Metastatic Osteosarcoma Cells Research Articles

Activation of TLR4 signaling inhibits progression of osteosarcoma by stimulating CD8-positive cytotoxic lymphocytes.

Osteosarcoma (OS) is the most common malignant bone tumor and the prognosis of advanced cases is still poor. Recently, there have been several reports suggesting the relationship between innate immunity and OS, but the detailed mechanism is unknown. We demonstrate the relationship between OS and Toll-like receptor 4 (TLR4) which is one of the most important factors in innate immunity. We established a syngenic mouse tumor model using C3H/HeN, C3H/HeJ mouse and a highly metastatic OS cell line, LM8. TLR4 activation with lipopolysaccharide (LPS) was performed on both mice and its influence on the progression of OS was evaluated. We also performed CD8 + cells depletion to examine the influence on TLR4 activation effects. Tumor volume of C3H/HeN mice was significantly smaller and overall survival of C3H/HeN mice was significantly longer than C3H/HeJ mice. We found more CD8+ cells infiltrating in lung metastases of C3H/HeN mice and depletion of CD8+ cells canceled the antitumor effects of LPS. TLR4 activation by LPS increased CD8+ cells infiltrating into lung metastases and suppressed OS progression in the mouse model. TLR4 activation may suppress the progression of OS via stimulating CD8+ cells and can be expected as a novel treatment for OS.

Mangiferin suppresses human metastatic osteosarcoma cell growth by down-regulating the expression of metalloproteinases-1/2 and parathyroid hormone receptor 1

The study evaluates the protective effect of mangiferin on osteosarcoma cell proliferation and metastasis. Saos-2 and U2OS cells were treated with mangiferin (25, 50, 75 and 100 µM) for 72 h. Mangiferin reduced the cell viability, invasion, and cell adhesion and migration rate. Matrix metalloproteinases-2/9 (MMP-2/9) mRNA expression was reduced significantly, while the levels of tissue inhibitors of metalloproteinases-1/2 (TIMP-1/2) were elevated in Saos-2 and U2OS cells. Mangiferin treatment significantly reduced parathyroid hormone receptor 1 (PTHR1) mRNA and protein expression by more than 0.5-fold in both osteosarcoma cells. In addition, the immunofluorescent analysis also showed decreased PTHR1 expression following treatment with mangiferin. In summary, we have demonstrated that treatment with mangiferin reduces cell viability, proliferation, invasion, adhesion and migration, and induces apoptosis of osteosarcoma cells. Therefore, treatment with mangiferin can be effective agent in inhibiting growth and inducing apoptosis in osteosarcoma cells. Our experimental results provide evidence for the therapeutic effect of mangiferin in osteosarcoma cells.

Acquisition of stem associated-features on metastatic osteosarcoma cells and their functional effects on mesenchymal stem cells

BackgroundOsteosarcoma (OS) is the most frequent malignant bone tumor, affecting predominantly children and young adults. Metastases are a major clinical challenge in OS. In this context, 20% of OS patients are diagnosed with metastatic OS, but near 80% of all OS patients could present non-detectable micrometastases at the moment of diagnosis. MethodsOsteogenic differentiation; doxorubicin exclusion assay; fluorescence microscopy; RT-qPCR; proteomic analysis. ResultsOur results suggest that metastatic OS cells possess a diminished osteoblastic differentiation potential with a gain of metastatic traits like the capacity to modify intracellular localization of chemodrugs and higher levels of expression of stemness-related genes. On the opposite hand, non-metastatic OS cells possess bone-associated traits like higher osteoblastic differentiation and also an osteoblastic-inducer secretome. OS cells also differ in the nature of their interaction with mesenchymal stem cells (MSCs), with opposites impacts on MSCs phenotype and behavior. ConclusionsAll this suggests that a major trait acquired by metastatic cells is a switch into a stem-like state that could favor its survival in the pulmonary niche, opening new possibilities for personalized chemotherapeutic schemes. General significanceOur work provides new insights regarding differences among metastatic and non-metastatic OS cells, with particular emphasis on differentiation potential, multidrug resistance and interaction with MSCs.

Exosomal communication by metastatic osteosarcoma cells modulates alveolar macrophages to an M2 tumor-promoting phenotype and inhibits tumoricidal functions

ABSTRACT Osteosarcoma metastasizes to the lung, and there is a link between the predominance of tumor-promoting immunosuppressive M2 macrophages in the metastases and poor patient survival. By contrast, M1 macrophage predominance correlates with longer survival. M2 macrophages can be induced by various stimuli in the tumor microenvironment, including exosomes, which are 40- to 150-nm vesicles that are involved in intercellular communication and contribute to tumor progression and immune evasion. Recognizing that tumor cells can influence the tumor microenvironment to make it more permissive and because of the link between M2 dominance and curtailed patient survival, we evaluated the effect of exosomes from non-metastatic K7 and Dunn osteosarcoma cells and the metastatic sublines K7M3 and DLM8 on macrophage phenotype and function. Incubating MHS mouse alveolar macrophages with K7M3 and DLM8 exosomes induced expression of IL10, TGFB2, and CCL22 mRNA (markers of M2 macrophages) and decreased phagocytosis, efferocytosis, and macrophage-mediated tumor cell killing. In contrast, exosomes from non-metastatic K7 or Dunn cells did not inhibit phagocytosis, efferocytosis, and macrophage-mediated cytotoxicity or induce increased expression of IL10, TGFB2 or CCL22 mRNA. In addition, metastatic osteosarcoma cell exosomes significantly increased the secretion of TGFB2, a key signaling pathway associated with tumor- mediated immune suppression. Finally, the inhibition of TGFB2 reversed the suppressive activity of alveolar macrophages exposed to metastatic osteosarcoma cell exosomes. Our data suggest that the exosomes from metastatic osteosarcoma cells can modulate cellular signaling of tumor-associated macrophages, thereby promoting the M2 phenotype and creating an immunosuppressive, tumor-promoting microenvironment through the production of TGFB2.

Inhibition of ATR-Chk1 signaling blocks DNA double-strand-break repair and induces cytoplasmic vacuolization in metastatic osteosarcoma.

Background:Ataxia-telangiectasia and Rad3 related protein kinase (ATR) is an essential regulator of the DNA damage response in various cancers; however, its expression and roles in osteosarcoma are unclear. We therefore chose to evaluate the significance and mechanism of ATR in metastatic osteosarcoma, as well as its potential to be a therapeutic target.Methods:The osteosarcoma tissue microarrays constructed from 70 patient specimens underwent immunohistochemistry to quantify ATR and activated phospho-ATR (pATR) expression and their correlation with clinical outcomes. ATR sublocalization within the metastatic osteosarcoma cells was confirmed by immunofluorescence assay. Cell proliferation, apoptosis, and migration were evaluated following treatment with ATR siRNA or the selective inhibitor Berzosertib. Antitumor effects were determined with ex vivo three-dimensional (3D) culture models, and the impacts on the DNA damage repair pathways were measured with Western blotting.Results:Elevated ATR and activated pATR expression correlated with shorter patient survival and less necrosis following neoadjuvant chemotherapy. Intranuclear sublocalization of ATR and pATR suggested a mechanism related to DNA replication. ATR knockdown with siRNA or inhibition with Berzosertib suppressed cell proliferation in a time- and dose-dependent manner and induced apoptosis. In addition, ATR inhibition decreased Chk1 phosphorylation while increasing γH2AX expression and PARP cleavage, consistent with the interference of DNA damage repair. The ATR inhibitor Berzosertib also produced the characteristic cytoplasmic vacuolization preceding cell death, and suppressed ex vivo 3D spheroid formation and cell motility.Conclusion:The faithful dependence of cells on ATR signaling for survival and progression makes it an emerging therapeutic target in metastatic osteosarcoma.

Receptor Tyrosine Kinases in Osteosarcoma: 2019 Update.

The primary conclusions of our 2014 contribution to this series were as follows: Multiple receptor tyrosine kinases (RTKs) likely contribute to aggressive phenotypes in osteosarcoma and, therefore, inhibition of multiple RTKs is likely necessary for successful clinical outcomes. Inhibition of multiple RTKs may also be useful to overcome resistance to inhibitors of individual RTKs as well as resistance to conventional chemotherapies. Different combinations of RTKs are likely important in individual patients. AXL, EPHB2, FGFR2, IGF1R, and RET were identified as promising therapeutic targets by our in vitro phosphoproteomic/siRNA screen of 42 RTKs in the highly metastatic LM7 and 143B human osteosarcoma cell lines. This chapter is intended to provide an update on these topics as well as the large number of osteosarcoma clinical studies of inhibitors of multiple tyrosine kinases (multi-TKIs) that were recently published.

PTEN loss activates a functional AKT/CXCR4 signaling axis to potentiate tumor growth and lung metastasis in human osteosarcoma cells.

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents. Loss of the tumor suppressor PTEN or activation of chemokine receptor CXCR4 has been demonstrated to associate with OS respectively. However, the signaling mechanism underlying PTEN-mediated antitumor effect remains largely unknown, and the crosstalk between PTEN and CXCR4 in OS has not been investigated. Here, we uncover a PTEN/AKT/CXCR4 pathway nexus in highly tumorigenic and metastatic human 143B OS cells. Loss of PTEN activates AKT/CXCR4 signaling axis and regulates a series of tumor cell behaviors. Notably, ERK is inversely regulated by PTEN and its activation occurs downstream of AKT but upstream of CXCR4, suggesting this kinase to be an important mediator between AKT and CXCR4. In vivo studies show that overexpression of PTEN dramatically attenuates bone destruction, and this inhibition is associated with reduced CXCR4 expression in tumors. CXCR4 inhibitor AMD3100 also markedly suppresses tumor growth in the bone. In addition, PTEN overexpression or AMD3100 substantially inhibits tumor expansion in the lung. Our studies highlight a novel PTEN/AKT/CXCR4 signaling nexus in OS tumor growth and lung metastasis, and provide a strong rationale to consider PTEN restoration or CXCR4 blockade for the treatment of aggressive OS in humans.

A unique multidrug nanomedicine made of squalenoyl-gemcitabine and alkyl-lysophospholipid edelfosine

Among anticancer nanomedicines, squalenoyl nanocomposites have obtained encouraging outcomes in a great variety of tumors. The prodrug squalenoyl-gemcitabine has been chosen in this study to construct a novel multidrug nanosystem in combination with edelfosine, an alkyl-lysophopholipid with proven anticancer activity. Given their amphiphilic nature, it was hypothesized that both anticancer compounds, with complementary molecular targets, could lead to the formation of a new multitherapy nanomedicine. Nanoassemblies were formulated by the nanoprecipitation method and characterized by dynamic light scattering, transmission electron microscopy and X-ray photoelectron spectroscopy. Because free edelfosine is highly hemolytic, hemolysis experiments were performed using human blood erythrocytes and nanoassemblies efficacy was evaluated in a patient-derived metastatic pediatric osteosarcoma cell line. It was observed that these molecules spontaneously self-assembled as stable and monodisperse nanoassemblies of 51 ± 1 nm in a surfactant/polymer free-aqueous suspension. Compared to squalenoyl-gemcitabine nanoassemblies, the combination of squalenoyl-gemcitabine with edelfosine resulted in smaller particle size and a new supramolecular conformation, with higher stability and drug content, and ameliorated antitumor profile.

Ex vivo screen identifies CDK12 as a metastatic vulnerability in osteosarcoma.

Despite progress in intensification of therapy, outcomes for patients with metastatic osteosarcoma (OS) have not improved in thirty years. We developed a system that enabled preclinical screening of compounds against metastatic OS cells in the context of the native lung microenvironment. Using this strategy to screen a library of epigenetically targeted compounds, we identified inhibitors of CDK12 to be most effective, reducing OS cell outgrowth in the lung by more than 90% at submicromolar doses. We found that knockout of CDK12 in an in vivo model of lung metastasis significantly decreased the ability of OS to colonize the lung. CDK12 inhibition led to defects in transcription elongation in a gene length- and expression-dependent manner. These effects were accompanied by defects in RNA processing and altered the expression of genes involved in transcription regulation and the DNA damage response. We further identified OS models that differ in their sensitivity to CDK12 inhibition in the lung and provided evidence that upregulated MYC levels may mediate these differences. Our studies provided a framework for rapid preclinical testing of compounds with antimetastatic activity and highlighted CDK12 as a potential therapeutic target in OS.

KMT2C modulates migration and invasion processes in osteosarcoma cell lines

In this study we investigated the role of KMT2C (a chromatin-modifying and remodelling protein) in osteosarcoma progression through cell migration and invasion assays in osteosarcoma primary and metastatic cell lines. Wound healing and transwell assays were used to detect changes of cell migration and matrigel assay was used to evaluate changes of cell invasion in primary and metastatic osteosarcoma cell lines after KMT2C siRNA transfection. We found that primary osteosarcoma cell lines showed the highest capacity of migration before mRNA KMT2C silencing and the highest capacity of invasion after mRNA KMT2C silencing; on the contrary, osteosarcoma metastatic cell line showed the highest capacity of migration after mRNA KMT2C silencing and the highest capacity of invasion before mRNA KMT2C silencing.Our study supports data in favour of selective enhancer changes, KMT2C-mediated, in metastatic osteosarcoma probably due to the different microenvironment between primary and metastatic sites.

Abstract 1995: Low-dose eribulin suppresses lung metastasis of osteosarcoma in vitro and in vivo

Abstract Osteosarcoma is the most common malignant bone tumor among children and adolescents. The prognosis of osteosarcoma remains poor in these thirty years because the effective therapeutic option for lung metastasis has not been established. Therefore, the development of effective metastatic therapy is required. Eribulin, a new microtubule targeting agent, has been indicated for recurrent breast cancer and malignant soft tissue sarcoma. Clinical trials of eribulin showed that it elongated overall survival period rather than disease-free survival period. These results motivated us to investigate the effect of eribulin on metastasis suppression of osteosarcoma. Here, we examined the inhibitory effect of eribulin on lung metastasis and its mechanism using murine osteosarcoma metastasis model. In vivo experiments: Eribulin was intravenously injected by two administration methods, (1) high dose administration on standard schedule; 1 mg/kg q7d × 2, (2) frequent low dose administration; 0.3 mg/kg q4d × 4. Both methods sufficiently reduced circulating tumor cells in blood and inhibited lung metastasis. Method (1) also inhibited primary tumor growth, but induced severe body weight loss. Method (2) had little effect on primary tumor growth and showed little body weight loss. In vitro experiments: The IC50 concentration of eribulin for metastatic osteosarcoma cell line, LM8, was about 20 nM. Pharmacokinetics studies have shown that eribulin intravenously administered at 1 mg/kg presents with a brief high-concentration phase which surges to ≥100 nM followed by a long low-concentration phase which stabilizes at ~10 nM for one week. These two phases sandwiched the IC50 concentration of eribulin. Thus, we separately investigated that the effect of high and low eribulin concentrations on LM8. High eribulin concentrations induced cell cycle arrest and apoptosis in LM8, whereas low eribulin concentrations changed cell morphology and decreased cell migration through reducing directionality and focal adhesion turnover. Reduced peripheral localization of adenomatous polyposis coli (APC) protein by eribulin might cause these effects. In a three-dimensional collagen culture system, low eribulin concentrations inhibited tumor cell infiltration and colony formation. The anti-metastatic effects at low concentrations backed-up the results of our in vivo experiments by frequent low dose administration method. Eribulin is a potential therapeutic option for lung metastasis of osteosarcoma. Frequent low dose administration method with fewer side effects enables the combination with other chemotherapeutic agents and broadens the application of eribulin as an anti-metastatic drug for osteosarcoma. Citation Format: Kenta Watanabe, Yoshihiro Yui, Satoru Sasagawa, Kayo Suzuki, Masahiko Kanamori, Taketoshi Yasuda, Tomoatsu Kimura. Low-dose eribulin suppresses lung metastasis of osteosarcoma in vitro and in vivo [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 1995.

Paradoxical effects of JZL184, an inhibitor of monoacylglycerol lipase, on bone remodelling in healthy and cancer-bearing mice

BackgroundCancer-associated bone disease is a serious complication in bone sarcomas and metastatic carcinomas of breast and prostate origin. Monoacylglycerol lipase (MAGL) is an enzyme of the endocannabinoid system, and is responsible for the degradation of the most abundant endocannabinoid in bone, 2-arachidonoyl glycerol (2AG). MethodsThe effects of the verified MAGL inhibitor on bone remodelling were assessed in healthy mice and in mouse models of bone disease caused by prostate and breast cancers and osteosarcoma. FindingsJZL184 reduced osteolytic bone metastasis in mouse models of breast and prostate cancers, and inhibited skeletal tumour growth, metastasis and the formation of ectopic bone in models of osteosarcoma. Additionally, JZL184 suppressed cachexia and prolonged survival in mice injected with metastatic osteosarcoma and osteotropic cancer cells. Functional and histological analysis revealed that the osteoprotective action of JZL184 in cancer models is predominately due to inhibition of tumour growth and metastasis. In the absence of cancer, however, exposure to JZL184 exerts a paradoxical reduction of bone volume via an effect that is mediated by both Cnr1 and Cnr2 cannabinoid receptors. InterpretationMAGL inhibitors such as JZL184, or its novel analogues, may be of value in the treatment of bone disease caused by primary bone cancer and bone metastasis, however, activation of the skeletal endocannabinoid system may limit their usefulness as osteoprotective agents.

Exosomal communication by metastatic osteosarcoma cells contributes to the modulation of alveolar macrophages to an M2 tumor-promoting phenotype

Abstract Osteosarcoma (OS) metastasizes to the lung and there is a link between tumor-promoting M2 macrophage content and poorer patient survival. Conversely, high M1 content correlates with improved survival. M2 macrophages are immunosuppressive, contribute to tumor progression and metastasis, and can be induced by various stimuli in the tumor microenvironment, including exosomes (Exo). Exo are small 40–150 nm vesicles that are involved in inter-cellular communication, and contribute to tumor progression, increased tumor survival and immune evasion. Because of the link between M2 content and inferior patient survival, we evaluated the effect of Exo from non-metastatic K7 OS cells and the metastatic subline K7M3 on macrophage phenotype and function. Incubating MHS mouse alveolar macrophage cells with K7M3 Exo induced mRNA expression of IL10, TGFB2, IL1rα, and CCL22, (markers of M2 macrophages), and decreased phagocytosis, efferocytosis and macrophage-mediated tumor cell killing. In contrast, Exo from non-metastatic K7 or 3T3 normal fibroblast cells did not inhibit phagocytosis, efferocytosis or macrophage-mediated tumor cell killing. Our data suggest that the Exo from metastatic OS cells can modulate cellular signaling of tumor associated macrophages, thereby promoting the M2 phenotype and inhibiting their phagocytic and cytotoxic abilities. Identifying the factors within Exo from metastatic OS cells and the mechanisms responsible for Exo communication between metastatic OS cells and macrophages could reveal new therapeutic targets and opportunities to improve the treatment of patients with metastatic disease in the lung.

Quercetin suppresses the proliferation and metastasis of metastatic osteosarcoma cells by inhibiting parathyroid hormone receptor 1

Osteosarcoma is an aggressive malignant neoplasm and cancerous bone tumor. Quercetin is a well-known flavonoid abundant in vegetables, fruits, grains, leaves, and red onions. In the present study, we evaluated the effects of quercetin-induced inhibition of parathyroid hormone receptor 1 (PTHR1) on proliferation, migration, and invasion in U2OS and Saos-2 cells. Following incubation with quercetin (20, 40, 60, 80, or 100 μM) for 48 h, the cell viability of U2OS and Saos-2 cells were significantly reduced in a dose-dependent manner. Additionally, there were significant decreases in cell adhesion, invasion, and migration as well as reduced cell viability at higher concentrations of quercetin. Furthermore, the mRNA expression levels of matrix metalloproteinases (MMP)-2 and -9 were attenuated, whereas the mRNA expression levels of tissue inhibitors of metalloproteinases (TIMP)-1 and -2 were elevated. Quercetin treatment also significantly reduced the mRNA expression levels of PTHR1 by 0.27-, and 0.55-fold at 80, and 100 μM, respectively, whereas 0.19 and 0.41 folds in Saos-2 cells. PTHR1 protein expression in U2OS cells was reduced by 0.19-, and 0.43-fold at 80, and 100 μM of quercetin, respectively (P < 0.05), whereas 0.17 and 0.35 folds in Saos-2 cells. Immunofluorescence analyses revealed reduced expression of PTHR1 following treatment with quercetin. PTHR1 expression in U2OS cells was reduced by 0.18-, and 0.41-fold at 80, and 100 μM, respectively, whereas 0.15 and 0.38 folds in Saos-2 cells. The knockdown of PTHR1enhanced quercetin-inhibited proliferation and invasion. Taken together, the present findings indicate that quercetin reduced human metastatic osteosarcoma cell invasion, adhesion, proliferation, and migration by inhibiting PTHR1.

Anticancer Potential of Oleuropein, the Polyphenol of Olive Oil, With 2-Methoxyestradiol, Separately or in Combination, in Human Osteosarcoma Cells.

Oleuropein belongs to the potent polyphenols of olive oil. Notably, it is considered as a potentially active anticancer agent. Herein, the anticancer efficiency of oleuropein, when used separately and in combination with the chemotherapeutic agent, 2-methoxyestradiol (2-ME), was investigated in highly metastatic osteosarcoma (OS) cells. Human OS cells (143B OS cell line) were incubated with oleuropein and 2-ME, alone or in combination. Cell viability was determined by the MTT assay. Cell migration assays were used in order to determine the anti-migratory potential of the compounds, while their impact on autophagy was evaluated via the LC3-antibody-based detection assay. The interaction between oleuropein and 2-ME was determined via the CalcuSyn software. Both anti-migratory and anti-proliferative effects of oleuropein were demonstrated on human OS cells. Anticancer effects of oleuropein were significantly enhanced after 2-ME addition. Treatment of 143B OS cell with oleuropein, alone or in combination with 2-ME resulted in induction of autophagy. The obtained data suggest an anticancer effect of oleuropein, alone and in combination with 2-ME, on highly metastatic 143B OS cells. Notably, a synergism between oleuropein and 2-ME towards 143B OS cells was detected. The exact mechanism of this synergism needs to be further investigated; nonetheless, induction of nitro-oxidative stress and/or induction of autophagy are suggested.

Tegavivint and the β-Catenin/ALDH Axis in Chemotherapy-Resistant and Metastatic Osteosarcoma

The Wnt/β-catenin pathway is closely associated with osteosarcoma (OS) development and metastatic progression. We investigated the antitumor activity of Tegavivint, a novel β-catenin/transducin β-like protein 1 (TBL1) inhibitor, against OS employing in vitro, ex vivo, and in vivo cell line and patient-derived xenograft (PDX) models that recapitulate high risk disease. The antitumor efficacy of Tegavivint was evaluated in vitro using established OS and PDX-derived cell lines. Use of an ex vivo three-dimensional pulmonary metastasis assay assessed targeting of β-catenin activity during micro- and macrometastatic development. The in vivo activity of Tegavivint was evaluated using chemoresistant and metastatic OS PDX models. Gene and protein expression were quantified by quantitative Reverse transcription polymerase chain reaction or immunoblot analysis. Bone integrity was determined via microCT. All statistical tests were two-sided. Tegavivint exhibited antiproliferative activity against OS cells in vitro and actively reduced micro- and macrometastatic development ex vivo. Multiple OS PDX tumors (n = 3), including paired patient primary and lung metastatic tumors with inherent chemoresistance, were suppressed by Tegavivint in vivo. We identified that metastatic lung OS cell lines (n = 2) exhibited increased stem cell signatures, including enhanced concomitant aldehyde dehydrogenase (ALDH1) and β-catenin expression and downstream activity, which were suppressed by Tegavivint (ALDH1: control group, mean relative mRNA expression = 1.00, 95% confidence interval [CI] = 0.68 to 1.22 vs Tegavivint group, mean = 0.011, 95% CI = 0.0012 to 0.056, P < .001; β-catenin: control group, mean relative mRNA expression = 1.00, 95% CI = 0.71 to 1.36 vs Tegavivint group, mean = 0.45, 95% CI = 0.36 to 0.52, P < .001). ALDH1high PDX-derived lung OS cells, which demonstrated enhanced metastatic potential compared with ALDHlow cells in vivo, were sensitive to Tegavivint. Toxicity studies revealed decreased bone density in male Tegavivint-treated mice (n = 4 mice per group). Tegavivint is a promising therapeutic agent for advanced stages of OS via its targeting of the β-catenin/ALDH1 axis.

The relationship between metastatic potential and in vitro mechanical properties of osteosarcoma cells.

Osteosarcoma is the most frequent primary tumor of bone and is characterized by its high tendency to metastasize in lungs. Although treatment in cases of early diagnosis results in a 5-yr survival rate of nearly 60%, the prognosis for patients with secondary lesions at diagnosis is poor, and their 5-yr survival rate remains below 30%. In the present work, we have used a number of analytical methods to investigate the impact of increased metastatic potential on the biophysical properties and force generation of osteosarcoma cells. With that aim, we used two paired osteosarcoma cell lines, with each one comprising a parental line with low metastatic potential and its experimentally selected, highly metastatic form. Mechanical characterization was performed by means of atomic force microscopy, tensile biaxial deformation, and real-time deformability, and cell traction was measured using two-dimensional and micropost-based traction force microscopy. Our results reveal that the low metastatic osteosarcoma cells display larger spreading sizes and generate higher forces than the experimentally selected, highly malignant variants. In turn, the outcome of cell stiffness measurements strongly depends on the method used and the state of the probed cell, indicating that only a set of phenotyping methods provides the full picture of cell mechanics.

Global analysis of osteosarcoma lipidomes reveal altered lipid profiles in metastatic versus nonmetastatic cells

Osteosarcoma (OS) is the most common form of primary bone cancer in humans. The early detection and subsequent control of metastasis has been challenging in OS. Lipids are important constituents of cells that maintain structural integrity that can be converted into lipid-signaling molecules and are reprogrammed in cancerous states. Here, we investigate the global lipidomic differences in metastatic (143B) and nonmetastatic (HOS) human OS cells as compared with normal fetal osteoblast cells (FOB) using lipidomics. We detect 15 distinct lipid classes in all three cell lines that included over 1,000 lipid species across various classes including phospholipids, sphingolipids and ceramides, glycolipids, and cholesterol. We identify a key class of lipids, diacylglycerols, which are overexpressed in metastatic OS cells as compared with their nonmetastatic or nontumorigenic counterparts. As a proof of concept, we show that blocking diacylglycerol synthesis reduces cellular viability and reduces cell migration in metastatic OS cells. Thus, the differentially regulated lipids identified in this study might aid in biomarker discovery, and the synthesis and metabolism of specific lipids could serve as future targets for therapeutic development.

Low-dose eribulin reduces lung metastasis of osteosarcoma in vitro and in vivo.

Lung metastasis markedly reduces the prognosis of osteosarcoma. Moreover, there is no effective treatment for lung metastasis, and a new treatment strategy for the treatment of osteosarcoma lung metastasis is required. Therefore, in this study, we investigated the suppressive effect of the microtubule inhibitor eribulin mesylate (eribulin) on lung metastasis of osteosarcoma. At concentrations >proliferation IC50, eribulin induced cell cycle arrest and apoptosis in a metastatic osteosarcoma cell line, LM8. However, at concentrations <proliferation IC50, (low dose), eribulin changed cell morphology and decreased LM8 migration. Low eribulin concentrations also reduced directionality during migration, peripheral localization of adenomatous polyposis coli protein, and turnover of focal adhesions. In a three-dimensional collagen culture system, low eribulin concentrations inhibited tumor cell proliferation and colony formation. Higher doses of eribulin administered on a standard schedule inhibited lung metastasis and primary tumor growth in a murine osteosarcoma metastasis model. Frequent low-dose eribulin administration (0.3 mg/kg every 4 days × 4) effectively inhibited lung metastasis but had little effect on primary tumor growth. Overall, our results indicate that eribulin could reduce osteosarcoma lung metastasis.

HACE1 is a potential tumor suppressor in osteosarcoma

Osteosarcoma is a malignant bone sarcoma characterized by extensive genomic disruption and a propensity for metastatic spread. Osteoid production suggests a close relationship with normal osteoblasts, and the latter are the presumptive cell of origin of this disease. The HACE1 gene, localized to human chromosome 6q21, encodes the HACE1 HECT E3 ligase, a tumor suppressor in diverse tumors that acts in part by targeting the activated form of RAC1 GTPase for proteasomal degradation. Disruption or loss of 6q21 is relatively common in osteosarcomas, and Hace1−/−/Tp53+/− mice frequently develop osteosarcomas, in contrast to Tp53+/− mice, which do not. This suggests an unexplored link between HACE1 loss and osteosarcoma. Here we compared HACE1 expression in normal osteoblasts and osteosarcoma cell lines in vitro by western blotting and quantitative RT-PCR, and in human osteosarcoma specimens by immunohistochemistry. Both HACE1 transcript and protein levels were reduced in osteosarcoma compared to osteoblasts in vitro. Reduced HACE1 expression in osteosarcoma tumors was observed in 76% of cases and associated with high-grade lesions. Further, clonally derived pairs of high and low metastatic osteosarcoma cell lines showed significant downregulation in the high compared to corresponding low metastatic cells. Ectopic expression of HACE1 markedly inhibited anchorage-independent growth and cell motility of HACE1 osteosarcoma cell lines, and was associated with reduced RAC1 activation and decreased reactive oxygen species (ROS). Finally, HACE1 overexpression blocked osteosarcoma xenograft growth and dramatically reduced pulmonary metastases. These findings point to a potential tumor suppressor function for HACE1 in osteosarcoma.