Rat Osteosarcoma Cells Research Articles

Characterization of the Rat Osteosarcoma Cell Line UMR-106 by Long-Read Technologies Identifies a Large Block of Amplified Genes Associated with Human Disease.

The rat osteosarcoma cell line UMR-106 is widely used for the study of bone cancer biology but it has not been well characterized with modern genomic methods. To better understand the biology of UMR-106 cells we used a combination of optical genome mapping (OGM), long-read sequencing nanopore sequencing and RNA sequencing.The UMR-106 genome was compared to a strain-matched Sprague-Dawley rat for variants associated with human osteosarcoma while expression data were contrasted with a public osteoblast dataset. Using the COSMIC database to identify the most affected genes in human osteosarcomas we found somatic mutations in Tp53 and H3f3a. OGM identified a relatively small number of differences between the cell line and a strain-matched control animal but did detect a ~45 Mb block of amplification that included Myc on chromosome 7 which was confirmed by long-read sequencing. The amplified region showed several blocks of non-contiguous rearranged sequence implying complex rearrangements during their formation and included 14 genes reported as biomarkers in human osteosarcoma, many of which also showed increased transcription. A comparison of 5mC methylation from the nanopore reads of tumor and control samples identified genes with distinct differences including the OS marker Cdkn2a. This dataset illustrates the value of long DNA methods for the characterization of cell lines and how inter-species analysis can inform us about the genetic nature underlying mutations that underpin specific tumor types. The data should be a valuable resource for investigators studying osteosarcoma, in general, and specifically the UMR-106 model.

Development, Optimization, and Validation of an in vitro Cell-Based Bioassay to Determine the Biological Activity of Teriparatide (PTH1–34)

AbstractThis study aimed to establish an efficient in vitro cell-based assay to measure the activity of teriparatide (PTH1–34). In this study, a rat osteosarcoma cell line (UMR-106) was treated with various concentrations of PTH1–34, and the biological activity of PTH1–34 was determined by quantitatively measuring intracellular cyclic adenosine monophosphate levels using a time-resolved fluoroimmunoassay. A four-parameter fitting analysis was used to calculate the relative potency of the samples. The experimental conditions were optimized. The method's specificity, relative accuracy, precision, and linearity were validated. Our data suggested that this method had good specificity, a relative bias of relative accuracy ranging from −0.8 to 1.4%, a correlation coefficient for the linear regression equation of 0.9953, a geometric coefficient of variation for intermediate precision ranges from 2.0 to 3.5%, and a linear range of 50 to 150%. This method significantly improves the quality control and release inspection efficiency of PTH1–34 and may be further developed and validated as an alternative to the existing United States Pharmacopeia and European Pharmacopoeia inclusion methods. This method also provides a platform for the high-throughput screening of PTH1–34 analogs.

3D bioprinted osteosarcoma model for experimental boron neutron capture therapy (BNCT) applications: Preliminary assessment.

Osteosarcoma is the most frequently primary malignant bone tumor characterized by infiltrative growth responsible for relapses and metastases. Treatment options are limited, and a new therapeutic option is required. Boron neutron capture therapy (BNCT) is an experimental alternative radiotherapy able to kill infiltrative tumor cells spearing surrounding healthy tissues. BNCT studies are performed on 2D in vitro models that are not able to reproduce pathological tumor tissue organization or on in vivo animal models that are expensive, time-consuming and must follow the 3R's principles. A 3D in vitro model is a solution to better recapitulate the complexity of solid tumors meanwhile limiting the animal's use. Objective of this study is to optimize the technical assessment for developing a 3D in vitro osteosarcoma model as a platform for BNCT studies: printing protocol, biomaterial selection, cell density, and crosslinking process. The best parameters that allow a fully colonized 3D bioprinted construct by rat osteosarcoma cell line UMR-106 are 6 × 106 cells/ml of hydrogel and 1% CaCl2 as a crosslinking agent. The proposed model could be an alternative or a parallel approach to 2D in vitro culture and in vivo animal models for BNCT experimental study.

The effects of vasoactive intestinal peptide on RANKL-induced osteoclast formation.

BackgroundCongenital pseudarthrosis of the tibia is a rare disease characterized by an imbalance in bone remodeling. Vasoactive intestinal peptide (VIP) has been proven to modulate bone resorption and the formation of osteoclasts. This study aimed to explore the effects of VIP on the homeostasis of bone metabolism in diverse in vitro systems.MethodsBone marrow-derived macrophages (BMMs) were differentiated into tartrate-resistant acid phosphatase-positive cells through incubation with receptor activator of nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). In vitro resorption pit detection was carried out to assess the effects of VIP on osteoclastic activity. Rat osteosarcoma cell line ROS 17/2.8 was cultured alone or co-cultured with rat BMMs in the presence or absence of VIP at various concentrations. The expression levels of RANKL, RANK, OPG, NF-κB, IL-6, ERK, CAII, and GAPDH were determined by qRT-PCR and WB assay.ResultsVIP was observed to repress osteoclast differentiation without affecting the number of osteoclast precursor cells. Furthermore, the modulation of the RANKL/osteoprotegerin (OPG), nuclear factor-κB (NF-κB), and extracellular signal-regulated kinase (ERK) signaling pathways were involved in the inhibitive influence of VIP upon bone erosion. Additionally, VIP affected the expression levels of osteoclastic factors including RANKL, OPG, and interleukin-6 in osteoblast cells. Furthermore, the expression levels of RANKL and RANK were increased, while OPG expression was reduced after treatment with VIP in the co-culture of ROS 17/2.8 and rat BMMs. ERK and NF-κB signal pathways were demonstrated to be involved in the effect of VIP in the co-culture system.ConclusionsVIP plays a critical role in bone remodeling and might serve as a potential target in the development of treatments for congenital pseudarthrosis of the tibia.

Mathematical Analysis of the Biophysical Response of EGCG‐treated yperglycemic HMEC‐1 and UMR Cells in vitro using Electric Cell‐Substrate Impedance Sensing Technology

Hyperglycemia, the hallmark of diabetes mellitus, is a major risk factor for endothelial dysfunction and compromised bone cell metabolism. Not many studies have measured the biophysical response of hyperglycemic human microvascular endothelial cells (HMEC‐1) and bone‐like rat osteosarcoma cells, UMR 106‐01 BSP (UMR cells), treated with (−)‐Epigallocatechin‐3‐gallate (EGCG), a natural antioxidant and the active ingredient in green tea, using electric cell‐substrate impedance sensing (ECIS) technology. The aim of this study was to determine the biophysical response of HMEC‐1 and UMR cells under hyperglycemic conditions when treated with EGCG. Moreover, the derivation of predictive mathematical formulas was sought to analyze the ECIS‐generated biophysical data. We hypothesized that ECIS technology can be used as a tool to evaluate the effects of glucose and EGCG on cell membrane resistance, spreading and attachment in HMEC‐1 and UMR cell cultures by mathematical analysis. HMEC‐1 and UMR cells were separately cultured and plated at 4.0×104 and 2.5×105 cells/mL, respectively, in eight‐well (8W) ECIS array plates. Designated plate wells were treated with glucose with or without EGCG. The cell cultures were incubated in a humidified chamber at 37° C with infusion of 5% CO2. Membrane resistance, time of complete cell attachment, and rate of spreading of the cells were monitored and measured in vitro, using the ECIS machine. The one‐tailed correlation analysis was employed to statistically study the relationships between glucose concentration and the following parameters: maximum resistance, spreading rate, and complete attachment time in both HMEC‐1 and UMR cells. To verify statistical significance of the results, p‐values were obtained for each set of experiments. The experimental data showed that the higher concentrations of glucose lowered the maximum cell membrane resistance, cell spreading rate, and time of cell attachment. The following mathematical formulas were derived based on the ECIS data: 1) dR/dt=kR(1−R/L), 2) R(t)=L/(1+be−kt), and 3) ln(π/1−π) = kt−ln(b), (where dR/dt=rate of spreading, R(t)=cell membrane resistance at any time point, L=maximum resistance, k=rate constant, b=(L−R0)/R0, t=time in hours, and π=r(R(t)−R0/L). The formulas were found to be of predictive value. The p‐values obtained from the correlation test of glucose concentration and two of the parameters tested indicated statistical significance (L for HMEC‐1 cells: 0.061; dR/dt for HMEC‐1 cells: 0.027; L for UMR cells: p=0.041; dR/dt for UMR cells: p=0.047). The results suggest that the ECIS technology is a useful tool in the mathematical analysis of biophysical phenomena in HMEC‐1 and UMR 106 cells in vitro.Support or Funding InformationFunding for this study was provided by the Ryckman Student Research Endowment Fund of the Biology Department of La Sierra University in Riverside, CA.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The effect of non-steroidal anti-inflammatory drugs on osteosarcoma cells.

Osteosarcoma (OS), an aggressive malignancy, is the most common primary bone tumor in children. Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used to reduce pain and inflammation. NSAIDs have shown to be toxic to certain malignancies such as colorectal, breast, and pancreatic cancers, but are not well-studied in OS. The purpose of this study is to assess whether ketorolac induces apoptosis in OS cells, compare this to indomethacin, which has been shown to inhibit OS proliferation, and explore the underlying mechanism. A rat OS cell line (UMR-108) was exposed to various concentrations of ketorolac and indomethacin. Cell viability, cytotoxicity, apoptosis induction, DNA fragmentation and the expression of apoptosis-related markers were examined by MTT assay, colony formation assay, flow cytometry, agarose gel electrophoresis, and Western blot respectively. The results indicated that ketorolac and indomethacin could induce apoptosis of rat OS cells in a dose- and time-dependent manner. Apoptosis was confirmed by cell morphology and annexin positivity. The molecular data showed that NSAIDs affected expression of Bcl-2, survivin, and Poly (ADP-ribose) polymerase-1 (PARP). These findings demonstrated that NSAIDs induced apoptosis in rat OS cells in vitro. Further research focusing on the potential cytotoxicity of NSAIDs in vivo is needed.

Selection of reference genes for quantitative real-time polymerase chain reaction studies in rat osteoblasts.

Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful tool to evaluate gene expression, but its accuracy depends on the choice and stability of the reference genes used for normalization. In this study, we aimed to identify reference genes for studies on osteoblasts derived from rat bone marrow mesenchymal stem cells (bone marrow osteoblasts), osteoblasts derived from newborn rat calvarial (calvarial osteoblasts), and rat osteosarcoma cell line UMR-106. The osteoblast phenotype was characterized by ALP activity and extracellular matrix mineralization. Thirty-one candidates for reference genes from a Taqman® array were assessed by qRT-PCR, and their expressions were analyzed by five different approaches. The data showed that several of the most traditional reference genes, such as Actb and Gapdh, were inadequate for normalization and that the experimental conditions may affect gene stability. Eif2b1 was frequently identified among the best reference genes in bone marrow osteoblasts, calvarial osteoblasts, and UMR-106 osteoblasts. Selected stable and unstable reference genes were used to normalize the gene expression of Runx2, Alp, and Oc. The data showed statistically significant differences in the expression of these genes depending on the stability of the reference gene used for normalization, creating a bias that may induce incorrect assumptions in terms of osteoblast characterization of these cells. In conclusion, our study indicates that a rigorous selection of reference genes is a key step in qRT-PCR studies in osteoblasts to generate precise and reliable data.

The Effect of (−)‐Epigallocatechin‐3‐gallate (EGCG) on Cell Viability and Protein Expression in Human Pancreatic Cancer Cells (Panc‐1) and Rat Osteosarcoma Cell Cultures (UMR 106‐01 BSP)

Traditional treatment of cancer includes surgery, radiation, and chemotherapy. There are many studies regarding how cancer cells respond to various treatments. However, not many studies have been done on how cancer cells respond to natural compounds such as (−)‐Epigallocatechin‐3‐gallate (EGCG), a potent ingredient of green tea. In this study, it was hypothesized that EGCG inhibits cell viability of both human pancreatic (Panc‐1) and rat osteosarcoma, UMR 106‐01 BSP (UMR) cell cultures, inhibits the expression of Panc‐1 CXCR‐4 and VEGF‐A proteins in Panc‐1 cell cultures, and the expression of the oncoproteins MYC and PD‐L1 in UMR cell cultures. Briefly, Panc‐1 and UMR cell cultures were routinely passaged, treated with different concentrations of EGCG (0, 20, 40, 60, 80 and 100 μM) in 6 well plates, then incubated for 48 hours at 37 degrees Celsius under humidification and 5% carbon dioxide infusion. At the end of the experiment, all cells were counted using the LUNA Automated Cell Counter (Logos Biosystems). Upon electrophoresis of experimental samples, Western blotting technique was employed to identify the expression of the Panc‐1 proteins (CXCR‐4, VEGFA) and the UMR oncoproteins (MYC and PD‐L1). F test ANOVA was used to compare variances and all values in the results were expressed as means ±SD. Cell viability results from the Panc‐1 and UMR cell lines showed a decrease at the higher concentrations (80 and 100 μM EGCG). The results of the Western blotting technique showed that the expression of Panc‐1 proteins and UMR oncoproteins were inhibited when treated with EGCG. The cell viability results suggest that EGCG may provide a therapeutic effect on both cancer cells lines at higher concentrations. Results of the Western blotting suggest that EGCG may exert its inhibitory effects on Panc‐1 and UMR cell viability through the regulation of Panc‐1 proteins and UMR oncoproteins.Support or Funding InformationThis work was made possible by support from the Ryckman Endowment Student Research Fund of the Biology Department of La Sierra University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Effects of Irrigants on the Survival of Human Stem Cells of the Apical Papilla, Including Endocyn

IntroductionEndocyn, a pH-neutral solution of hypochlorous acid and hypochlorite has been developed for use as an endodontic irrigant. The purpose of this study was to evaluate the effect of Endocyn on human periodontal ligament (PDL) fibroblasts, rat osteosarcoma cells (UMR-106), and stem cells of the apical papilla (SCAP) compared with other commonly used endodontic irrigants. MethodsTo determine cytotoxicity, cells were exposed to various concentrations of Endocyn, 6% sodium hypochlorite (NaOCl), 17% EDTA, and 2% chlorhexidine for 10 minutes, 1 hour, or 24 hours. Cell survival was measured fluorescently using calcein AM. Endocyn also was tested for its ability to inhibit SCAP proliferation and alkaline phosphatase activity. Finally, SCAP transcript expression was examined via reverse-transcriptase polymerase chain reaction. ResultsEndocyn was no more toxic to PDL and UMR cells than water for up to 24 hours. Endocyn concentrations of 50% were toxic to SCAP after 1 hour of exposure. Endocyn concentrations of >20% inhibited SCAP proliferation, whereas concentrations of ≥10% inhibited alkaline phosphatase activity. Exposure of SCAP to 10% Endocyn for 3 days did not alter most transcript expression, but did significantly reduce the expression of alkaline phosphatase, fibromodulin, and osteomodulin. ConclusionEndocyn was significantly less cytotoxic to PDL, UMR-106, and SCAP cells compared with other commonly used endodontic irrigants. High concentrations of Endocyn did inhibit some transcript expression and alkaline phosphatase activity, indicating a potential reduction in the osteogenic potential of stems cells exposed to Endocyn.

Bone-targeting parathyroid hormone conjugates outperform unmodified PTH in the anabolic treatment of osteoporosis in rats.

Synthetic parathyroid hormone (PTH) is clinically indicated for the treatment of osteoporosis, through its anabolic effects on parathyroid hormone receptors (PTHRs), located on osteoblast cells. However, the bioavailability of PTH for bone cells is restricted by the short half-life of PTH and the widespread distribution of PTHRs in non-skeletal tissues. To impart affinity for mineralized bone surfaces, bisphosphonate (BP)-mediated PTH analogues were synthesized, characterized, and evaluated in vitro and in vivo. The successful synthesis of PTH-PEG-BP was identified on MALDI-ToF mass spectra; bone-targeting potential was evaluated by hydroxyapatite binding test; and receptor bioactivity was assessed in UMR-106 (rat osteosarcoma) cells that constitutively express PTHRs. Therapeutic efficacy was evaluated using ovariectomized rats that remained untreated for 8weeks to allow development of osteopenia. Those rats then received daily subcutaneous injections of PTH-PEG-BP, thiol-BP vehicle, or unmodified PTH, and compared to sham-operated healthy rats at 0, 4, 8, 12, and 16weeks. In vivo micro-CT was conducted on the proximal tibial metaphysis to measure microstructural bone parameters, and new bone formation was detected using dynamic labeling. Bone strength was assessed using three-point bending mechanical testing. Our study determined that PTH-PEG-BP conjugates significantly enhanced PTH targeting to the bone matrix while retaining full PTH bioactivity. Moreover, PTH-PEG-BP conjugates significantly increased trabecular bone quality, anabolic bone formation, and improved bone strength over systemically administered PTH alone. We highlight the promise of a novel class of bone-targeting anabolic compound for the treatment of osteoporosis and related bone disorders.

Metabolism of selective 20-epi-vitamin D3 analogs in rat osteosarcoma UMR-106 cells: Isolation and identification of four novel C-1 fatty acid esters of 1α,25-dihydroxy-16-ene-20-epi-vitamin D3

Analogs of 1α,25-dihydroxyvitamin D3 (S1) with 20-epi modification (20-epi analogs) possess unique biological properties. We previously reported that 1α,25-dihydroxy-20-epi-vitamin D3 (S2), the basic 20-epi analog is metabolized into less polar metabolites (LPMs) in rat osteosarcoma cells (UMR-106) but not in a perfused rat kidney. Furthermore, we also noted that only selective 20-epi analogs are metabolized into LPMs. For example, 1α,25-dihydroxy-16-ene-20-epi-vitamin D3 (S4), but not 1α,25-dihydroxy-16-ene-23-yne-20-epi-vitamin D3 (S5) is metabolized into LPMs. In spite of these novel findings, the unequivocal identification of LPMs has not been achieved to date. We report here on a thorough investigation of the metabolism of S4 in UMR-106 cells and isolated two major LPMs produced directly from the substrate S4 itself and two minor LPMs produced from 3-epi-S4, a metabolite of S4 produced through C-3 epimerization pathway. Using GC/MS, ESI-MS and 1H NMR analysis, we identified all the four LPMs of S4 as 25-hydroxy-16-ene-20-epi-vitamin D3-1-stearate and 25-hydroxy-16-ene-20-epi-vitamin D3-1-oleate and their respective C-3 epimers. We report here for the first time the elucidation of a novel pathway of metabolism in UMR-106 cells in which both 1α,25(OH)2-16-ene-20-epi-D3 and 1α,25(OH)2-16-ene-20-epi-3-epi-D3 undergo C-1 esterification into stearic and oleic acid esters.

A New Modality for Cancer Treatment--Nanoparticle Mediated Microwave Induced Photodynamic Therapy.

Photodynamic therapy (PDT) has attracted ever-growing attention as a promising modality for cancer treatment. However, due to poor tissue penetration by light, photodynamic therapy has rarely been used for deeply situated tumors. This problem can be solved if photosensitizers are activated by microwaves (MW) that are able to penetrate deeply into tissues. Here, for the first time, we report microwave-induced photodynamic therapy and exploit copper cysteamine nanoparticles as a new type of photosensitizer that can be activated by microwaves to produce singlet oxygen for cancer treatment. Both in vitro and in vivo studies on a rat osteosarcoma cell line (UMR 106-01) have shown significant cell destruction using copper cysteamine (Cu-Cy) under microwave activation. The heating effects and the release of copper ions from Cu-Cy upon MW stimulation are the main mechanisms for the generation of reactive oxygen species that are lethal bullets for cancer destruction. The copper cysteamine nanoparticle-based microwave-induced photodynamic therapy opens a new door for treating cancer and other diseases.

Rat Osteosarcoma Cells as a Therapeutic Target Model for Osteoregeneration via Sclerostin Knockdown

There are various conceptually different strategies to improve bone regeneration and to treat osteoporosis, each with distinct inherent advantages and disadvantages. The use of RNA interference strategies to suppress the biological action of catabolic factors or antagonists of osteogenic proteins is promising, and such strategies can be applied locally. They are comparably inexpensive and do not suffer from stability problems as protein-based approaches. In this study, we focus on sclerostin, encoded by the SOST gene, a key regulator of bone formation and remodeling. Sclerostin is expressed by mature osteocytes but also by late osteogenically differentiated cells. Thus, it is difficult and requires long-term cultures to investigate the effects of SOST silencing on the expression of osteogenic markers using primary cells. We, therefore, selected a rat osteosarcoma cell line, UMR-106, that has been shown to express SOST and secrete sclerostin in a comparable fashion as late osteoblasts and osteocytes. We investigated the effects of differentiating supplements on SOST expression and sclerostin secretion in UMR-106 cells and found that addition of 100 ng/ml of bone morphogenetic protein (BMP)-2 strongly induced sclerostin secretion, whereas dexamethasone inhibited secretion. Effects of silencing SOST in UMR-106 cells cultured in various differentiation media including BMP-2 and/or dexamethasone were determined next with the aim to find promising test conditions for a readout system for the evaluation of future small interfering RNA release formulations for local induction of bone formation. We found a direct correlation between attenuated SOST expression and an increase in the osteogenic potential of UMR-106 cells. The combination of SOST silencing and BMP-2 could synergistically improve osteogenic factors. A lowered proliferation rate in silenced groups may indicate a faster switch to differentiation.

Communication of cAMP by connexin43 gap junctions regulates osteoblast signaling and gene expression

Connexin43 (Cx43) containing gap junctions play an important role in bone homeostasis, yet little is known about the second messengers communicated by Cx43 among bone cells. Here, we used MC3T3-E1 pre-osteoblasts and UMR106 rat osteosarcoma cells to test the hypothesis that cAMP is a second messenger communicated by bone cells through Cx43 containing gap junctions in a manner that is sufficient to impact osteoblast function. Overexpression of Cx43 markedly enhanced the activity of a cAMP-response element driven transcriptional luciferase reporter (CRE-luc) and increased phospho-CREB and phospho-ERK1/2 levels following expression of a constitutively active Gsα or by treatment with prostaglandin E2 (PGE2), 3-Isobutyl–1-methyl xanthine (IBMX) or forskolin. The Cx43-dependent potentiation of signaling in PGE2 treated cells was not accompanied by a further increase in cAMP levels, suggesting that the cAMP was shared between cells rather than Cx43 enhancing cAMP production. To support this, we developed a novel assay in which one set of cells expressing constitutively active Gsα (donor cells) were co-cultured with a second set of cells expressing a CRE-luc reporter (acceptor cells). Using this assay, activation of a CRE-luc reporter in the acceptor cells was both Cx43- and cell contact-dependent, indicating communication of cAMP among cells. Finally, we showed that Cx43 increased the cAMP-dependent mRNA expression of receptor activator of nuclear factor kappa B ligand (RANKL) and enhanced the repression of the sclerostin mRNA, implying a potential mechanism for the modulation of tissue remodeling. In total, these data demonstrate that Cx43 can communicate cAMP between cells and, more importantly, that the communicated cAMP is sufficient to impact signal transduction cascades and the expression of key bone effector molecules between interconnected cells.

Preparation of Composite Electrospun Membranes Containing Strontium‐Substituted Bioactive Glasses for Bone Tissue Regeneration

Barrier membranes used for the treatment of bone tissue defects caused by periodontitis lack the ability to promote new bone tissue regeneration. However, the addition of an osteogenic component to membranes may enhance their regenerative potential. Here the manufacturing of composite membranes made of poly(caprolactone) and strontium‐substituted bioactive glass is described using the solution‐electrospinning technique, with particles located both inside and on the surface of the fibers. All membranes are characterized using scanning electron microscopy and energy dispersive X‐ray spectroscopy, and glass dissolution from within the fibers is investigated in water. In vitro material cytotoxicity is determined using a rat osteosarcoma cell line. Electrospun fibers exhibit porous surfaces and regions of increased diameter where the particles are accumulated. The glass dissolves after immersion in water, releasing dissolution products that are associated with increased pH. Further evidence suggests accelerated polymer degradation due to interactions between both components, which may provide the additional benefit of reducing the pH changes associated with glass dissolution. All compositions are biocompatible in vitro, with the exception of membranes with >50 μg of glass on their surface. In conclusion, these membranes show great potential for bone healing applications, including guided bone regeneration and scaffolds for musculoskeletal tissue engineering. image

Development of three-dimensional tissue engineered bone-oral mucosal composite models.

Tissue engineering of bone and oral mucosa have been extensively studied independently. The aim of this study was to develop and investigate a novel combination of bone and oral mucosa in a single 3D in vitro composite tissue mimicking the natural structure of alveolar bone with an overlying oral mucosa. Rat osteosarcoma (ROS) cells were seeded into a hydroxyapatite/tri-calcium phosphate scaffold and bone constructs were cultured in a spinner bioreactor for 3 months. An engineered oral mucosa was fabricated by air/liquid interface culture of immortalized OKF6/TERET-2 oral keratinocytes on collagen gel-embedded fibroblasts. EOM was incorporated into the engineered bone using a tissue adhesive and further cultured prior to qualitative and quantitative assessments. Presto Blue assay revealed that ROS cells remained vital throughout the experiment. The histological and scanning electron microscope examinations showed that the cells proliferated and densely populated the scaffold construct. Micro computed tomography (micro-CT) scanning revealed an increase in closed porosity and a decrease in open and total porosity at the end of the culture period. Histological examination of bone-oral mucosa model showed a relatively differentiated parakeratinized epithelium, evenly distributed fibroblasts in the connective tissue layer and widely spread ROS cells within the bone scaffold. The feasibility of fabricating a novel bone-oral mucosa model using cell lines is demonstrated. Generating human ‘normal’ cell-based models with further characterization is required to optimize the model for in vitro and in vivo applications.

Abstract 2222: Possible involvement of senescence bypass in mesenchymal stem cells for sarcomagenesis identified through a comparative gene expression profiling in rat sarcoma model

Abstract INTRODUCTION. Mesenchymal stem cells (MSCs) exist in a variety of tissues such as bone marrow and adipose tissue contributing tissue repair and maintenance, and have been used in clinical cell transplantation therapy. On the other hand, MSCs are also believed to be one of the cell of origins for certain sarcomas including osteosarcoma and malignant fibrous histiocytoma/undifferentiated pleomorphic sarcoma (MFH/UPS). To identify the signaling pathways that are involved in sarcoma pathogenesis, we compared gene expression profiles in rat osteosarcoma and MFH cells with those in syngeneic rat MSCs. MATERIALS & METHODS. Rat osteosarcoma cell line COS1NR and MFH cell line MFH1NR, that were established in our laboratory from chemically induced tumors by 4-hydroxy(amino)-quinolin 1-oxide, and syngeneic rat MSCs isolated from rat femur bone marrow were used in this study. To investigate the difference of gene expression between sarcoma cells and MSCs, gene expression profiling was performed by Agilent array analysis. Data analysis was carried out using Agilent Feature Extraction software, analyzing pathways that were differentially expressed in sarcoma cells compared to MSCs. RESULTS & DISCUSSION. Analysis of genes that characterize MSCs such as CD44, CD105, CD73 and CD90 showed higher expression in MSCs compared to sarcomas. Pathways involved in focal adhesion, cytokines, extracellular matrix-receptors and Wnt signaling were down-regulated in both sarcomas. In those factors, the genes involved in senescence acceleration in MSCs including Wnt4 and 5a, Lef1/tcf, β-catenin, p21Cip1, lysophosphatidic acid receptor 1 (LPAR1) are significantly down-regulated in both sarcomas. Meanwhile, DNA replication, cell cycle, mismatch repair, hedgehog signaling and metabolic pathways were up-regulated in both sarcomas and those include the genes involved in inhibiting senescence of MSCs such as DKK1, SOD1, FOXO4 and SIRT1. These results indicated that senescence factors for MSCs, which several previous reports suggested, such as Wnt pathways, p21Cip1 and CDKs-cyclins expression are mal-functionally regulated in sarcomas, and those have been suggested the involvement of cellular senescence in MSCs as the responses to oxidative stress and DNA damages. Taken together, acquiring the ability to bypassing senescence in MSCs is possibly involved in sarcomagenesis. CONCLUSIONS. The current study suggests that the loss of senescence mechanism in MSCs might be involved in sarcomagenesis. Further study will clarify the critical pathways containing the driver genes in sarcoma development upon the data that current results indicated and targeting those genes and pathways will be beneficial to develop novel treatment for sarcomas. Citation Format: Kanya Honoki, Hiromasa Fujii, Akira Kido, Shinji Tsukamoto, Toshio Mori, Yasuhito Tanaka, Toshifumi Tsujiuchi. Possible involvement of senescence bypass in mesenchymal stem cells for sarcomagenesis identified through a comparative gene expression profiling in rat sarcoma model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2222. doi:10.1158/1538-7445.AM2015-2222

Nanoparticle-conjugated aptamer targeting hnRNP A2/B1 can recognize multiple tumor cells and inhibit their proliferation

In this study, we further investigated a previously developed aptamer targeting ROS 17/2.8 (rat osteosarcoma) cells. We found that this C6-8 aptamer specifically binds to heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 and that it specifically labeled multiple tumor-cell lines as effectively as hnRNP A2/B1 monoclonal antibodies. When conjugated with fluorescent carbon nanodots (CDots) it could freely enter multiple living tumor cell lines (HepG2, MCF-7, H1299, and HeLa), whose growth it inhibited by targeting hnRNP A2/B1. Similar inhibitory effects were observed when the GFP-HepG2 hepatocarcinoma cells treated with C6-8-conjugated CDots were implanted in nude mice. Our work provides a new aptamer for targeting/labeling multiple tumor cell types, and its nanoparticle conjugates bring further advantages that increase its potential for use in cancer diagnosis and therapy.

Ubiquilin 2 enhances osteosarcoma progression through resistance to hypoxic stress.

Ubiquilin 2 (UBQLN2), a member of the ubiquitin-like protein family (ubiquilins), maintains protein homeostasis. Although UBQLN2 has been implicated in the pathogenesis of neurodegenerative diseases, it is also associated with malignant tumors. Therefore, we examined whether UBQLN2 plays a role in human osteosarcoma. The human osteosarcoma cell line MG63 was transfected with UBQLN2 siRNA and cultured under hypoxic conditions. The rat osteosarcoma cell line COS1NR was inoculated into Fischer 344 rats, followed by injection of UBQLN2 siRNA with atelocollagen. An immunohistochemical analysis of UBQLN2 was performed using 34 cases of human high-grade osteosarcomas, and metastasis-free survival was estimated by the Kaplan-Meier method. Silencing of UBQLN2 by siRNA transfection under hypoxia led to activation of JNK and p38, resulting in induction of apoptosis in the osteosarcoma cell line MG63. Injection of UBQLN2 siRNA suppressed tumor growth in the rat osteosarcoma model, followed by apoptosis induction. The immunohistochemical examination revealed that high UBQLN2 expression was significantly associated with the unfavorable metastasis-free survival of osteosarcoma patients. UBQLN2 plays an important role in resistance to hypoxic stress and enhances tumor progression in osteosarcoma. UBQLN2 may be a new molecular target for chemotherapeutics and a useful clinicopathological marker in human osteosarcoma.

Mechanism of rat osteosarcoma cell apoptosis induced by a combination of low-intensity ultrasound and 5-aminolevulinic acid in vitro

We investigated the killing effect of low-intensity ultrasound combined with 5-aminolevulinic acid (5-ALA) on the rat osteosarcoma cell line UMR-106. Logarithmic-phase UMR-106 cells were divided into a control group, ultrasound group and 5-ALA group. The cell apoptotic rate, production of reactive oxygen species, and the change in mitochondrial membrane potential were analyzed by flow cytometry; ultrastructural changes were observed by transmission electron microscopy. Using low-intensity ultrasound at 1.0 MHz and 2.0 W/cm(2) plus 5-ALA at a concentration of 2 mM, the apoptotic rate of the sonodynamic therapy group was 27.2 ± 3.4% which was significantly higher than that of the control group, ultrasound group, and 5-ALA group (P < 0.05). The production of reactive oxygen species was 32.6 ± 2.2% and the decrease in mitochondrial membrane potential was 39.5 ± 2.5%. The 33342 staining showed nuclear condensation and fragmentation in the ultrasound group and 5-ALA group. Structural changes in the cell membrane, mitochondria, Golgi apparatus, and other organelles observed by transmission electron microscopy included formation of apoptotic bodies. The killing effect of low-intensity ultrasound combined with 5-ALA on UMR-106 cells was significant. Cell apoptosis played a vital role in the killing effect, and the mitochondria pathway contributed to the apoptosis of UMR-106 cells.