for the clinical treatment of patients with GCTB, to release the tumor burden and reduce the recurrence rate and metastasis of patients with this condition.MethodsThe expression of RUNX2 in the tumors was verified by Western Blot, qRT‐PCR and immunohistochemistry, compared with the normal tissues’ adjacent tumors. Subsequently, the plasmids expressing RUNX2 were constructed, amplified and transfected into the 0404 cell line through transfection kits (0.4, 0.8, 1.6, 2.4 ng/μl). After that, the proliferation, migration, invasion, cellular viability and apoptosis of 0404 cell lines were examined by EDU assay, wound healing assay, transwell assay, annexin v staining, and CCK8 assay, respectively.ResultsThe messenger RNA (mRNA) level of RUNX2 in tumors was over 100 folds more than the normal tissues. The protein level of tumors upregulated 8.32(±4.41) folds relatively. After the transfection of RUNX2 overexpressed plasmids into the 0404 cell line, the mRNA level of RUNX2 increased approximately 530.11(±24.87), 1117.96(±77.68), 2835.09(±45.22) and 4781.51(±79.37) folds respectively, and the protein level was upregulated about 4.12(±1.15), 16.73(±1.63), 21.53(±2.41) and 23.39(±0.85) folds respectively. The proliferation of 0404 cells was inhibited by 2.13(±1.02)% of 1.6 ng/μl group and 3.03(±1.76)% of 2.4 ng/μl group. And the migration was inhibited about 45.56(±6.13)%, 50.79(±5.27)%, 63.15(±8.62)% and 93.90(±3.65)% respectively. The invasion was decreased about 14.49(±5.4)%, 37.02(±6.52)%, 42.24(±2.59)% and 48.97(±10.61)% respectively. Meanwhile, FITC Annexin V/PI apoptosis assay demonstrated that RUNX2 plasmids could promote apoptosis rate around 4.15(±0.27)%, 5.07(±0.27)%, 7.61(±0.45)% and 11.32(±1.02)% respectively, and CCK8 proved these plasmids could weaken cellular viability in a concentration‐dependent manner with the time passing.ConclusionsRUNX2 is highly expressed in giant cell tumors of bone. The RUNX2 overexpressed plasmids we constructed could be successfully transfected into 0404 cell line. Far more importantly, the exogenous RUNX2 can seriously block the biological functions of 0404 cell line in a concentration‐dependent manner, including proliferation, translocation, invasion, cellular viability, and apoptosis. Meanwhile, the mechanism was hypothesized and discussed in the article.

Simvastatin Possesses Antitumor and Differentiation‐Promoting Properties That Affect Stromal Cells in Giant Cell Tumor of Bone

Giant cell tumor of bone (GCTB) is a locally aggressive destructive bone lesion. The management of pulmonary metastasis and local recurrence after the surgical treatment of GCTB remains a challenge. Pathologically, stromal cells in GCTB are known as primary neoplastic cells and are recognized as incompletely differentiated preosteoblasts. Therefore, inducing GCTB stromal cells to differentiate into cells with a mature osteoblastic phenotype may stop tumor growth and recurrence. In this study, we aimed to investigate how simvastatin, a clinically approved and commonly used statin that has been known to promote the maturation of cells of the osteogenic lineage, affects GCTB stromal cells. We found that simvastatin effectively inhibited cell viability by suppressing proliferation and by inducing apoptosis in GCTB stromal cells. Moreover, simvastatin treatment upregulated the expression of genes related to osteogenic maturation, such as runt-related transcription factor 2, osteopontin, and osteocalcin, and increased the mineralization of the extracellular matrix in GCTB stromal cells. Ingenuity pathway analysis was used to discover that the vitamin D receptor pathway was involved in the simvastatin-induced osteogenic differentiation of GCTB stromal cells by upregulating the 1,25-dihydroxyvitamin D metabolism. Taken together, this in vitro study demonstrates the antitumor and differentiation-promoting effects of simvastatin on GCTB stromal cells and suggests the possibility of using simvastatin as an adjuvant therapy for GCTB. These findings support further clinical investigation of the efficacy of using simvastatin as an adjuvant therapy for GCTB to reduce recurrence and distant metastasis after surgical treatment. © 2019 Orthopedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:297-310, 2020.

Abstract 1486: The immune atlas and chemokine profile of giant cell tumor of bone

Abstract Background and purpose: Giant cell tumor of bone (GCTB) is a locally aggressive tumor with metastatic potential. Given that GCTBs are composed of three major cell populations and the bone-resorption activity is attributed to the multinucleated giant cells which are conjectured to be recruited by “real” tumor cells, it is still elusive that how and what are these immune cells recruited. The purpose of this study is to present the immune landscape of GCTB, as well as to find out responsible chemokines during immune cell recruiting. Experimental procedures: We collected fresh GCTB tissues from surgical excisions. To identify immune cell composition within the tumor microenvironment, we performed mass cytometry (CyTOF) and conventional multicolor cytometry. Some fresh osteosarcoma and chondrosarcoma samples were also used to compare the immune atlas. For chemokine profiling, chemokine arrays were utilized. Further analysis on potential association between chemokines and immune cells were conducted. Results: For last updated, 47 GCTB fresh samples were collected, of which 12 cases received CyTOF analysis and the rest 35 samples were analyzed by multicolor cytometry. All GCTB samples were analyzed by chemokine array In-depth interrogation of the immune landscapes showed that compared with osteosarcoma and chondrosarcoma, GCTBs had some unique immune cell subgroups. Some CD163+ CD206+ macrophage clusters which expressed various immune suppressive markers were significantly enriched in GCTBs, while CD4+ CD25+ Treg cells were fewer in GCTB. In addition, some macrophage-like mononuclear cells (CD14- CD68+ CD163- CD206+ HLA-DR+) were only seen in GCTBs.Chemokine array revealed that higher levels of CXCL12, CCL22, and CCL7 might be associated with moremacrophage-like mononuclear cells in GCTBs. Conclusion: GCTB have very different immune features from other malignant bone tumors. Multiple chemokines may play important roles in recruiting “functional” monocytes. Such in-depth understanding has important implications in appropriate therapeutics for GCTB. Bigger sample size will further help in studying the immune and chemokine features of GCTB. Note: This abstract was not presented at the meeting. Citation Format: Binghao Li, Zhan Wang, Zhaoming Ye. The immune atlas and chemokine profile of giant cell tumor of bone [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1486.

Characterization of Three Novel H3F3A-mutated Giant Cell Tumor Cell Lines and Targeting of Their Wee1 Pathway

The giant cell tumor of bone (GCTB) is a locally aggressive primary bone tumor that is composed of mononuclear stroma cells, scattered macrophages, and multinucleated osteoclast-like giant cells which cause pathologic osteolysis. The stroma cells represent the neoplastic population of the tumor and are characterized by the H3F3A mutation G34W. This point mutation is regarded as the driver mutation of GCTB. We have established three new stable H3F3A mutated GCTB cell lines: U-GCT1, U-GCT2, and U-GCT3M. MK-1775 is a Wee1-kinase inhibitor which has been used for blocking of sarcoma growth. In the cell lines we detected Wee1, Cdk1, Cyclin B1, H3K36me3, and Rrm2 as members of the Wee1 pathway. We analyzed the effect of MK-1775 and gemcitabine, alone and in combination, on the growth of the cell lines. The cell lines showed a significant reduction in cell proliferation when treated with MK-1775 or gemcitabine. The combination of both agents led to a further significant reduction in cell proliferation compared to the single agents. Immunohistochemical analysis of 13 GCTB samples revealed that Wee1 and downstream-relevant members are present in GCTB tissue samples. Overall, our work offers valuable new tools for GCTB studies and presents a description of novel biomarkers and molecular targeting strategies.

Intratibial injection of patient-derived tumor cells from giant cell tumor of bone elicits osteolytic reaction in nude mouse.

There have been various reports in the literature of an in vivo model for giant cell tumor of bone (GCTB). However, few suitable animal models of GCTB have been established, due to the fact that GCTB contains three histologically different cell types. To the best of our knowledge, injection of patient-derived GCTB cells into bone environment has not been reported until now. In the present study, the biological behavior of GCTB cells in nude mice was investigated through intratibial injection of patient-derived GCTB cells. Patient-derived GCTB cells were obtained from 5 patients who had not undergone chemo- and radiotherapy. Once isolated, the cell suspension was injected into the tibias of nude mice. The growth process was monitored by weekly observation and photographic documentation using X-ray. Four months after injection, nude mice were sacrificed and the injected tibial samples were fixed, and further analyzed using micro-computed tomography (micro-CT), standard histology, tartrate-resistant acid phosphatase (TRAP) staining and mitochondrial immunofluorescence staining. X-ray, micro-CT and standard histology revealed osteolytic destruction in the proximal end of the tibia. TRAP staining identified TRAP-positive, osteoclast-like cells distributed in the bone marrow interface of the lesion area. Anti-human mitochondrial immunofluorescence staining confirmed that the surviving cells in the osteolytic destruction were of human GCTB cell origin. These findings indicate that intratibial injection of patient-derived GCTB cells may elicit osteolytic destruction in nude mice. The results of the current study present a novel animal model for GCTB, opening new perspectives to investigate this disease and develop therapeutic agents.

Giant cell tumor of bone: updated molecular pathogenesis and tumor biology

Giant cell tumor of bone (GCTB)-related clonal aberrations occur in a background of epigenetic histone modifications (especially the G34W mutation of H3F3A gene) that induce cytogenetic abnormalities. Clonal aberrations are closely linked to the aggressiveness of GCTB. The "neoplastic" mononuclear stromal cells in GCTB express fundamental RANKLs and various chemokines and cytokines associated with monocyte recruitment and "reactive" multinucleated giant cells (osteoclastogenesis). The reciprocal and orchestrated actions between mononuclear stromal cells and multinucleated giant cells help in the understanding of the molecular pathogenesis and tumor biology of GCTB. In the future, novel targets in the updated tumor biology and molecular pathogenesis of GCTB should be explored and scrutinized for the development of systemic therapy.