Metastatic Osteosarcoma Cells Research Articles

An efficient methodological approach for synthesis of selenopyridines: generation, reactions, anticancer activity, EGFR inhibitory activity and molecular docking studies.

In the present work, we successfully synthesized Se-alkyl selenopyridines 1 and 3, selenopheno[2,3-b]pyridine 2, and bis-selenopyridine 4 derivatives using an eco-friendly method by utilizing NaHSe instead of toxic hydrogen selenide. The effect of the temperature on the reaction was screening at various temperatures. The regiospecific reaction of selenopyridine 1 with bromine afforded an unexpected product 4,6-diamino-5-bromo-2-[(cyanomethyl)selenyl]-pyridine-3-carbonitrile (5), which was cyclized to selenopheno[2,3-b]pyridine (7) by refluxing in the presence of TEA. While its treatment with thiophenol and/or p-chlorothiophenol gave 8a, b. On the other hand, its reaction with aminothiophenol afforded 2-(benzo[d]-thiazol-2-yl)-5-bromoselenopheno[2,3-b]pyridine-3,4,6-triamine (9). Also, N-(2-cyano-4-methyl-5H-1-seleno-3,5,8-triazaacenaphthylen-7-yl)acetamide (11) and a novel series of selenoazo dyes 12a-d were synthesized by treatment of selenopheno[2,3-b]pyridine 2 with acetic anhydride and/or diazonium chlorides of aromatic amines, respectively. Then, we ascertained the potential activity of synthesized compounds against highly metastatic prostate cancer cells (PC-3) and osteosarcoma cells (MG-63) and found that 12a, 12b, 12c, and 12d were more cytotoxic than doxorubicin in both tested cell lines, showing nearly the same anticancer activity with IC50 values ranging from 2.59 ± 0.02µM to 3.93 ± 0.23µM. Mechanistically, the most potent compounds 12a and 12b proved to be potent EGFR inhibitors with IC50 values of 0.301 and 0.123µM, respectively, compared to lapatinib as a positive reference (IC50 = 0.049µM). Moreover, the docking results are in good agreement with the anticancer activity as well as the EGFR inhibitory activity, suggesting these two compounds as promising EGFR anticancer candidates.

Accelerating Cellular Uptake with Unnatural Amino Acid for Inhibiting Immunosuppressive Cancer Cells.

Targeting immunosuppressive metastatic cancer cells is a key challenge in therapy. We recently have shown that a rigid-rod aromatic, pBP-NBD, that responds to enzymes and kill immunosuppressive metastatic osteosarcoma (mOS) and castration resistant prostate cancer (CRPC) cells in mimetic bone microenvironment. However, pBP-NBD demonstrated moderate efficacy against CRPC cells. To enhance activity, we incorporated the unnatural amino acid L- or D-4,4'-biphenylalanine (L- or D-BiP) into pBP-NBD, drastically increasing cellular uptake and CRPC inhibition. Specifically, we inserted BiP into pBP-NBD to target mOS (Saos2 and SJSA1) and CRPC (VCaP and PC3) cells with overexpressed phosphatases. Our results show that the D-peptide backbone with an aspartate methyl diester at the C-terminal offers the highest activity against these immunosuppressive mOS and CRPC cells. Importantly, imaging shows that the peptide assemblies almost instantly enter the cells and accumulate primarily within the endoplasmic reticulum of Saos2, SJSA1, and PC3 cells and at the lysosomes of VCaP cells. By using BiP to boost cellular uptake and self-assembly within cancer cells, this work illustrates an unnatural hydrophobic amino acid as a versatile and effective residue to boost endocytosis of synthetic peptides for intracellular self-assembly.

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.

Osteosarcoma cells exhibit functional interactions with stromal cells, fostering a lung microenvironment conducive to the establishment of metastatic tumor cells.

Osteosarcoma (OS) stands out as the most common bone tumor, with approximately 20% of the patients receiving a diagnosis of metastatic OS at their initial assessment. A significant challenge lies in the frequent existence of undetected metastases during the initial diagnosis. Mesenchymal stem cells (MSCs) possess unique abilities that facilitate tumor growth, and their interaction with OS cells is crucial for metastatic spread. We demonstrated that, in vitro, MSCs exhibited a heightened migration response toward the secretome of non-metastatic OS cells. When challenged to a secretome derived from lungs preloaded with OS cells, MSCs exhibited greater migration toward lungs colonized with metastatic OS cells. Moreover, in vivo, MSCs displayed preferential migratory and homing behavior toward lungs colonized by metastatic OS cells. Metastatic OS cells, in turn, demonstrated an increased migratory response to the MSCs' secretome. This behavior was associated with heightened cathepsin D (CTSD) expression and the release of active metalloproteinase 2 (MMP2) by metastatic OS cells. Our assessment focused on two complementary tumor capabilities crucial to metastatic spread, emphasizing the significance of inherent cell features. The findings underscore the pivotal role of signaling integration within the niche, with a complex interplay of migratory responses among established OS cells in the lungs, prometastatic OS cells in the primary tumor, and circulating MSCs. Pulmonary metastases continue to be a significant factor contributing to OS mortality. Understanding these mechanisms and identifying differentially expressed genes is essential for pinpointing markers and targets to manage metastatic spread and improve outcomes for patients with OS.

Abstract 5527: Spatiotemporal analysis of growth factor regulation of osteosarcoma AKT and ERK signaling dynamics in an ex vivo lung metastasis model

Abstract In the present study, we investigated the relationships between spatiotemporal distribution of growth factors in the lung microenvironment, metastatic osteosarcoma cell signaling, and intratumoral gene expression heterogeneity. Osteosarcoma is an aggressive bone malignancy that commonly metastasizes to the lungs, resulting in significant patient mortality. During metastasis, disseminated cancer cells are exposed to microenvironment-derived growth factors that result in signaling pathway activation. Two such pathways are MAPK/ERK and PI3K/AKT, which regulate complex transcriptional programs underlying cell proliferation and survival decisions. In this study, we utilized precision cut lung slices (PCLS) obtained from mice with established osteosarcoma metastases to ex vivo model associations between the spatiotemporal distributions of growth factors in the lung, osteosarcoma signaling dynamics, and intratumoral gene expression heterogeneity, with single cell precision. To accomplish this, we engineered osteosarcoma cells that co-expressed genetically-encoded ERK and AKT sensors, engrafted them into PCLS tissues, then recorded sensor dynamics via live-cell microscopy. These tissues were subsequently fixed and immuno-stained for measurement of growth factor expression in resident lung cells and spatial correlation with observed tumor cell ERK-AKT signaling dynamics. Using this approach, we found that growth factor expression was diffusely increased within areas bordering osteosarcoma metastases. FGF2 and IGF1, which we have previously demonstrated to potently induce ERK signaling in osteosarcoma cells, were both significantly upregulated in these regions. However, despite relatively uniform overexpression of growth factors adjacent to tumors, individual osteosarcoma cells displayed stochastics patterns of ERK and AKT signaling, and downstream gene targets. These findings revealed that upregulation of lung growth factors, and the inherent systems-level properties of osteosarcoma signaling networks, can converge to promote intratumoral signaling and gene expression heterogeneity. Together, our results, using a novel PCLS and live-cell biosensor imaging approach, provide new insight into how spatial and temporal changes in microenvironmental signaling composition are integrated by metastatic osteosarcoma cells to promote heterogeneity. Citation Format: Rawan Makkawi, Ryan D. Roberts, Jeremy Copperman, Alexander E. Davies. Spatiotemporal analysis of growth factor regulation of osteosarcoma AKT and ERK signaling dynamics in an ex vivo lung metastasis model [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 5527.

Abstract 5457: Inflammatory paracrine loops established between metastasis-initiating cells and alveolar epithelium drive interactions between tumor cell subtypes that are essential for osteosarcoma lung colonization

Abstract Introduction: The development of lung metastasis drives the poor outcomes experienced by children and young adults with osteosarcoma. The mechanisms that facilitate metastatic colonization remain poorly understood. We previously determined that IL6 and CXCL8 are two osteosarcoma derived cytokines that are required for metastasis, though roles played by these cytokines have not been identified. Methods: To explore the interactions that drive cytokine expression, the role these factors play in tumor cell trafficking, and the mechanisms that facilitate metastatic niche development, we utilized transwell and co-culture systems together with a panel of metastatic osteosarcoma mouse models that allowed tumor cell manipulation via lentiviruses, CRISPR-based gene editing, and pre-clinical therapeutic evaluation approaches. Results: While metastatic osteosarcoma cells produced small amounts of IL6 and CXCL8 at baseline, lung epithelial cells markedly upregulated IL6/CXCL8 production. Ligand-receptor analysis utilizing single-cell transcriptomic data identified IL1 as an epithelial derived ligand that drives tumor IL6/CXCL8 production. Interestingly, IL6/CXCL8 production was limited to a small subset of hypo-proliferative osteosarcoma cells that have metastasis-initiating capacity. Utilizing CRISPR-mediated ablation of IL1R1 signaling within osteosarcoma cells and pharmacological inhibition with recombinant IL1R antagonist (anakinra), we elucidated that formation of pulmonary lesions in multiple mouse models of metastatic osteosarcoma required IL1 signaling. Recent work from our lab has demonstrated that osteosarcoma metastases are defined by marked phenotypic heterogeneity, though the mechanisms by which metastatic heterogeneity occurred were unclear. We hypothesized that hypo-proliferative metastasis-initiating cells may recruit circulating cells with higher proliferative capacity to establish phenotypic heterogeneity. Transwell migration assays showed that tumor cells induced the chemotactic recruitment of other tumor cells in an IL6/CXCL8-dependent manner. To test this hypothesis in vivo, we injected mice with GFP-tagged osteosarcoma cells, then injected the same mice 14 days later with RFP-labeled tumor cells from the same culture. Lungs harvested 14 days later exhibited RFP-dominant lesions surrounding a small core of GFP-labeled cells. Conclusions: Collectively, these data suggest that cytokines expressed by a subgroup of hypo-proliferative tumor cells upon interaction with lung epithelium drive the trafficking of highly proliferative circulating tumor cells into developing metastatic lesions and that disrupting these signaling networks can prevent metastatic colonization. Citation Format: Amy C. Gross, James B. Reinecke, Amanda Saraf, John Hinckley, Maren Cam, Matthew V. Cannon, Sophia Vatelle, Ryan D. Roberts. Inflammatory paracrine loops established between metastasis-initiating cells and alveolar epithelium drive interactions between tumor cell subtypes that are essential for osteosarcoma lung colonization [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 5457.

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.

Abstract A026: Administration of preoperative ketorolac prevents osteosarcoma recurrence by promoting T cell memory and inhibiting immunosuppression

Abstract Introduction: Surgery induces an inflammatory response that is important for wound healing, but can also promote the survival of cancer cells and future tumor recurrence. Surgical injury activates the cyclooxygenase pathway and the production of downstream mediators such as prostaglandin E2 (PGE2) which has tumor-promoting and immunosuppressive activity. PGE2 stimulates regulatory T cells (Tregs), inhibits antigen presentation, and suppresses natural killer cells. Using a murine model of metastatic osteosarcoma, we investigated if preoperative blockade of this inflammatory cascade with ketorolac, a nonsteroidal anti-inflammatory drug, can boost anticancer T cell immunity. We also assessed if ketorolac had a synergistic effect when administered with a Programmed cell death protein-1 (PD-1) inhibitor. Methods: C3H/HeJ mice were inoculated with LM8 metastatic osteosarcoma cells. Tumors were resected at 50-150 mm3 and 2 hours before surgery, mice received an intraperitoneal injection containing either PBS, ketorolac, or ketorolac and antibodies to PD-1. All mice who survived beyond 100 days were considered long-term survivors and rechallenged with LM8 cells to evaluate immunological memory. Peripheral blood and spleen were collected and processed to perform flow cytometric analysis and single-cell sequencing of immune cell populations. Results: Following resection of primary tumors, 24 out of 25 control mice succumbed to lung metastasis by day 36 after resection while 4 out of 25 mice administered preoperative ketorolac exhibited long-term survival (defined as >90 days after tumor resection). All long-term survivors who received preoperative ketorolac were resistant to a rechallenge with LM8 cells. There was no difference in survival between mice who did and did not receive PD-1 inhibition. Flow cytometry analysis showed that the ketorolac-treated mice had increased T cell activity with a higher percent of effector and effector memory CD4+ and CD8+ T cells. In addition, a lower percent of myeloid derived suppressor cells (MDSCs) and Tregs were found in the spleens of mice treated with preoperative ketorolac. Single cell sequencing revealed that ketorolac-treated mice exhibited an expansion in B cells, specifically follicular B cells that expressed genes related to antigen presentation and cell proliferation. Conclusions: Administration of preoperative ketorolac in mice with osteosarcoma resulted in long-term survival after tumor resection and prevention of tumor recurrence after secondary challenge. Further, treatment with ketorolac reduced two cell populations of immunosuppressive significance, while enhancing immune cell memory. Thus, targeting surgery-induced inflammation with preoperative ketorolac should be further explored as a strategy to bolster a tumor-specific, T cell mediated immune response. Citation Format: Rebecca Pankove, Ramya Ganesan, Swati S Bhasin, Marina Michaud, Manoj Bhasin, Vikas P Sukhatme. Administration of preoperative ketorolac prevents osteosarcoma recurrence by promoting T cell memory and inhibiting immunosuppression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A026.

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.

Endogenous Extracellular Matrix Regulates the Response of Osteosarcoma 3D Spheroids to Doxorubicin

The extracellular matrix (ECM) modulates cell behavior, shape, and viability as well as mechanical properties. In recent years, ECM disregulation and aberrant remodeling has gained considerable attention in cancer targeting and prevention since it may stimulate tumorigenesis and metastasis. Here, we developed an in vitro model that aims at mimicking the in vivo tumor microenvironment by recapitulating the interactions between osteosarcoma (OS) cells and ECM with respect to cancer progression. We long-term cultured 3D OS spheroids made of metastatic or non-metastatic OS cells mixed with mesenchymal stromal cells (MSCs); confirmed the deposition of ECM proteins such as Type I collagen, Type III collagen, and fibronectin by the stromal component at the interface between tumor cells and MSCs; and found that ECM secretion is inhibited by a neutralizing anti-IL-6 antibody, suggesting a new role of this cytokine in OS ECM deposition. Most importantly, we showed that the cytotoxic effect of doxorubicin is reduced by the presence of Type I collagen. We thus conclude that ECM protein deposition is crucial for modelling and studying drug response. Our results also suggest that targeting ECM proteins might improve the outcome of a subset of chemoresistant tumors.

Abstract A024: A potent eIF4A1/2 inhibitor CR-1-31B down-modulates the antioxidant stress response in osteosarcoma cells and inhibits in vivo lung metastases

Abstract Background: Effective treatment of metastatic disease remains a major challenge in the improvement of patient outcomes in osteosarcoma (OS). Novel anti-metastatic therapies are needed to treat distant metastases. To this end, the current research evaluates whether targeting the dysregulated mRNA translation machinery in OS can inhibit metastases. Hypothesis: We hypothesize that mRNA translation factors present at an abnormally high abundance in OS cells support the rapid synthesis of cytoprotective proteins that are needed to survive in the oxidative stress-rich microenvironment of the lung. Experimental Approach: Databases of OS cell lines and patient tumor data (A. Sweet-Cordero, UCSF) were queried to identify mRNA translation factors with abnormal transcript levels. A limited drug screen of small molecule inhibitors (SMIs) against identified candidates was carried out to evaluate IC50 values in metastatic OS cells. A candidate inhibitor identified from these data was further characterized for synergy with chemical inducers of oxidative stress (e.g. tert-butylhydroquinone [tBHQ]) that mimics conditions encountered in the lung. Drug combination studies examined 2D and 3D tumor spheroid growth, cellular oxidative stress, and PARP-cleavage, under +/- inhibitor and +/- oxidative stress conditions. Metastatic OS cells were engineered to express an antioxidant response element (ARE)-mCherry fluorescent reporter to directly monitor the antioxidant response by fluorescence microscopy. Polysome profiling was used to assess inhibitor-mediated changes in global mRNA translation. The anti-metastatic activity of the inhibitor was tested in the ex vivo pulmonary metastasis assay (PuMA) and in in vivo metastasis models. Results: From cell and patient sample screening, eIF4A1/2 was identified as being abnormally regulated in metastatic OS cells. The SMI, CR-1-31B, specifically targets eIF4A1/2 and was found to have an IC50 of just 8 nM. CR-1-31B was found to inhibit tumor cell growth in 2D and 3D, increase cellular oxidative stress, and enhance PARP-cleavage, but only under oxidative stress conditions. Western analysis of tBHQ-treated metastatic OS cells with the ARE-mCherry reporter confirmed that the temporal expression of mCherry correlated with the upregulation of Nuclear factor erythroid 2-related factor-2 (Nrf2), a key transcriptional regulator of the antioxidant response. CR-1-31B blunted the upregulation of the antioxidant response in 2D and 3D tumor growth conditions with oxidative stress. CR-1-31B, in a dose-dependent manner, decreased the amount of polysomal mRNAs. CR-1-31B reduced the lung tumor burden in the ex vivo PuMA model, delayed primary tumor growth, and reduced lung metastases in in vivo xenograft OS models. Conclusions: Our data demonstrates that dysregulated mRNA translation is a metastatic vulnerability that can be exploited with SMIs. Altogether, these data support the inhibition of metastatic OS by CR-1-31B, highlighting the potential therapeutic utility of this selective translation inhibitor. Citation Format: Michael M. Lizardo, Christopher Hughes, Yue Zhou Huang, Alberto Delaidelli, Taras Shyp, Haifeng Zhang, Sol Snir Shaool, Poul H. Sorensen. A potent eIF4A1/2 inhibitor CR-1-31B down-modulates the antioxidant stress response in osteosarcoma cells and inhibits in vivo lung metastases [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A024.

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.

Osteosarcoma-Derived Exosomes as Potential PET Imaging Nanocarriers for Lung Metastasis.

Lung metastases represent the most adverse clinical factor and rank as the leading cause of osteosarcoma-related death. Nearly 80% of patients present lung micrometastasis at diagnosis not detected with current clinical tools. Herein, an exosome (EX)-based imaging tool is developed for lung micrometastasis by positron emission tomography (PET) using osteosarcoma-derived EXs as natural nanocarriers of the positron-emitter copper-64 (64 Cu). Exosomes are isolated from metastatic osteosarcoma cells and functionalized with the macrocyclic chelator NODAGA for complexation with 64 Cu. Surface functionalization has no effect on the physicochemical properties of EXs, or affinity for donor cells and endows them with favorable pharmacokinetics for in vivo studies. Whole-body PET/magnetic resonance imaging (MRI) images in xenografted models show a specific accumulation of 64 Cu-NODAGA-EXs in metastatic lesions as small as 2-3mm or in a primary tumor, demonstrating the exquisite tropism of EXs for homotypic donor cells. The targetability for lung metastasis is also observed by optical imaging using indocyanine green (ICG)-labeled EXs and D-luciferin-loaded EXs. These findings show that tumor-derived EXs hold great potential as targeted imaging agents for the noninvasive detection of small lung metastasis by PET. This represents a step forward in the biomedical application of EXs in imaging diagnosis with increased translational potential.

Delivery of mGluR5 siRNAs by Iron Oxide Nanocages by Alternating Magnetic Fields for Blocking Proliferation of Metastatic Osteosarcoma Cells.

Although osteosarcoma is the most common primary malignant bone tumor, chemotherapeutic drugs and treatment have failed to increase the five-year survival rate over the last three decades. We previously demonstrated that type 5 metabotropic glutamate receptor, mGluR5, is required to proliferate metastatic osteosarcoma cells. In this work, we delivered mGluR5 siRNAs in vitro using superparamagnetic iron oxide nanocages (IO-nanocages) as delivery vehicles and applied alternating magnetic fields (AMFs) to improve mGluR5 siRNAs release. We observed functional outcomes when mGluR5 expression is silenced in human and mouse osteosarcoma cell lines. The results elucidated that the mGluR5 siRNAs were successfully delivered by IO-nanocages and their release was enhanced by AMFs, leading to mGluR5 silencing. Moreover, we observed that the proliferation of both human and mouse osteosarcoma cells decreased significantly when mGluR5 expression was silenced in the cells. This novel magnetic siRNA delivery methodology was capable of silencing mGluR5 expression significantly in osteosarcoma cell lines under the AMFs, and our data suggested that this method can be further used in future clinical applications in cancer therapy.

Radiation therapy enhances systemic antitumor efficacy in PD-L1 therapy regardless of sequence of radiation in murine osteosarcoma.

Recent studies demonstrate that immune checkpoint blockade (ICB) increases the chances of the abscopal effect, an anti-tumor effect outside the radiation field in radiation therapy. However, the optimal sequence between radiation and ICB remains unclear. To investigate the impact of sequence of radiation in anti-PD-L1 antibody (P1) therapy on immune microenvironments and antitumor efficacies in local and abscopal tumors, metastatic LM8 osteosarcoma cells were inoculated into both legs of C3H mice. For irradiation, only one side leg was irradiated at 10 Gy. Then mice were divided into four groups: administrated anti-PD-L1 antibody three times (P1 monotherapy), receiving radiation 3 days prior to P1 therapy (P1+pre-Rad), and receiving concurrent radiation with P1 therapy (P1+conc-Rad). Thereafter, tumor immune microenvironment and tumor volume changes were analyzed in irradiated and unirradiated tumors. The P1+pre-Rad regimen increased the proportion of CD8+ programmed cell death 1 (PD-1)+ granzyme B (GzmB)+ reinvigorated T cells and decreased the proportion of CD8+ PD-1+ GzmB- exhausted T cells than P1+conc-Rad regimen in unirradiated tumors. Combination regimens suppressed tumor growth in irradiated tumors compared with that in P1 monotherapy. In both irradiated and unirradiated tumors, significant tumor growth suppression and prolonged overall survival were observed under both combination treatment regimens compared with P1 monotherapy. However, no distinct differences in unirradiated tumor volume and survival were observed between P1+pre-Rad and P1+conc-Rad groups. These results suggest that local irradiation is necessary to improve systemic treatment efficacy in P1 therapy regardless of sequence of local irradiation.

Abstract 6325: Identification of genes responsible for osteosarcomas metastatic evolution by active super enhancers profiling

Abstract Super enhancers are strong spatiotemporal transcriptional regulators. They can be mapped on genomes thanks to their astounding enrichment for well-known enhancers markers, such as acetylation of the lysine 27 of type 3 histones (H3K27Ac). Their target genes, confined in their so-called topologically associated domains, present exceptionally high expression levels, depending on associated super enhancers activation status, and are responsible for defining cells identity whether physiological or pathological. Osteosarcomas are the most common primary malignant bone tumors. Patients suffering from osteosarcomas, on average 15 years old, mostly develop pulmonary metastasis, responsible for drastic decrease in their survival. We have conducted chromatin immunoprecipitation sequencing against H3K27Ac and RNA sequencing experiments on osteosarcoma cells presenting high propensity to metastatic dissemination or devoid of this capacity, in order to unveil genes implicated in metastasis development through super enhancers genomic mapping. We have highlighted exclusive active super enhancers profiles between metastatic and non-metastatic osteosarcoma cells. Among target genes of spotted super enhancers, three of them caught our attention. Implication of these genes in metastatic development have already been demonstrated in various cancers evolution, except in osteosarcomas. Osteosarcoma cells having high capacity for metastatic development present identified genes over expressions compared to cells incompetent in this pathological process. Artificial down expressions of identified genes decrease osteosarcoma cells metastatic capacity in vitro. In vivo experiments, that correspond to paratibial implantation of modified osteosarcoma cells and analysis of primary tumor metastatic development, are currently under way. Our strategy may allow identification of new therapeutic targets to fight against osteosarcoma metastatic evolution and open new perspectives to improve patients prognostic. This work is funded by Ligue contre le cancer, ANR, SFCE and Région Pays de la Loire. Citation Format: Mélanie Lavaud, Robel Tesfaye, Céline Charrier, Rose Anne Thépault, Maxime Golias, Sarah Le Page, Mathilde Mullard, Bénédicte Brounais, Franck Verrecchia, Marc Baud'huin, François Lamoureux, Steven Georges, Benjamin Ory. Identification of genes responsible for osteosarcomas metastatic evolution by active super enhancers profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6325.

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.

MiRNA-34c Suppresses Osteosarcoma Progression In Vivo by Targeting Notch and E2F.

ABSTRACTThe expression of microRNAs (miRNAs) is dysregulated in many types of cancers including osteosarcoma (OS) due to genetic and epigenetic alterations. Among these, miR‐34c, an effector of tumor suppressor P53 and an upstream negative regulator of Notch signaling in osteoblast differentiation, is dysregulated in OS. Here, we demonstrated a tumor suppressive role of miR‐34c in OS progression using in vitro assays and in vivo genetic mouse models. We found that miR‐34c inhibits the proliferation and the invasion of metastatic OS cells, which resulted in reduction of the tumor burden and increased overall survival in an orthotopic xenograft model. Moreover, the osteoblast‐specific overexpression of miR‐34c increased survival in the osteoblast specific p53 mutant OS mouse model. We found that miR‐34c regulates the transcription of several genes in Notch signaling (NOTCH1, JAG1, and HEY2) and in p53‐mediated cell cycle and apoptosis (CCNE2, E2F5, E2F2, and HDAC1). More interestingly, we found that the metastatic‐free survival probability was increased among a patient cohort from Therapeutically Applicable Research to Generate Effective Treatments (TARGET) OS, which has lower expression of direct targets of miR‐34c that was identified in our transcriptome analysis, such as E2F5 and NOTCH1. In conclusion, we demonstrate that miR‐34c is a tumor suppressive miRNA in OS progression in vivo. In addition, we highlight the therapeutic potential of targeting miR‐34c in OS. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Proteomic Assessment of Extracellular Vesicles from Canine Tissue Explants as a Pipeline to Identify Molecular Targets in Osteosarcoma: PSMD14/Rpn11 as a Proof of Principle.

Osteosarcoma (OS) is a highly malignant bone tumour that has seen little improvement in treatment modalities in the past 30 years. Understanding what molecules contribute to OS biology could aid in the discovery of novel therapies. Extracellular vesicles (EVs) serve as a mode of cell-to-cell communication and have the potential to uncover novel protein signatures. In our research, we developed a novel pipeline to isolate, characterize, and profile EVs from normal bone and osteosarcoma tissue explants from canine OS patients. Proteomic analysis of vesicle preparations revealed a protein signature related to protein metabolism. One molecule of interest, PSMD14/Rpn11, was explored further given its prognostic potential in human and canine OS, and its targetability with the drug capzimin. In vitro experiments demonstrated that capzimin induces apoptosis and reduces clonogenic survival, proliferation, and migration in two metastatic canine OS cell lines. Capzimin also reduces the viability of metastatic human OS cells cultured under 3D conditions that mimic the growth of OS cells at secondary sites. This unique pipeline can improve our understanding of OS biology and identify new prognostic markers and molecular targets for both canine and human OS patients.