Development of deep learning algorithms to discriminate giant cell tumors of bone from adjacent normal tissues by confocal Raman spectroscopy.

Raman spectroscopy is a non-destructive analysis technique that provides detailed information about the chemical structure of tumors. Raman spectra of 52 giant cell tumors of bone (GCTB) and 21 adjacent normal tissues of formalin-fixed paraffin embedded (FFPE) and frozen specimens were obtained using a confocal Raman spectrometer and analyzed with machine learning and deep learning algorithms. We discovered characteristic Raman shifts in the GCTB specimens. They were assigned to phenylalanine and tyrosine. Based on the spectroscopic data, classification algorithms including support vector machine, k-nearest neighbors and long short-term memory (LSTM) were successfully applied to discriminate GCTB from adjacent normal tissues of both the FFPE and frozen specimens, with the accuracy ranging from 82.8% to 94.5%. Importantly, our LSTM algorithm showed the best performance in the discrimination of the frozen specimens, with a sensitivity and specificity of 93.9% and 95.1% respectively, and the AUC was 0.97. The results of our study suggest that confocal Raman spectroscopy accomplished by the LSTM network could non-destructively evaluate a tumor margin by its inherent biochemical specificity which may allow intraoperative assessment of the adequacy of tumor clearance.

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.

Genome-Wide Transcriptome Profiling of the Neoplastic Giant Cell Tumor of Bone Stromal Cells by RNA Sequencing.

Giant cell tumor of bone (GCTB) is the most common non-malignant primary bone tumor reported in Hong Kong. Failure of treatment in advanced GCTB with aggressive local recurrence remains a clinical challenge. In order to reveal the molecular mechanism underlying the pathogenesis of this tumor, we aimed to examine the transcriptome profiling of the neoplastic stromal cells of GCTB in this study. RNA-sequencing was performed on three GCTB stromal cell samples and one bone marrow-derived MSC sample and 174 differentially expressed genes (DEGs) were identified between these two cell types. The top five up-regulated genes are SPP1, F3, TSPAN12, MMP13, and LGALS3BP and further validated by qPCR and Western Blotting. Knockdown of SPP1 was found to induce RUNX2 and OPG expression in GCTB stromal cells but not the MSCs. Ingenuity pathway analysis (IPA) of the 174 DEGs revealed significant alternations in 23 pathways; variant calling analysis revealed 1915 somatic variants of 384 genes with high or moderate impacts. Interestingly, four canonical pathways were found overlapping in both analyses; from which VEGFA, CSF1, PLAUR, and F3 genes with somatic mutation were found up-regulated in GCTB stromal cells. The STRING diagram showed two main clusters of the DEGs; one cluster of histone genes that are down-regulated in GCTB samples and another related to osteoblast differentiation, angiogenesis, cell cycle progression, and tumor growth. The DEGs and somatic mutations found in our study warrant further investigation and validation, nevertheless, our study add new insights in the search for new therapeutic targets in treating GCTB. J. Cell. Biochem. 118: 1349-1360, 2017. © 2016 Wiley Periodicals, Inc.

A mouse model of luciferase-transfected stromal cells of giant cell tumor of bone.

A major barrier towards the study of the effects of drugs on Giant Cell Tumor of Bone (GCT) has been the lack of an animal model. In this study, we created an animal model in which GCT stromal cells survived and functioned as proliferating neoplastic cells. A proliferative cell line of GCT stromal cells was used to create a stable and luciferase-transduced cell line, Luc-G33. The cell line was characterized and was found that there were no significant differences on cell proliferation rate and recruitment of monocytes when compared with the wild type GCT stromal cells. We delivered the Luc-G33 cells either subcutaneously on the back or to the tibiae of the nude mice. The presence of viable Luc-G33 cells was assessed using real-time live imaging by the IVIS 200 bioluminescent imaging (BLI) system. The tumor cells initially propagated and remained viable on site for 7 weeks in the subcutaneous tumor model. We also tested in vivo antitumor effects of Zoledronate (ZOL) and Geranylgeranyl transferase-I inhibitor (GGTI-298) alone or their combinations in Luc-G33-transplanted nude mice. ZOL alone at 400 µg/kg and the co-treatment of ZOL at 400 µg/kg and GGTI-298 at 1.16 mg/kg reduced tumor cell viability in the model. Furthermore, the anti-tumor effects by ZOL, GGTI-298 and the co-treatment in subcutaneous tumor model were also confirmed by immunohistochemical (IHC) staining. In conclusion, we established a nude mice model of GCT stromal cells which allows non-invasive, real-time assessments of tumor development and testing the in vivo effects of different adjuvants for treating GCT.

Immortalized human fetal bone marrow-derived mesenchymal stromal cell expressing suicide gene for anti-tumor therapy in vitro and in vivo.

BACKGROUND AIMS: Cancer is one of the greatest health challenges facing the world today with >10 million new cases of cancer every year. The self-renewal, tumor-homing ability and low immunogenicity of mesenchymal stromal cells (MSCs) make them potential delivery candidates for suicide genes for anti-tumor therapy. However, unstable supply and short life span of adult MSCs in vitro have limited this therapeutic potential. In this study, we aimed to evaluate if immortalization of human fetal bone marrow-derived mesenchymal stromal cells by simian virus 40 (SV40-hfBMSCs) could be a stable source of MSCs for clinical application of suicide gene therapy. METHODS AND RESULTS: Transduction of SV40 and herpes simplex virus thymidine kinase-IRES-green fluorescent protein (TK-GFP) did not cause significant change in the stem cell properties of hfBMSCs. The anti-tumor effect of SV40-TK-hfBMSCs in the presence of the prodrug ganciclovir was demonstrated in vitro and in nude mice bearing human prostate cancer cells, DU145 and PC3, which had been transduced with luciferase and GFP for imaging evaluation by an in vivo live imaging system (IVIS 200 imaging system; Caliper Life Sciences). Repeated injection of low doses (1 × 10(6) cells/kg) of SV40-TK-hfBMSCs was as effective as previously reported and did not cause observable harmful side effects in multiple organs. Mixed lymphocyte reaction showed that SV40-TK-hfBMSCs did not induce significant proliferation of lymphocytes isolated from healthy adults. CONCLUSIONS: Taken together, immortalized hfBMSCs represent a reliable and safe source of MSCs for further clinical translational study.

Comparison of the anti-tumor effects of denosumab and zoledronic acid on the neoplastic stromal cells of giant cell tumor of bone.

Denosumab and Zoledronic acid (ZOL) are two antiresorptive drugs currently in use for treating osteoporosis. They have different mechanisms of action but both have been shown to delay the onset of skeletal-related events in patients with giant cell tumor of bone (GCT). However, the anti-tumor mechanisms of denosumab on the neoplastic GCT stromal cells remain unknown. In this study, we focused on the direct effects of denosumab on the neoplastic GCT stromal cells and compared with ZOL. The microscopic view demonstrated a reduced cell growth in ZOL-treated but not in denosumab-treated GCT stromal cells. ZOL was found to exhibit a dose-dependent inhibition in cell growth in all GCT stromal cell lines tested and cause apoptosis in two out of three cell lines. In contrast, denosumab only exerted a minimal inhibitory effect in one cell line and did not induce any apoptosis. ZOL significantly inhibited the mRNA expression of receptor activator of nuclear factor kappa-B ligand (RANKL) and osteoprotegerin (OPG) in two GCT stromal cell lines whereas their protein levels remained unchanged. On the contrary, denosumab did not regulate RANKL and OPG expression at both mRNA and protein levels. Moreover, the protein expression of Macrophage Colony-Stimulating Factor (M-CSF), Alkaline Phosphatase (ALP), and Collagen α1 Type I were not regulated by denosumab and ZOL either. Our findings provide new insights in the anti-tumor effect of denosumab on GCT stromal cells and raise a concern that tumor recurrence may occur after the withdrawal of the drug.

Effect of water-soluble P-chitosan and S-chitosan on human primary osteoblasts and giant cell tumor of bone stromal cells.

Water-soluble phosphorylated chitosan (P-chitosan) and disodium (1 → 4)-2-deoxy-2-sulfoamino-ß-D-glucopyranuronan (S-chitosan) are two chemically modified chitosans. In this study, we found that P-chitosan significantly promotes cell proliferation of both human primary osteoblasts (OBs) and the OB like stromal cell component of the giant cell tumor of bone (GCTB) cells at the concentration from 125 to 1000 µg ml⁻¹ at all time points of 1, 3, 5 and 7 days after treatment. Further investigation of the osteogenic effect of the P-chitosan suggested that it regulates the levels of osteoclastogenic factors, receptor activator of nuclear factor kappa B ligand and osteoprotegerin expression. An interesting finding is that S-chitosan at lower concentration (100 µg ml⁻¹) stimulates cell proliferation while a higher dose (1000 µg ml⁻¹) of S-chitosan inhibits it. The inhibitory effect of S-chitosan on human primary GCT stromal cells was greater than that of OBs (p < 0.05). Taken together, our findings elucidated the osteogenic effect of P-chitosan and the varying effects of S-chitosan on the proliferation of human primary OBs and GCT stromal cells and provided us the rationale for the construction of novel bone repair biomaterials with the dual properties of bone induction and bone tumor inhibition.

Pamidronate, farnesyl transferase, and geranylgeranyl transferase-I inhibitors affects cell proliferation, apoptosis, and OPG/RANKL mRNA expression in stromal cells of giant cell tumor of bone.

Giant cell tumor (GCT) is the most common nonmalignant primary bone tumor reported in Hong Kong. It usually affects young adults between the ages of 20 and 40. This tumor is well known for its potential to recur following treatment. To date no effective adjuvant therapy exists for GCT. Our project aimed to study the effects of pamidronate (PAM), farnesyl transferase inhibitor (FTI-277), geranylgeranyl transferase inhibitor (GGTI-298), and their combinations on GCT stromal cells (SC). Individual treatment with PAM, FTI-277, and GGTI-298, inhibited the cell viability and proliferation of GCT SC in a dose-dependent way. Combination of FTI-277 with GGTI-298 caused synergistic effects in reducing cell viability, and its combination index was 0.49, indicating a strong synergism. Moreover, the combination of FTI-277 with GGTI-298 synergistically enhanced cell apoptosis and activated caspase-3/7, -8, and -9 activities. PAM induced cell-cycle arrest at the S-phase. The combination of PAM with GGTI-298 significantly increased OPG/RANKL mRNA ratio and activated caspase-3/7 activity. Our findings support that the combination of bisphosphonates with GGTIs or FTIs with GGTIs may be used as potential adjuvants in the treatment of GCT of bone.

Hybrid origin of "Bauhinia blakeana" (Leguminosae: Caesalpinioideae), inferred using morphological, reproductive, and molecular data.

Bauhinia blakeana (Leguminosae subfam. Caesalpinioideae tribe Cercideae), or the Hong Kong Orchid Tree, is of great horticultural value. It is completely sterile and is shown here to be the result of hybridization between the largely sympatric species, B. purpurea and B. variegata. Although the analysis of patterns of morphological variation revealed only a few examples of phenotypic intermediacy, study of intersimple sequence repeat (ISSR) markers enabled unequivocal identification of the parental species due to the presence of additive inheritance of alleles and the absence of any bands that are unique to B. blakeana. Investigation of aspects of the reproductive biology of the taxa furthermore revealed that the parental species are largely xenogamous, have flowering periods that overlap seasonally and temporally, and share common pollinators. Evidence is provided to show that B. blakeana is not naturally stabilized and is only maintained horticulturally by artificial propagation. It is therefore recommended that the hybrid be regarded as a horticultural cultivar rather than a naturally occurring species; a new cultivar name, Bauhinia 'Blakeana', is accordingly validated.