Metastatic Osteosarcoma Cell Lines Research Articles

Inhibition of osteosarcoma metastasis in vivo by targeted downregulation of MMP1 and MMP9

Osteosarcoma (OS) mortality stems from lung metastases. Matrix metalloproteinases (MMPs) facilitate metastatic dissemination by degrading extracellular matrix components. Herein we studied the impact of targeted MMP downregulation on OS metastasis. Differential gene expression analysis of human OS cell lines revealed high MMP9 expression in the majority of OS cell lines. Furthermore, 143B, a metastatic OS cell line, exhibited increased MMP1 and MMP9 mRNA levels. Gene set enrichment analysis on metastatic and non-metastatic OS patient specimens indicated epithelial-mesenchymal transition as the most enriched gene set, with MMP9 displaying strong association to genes in this network. Using the same dataset, Kaplan-Meier analysis revealed a correlation between MMP1 expression and dismal patient survival. Hence, we undertook targeted suppression of MMP1 and MMP9 gene expression in OS cell lines. The ability of OS cells to migrate and form colonies was markedly reduced upon MMP1 and MMP9 downregulation, whereas their cell proliferation capacity remained intact. MMP9 downregulation decreased tumor growth and lung metastases area in an orthotopic mouse OS model. Consistently, human OS lung metastasis specimens displayed marked MMP9 protein expression. Our findings highlight the role of MMP1 and MMP9 in OS metastasis, warranting further exploration of simultaneous inhibition of MMPs for future OS therapeutics.

Pharmacologic inhibition of eIF4A blocks NRF2 synthesis to prevent osteosarcoma metastasis.

Effective therapies for metastatic osteosarcoma (OS) remain a critical unmet need. Targeting mRNA translation in metastatic OS offers a promising option, as selective translation drives synthesis of cytoprotective proteins under harsh microenvironmental conditions to facilitate metastatic competence. We assessed expression levels of eukaryotic translation factors in OS, revealing high expression of the eIF4A1 initiation factor. Using a panel of metastatic OS cell lines and PDX models, eIF4A1 inhibitors were evaluated for their ability to block proliferation and reduce survival under oxidative stress, mimicking harsh conditions of the lung microenvironment. Inhibitors were also evaluated for their anti-metastatic activity using the ex vivo pulmonary metastasis assay (PuMA) and in vivo metastasis models. Proteomics were performed to catalog which cytoprotective proteins or pathways were affected by eIF4A1 inhibition. CR-1-31B, a rocaglate-based eIF4A1 inhibitor, exhibited nanomolar cytotoxicity against all metastatic OS models tested. CR-1-31B exacerbated oxidative stress and apoptosis when OS cells were co-treated with a tert-butylhydroquinone (tBHQ), a chemical oxidative stress inducer. CR-1-31B potently inhibited OS growth in the PuMA model and in experimental and spontaneous models of OS lung metastasis. Proteomic analysis revealed that tBHQ-mediated upregulation of the NRF2 antioxidant factor was blocked by co-treatment with CR-1-31B. Genetic inactivation of NRF2 phenocopied the anti-metastatic activity of CR-1-31B. Finally, the clinical grade eIF4A1 phase 1-2 inhibitor, Zotatifin, similarly blocked NRF2 synthesis and the OS metastatic phenotype. Collectively, our data reveal that pharmacologic targeting of eIF4A1 is highly effective in blocking OS metastasis by blunting the NRF2 antioxidant response.

Extracellular Vesicles from Highly Metastatic Osteosarcoma Cells Induce Pro-Tumorigenic Macrophage Phenotypes.

Metastasis is the principal factor in poor prognosis for individuals with osteosarcoma (OS). Understanding the events that lead to metastasis is critical to develop better interventions for this disease. Alveolar macrophages are potentially involved in priming the lung microenvironment for OS metastasis, yet the mechanisms involved in this process remain unclear. Since extracellular vesicles (EVs) are a known actor in primary tumor development, their potential role in OS metastagenesis through macrophage modulationis explored here. The interaction of EVs isolated from highly metastatic (K7M2) and less metastatic (K12) osteosarcoma cell lines is compared with a peritoneal macrophage cell line. An EV concentration that reproducibly induced macrophage migration is identified first, then used for later experiments. By confocal microscopy, both EV types associated with M0 or M1 macrophages; however, only K7M2-EVs are associated with M2 macrophages, an interaction that is abrogated by EV pre-treatment with anti-CD47 antibody. Interestingly, all interactions appeared to be surface binding, not internalized. In functional studies, K7M2-EVs polarized fewer macrophages to M1. Together, these data suggest that K7M2-EVs have unique interactions with macrophages that can contribute to the production of a higher proportion of pro-tumor type macrophages, thereby accelerating metastasis.

Abstract 7554: The histone deacetylase inhibitor romidepsin is a potential therapeutic for metastatic osteosarcoma

Abstract Osteosarcoma predominately affects adolescents, young adults, and canines. For human patients, the five-year survival rate of those with detectable lung metastases is only 30% while most canine patients with metastatic disease die within 1-2 years. Standard therapy is not effective for this patient group, so it is therefore necessary to identify novel therapies that target the progression of lung metastases. Our lab previously screened 114 FDA-approved anti-cancer drugs to identify agents that decrease the growth of 3D spheroids (sarcospheres) generated from highly metastatic osteosarcoma cell lines. The top hits included both histone deacetylase inhibitors (HDIs) that were tested. In follow-up experiments measuring potency on sarcospheres and toxicity on non-transformed cells, romidepsin was the top hit of the 114-drug panel as well of the two additional FDA-approved HDIs and seven HDIs that are in clinical oncology trials. Our goal was therefore to further evaluate romidepsin as a potential therapy for metastatic osteosarcoma. Romidepsin potently decreased viability in sarcospheres from established highly metastatic osteosarcoma cell lines (relative ED50s = 3-36nM). About 50% of the patients (both canine and human) had similar responses to romidepsin (relative ED50s = 1- 18nM) suggesting that those patients might be good candidates for romidepsin therapy. This includes patients who had been heavily pretreated with standard-of-care osteosarcoma chemotherapeutics. Mechanistically, romidepsin blocks proliferation and causes a G2/M cell cycle arrest in fast growing sarcospheres (143B and MG63.3) while in slow growing sarcospheres (LM7 and K7M2) where primarily induces cytotoxicity and there is no cell cycle arrest. Patient-derived sarcospheres are slow growing, so we predict that the primary mechanism will be cytotoxicity. Using a tail vein injection model of osteosarcoma lung metastases, romidepsin dose dependently prolonged survival and reduced the area and number of lung metastases in immunodeficient mice injected with 143B cells and a syngeneic model using K7M2 cells. Romidepsin will be evaluated in mice with metastases from patient-derived cells and if effective, will be repurposed for canine and human clinical trials to improve outcomes in metastatic osteosarcoma without the associated costs and extensive timeframe of traditional drug discovery. Citation Format: Emily E. Seiden, Spencer M. Richardson, Shrey Ramnath, Tia Whiteside, Kathryn L. Coy, Anthony L. Sinn, Maegan L. Capitano, Karen E. Pollok, Chris D. Collier, Ed M. Greenfield. The histone deacetylase inhibitor romidepsin is a potential therapeutic for metastatic osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7554.

1-DEOXYNOJIRIMYCIN, A GLYCOSYLATION INHIBITOR INFLUENCES THE EXPRESSION OF EXTRACELLULAR MATRIX PROTEINS IN OSTEOSARCOMA CELLS

Malignant tumour cells secrete extracellular matrix (ECM) proteins with growth factors and cytokines to promote cell motility and adhesion for metastasis. The cell-ECM adhesion sites are comprised of integrin-bound ECM proteins, which are glycoconjugates, susceptible to the aberrant glycosylation resulting in alterations of cell-ECM adhesion dynamics. Inhibition of glycosylation could decrease the rate of invasiveness in metastatic tumour cells like osteosarcoma (OS), of which the underlying mechanism is still unknown. Hence, this study aims to determine the effect of glucosidase inhibitors towards the expression of ECM proteins in metastatic OS cell lines. OS cells (MG63) were treated with 0.5 mM of 1-deoxynojirimycin (1-DNJ). Analyses such as the glycosylation assay, ECM-cell adhesion assay, scratch assay, and Western blot analysis were performed after a 24-h treatment for each respective sample. The expression level of glycosylated proteins was the highest in non-treated MG63 cells. The cell migration rate was increased in MG63 cells treated with 1-DNJ compared to the normal osteoblast cells. The cell adhesion assay results showed an increased rate of cell adhesion towards vitronectin but decreased adhesion rate towards collagen (types I, II, and IV), fibronectin, laminin, and tenascin in 1-DNJ-treated OS cells. Western blot analysis presented a high expression level of fibronectin, vitronectin, and collagen type II in both non-treated and treated groups of MG63 cells. The inhibitory effect of 1-DNJ upregulates ECM remodelling by disrupting integrin from binding to the ECM proteins, subsequently increasing the migration rate of metastatic OS cells. Keyword(s) : Extracellular matrix protein, 1-Deoxynojirimycin, integrin glycosylation, cell adhesion rate, migration assay.

A Novel Approach to Reducing Lung Metastasis in Osteosarcoma: Increasing Cell Stiffness with Carbenoxolone

Primary malignant bone tumor osteosarcoma can metastasize to the lung. Diminishing lung metastasis would positively affect the prognosis of patients. Our previous studies demonstrated that highly metastatic osteosarcoma cell lines are significantly softer than low-metastasis cell lines. We therefore hypothesized that increasing cell stiffness would suppress metastasis by reducing cell motility. In this study, we tested whether carbenoxolone (CBX) increases the stiffness of LM8 osteosarcoma cells and prevents lung metastasis in vivo. We evaluated the actin cytoskeletal structure and polymerization of CBX-treated LM8 cells using actin staining. Cell stiffness was measured using atomic force microscopy. Metastasis-related cell functions were analyzed using cell proliferation, wound healing, invasion, and cell adhesion assays. Furthermore, lung metastasis was examined in LM8-bearing mice administered with CBX. Treatment with CBX significantly increased actin staining intensity and stiffness of LM8 cells compared with vehicle-treated LM8 cells (p < 0.01). In Young's modulus images, compared with the control group, rigid fibrillate structures were observed in the CBX treatment group. CBX suppressed cell migration, invasion, and adhesion but not cell proliferation. The number of LM8 lung metastases were significantly reduced in the CBX administration group compared with the control group (p < 0.01). In this study, we demonstrated that CBX increases tumor cell stiffness and significantly reduces lung metastasis. Our study is the first to provide evidence that reducing cell motility by increasing cell stiffness might be effective as a novel anti-metastasis approach in vivo.

Superenhancers activate the autophagy-related genes Beclin1 and LC3B to drive metastasis and drug resistance in osteosarcoma.

Metastasis and drug resistance are the leading causes of poor prognosis in patients with osteosarcoma. Identifying the relevant factors that drive metastasis and drug resistance is the key to improving the therapeutic outcome of osteosarcoma. Here, we reported that autophagy was highly activated in metastatic osteosarcoma. We found increased autophagolysosomes in metastatic osteosarcoma cell lines by using electron microscopy, Western blot, and immunofluorescence experiments. We further examined the expression of the autophagy-related genes Beclin1 and LC3B in 82 patients through immunohistochemistry and found that Beclin1 and LC3B were highly related to unfavorable prognosis of osteosarcoma. Knockdown of Beclin1 and LC3B reduced invasion, metastasis, and proliferation in metastatic osteosarcoma cells. In vitro and in vivo studies also demonstrated that inhibiting by 3-MA inhibited cell growth and metastasis. Moreover, we demonstrated that autophagy-related genes were activated by SEs and that the inhibition of SEs by JQ-1 decreased the metastasis of osteosarcoma. Overall, our findings highlighted the association of autophagy with osteosarcoma progression and shed new light on autophagy-targeting therapy for osteosarcoma.

Identifying Key Differences Between Responders vs. Non-Responders to Immune Checkpoint Blockade for Metastatic Osteosarcoma (mOS)

Abstract Osteosarcoma is the most common childhood bone malignancy, with a sharp decline in survival rates upon metastasis. We have previously shown immune checkpoint blockade (ICB) of anti-CTLA-4/anti-PD-L1 for mice inoculated with a K7M2 metastatic osteosarcoma (mOS) cell line resulted in ~50% survival with complete tumor clearance, including recent analogous results upon replication of this treatment regimen. Differences in response rates to ICB are common among patients with the same malignancy across various cancers. Unlike inbred lab mice, human patients have diversity in genetic makeup and other factors, causing the discrepancy in ICB response to remain poorly understood. A culprit for these discrepancies is variance in circulating antibodies (Abs), which can indicate differences in disease presence, microbiome composition, or immune-modulating factors. Recent studies have characterized Ab patterns in sera by using microarrays with thousands of randomly sequenced peptides and incubating on them diluted sera. The Ab binding pattern on the microarray is termed an “immunosignature.” We have found that before inoculation with mOS or ICB, blood from mice displays distinct differences in immunosignatures between responders and non-responders, suggesting that the presence or absence of particular Abs may influence the outcome of ICB for mOS. Additionally, recent studies have discovered that ICB’s effectiveness can be associated with or even reliant on ICB recipients’ microbiome composition and that changes in microbiome composition can result in subsequent changes to circulating Abs. Here we analyze both differences in Ab composition and microbiome composition, highlighting their impact on ICB efficacy for mOS. Funding support for this work has been provided by a grant through the Sarcoma Foundation of America (SFA). Additional funding support for this work has been provided by a grant through the Graduate and Professional Student Association (GPSA) at Arizona State University.

Transcription factor c-Myb: novel prognostic factor in osteosarcoma

The transcription factor c-Myb is an oncoprotein promoting cell proliferation and survival when aberrantly activated/expressed, thus contributing to malignant transformation. Overexpression of c-Myb has been found in leukemias, breast, colon and adenoid cystic carcinoma. Recent studies revealed its expression also in osteosarcoma cell lines and suggested its functional importance during bone development. However, the relevance of c-Myb in control of osteosarcoma progression remains unknown. A retrospective clinical study was carried out to assess a relationship between c-Myb expression in archival osteosarcoma tissues and prognosis in a cohort of high-grade osteosarcoma patients. In addition, MYB was depleted in metastatic osteosarcoma cell lines SAOS-2 LM5 and 143B and their growth, chemosensitivity, migration and metastatic activity were determined. Immunohistochemical analysis revealed that high c-Myb expression was significantly associated with poor overall survival in the cohort and metastatic progression in young patients. Increased level of c-Myb was detected in metastatic osteosarcoma cell lines and its depletion suppressed their growth, colony-forming capacity, migration and chemoresistance in vitro in a cell line-dependent manner. MYB knock-out resulted in reduced metastatic activity of both SAOS-2 LM5 and 143B cell lines in immunodeficient mice. Transcriptomic analysis revealed the c-Myb-driven functional programs enriched for genes involved in the regulation of cell growth, stress response, cell adhesion and cell differentiation/morphogenesis. Wnt signaling pathway was identified as c-Myb target in osteosarcoma cells. Taken together, we identified c-Myb as a negative prognostic factor in osteosarcoma and showed its involvement in the regulation of osteosarcoma cell growth, chemosensitivity, migration and metastatic activity.

Analysis and identification of m6A RNA methylation regulators in metastatic osteosarcoma

Osteosarcoma (OS) is characterized by rapid growth and early metastasis. However, its mechanism remains unclear. N6-methyladenosine (m6A) modification and its regulatory factors play essential roles in most cancers, including OS. In this study, we screened out 21 m6A modifiers using the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database, followed by the identification of the critical m6A methylation modifiers. The results revealed that the expression levels of three m6A methylation regulators, namely RBM15, METTL3, and LRPPRC, were associated with the low survival rate of patients with OS. We further studied the independent prognostic factors by performing univariate and multivariate Cox analyses and found that metastasis was an independent prognostic factor for patients with OS. Furthermore, we found for the first time that RBM15 was specific for metastatic OS rather than non-metastatic OS. Moreover, the significant overexpression of RBM15 was validated in metastatic OS cell lines and in actual human clinical specimens. We also revealed that RBM15 promoted the invasion, migration, and metastasis of OS cells through loss-functional and gain-functional experiments and an animal metastatic model. In conclusion, RBM15 has a high correlation with OS metastasis formation and the decreased survival rate of patients with OS, and this may serve as a useful biomarker for predicting metastasis and prognosis of patients with OS.

Impact of limb amputation and cisplatin chemotherapy on metastatic progression in mouse models of osteosarcoma

Development of animal models that accurately recapitulate human cancer is an ongoing challenge. This is particularly relevant in the study of osteosarcoma (OS), a highly malignant bone tumor diagnosed in approximately 1000 pediatric/adolescent patients each year. Metastasis is the leading cause of patient death underscoring the need for relevant animal models of metastatic OS. In this study, we describe how existing OS mouse models can be interrogated in a time-course context to determine the kinetics of spontaneous metastasis from an orthotopically implanted primary tumor. We evaluated four highly metastatic OS cell lines (3 human, 1 mouse) to establish a timeline for metastatic progression in immune deficient NSG mice. To discern the effects of therapy on tumor development and metastasis in these models, we investigated cisplatin therapy and surgical limb amputation at early and late timepoints. These data help define the appropriate observational periods for studies of metastatic progression in OS and further our understanding of existing mouse models. Efforts to advance the study of metastatic OS are critical for facilitating the identification of novel therapeutics and for improving patient survival.

Establishment and characterization of a highly metastatic human osteosarcoma cell line from osteosarcoma lung metastases

OS (Osteosarcoma) is the most common malignant tumor in adolescents, and lung metastasis limits its therapeutic outcome. The present study aimed to establish a highly metastatic human OS cell line directly from lung metastases and characterize its biological functions. In this study, epithelioid tumor cells with large nucleo-cytoplasmic ratio and abundant organelles were obtained by the tissue mass adherent and repeated digestion adherent method and named ZOSL-1 cells. ZOSL-1 cells had the potential to proliferate in vitro with a doubling time of 39.28 ± 3.04 h and migrate with or without a matrix. ZOSL-1 cells were tumorigenic in vivo, and had the ability to develop lung metastasis after intratibial injection. ZOSL-1 cells expressed the osteogenic-related genes osteocalcin and osteopontin. In addition, the expression of ZOSL-1 in Fas cell surface death receptor (FAS), CD44 molecule (CD44), GNAS complex locus (GNAS), scavenger receptor class B member 1 (SCARB1), C-X-C motif chemokine receptor 4 (CXCR4), cadherin 11 (CDH11), neurofibromin 2 (NF2) and ezrin (EZR) genes may be related to its transfer efficiency. Taken together, these results indicated the high metastatic capability and important biological functions of ZOSL-1 cells. ZOSL-1 establishment provided a relevant model for the study of osteosarcoma lung metastasis.

Abstract PO-035: A novel bioengineered model of osteosarcoma recapitulates critical tumor phenotypes within a native bone microenvironment

Abstract Traditional in-vitro models of solid bone tumors used in basic and preclinical research are unable to faithfully recapitulate human physiology. Specifically, monolayer cultures of osteosarcoma fail to recapitulate various features of 3D tumors, and they lack the native bone milieu, where tumor growth, metastasis, and response to therapy are critically dependent on cancer cell interactions with the bone matrix, supporting cells, and secreted regulatory factors. Based on our previous success in developing a biomimetic tissue model of Ewing sarcoma, we extended our approach towards bioengineering a novel human 3D tumor model of osteosarcoma within a bone microenvironment that can overcome some of these limitations. Briefly, osteosarcoma cell lines were used to generate tumor-like aggregates which were subsequently introduced into, and cultured within, engineered bone tissue scaffolds. Our bioengineered osteosarcoma model was able to recapitulate several aspects of native tumors and their microenvironment missing in monolayer cultures. Significantly higher expression of cancer-related genes (as seen in patients), re-activation of the hypoxic and glycolytic tumor phenotype, re-activation of hypoxia-mediated expression of vascular endothelial growth factor VEGF-α (a critical component of tumor angiogenesis), induction of vasculogenic mimicry, more physiologically relevant growth rates, and co-culture with adjacent functional osteoblasts within a demineralized native bone scaffold were all achieved. Additionally, we were able to capture a degree of both intra-tumor heterogeneity, with the presence of a necrotic core and variability in proliferation across the tumor aggregates, as well as inter-tumor heterogeneity, with notable differences observed between our models generated from metastatic and non-metastatic osteosarcoma cell lines. Finally, the osteosarcoma cell lines were transduced with a GFP-luciferase reporter, allowing us to visualize the engineered tumors in situ within the bone scaffolds, and to evaluate tumor cell responses to various canonical and experimental drug treatments. Notably, unlike monolayers, our bioengineered osteosarcoma model could be cultured stably for several weeks. This allowed us to apply a clinical treatment regimen cycle of linsitinib, an experimental drug in an ongoing phase II clinical trial for Ewing sarcoma, in also being effective at preventing the growth of non-metastatic osteosarcoma bioengineered tumors. Overall, we were able to successfully generate a new, more native-like 3D bioengineered model of osteosarcoma with significant advantages over traditional in-vitro culture that has the potential to explore novel questions regarding tumor biology, the tumor microenvironment, and translational applications. Citation Format: Alan Chramiec, Alessandro Marturano-Kruik, Keith Yeager, Luke Hao, Roberta Lock, Gordana Vunjak-Novakovic. A novel bioengineered model of osteosarcoma recapitulates critical tumor phenotypes within a native bone microenvironment [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-035.

Identification of potential modulators of osteosarcoma metastasis by high-throughput cellular screening of natural products.

A high-throughput screening assay was developed and applied to a large library of natural product extract samples, in order to identify compounds which preferentially inhibited the in vitro 2D growth of a highly metastatic osteosarcoma cell line (MG63.3) compared to a cognate parental cell line (MG63) with low metastatic potential. Evaluation of differentially active natural product extracts with bioassay-guided fractionation led to the identification of lovastatin (IC50 =11µm) and the limonoid toosendanin (IC50 =26nm). Other statins and limonoids were then tested, and cerivastatin was identified as a particularly potent (IC50 <0.1µm) and selective agent. These compounds potently and selectively induced apoptosis in MG63.3 cells, but not MG63. Assays with other cell pairs were used to examine the generality of these results. Statins and limonoids may represent unexplored opportunities for development of modulators of osteosarcoma metastasis. As cerivastatin was previously approved for clinical use, it could be considered for repurposing in osteosarcoma, pending validation in further models.

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.

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.

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.

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.

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.