Mouse Model Of Osteosarcoma Research Articles

Translational nanomedicine potentiates immunotherapy in sarcoma by normalizing the microenvironment.

Nanocarrier-based chemo-immunotherapy has succeeded in clinical trials and understanding its effect on the tumor microenvironment could facilitate development of strategies to increase efficacy of these regimens further. NC-6300 (epirubicin micelle) demonstrates anti-tumor activity in sarcoma patients, but whether it is combinable with immune checkpoint inhibition is unclear. Here, we tested NC-6300 combined with anti-PD-L1 antibody in mouse models of osteosarcoma and fibrosarcoma. We found that sarcoma responds to NC-6300 in a dose-dependent manner, while anti-PD-L1 efficacy is potentiated even at a dose of NC-6300 less than 10% of the maximum tolerated dose. Furthermore, NC-6300 is more effective than the maximum tolerated dose of doxorubicin in increasing the tumor growth delay induced by anti-PD-L1 antibody. We investigated the mechanism of action of this combination. NC-6300 induces immunogenic cell death and its effect on the efficacy of anti-PD-L1 antibody is dependent on T cells. Also, NC-6300 normalized the tumor microenvironment (i.e., ameliorated pathophysiology towards normal phenotype) as evidenced through increased blood vessel maturity and reduced fibrosis. As a result, the combination with anti-PD-L1 antibody increased the intratumor density and proliferation of T cells. In conclusion, NC-6300 potentiates immune checkpoint inhibition in sarcoma, and normalization of the tumor microenvironment should be investigated when developing nanocarrier-based chemo-immunotherapy regimens.

Precise and Facile Endotracheal Lung-tumor-implantation Mouse Model Visualized by GFP Expression.

In osteosarcoma, lung metastasis is a major cause of cancer-related death, as the 5-year survival rate for patients with metastases is approximately only 20-30%. To develop improved therapeutic strategies against lung-metastatic osteosarcoma, an experimental lung-tumor-implantation mouse model is needed for basic research. In the present study, we developed a precise and facile endotracheal lung-tumor-implantation technique. For establishment of the lung-tumor-implantation mouse model of metastatic osteosarcoma, 5 mice were used. A 15-mm longitudinal incision was made in the center of the neck to expose the salivary glands. The salivary glands were then split, exposing the trachea covered by the sternohyoid muscles. The trachea was then clearly exposed by cutting the sternohyoid muscles longitudinally. A 22 G gavage needle was tilted slightly toward the left side of the mouse and inserted from the oral cavity into the bronchus, with confirmation of the position of the tip of the gavage needle visualized through the tracheal wall, followed by injection of 0.5% crystal violet to first confirm the accuracy of endotracheal injection in the lung. A 143B-GFP cell suspension (2.0×106 cells/50 μl PBS) was then injected endotracheally in other mice. The procedure, including anesthesia and suturing, took approximately 10 minutes. The left lobe of the lung, in which crystal violet was injected endotracheally, was stained in 3 out of 3 mice (100%). 143B-GFP-osteosarcoma tumors were detected with GFP fluorescence in the left lobe of the lung in 3 out of 4 mice (75%), 5 weeks after endotracheal injection. One mouse died 4 weeks after 143B-GFP-cell implantation. This novel technique of establishing tumors in the lung via endotracheal injection of cancer cells is precise and facile and can be used widely, since neither a surgical microscope nor X-ray imaging are needed.

Enhancement of the therapeutic efficacy of the MAP regimen using thiamine pyrophosphate-decorated albumin nanoclusters in osteosarcoma treatment.

Recent studies on osteosarcoma regimens have mainly focused on modifying the combination of antineoplastic agents rather than enhancing the therapeutic efficacy of each component. Here, an albumin nanocluster (NC)-assisted methotrexate (MTX), doxorubicin (DOX), and cisplatin (MAP) regimen with improved antitumor efficacy is presented. Human serum albumin (HSA) is decorated with thiamine pyrophosphate (TPP) to increase the affinity to the bone tumor microenvironment (TME). MTX or DOX (hydrophobic MAP components) is adsorbed to HSA-TPP via hydrophobic interactions. MTX- or DOX-adsorbed HSA-TPP NCs exhibit 20.8- and 1.64-fold higher binding affinity to hydroxyapatite, respectively, than corresponding HSA NCs, suggesting improved targeting ability to the bone TME via TPP decoration. A modified MAP regimen consisting of MTX- or DOX-adsorbed HSA-TPP NCs and free cisplatin displays a higher synergistic anticancer effect in HOS/MNNG human osteosarcoma cells than conventional MAP. TPP-decorated NCs show 1.53-fold higher tumor accumulation than unmodified NCs in an orthotopic osteosarcoma mouse model, indicating increased bone tumor distribution. As a result, the modified regimen more significantly suppresses tumor growth in vivo than solution-based conventional MAP, suggesting that HSA-TPP NC-assisted MAP may be a promising strategy for osteosarcoma treatment.

Thermo-sensitive hydrogel-mediated locally sequential release of doxorubicin and palbociclib for chemo-immunotherapy of osteosarcoma

Traditional cytotoxic drugs induce immunogenic cell death (ICD) of tumor cells to trigger anti-tumor immunity. Unfortunately, the presence of immunosuppressive cells such as regulatory T cells (Tregs) in tumor microenvironment offsets part of anti-tumor immunity. In this work, we engineered a methoxy poly(ethylene glycol)-block-poly(l-alanine) (mPEG − PLAla)-based thermo-sensitive hydrogel by ring-opening polymerization as a vehicle for locally sequential delivery of doxorubicin (DOX) and palbociclib (PAL) to induce the antitumor immunity in osteosarcoma. The copolymer exhibited an appropriate sol–gel transition behavior triggered by physiological temperature. Meanwhile, the superior rheological properties, the uniform morphology and the suitable biodegradability were also investigated in the hydrogel. Interestingly, codelivery of gel and DOX + PAL displayed sequential drug release behavior and exhibited improved antitumor effect toward the K7 orthotopic osteosarcoma mouse model. The DOX-induced immunogenic cell death (ICD) and PAL-mediated suppression of regulatory T cells jointly facilitate antitumor immunity in osteosarcoma. Furthermore, the dual drug codelivery hydrogel showed reduced lung metastasis and enhanced biosafety than free drugs in mouse models of osteosarcoma. Therefore, the locally sequential drug delivery system to induce antitumor immunity will have great potential in future tumor therapy.

FLASH with carbon ions: Tumor control, normal tissue sparing, and distal metastasis in a mouse osteosarcoma model.

The FLASH effect is a potential breakthrough in radiotherapy because ultra-high dose-rate irradiation can substantially widen the therapeutic window. While the normal tissue sparing at high doses and short irradiation times has been demonstrated with electrons, photons, and protons, so far evidence with heavy ions is limited to in vitro cell experiments. Here we present the first in vivo results with high-energy 12C-ions delivered at an ultra-high dose rate. LM8 osteosarcoma cells were subcutaneously injected in the posterior limb of female C3H/He mice 7days before radiation exposure. Both hind limbs of the animals were irradiated with 240MeV/n 12C-ions at ultra-high (18Gy in 150ms) or conventional dose rate (∼18Gy/min). Tumor size was measured until 28days post-exposure, when animals were sacrificed and lungs, limb muscles, and tumors were collected for further histological analysis. Irradiation with carbon ions was able to control the tumour both at conventional and ultra-high dose rate. FLASH decreases normal tissue toxicity as demonstrated by the reduced structural changes in muscle compared to conventional dose-rate irradiation. Carbon ion irradiation in FLASH conditions significantly reduced lung metastasis compared to conventional dose-rate irradiation and sham-irradiated animals. We demonstrated the FLASH effect in vivo with high-energy carbon ions. In addition to normal tissue sparing, we observed tumor control and a substantial reduction of lung metastasis in an osteosarcoma mouse model.

Pramlintide: A Novel Therapeutic Approach for Osteosarcoma through Metabolic Reprogramming.

Simple SummaryOutcomes for patients with osteosarcoma have remained stagnant for more than 3 decades. Novel therapeutic agents are desperately needed. Osteosarcoma cells often express abnormal form of the p53, p63, and p73 genes, which promote glucose uptake, cell glycolysis, and rapid proliferation. Pramlintide, an FDA-approved drug for type 2 diabetes, interfered with tumor glycolysis leading to decreased cell growth and increased cell death. When we injected Pramlintide into osteosarcoma tumor nodules of the mice, the tumors were significantly smaller after 21 days while the control tumors continued to grow. Tumor hypoxia was also decreased. This is the first report showing the potential efficacy of Pramlintide against osteosarcoma, indicating that Pramlintide may be a novel therapeutic approach for patients with relapsed osteosarcoma.Despite aggressive combination chemotherapy and surgery, outcomes for patients with osteosarcoma have remained stagnant for more than 25 years, and numerous clinical trials have identified no new therapies. p53 deletion or mutation is found in more than 80% of osteosarcoma tumors. In p53-deficient cancers with structurally altered p63 and p73, interfering with tumor cell metabolism using Pramlintide (an FDA-approved drug for type 2 diabetes) results in tumor regression. Pramlintide response is mediated through upregulation of islet amyloid polypeptide (IAPP). Here, we showed that osteosarcoma cells have altered p63, p73, and p53, and decreased IAPP expression but have the two main IAPP receptors, CalcR and RAMP3, which inhibit glycolysis and induce apoptosis. We showed that in osteosarcoma cells with high- or mid-range glycolytic activity, Pramlintide decreased cell glycolysis, resulting in decreased proliferation and increased apoptosis in vitro. In contrast, Pramlintide had no effect in osteosarcoma cells with low glycolytic activity. Using a subcutaneous osteosarcoma mouse model, we showed that intratumoral injection of Pramlintide-induced tumor regression. Tumor sections showed increased apoptosis and a decrease in Ki-67 and HIF-1α. These data suggest that in osteosarcoma cells with altered p53, p63, and p73 and a high glycolytic function, Pramlintide therapy can modulate metabolic programming and inhibit tumor growth.

Application value of a selenium-hydroxyapatite nanodelivery system as osteosarcoma treatment

To investigate the anticancer and osteogenic properties and mechanism of action of a selenium (Se4+) hydroxyapatite (HA) nanodelivery system for osteosarcoma (OS) therapy. We loaded different concentrations of Na2SeO3 (3% and 6%) into HA nanoparticles (HANP) for OS therapy. Simultaneously, we tested the stability and pH responsiveness of Se-HANP in vitro experiments, and OS MG-63 cells and mice BMSCs were cultured to further evaluate its permeability, anticancer ability and mineralization ability [alkaline phosphatase (ALP), osteocalcin (OCN)]. In addition, We conducted in vivo experiments by constructing OS mouse models to further investigate the anticancer mechanism of Se-HANP. Se-HANP showed good stability in blood; an acidic environment (pH 5) was more conducive to Se4+ ion release, and could effectively penetrate into OS MG-63 cells. In cell experiments, compared with HANP, Se-HANP could more effectively improve BMSCs viability and enhanced their mineralization performance, such as improving ALP and OCN levels. In addition, Se-HANP could effectively inhibit the activity and invasiveness of MG-63 cells; 6% Se-HANP showed stronger anticancer effects. In OS in vivo mouse experiments, Se-HANP could effectively inhibit tumor tissue growth (volume and weight) in a concentration dependent manner. In addition, Se4+ activated endogenous and exogenous apoptotic pathways by increasing the level of reactive oxygen species (8-OHdG) in tumor cells, leading to tumor cell apoptosis. Se-HANP could effectively penetrate OS cells, and its good stability and pH responsiveness could improve the anticancer efficiency of Se4+. Moreover, its excellent mineralization ability could effectively repair the bone defects caused by OS. Accordingly, Se-HANP can have great potential in OS treatment.

Self-Assembling Imageable Silk Hydrogels for the Focal Treatment of Osteosarcoma.

Background: The standard treatment for osteosarcoma comprises complete surgical resection and neoadjuvant chemotherapy, which may cause serious side effects and partial or total limb loss. Therefore, to avoid the disadvantages of traditional treatment, we developed self-assembling imageable silk hydrogels for osteosarcoma. Methods: We analysed whether iodine induced apoptosis in MG-63 and Saos-2 cells by using CCK-8 and flow cytometry assays and transmission electron microscopy. Western blotting was used to analyse the pathway of iodine-induced apoptosis in osteosarcoma cells. PEG400, silk fibroin solution, polyvinylpyrrolidone iodine (PVP-I), and meglumine diatrizoate (MD) were mixed to produce an imageable hydrogel. A nude mouse model of osteosarcoma was established, and the hydrogel was injected locally into the interior of the osteosarcoma with X-ray guidance. The therapeutic effect and biosafety of the hydrogel were evaluated. Results: Iodine treatment at 18 and 20 µM for 12 h resulted in cell survival rate reduced to 50 ± 2.1% and 50.5 ± 2.7% for MG-63 and Sao-2 cells, respectively (p < 0.01). The proportion of apoptotic cells was significantly higher in the iodine-treatment group than in the control group (p < 0.05), and apoptotic bodies were observed by transmission electron microscopy. Iodine could regulate the death receptor pathway and induce MG-63 and Saos-2 cell apoptosis. The hydrogels were simple to assemble, and gels could be formed within 38 min. A force of less than 50 N was required to inject the gels with a syringe. The hydrogels were readily loaded and led to sustained iodine release over 1 week. The osteosarcoma volume in the PEG-iodine-silk/MD hydrogel group was significantly smaller than that in the other three groups (p < 0.001). Caspase-3 and poly (ADP-ribose) polymerase (PARP) expression levels were significantly higher in the PEG-iodine-silk/MD hydrogel group than in the other three groups (p < 0.001). Haematoxylin and eosin (H&E) staining showed no abnormalities in the heart, liver, spleen, lung, kidney, pancreas or thyroid in any group. Conclusions: Self-assembling imageable silk hydrogels could be injected locally into osteosarcoma tissues with X-ray assistance. With the advantages of good biosafety, low systemic toxicity and minimal invasiveness, self-assembling imageable silk hydrogels provide a promising approach for improving the locoregional control of osteosarcoma.

Abstract 6188: Targeted inhibition of the E3 ligase SCFSkp2/Cks1 has survival benefit and antitumor activity in osteosarcoma

Abstract Background: Osteosarcoma (OS) is a highly aggressive bone malignancy for which treatment has remained unchanged for years. Our previous studies indicate that the F-box protein SKP2 is overexpressed in OS and acts as a proto-oncogene. We recently reported that abolishing the interaction between SKP2 and its downstream substrate p27 using a Thr187Ala knock-in mutation leads to impaired tumor growth in an RB1/TP53 deficient transgenic OS mouse model. These data suggest that inhibiting the function of SKP2 may yield therapeutic benefits in OS. However, the precise mechanisms by which SKP2 promotes tumorigenesis in OS are not fully understood. Purposes: To determine the role of SCFSkp2/Cks1 in OS tumorigenesis and as a potential therapeutic target. Methods: We generated a genetically engineered mouse model of OS with a double knockout of Rb1 and p53 only within cells of the osteoblastic lineage (Osx1-Cre;Rb1lox/lox;Trp53lox/lox, DKO). The SKP2-knockout OS mouse model was further developed in the DKO background (Osx1-Cre;Rb1lox/lox;Trp53lox/lox; Skp2-/-, TKO). Mice of both genotypes were monitored for tumor growth, overall survival, and in vivo drug response. Early passaged OS cells and OS organoid culture was derived from mice tumors and used for in vitro analysis. Western Blot and IHC were used for protein level determination. RT-qPCR and RNA-seq were used for transcriptional level determination. Two small-molecule SCFSkp2/Cks1 inhibitors (C1 and Pevonedistat) were used for in vivo and in vitro testing. Results: All genotypes were born at the expected ratios and developmentally normal. DKO animals developed OS tumors with nearly full penetrance. SKP2 deletion significantly prolongs the overall survival (p&amp;lt;0.0001) and reduced the average tumor occurrence in OS mice (p&amp;lt;0.001). The in vivo tumor proliferation was also significantly delayed in the SKP2 deletion OS tumorigenesis (p=0.007). A histological analysis comparing tumors from two groups showed a decrease in proliferation (PCNA) and an increase in pro-apoptotic markers (TUNEL,etc) expression in SKP2-deleted OS. RT-qPCR and RNA-seq revealed upregulation of E2F1 downstream apoptotic-related target genes in Skp2-/- OS tumors. The transcriptional level analysis also revealed inhibition of cancer stemness markers and pathways in SKP2 deleted tumors and further proved by functional analyses. Two SCFSkp2/Cks1 inhibitors not only showed anti-tumoral activities both in vivo (p&amp;lt;0.05) and in organoid culture, but also synergistic with chemotherapy agents (ZIP synergy Score:9.34±1.24). Conclusions: Using genetic and pharmacologic approaches, we show that inhibiting SKP2 leads to significant antitumor activities in a clinically relevant OS mouse model. The strong survival benefit highlights SKP2 as a downstream actionable target in TP53/RB1-deficient OS, for which the targeted therapies have been disappointing to date. Citation Format: Jichuan Wang, Alexander Ferrena, Ranxin Zhang, Swapnil Singh, Hongling Zhao, Deyou Zheng, Hasibagan Borjihan, Waleed Al-Harden, Janet Tingling, Rui Yang, David S. Geller, Bang H. Hoang. Targeted inhibition of the E3 ligase SCFSkp2/Cks1 has survival benefit and antitumor activity in osteosarcoma [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 6188.

Abstract 1668: Development and characterization of a c-Myc-driven preclinical mouse model of osteosarcoma to investigate the tumor immune microenvironment

Abstract Osteosarcoma (OS) is the most common bone tumor in pediatric patients, particularly in adolescents and young adults. Currently, no effective molecular targeted therapies are available for the OS, however, many OS patients possess genetically defined somatic DNA copy number alterations, including amplification of chromosome 8q24, which harbors the oncogene Myc, and correlates with a worse prognosis. To better understand the molecular pathogenesis, and identify targeted therapy for OS, a robust and reliable preclinical model is needed both for the primary and metastatic disease. We have developed a novel conditional, osteoblast-specific Myc knock-in murine model to understand the mechanisms that underlie the ability of Myc to drive the development and progression of OS and utilize it for the identification of Myc-dependent intrinsic and extrinsic molecular vulnerabilities. A tissue-specific Myc-in genetically engineered murine model was generated by crossing Col2.3-Cre; TP53Flox/+ mice with LSL-MycT58A mice and characterized integrating RNA-seq and Mass Spectrometry. Syngeneic murine OS cell lines were also generated and characterized. The molecular characterization of the GEMM model was compared with high and low Myc expressing human tumors using the OS-TARGET dataset. Differential gene expression in Myc-driven and non-driven-tumor tissue samples were quantified by RNA sequencing. The immune landscape of OS was evaluated using SymphonyFACS and IHC staining. Murine OS tumor type was confirmed by H&amp;E staining. OS tumor was highly aggressive and metastatic in more than 60% of the mice. Myc expression was significantly higher both at the transcriptional and protein levels in the Myc-in tissue/cells compared to the non-Myc-driven model. Expression of 1479 genes (out of 13781 genes) were significantly altered in which 543 were upregulated and 936 were downregulated at transcriptional levels in Myc-in tumor tissue samples as compared to the low-Myc-driven OS tumor model analyzed by RNA sequencing. Myc-target-genes, splicing factor-related genes, E2F, G2M check-point, and DNA repair associated genes were significantly upregulated whereas the immune-related genes, oxidative phosphorylation, adipogenesis, and E2M transition genes were significantly downregulated when compared between two groups, which correlates to differential gene ontologies noted in high Myc human tumors. The total immune cell population, specifically the myeloid and macrophage populations were significantly diminished in the Myc-in tumor models, which could contribute to the poor prognosis in OS.Our murine model closely resembles human OS and will provide new opportunities for dissecting the molecular pathogenesis, identification of novel therapeutic strategies, and pre-clinical modeling for direct translational applications and targeting of Myc-driven OS. Citation Format: Bikesh K. Nirala, Lyazat Kurenbekova, Ryan L. Shuck, Tajhal Patel, Kimal Rajapakshe, Jason T. Yustein. Development and characterization of a c-Myc-driven preclinical mouse model of osteosarcoma to investigate the tumor immune microenvironment [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 1668.

Combined IL-6 and IL-8 inhibition to overcome mesenchymal stem cell (MSC)-induced resistance to antimetastatic drugs in osteosarcoma.

10037 Background: Mesenchymal stem cells (MSCs) play a key role in the progression of osteosarcoma (OS). In response to tumor-associated signals, including tumor-secreted extracellular vesicles (EVs), MSCs increase the expression of inflammatory cytokines promoting tumor cell aggressiveness. Previous data has demonstrated that IL-6 and IL-8 signaling mediate metastasis in OS. The aim of this study was to evaluate tumor EV-induced alterations of MSCs and identify combination therapies that can counteract MSC-induced resistance to antimetastatic drugs. Methods: Tumor EV-induced alterations of the MSC transcriptome were analyzed by RNA-seq. Gene set enrichment analysis (GSEA) was applied to discriminate TGFβ-dependent and independent pathways. EV RNA-induced expression changes were identified by transfecting purified EV-RNA in MSCs and by using a selective dsRNA antagonist. We selected candidate targets to block MSC-induced drug resistance and evaluated their effect in an orthotopic xenograft model of OS. Ladarixin (an allosteric inhibitor of the CXCL8/IL-8 receptors CXCR1 and CXCR2; 30 mg/kg 6x per week i.p.) and tocilizumab (anti-IL6 receptor antibody; 100 µg/mouse every other day i.p.) were administered starting from day one until the experimental endpoint. Metastasis were quantified by histological examination. This study was funded by the Dutch Cancer Society (KWF), POR FESR Campania 2014‐2020 and Dompé Farmaceutici SpA. Results: EVs from aggressive cancer cell lines induced an inflammatory MSC (iMSC) phenotype, characterized by increased expression of chemokines, including IL-8 as the most upregulated. Apart from IL-6, these alterations were mostly independent from TGFβ signaling and related to pattern recognition receptor (PRR) activation. We demonstrated that tumor EV-associated non-coding RNAs trigger TLR3 signaling in MSCs activating an innate immune response leading to high induction of IL-8 and other chemokines. Ladarixin and tocilizumab combination significantly reduced metastasis formation in a spontaneous metastasis model and overcame iMSC-induced resistance observed with single antimetastatic treatments. No effect was observed on primary tumor growth. Conclusions: EV-associated TGFβ together with EV-RNA induce iMSCs development in OS. Ladarixin in combination with tocilizumab reduced metastasis formation in a xenograft mouse model of OS, and, importantly, may prevent the occurrence of iMSC-induced tumor resistance to antimetastatic drugs.

Acacetin inhibits the tumor growth of human osteosarcoma cells through regulating Wnt/β-catenin and JNK signaling pathways

• Acacetin weakens the proliferation, migration and invasion of osteosarcoma cells. • Acacetin promotes the apoptosis of osteosarcoma cells. • Xenograft mouse osteosarcoma model confirms the inhibitory effect in vivo . • The signal pathway is predicted by network pharmacology and confirmed by the experiments. Osteosarcoma (OS) is the most common primary malignant bone tumor with high lung metastasis and mortality rate. Neo-adjuvant chemotherapy combined with limb salvage is the main treatment strategy for OS. However, because of the tolerance to conventional chemotherapy and the cell toxicity of chemotherapy drugs, new drugs with lower toxicity for OS are urgently needed. Acacetin is a natural flavonoid compound that has inhibitory effects on many kinds of tumors. Herein, we explored the impact of acacetin on OS cell growth in vitro and in vivo . We discovered that acacetin suppressed the proliferation, migration and invasion, while promoted the apoptosis of OS cells. Notably, acacetin had little impact on proliferation and apoptosis of human normal cells. Furthermore, we confirmed that acacetin repressed the growth and metastasis of OS in xenograft mouse model in vivo . Mechanically, we discovered that acacetin activated JNK pathway and inactivated Wnt/β-catenin pathway. β-catenin over-expression or JNK inhibitor SP600125 was able to partially neutralize acacetin-induced inhibitory effect on OS cells. To sum up, our study implies that acacetin may inhibit tumor growth of human OS cells through regulating Wnt/β-catenin and JNK signaling pathways.

Identification of immunogenic peptide neoantigens expressed in sarcomas and their therapeutic potential

Abstract T cells recognize tumor neoantigens (TNag) in the context of MHC and eliminate cells which express these antigens, leaving those lacking immunodominant epitopes. Central to investigating tumor immunoediting is the ability to define immunogenic TNag. Differential aggretope index (DAI) is a measure of the difference in MHC binding affinity between mutated and unmutated peptides and has emerged as a highly efficient way of identifying immunogenic TNag. Our previous studies have demonstrated that in mice, TNag with a DAI &amp;gt;8 are considered immunogenic and elicit T cells that lead to the tumor elimination. Thus, tumors expressing TNag of DAI &amp;gt;8 are only found in immunodeficient mice. Given these observations, we investigated the mutational burden and immune phenotype of bone proximal tumors in 122 patients. Tumors included are of bone, surrounding tissue and distal metastases, and were analyzed by flow cytometry, whole exome and TCR-Vβ sequencing. We hypothesized that tumors with high DAI TNag would be immunoedited in patients with robust T cell responses as measured by T cell clonality. Similar to observations in mice, this correlation was only observed in TNag with DAI &amp;gt;8. In contrast, there was no correlation between DAI and frequency of CD8 TILs indicating that the repertoire of TILs is a better indicator of immune reactivity than their frequency. Among tumor types analyzed, osteosarcomas were the least immunoedited, suggesting greatest potential for immunotherapy. We confirmed this potential in a mouse model of osteosarcoma, where we show that therapeutic HSP-mediated responses, augmented by PD-1 blockade, conferred the best survival. These studies provide compelling rationale for novel immunotherapies in patients with osteosarcoma. Supported by NIH R01 CA233803 and an HCC UPMC Developmental Award

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.

Inhibiting Endothelin Receptors with Macitentan Strengthens the Bone Protective Action of RANKL Inhibition and Reduces Metastatic Dissemination in Osteosarcoma.

Simple SummaryThe efficacy of current osteosarcoma therapy is diminished by two adverse events, namely resistance to chemotherapy and metastatic dissemination. In recent decades, research has been devoted to reducing these adverse events. Inhibiting bone resorption has shown promising effects on metastatic dissemination and tumor growth, with, however, the formation of significant tumoral mineralized tissue. Endothelin signaling is implicated in activating the cell that forms the mineralized tissues, consequently the impact of inhibiting it alone and in combination with the inhibition of bone resorption was evaluated using osteosarcoma models. The results obtained showed that inhibiting endothelin signaling significantly reduced the formation of mineralized tumor tissue concomitantly to metastatic dissemination without affecting sensitivity to chemotherapy. This inhibition appears to be a promising new therapeutic tool in the fight against osteosarcoma.Current treatments for osteosarcoma, combining conventional polychemotherapy and surgery, make it possible to attain a five-year survival rate of 70% in affected individuals. The presence of chemoresistance and metastases significantly shorten the patient’s lifespan, making identification of new therapeutic tools essential. Inhibiting bone resorption has been shown to be an efficient adjuvant strategy impacting the metastatic dissemination of osteosarcoma, tumor growth, and associated bone destruction. Unfortunately, over-apposition of mineralized matrix by normal and tumoral osteoblasts was associated with this inhibition. Endothelin signaling is implicated in the functional differentiation of osteoblasts, raising the question of the potential value of inhibiting it alone, or in combination with bone resorption repression. Using mouse models of osteosarcoma, the impact of macitentan, an endothelin receptor inhibitor, was evaluated regarding tumor growth, metastatic dissemination, matrix over-apposition secondary to RANKL blockade, and safety when combined with chemotherapy. The results showed that macitentan has no impact on tumor growth or sensitivity to ifosfamide, but significantly reduces tumoral osteoid tissue formation and the metastatic capacity of the osteosarcoma. To conclude, macitentan appears to be a promising therapeutic adjuvant for osteosarcoma alone or associated with bone resorption inhibitors.

Nigericin exerts anticancer effects through inhibition of the SRC/STAT3/BCL-2 in osteosarcoma

The treatment of osteosarcoma has reached a bottleneck period in recent 30 years, there is an urgent need to find new drugs and treatment methods. Nigericin, an antibiotic derived from Streptomyces hygroscopicus, has exerted promising antitumoral effect in various tumors. The anticancer effect of Nigericin in human osteosarcoma has never been reported. In the present study, we explored the anticancer effects of Nigericin in osteosarcoma in vitro and in vivo. Our results showed that nigericin treatment significantly reduced tumor cell proliferation in dose-dependent and time-dependent in human osteosarcoma cells. Nigericin can inhibit cell growth of osteosarcoma cells, in addition to S-phase cycle arrest, the nigericin induces apoptosis. Furthermore, bioinformatics predicted that Nigericin exerts anticancer effects through inhibiting SRC/STAT3 signaling pathway in osteosarcoma. The direct binding between SRC and activator of transcription 3 (STAT3) was confirmed by Western blot. Nigericin can down regulate STAT3 and Bcl-2. In order to further elucidate the inhibitory effect of nigericin on SRC/STAT3/Bcl-2 signal transduction mechanism, we established human osteosarcoma cancer cells stably expressing STAT3. Western blot confirmed that nigericin exerts anticancer effects on human osteosarcoma cancer cells by directly targeting STAT3. In addition, Nigericin can significantly inhibit tumor migration and invasion. Finally, Nigericin inhibits tumor growth in a mouse osteosarcoma model. The nigericin targeting the SRC/STAT3/BCL-2 signaling pathway may provide new insights into the molecular mechanism of nigericin on cancer cells and suggest its possible clinical application in osteosarcoma.

Oral-recombinant Methioninase Lowers the Effective Dose and Eliminates Toxicity of Cisplatinum for Primary Osteosarcoma of the Mammary Gland in a Patient-derived Orthotopic Xenograft Mouse Model.

Primary osteosarcoma of the mammary gland is a very rare disease, accounting for under 1% of all mammary gland malignancies. There is no established first-line treatment, and prognosis is poor compared to conventional breast cancer. We previously demonstrated the efficacy of cisplatinum and eribulin in a patient-derived orthotopic xenograft (PDOX) mouse model of primary breast osteosarcoma. However, these drugs show significant clinical toxicity. All cancers are addicted to methionine (Hoffman effect). In the present study, we determined whether methionine restriction with oral recombinant methioninase (o-rMETase) would lower the effective dose of cisplatinum in a PDOX model of primary osteosarcoma of the mammary gland, thereby reducing its toxicity. Mouse PDOX models of primary osteosarcoma of the breast were randomized into the following groups: control; cisplatinum (weekly at 3 or 6 mg/kg); twice-daily o-rMETase; or o-rMETase combined with 3 mg/kg cisplatinum, with treatment for 2 weeks. Cisplatinum at 6 mg/kg significantly inhibited breast osteosarcoma growth compared with the untreated control and mice treated with 3 mg/kg cisplatinum (p=0.01 and 0.009, respectively). There was no significant difference in tumor growth between mice treated with cisplatinum at 3 mg/kg and the control (p=0.16). Combination therapy with cisplatinum at 3 mg/kg and twice daily o-rMETase regressed the osteosarcoma of the mammary gland (p=0.009), similar to the inhibition by cisplatinum at 6 mg/kg alone. Cisplatinum at 6 mg/kg caused a significant loss of mouse body weight, compared to the control (p=0.02). There was no significant body-weight loss with the combination therapy of o-rMETase and cisplatinum at 3 mg/kg, compared to the untreated control. o-rMETase halved the effective dose of cisplatinum, thereby eliminating cisplatinum toxicity, demonstrating a future clinical strategy for therapy of osteosarcoma of the breast.

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.

Peiminine Induces G0/G1-Phase Arrest, Apoptosis, and Autophagy via the ROS/JNK Signaling Pathway in Human Osteosarcoma Cells in Vitro and in Vivo.

Aims: Peiminine has been reported to have various pharmacological properties, including anticancer activity. In this study, we investigated the effect of this alkaloid on osteosarcoma and explored the underlying mechanisms. Methods: To evaluate the antiosteosarcoma effects of peiminine in vitro, cell viability was assessed by CCK-8 and live/dead assays; the effects of the drug on apoptosis and the cell cycle were examined by flow cytometry; the effects on cell migration and invasion were detected by wound healing and Transwell assays, respectively, while its effects on autophagy were observed by transmission electron microscopy and an LC3 fluorescent puncta formation assay. The role of autophagy in the peiminine-mediated effects in osteosarcoma cells was evaluated by CCK-8 assay and western blotting after the application of the autophagy inhibitor chloroquine. The effect of peiminine on reactive oxygen species (ROS) production was analyzed using fluorescence confocal microscopy and spectrophotometry. Additionally, peiminine-treated osteosarcoma cells were exposed to SP600125, a JNK inhibitor, and N-acetylcysteine, a ROS scavenger, after which the contribution of the ROS/JNK signaling pathway to osteosarcoma was assessed using cell viability and LC3 fluorescent puncta formation assays, flow cytometry, and western blotting. A xenograft mouse model of osteosarcoma was generated to determine the antitumor effects of peiminine in vivo. Results: Peiminine suppressed proliferation and metastasis and induced cell cycle arrest, apoptosis, and autophagy in osteosarcoma cells. These anticancer effects of peiminine were found to be dependent on intracellular ROS generation and activation of the JNK pathway. In line with these results, peiminine significantly inhibited xenograft tumor growth in vivo. Conclusions: Peiminine induced G0/G1-phase arrest, apoptosis, and autophagy in human osteosarcoma cells via the ROS/JNK signaling pathway both in vitro and in vivo. Our study may provide an experimental basis for the evaluation of peiminine as an alternative drug for the treatment of osteosarcoma.

β2-and β3-Adrenergic Receptors Contribute to Cancer-Evoked Pain in a Mouse Model of Osteosarcoma via Modulation of Neural Macrophages.

The mechanisms involved in the development and maintenance of cancer pain remain largely unidentified. Recently, it has been reported that β-adrenergic receptors (β-ARs), mainly β2-and β3-ARs, contribute to tumor proliferation and progression and may favor cancer-associated pain and neuroinflammation. However, the mechanism underlying β-ARs in cancer pain is still unknown. Here, we investigated the role of β1-, β2-and β3-ARs in a mouse model of cancer pain generated by the para-tibial injection of K7M2 osteosarcoma cells. Results showed a rapid tumor growth in the soft tissue associated with the development of mechanical allodynia in the hind paw ipsilateral to the injected site. In addition to reduce tumor growth, both propranolol and SR59230A, β1-/β2-and β3-AR antagonists, respectively, attenuated mechanical allodynia, the number of macrophages and an oxidative stress by-product accumulated in the ipsilateral tibial nerve. The selective β1-AR antagonist atenolol was able to slightly reduce the tumor growth but showed no effect in reducing the development of mechanical allodynia. Results suggest that the development of the mechanical allodynia in K7M2 osteosarcoma-bearing mice is mediated by oxidative stress associated with the recruitment of neural macrophages, and that antagonism of β2-and β3-ARs contribute not solely to the reduction of tumor growth, but also in cancer pain. Thus, the targeting of the β2-and β3-ARs signaling may be a promising therapeutic strategy against both tumor progression and the development of cancer-evoke pain in osteosarcoma.