Mouse Model Of Osteosarcoma Research Articles

Chromatin remodeling protein HELLS is upregulated by inactivation of the RB-E2F pathway and is nonessential for osteosarcoma tumorigenesis.

Osteosarcoma is the most common primary bone malignancy in children and adolescents. Among the various molecular mechanisms implicated in osteosarcomagenesis, the RB-E2F pathway is of particular importance as virtually all cases of osteosarcoma display alterations in the RB-E2F pathway. In this study, we examined the transcription factor E2F family members that are associated with increased malignancy in Rb1-null osteosarcoma tumors. Using genetically engineered mouse models of osteosarcoma, we found that loss of activator E2Fs, E2F1 and E2F3, significantly delays tumor progression and increases the overall survival of the p53/Rb1-deficient osteosarcoma mouse model. We also studied the role of helicase, lymphoid specific (HELLS), a chromatin remodeling protein identified as a critical downstream effector of the RB-E2F signaling pathway in various cancers. In this study, we confirmed that the RB-E2F pathway directly regulates HELLS gene expression. We also found that HELLS mRNA is upregulated and its protein overexpressed in osteosarcoma. Using loss-of-function assays to study the role of HELLS in human osteosarcoma, we observed that HELLS has no effect on tumor proliferation and migration. Further, we pioneered the study of Hells in developmental tumor models by generating Hells conditional knockout osteosarcoma mouse models to examine the role of HELLS in osteosarcoma tumor development. We found that loss of Hells in osteosarcoma has no effect in tumor initiation and overall survival of mice. This suggests that while HELLS may serve as a biomarker for tumorigenesis and for RB-E2F pathway status, it is unlikely to serve as a relevant target for therapeutics in osteosarcoma.

Abstract 2731: Checkpoint inhibitor therapy in combination with the implantation of agarose encapsulated cancer cells inhibits tumor growth in a mouse model of osteosarcoma

Abstract We have previously shown that murine renal adenocarcinoma (RENCA) cells encapsulated in agarose macrobeads (MB) secrete factors that inhibit the proliferation of freely growing tumor cells outside the MB, both in vitro and in vivo. This effect is being investigated in ongoing clinical trials with colorectal patients who underwent laparoscopic intraperitoneal implantations of RENCA MBs (NCT01053013, NCT02046174). In the current study, we report the effect of immune checkpoint inhibitors (anti-PD-1 and anti-PD-L1 monoclonal antibodies) in combination with RENCA MBs on tumor burden in BALB/cJ mice induced with K7M2 murine osteosarcoma tumors. Mice that developed tumors (2-5 mm) on subcutaneous injection of K7M2 cells were randomly enrolled in the following study groups: sham, RENCA MBs only, anti-PD-1 only, RENCA MBs + anti-PD-1, anti-PD-L1 only, RENCA MBs + anti-PD-L1. Anti-PD-1 and anti-PD-L1 antibodies were administered ip bi-weekly for 3-6 weeks. RENCA MBs were implanted in the peritoneal cavity on the day of enrollment. Mean tumor volumes (mm3) throughout days 0-24 were: RENCA MB (199.17 ± 125.57), anti-PD-1 only (278.87 ± 180.34), anti-PD-L1 only (332.46 ± 185.13) and sham treated mice (512.61 ± 230.12). RENCA MB and anti-PD-L1 combination therapy did not inhibit tumor growth (375.00 ± 511.61). PD-L1 expression was absent in primary tumors as observed by immunostaining. Treatment with RENCA MBs plus anti-PD-1 treatment demonstrated an additive effect (116.94 ± 83.77) compared to RENCA MB only or anti-PD-1 alone. Moreover, tumors in 4 out of 16 mice from the RENCA MB plus anti-PD-1 group receded and were undetectable (3 in less than 1 week and 1 at 39 days) following treatment. Tumors also regressed and were not palpable in 1 mouse each in the anti-PD-1 and RENCA MB plus anti-PD-L1 groups at day 14 and 9, respectively. Discontinuation of anti-PD-1 and anti-PD-L1 treatment after 3 weeks of treatment resulted in loss of tumor growth regulation. Preliminary data suggests that continued treatment with these immune checkpoint inhibitors beyond 3 weeks prolongs the regulation of tumor growth in this model. These data support the use of anti-PD-1 in combination with the implantation of RENCA macrobeads in this mouse model of osteosarcoma, and the possibility of similar approaches in the clinic. Citation Format: Pradeep R. Dumpala, Prithy C. Martis, Melissa A. Bemrose, Atira Dudley, Barry H. Smith, Lawrence S. Gazda. Checkpoint inhibitor therapy in combination with the implantation of agarose encapsulated cancer cells inhibits tumor growth in a mouse model of osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2731.

Abstract 4128: Loss of p53 and Rb enhances ligand-dependent hedgehog signaling in development and cancer

Abstract The evolutionary conserved Hedgehog (Hh) signaling pathway plays fundamental morphogenic and mitogenic roles in tissue development and homeostasis, as well as in the initiation and progression of various cancers. Signaling is driven by the binding of Sonic hedgehog (Shh) ligand to the receptor Patched (Ptch1), to initiate primary cilia dependent Smoothened (Smo)-Gli activation. Although mutations in Hh pathway components are described in some cancers, the majority of Hh tumors exhibit ligand-dependent Hh signaling, where Hh ligand is produced by the stroma and/or tumor cells to maintain pathway activation and self-renewal. Here, we have utilized various Hh-dependent developmental models to better understand the tumorigenic mechanisms of Hh ligand-dependent signaling in cancer. During cerebellar development, Shh is secreted from cerebellar purkinje cells for rapid expansion of the cerebellar granule cell precursor (GCP) population in the external granule layer (EGL). Inactivation of Shh in purkinje cells (Pcp2Cre;Shhfl/fl) leads to reduced GCP proliferation, premature eradication of the EGL and reduced cerebellar size. In contrast, Shh overexpression by purkinje cells (Pcp2Cre;ShhTg) results in increased GCP expansion, a sustained EGL and increased cerebellar size. Indeed, signs of remnant EGL persist into adulthood, similar to the pre-neoplastic lesions observed in Ptch1+/- mice, believed to be the origin of SHH medulloblastoma. During skeletal development, Indian hedgehog secreted from pre-hypertrophic chondroblasts is required for osteoblast progenitor specification from multipotent mesenchymal stem cells. Sustained Hh signaling in osteoblast precursors (OsxCre;SmoM2 or OsxCre;ShhTg) results in reduced ossification of the calvaria, maxilla and mandible, reduced long bone width, and reduced expression of mature osteoblast markers. Together, these models implicate sustained Hh expression in the maintenance of an immature cell state at the expense of differentiation. Moreover, in a mouse model of osteosarcoma, inactivation of Smo (OsxCre;p53fl/fl;Rbfl/fl;Smofl/fl) abolished the malignant phenotype of OsxCre;p53fl/fl;Rbfl/fl mice, leading to benign osteoid osteoma consistent with osteoblast differentiation. Increased Hh signaling in cancers with prevalent p53 and Rb mutations led us to investigate a role for p53 and Rb in Hh ligand-dependent signaling. Genetic inactivation of p53 and/or Rb in the developing mouse neural tube resulted in expansion of the Hh-dependent Nkx2.2 ventral domain, consistent with a Hh gain of function phenotype. Similarly, siRNA knockdown of p53 and/or Rb in mouse mesenchymal stem cell line C3H10T1/2 markedly increased Hh-dependent osteoblast specification in response to Hh ligand. Taken together, we propose that p53 and Rb mutations enhance ligand-dependent Hh pathway activation that maintains cells in a primitive state and promotes tumorigenesis. Citation Format: Catherine R. Cochrane, Anette Szczepny, Samantha W. Jayasekara, Vijesh Vaghjiani, D. Watkins, Jason E. Cain. Loss of p53 and Rb enhances ligand-dependent hedgehog signaling in development and cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4128.

Irradiation enhanced the effects of PD-1 blockade in brain metastatic osteosarcoma

Brain metastasis of osteosarcoma are rare but carry a dismal prognosis. Despite the advances in both systemic immunotherapy and localized radiation, it is still difficult to treat brain metastasis, with less than 12 months of survival from the time of diagnosis for most patients. Currently, there is interest in combining strategies to take advantage of the potential synergy. In this study, the mouse model of metastatic osteosarcoma to brain was used to explore the ability of local radiation and anti-PD-1 blockade to induce beneficial anti-tumor immune responses against distant, unirradiated brain metastatic tumors. Immune markers from the peripheral blood and tumor tissue were analyzed by flow cytometry, real-time PCR and western blot. The combination treatment produced a stronger systemic anti-tumor response than either treatment alone, shown by the reduced tumor burden and larger numbers of cytotoxic CD8+ T cells in the unirradiated tumors, indicating an abscopal effect. These data suggested that combination treatment of irradiation with anti-PD-1 immunotherapy can induce abscopal anti-tumor responses and improve both local and distant control.

Sox2 is required for tumor development and cancer cell proliferation in osteosarcoma.

The stem cell transcription factor Sox2 is highly expressed in many cancers where it is thought to mark cancer stem cells (CSC). In osteosarcomas, the most common bone malignancy, high Sox2 expression marks and maintains a fraction of tumor initiating cells that show all the properties of CSC. Knock down of Sox2 expression abolishes tumorigenicity and suppresses the CSC phenotype. Here we show that, in a mouse model of osteosarcoma, osteoblast-specific Sox2 conditional knockout (CKO) causes a drastic reduction in the frequency and onset of tumors. The rare tumors detected in the Sox2 CKO animals were all Sox2 positive, indicating that they arose from cells that had escaped Sox2 deletion. Furthermore Sox2 inactivation in cultured osteosarcoma cells by CRISPR/CAS technology leads to a loss of viability and proliferation of the entire cell population. Inactivation of the YAP gene, a major Hippo Pathway effector which is a direct Sox2 target, causes similar results and YAP overexpression rescues cells from the lethality caused by Sox2 inactivation. These effects were osteosarcoma-specific, suggesting a mechanism of cell “addiction” to Sox2 initiated pathways. The requirement for Sox2 for osteosarcoma formation as well as for the survival of the tumor cells suggests that disruption of Sox2-initiated pathways could be an effective strategy for the treatment of osteosarcoma.

Migration-inducing gene-7 independently predicts poor prognosis of human osteosarcoma and is associated with vasculogenic mimicry

Vasculogenic mimicry (VM) is a special type of vascular channel formed by tumor cells without endothelial cell participation. Migration-inducing gene 7 (MIG-7) plays an important role in regulating VM. In this study, immunohistochemical staining was used to detect MIG-7 in tissue specimens from 141 primary osteosarcoma patients, and the relationship between MIG-7 and VM was examined. Survival analysis were performed to evaluate the prognoses. MIG-7 knockdown osteosarcoma cells were used for cell proliferation, apoptosis, migration, invasiveness and VM formation assays. A spontaneously metastasizing cell line-derived orthotopic xenograft mouse model was established to evaluate the effect of MIG-7 knockdown on tumorigenesis, VM formation and lung metastasis. MIG-7 expression was associated with VM formation. There were significant differences in overall and metastasis-free survival between the MIG-7-positive and MIG-7-negative groups. The MIG-7 expression was shown to be an independent indicator of both overall and metastasis-free survival. In vitro knockdown of MIG-7 dramatically reduced migration, invasion and VM formation in osteosarcoma cells without any significant effect on cell proliferation and apoptosis. MIG-7 knockdown also exhibited potent antitumor, antimetastasis and anti-VM effects in the orthotopic mouse model of 143B osteosarcoma. Therefore, MIG-7 serves as an independent unfavorable prognostic indicator in osteosarcoma patients and MIG-7 is an important mediator of osteosarcoma VM formation.

Metastatic tumor cells detection and anti-metastatic potential with vesicular stomatitis virus in immunocompetent murine model of osteosarcoma.

Sarcomas are associated with a high incidence of lung metastasis, which leads to a high-risk of cancer death. This study was performed to explore the pre-clinical theranostic potential of a novel fully functional recombinant vesicular stomatitis virus carrying imaging gene Katushka (rVSV-K), as virotherapy and circulating tumor cells (CTCs) detection in the syngeneic mouse model of osteosarcoma with spontaneous pulmonary metastases. Recombinant VSV-K was generated and evaluated in vitro on human and murine osteosarcoma cells. Spontaneous osteosarcoma metastases were established in immune-competent mice by implanting subcutaneously syngeneic osteosarcoma LM8 cells. The vector was injected into the tumor-bearing mice via jugular vein either once or repeatedly. To assess effectiveness, primary tumor growth and development of lung metastasis as well as survival were evaluated. We found that rVSV-K efficiently replicated in and killed all osteosarcoma cell lines in time-dependent manners. Both single or repeated systemic injections of the virus did not inhibit the growth of the primary tumor, but the repeated administration could effectively suppress the development of lung metastases and was likely responsible for the observed increase in survival. Furthermore, we demonstrated, for the first time, that CTCs in blood samples from syngeneic osteosarcoma-bearing mice were successfully detected by utilizing rVSV-K ex vivo. Our results show that repeated systemic injections of rVSV-K are an effective anti-metastatic agent against osteosarcoma in immune-competent mice and this virus to be a useful tool for detection of osteosarcoma CTCs, suggesting that further development of future viral-based theranostic approach in patients with osteosarcoma is warranted. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2562-2569, 2018.

Anti-PD-1 therapy redirects macrophages from an M2 to an M1 phenotype inducing regression of OS lung metastases.

Osteosarcoma (OS) pulmonary metastasis translates into poor patient survival. The implication of PD‐1‐PD‐L1 pathway in the context of NK cells and/or macrophages in OS is unknown. We investigated the effect of anti‐PD‐1 in OS lung metastasis and the role of NK cells and/or macrophages in anti‐PD‐1 responses. A human LM7 OS mouse model was used. Immunohistochemistry for tissues (PD‐L1, caspase‐3, Ki‐67, NK cells, macrophages), and Western blotting for OS lung tumors (p‐Stat3, p‐Erk1/2) was performed. NK and macrophages were assessed using flow cytometry. NK cell and macrophage depletion were conducted using anti‐asialo GM1 and clodrosome, respectively. PD‐L1 expression was observed in human OS cells and OS patient lung metastases. Anti‐PD1 antibody led to a significant decrease in the number of OS lung metastases, enhanced tumor apoptosis, decreased tumor cell proliferation, and p‐STAT‐3/p‐Erk1/2 signaling blockade in OS lung tumors. NK cells and macrophages in OS lung tumors expressed PD‐1 and anti‐PD1 increased NK cell and macrophage tumor infiltration. Increased numbers of antitumor M1 macrophages and decreased pro‐inflammatory M2 macrophages were seen. NK depletion did not affect therapeutic effect of anti‐PD‐1, suggesting that NK cells were not directly involved. However, macrophage depletion significantly compromised anti‐PD1 efficacy, confirming their role in efficacy of anti‐PD‐1 against OS lung metastasis. Our findings suggest that OS lung metastases regression by anti‐PD1 can be attributed to activated tumor M1 macrophages and reduced M2 macrophages. Owing to the co‐relation of M1 macrophages with OS patient outcome, we provide a novel mechanism of PD‐1 blockade and a basis for future clinical trials for anti‐PD‐1 antibodies in OS.

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells.

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under the influence of specific environmental signals, these adult stem cells can release paracrine mediators leading to accelerated tumor growth and metastasis. Defining the crosstalk between tumor and MSCs is of primary importance to understand the mechanisms underlying cancer progression and identify novel targets for therapeutic intervention. Cancer cells produce high amounts of extracellular vesicles (EVs), which can profoundly affect the behavior of target cells in the tumor microenvironment or at distant sites. Tumor EVs enclose functional biomolecules, including inflammatory RNAs and (onco)proteins, that can educate stromal cells to enhance the metastatic behavior of cancer cells or to participate in the pre-metastatic niche formation. In this article, we describe the development of a preclinical cancer mouse model that enables specific evaluation of the EV-mediated crosstalk between tumor and mesenchymal stem cells. First, we describe the purification and characterization of tumor-secreted EVs and the assessment of the EV internalization by MSCs. We then make use of a multiplex bead-based immunoassay to evaluate the alteration of the MSC cytokine expression profile induced by cancer EVs. Finally, we illustrate the generation of a bioluminescent orthotopic xenograft mouse model of osteosarcoma that recapitulates the tumor-MSC interaction, and show the contribution of EV-educated MSCs to tumor growth and metastasis formation. Our model provides the opportunity to define how cancer EVs shape a tumor-supporting environment, and to evaluate whether blockade of the EV-mediated communication between tumor and MSCs prevents cancer progression.

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

A Preclinical Mouse Model Of Osteosarcoma To Define The Extracellular Vesicle-mediated Communication Between Tumor And Mesenchymal Stem Cells - Video. JoVE publishes peer-reviewed scientific video protocols to accelerate biological, medical, chemical and physical research. Watch our scientific video articles. JoVE is the world-leading producer and provider of science videos with the mission to improve scientific research, scientific journals, and education. Millions of scientists, educators and students at thousands of universities, colleges, hospitals and biopharmaceutical companies worldwide use JoVE for their research, teaching and learning.

Regulatory mechanism of microRNA-128 in osteosarcoma tumorigenesis and evolution through targeting SASH1.

Osteosarcoma, which commonly occurs in young individuals, is a type of malignant tumor of growing bones. MicroRNAs (miRNAs) have been found to be involved in various cancer-related processes. In the present study, it was reported that miRNA-128 (miR-128) was overexpressed in pathological tissues from patients with osteosarcoma. The present study investigated the possible regulatory mechanism of miR-128 on the progression of osteosarcoma and offered a foundation for clinical therapeutics in osteosarcoma. First, the expressions levels of miR-128 and its target gene, SAM and SH3 domain-containing 1 (SASH1), were measured in tissues from patients with osteosarcoma, and their correlation with osteosarcoma in terms of the pathological level were examined. The effects of miR-128 on osteosarcoma cell proliferation and apoptosis were examined, and its regulation of the expression levels of SASH1 and associated proteins was analyzed. Subsequently, the association between SASH1 and miR-128 was evaluated using a dual luciferase gene reporter assay. Finally, an in vivo xenograft tumor mouse model of osteosarcoma was established to confirm the in vitro results. The results demonstrated a higher expression of miR-128 in pathological tissues, compared with that in normal tissues. From examining the patient osteosarcoma tissues, marked correlations were found between the expression of miR-128 and that of SASH1, particularly with tumor size, invasion depth, lymph node metastasis, and tumor-node-metastasis stage. Compared with the negative control group and blank control group, the results showed that the inhibition of miR-128 led to a lower cell proliferation rate and higher apoptotic rate in MG-63 cells (P<0.05). Additionally, the expression of B-cell lymphoma 2 (Bcl-2) was downregulated in the miR-128-inhibited group, compared with that in the control group, whereas the expression levels of SASH1, Bcl-2-associated X protein and caspase-3 were upregulated in the group with miR-128 inhibition (P<0.05). SASH1 was confirmed as a direct target of miR-128 using a dual luciferase gene reporter assay. Finally, the downregulation of miR-128 was found to induce tumor suppressive effects on xenograft tumor models of osteosarcoma in mice in vivo. The results of the present study suggested that miR-128 may regulate the tumorigenesis and evolution of osteosarcoma through targeting SASH1.

Murine models of osteosarcoma: A piece of the translational puzzle

Osteosarcoma (OS) is the most common cancer of bone in children and young adults. Despite extensive research efforts, there has been no significant improvement in patient outcome for many years. An improved understanding of the biology of this cancer and how genes frequently mutated contribute to OS may help improve outcomes for patients. While our knowledge of the mutational burden of OS is approaching saturation, our understanding of how these mutations contribute to OS initiation and maintenance is less clear. Murine models of OS have now been demonstrated to be highly valid recapitulations of human OS. These models were originally based on the frequent disruption of p53 and Rb in familial OS syndromes, which are also common mutations in sporadic OS. They have been applied to significantly improve our understanding about the functions of recurrently mutated genes in disease. The murine models can be used as a platform for preclinical testing and identifying new therapeutic targets, in addition to testing the role of additional mutations in vivo. Most recently these models have begun to be used for discovery based approaches and screens, which hold significant promise in furthering our understanding of the genetic and therapeutic sensitivities of OS. In this review, we discuss the mouse models of OS that have been reported in the last 3-5 years and newly identified pathways from these studies. Finally, we discuss the preclinical utilization of the mouse models of OS for identifying and validating actionable targets to improve patient outcome.

Olaratumab Exerts Antitumor Activity in Preclinical Models of Pediatric Bone and Soft Tissue Tumors through Inhibition of Platelet-Derived Growth Factor Receptor α.

Purpose: Platelet-derived growth factor receptor α (PDGFRα) is implicated in several adult and pediatric malignancies, where activated signaling in tumor cells and/or cells within the microenvironment drive tumorigenesis and disease progression. Olaratumab (LY3012207/IMC-3G3) is a human mAb that exclusively binds to PDGFRα and recently received accelerated FDA approval and conditional EMA approval for treatment of advanced adult sarcoma patients in combination with doxorubicin. In this study, we investigated olaratumab in preclinical models of pediatric bone and soft tissue tumors.Experimental Design: PDGFRα expression was evaluated by qPCR and Western blot analysis. Olaratumab was investigated in in vitro cell proliferation and invasion assays using pediatric osteosarcoma and rhabdoid tumor cell lines. In vivo activity of olaratumab was assessed in preclinical mouse models of pediatric osteosarcoma and malignant rhabdoid tumor.Results:In vitro olaratumab treatment of osteosarcoma and rhabdoid tumor cell lines reduced proliferation and inhibited invasion driven by individual platelet-derived growth factors (PDGFs) or serum. Furthermore, olaratumab delayed primary tumor growth in mouse models of pediatric osteosarcoma and malignant rhabdoid tumor, and this activity was enhanced by combination with either doxorubicin or cisplatin.Conclusions: Overall, these data indicate that olaratumab, alone and in combination with standard of care, blocks the growth of some preclinical PDGFRα-expressing pediatric bone and soft tissue tumor models. Clin Cancer Res; 24(4); 847-57. ©2017 AACR.

Abstract LB-B01: The anti-platelet-derived growth factor receptor α antibody olaratumab (Lartruvo) increases overall survival in metastatic mouse models of human osteosarcoma

Abstract Platelet-derived growth factor receptor alpha (PDGFRα) is implicated in several types of adult and pediatric malignancies, where its aberrant expression and/or activation in tumor cells and/or tumor-associated stromal cells promote primary tumor growth and metastasis. Therefore, PDGFRα signaling may regulate disease progression via autocrine and paracrine modes of activation and facilitating crosstalk between the tumor and stroma. Olaratumab is a fully humanized monoclonal antibody that selectively binds human PDGFRα and blocks signaling initiated by ligand binding. We evaluated the efficacy of olaratumab and 1E10, a high affinity anti-mouse PDGFRα antibody in preclinical metastatic models of human osteosarcoma. A metastatic derivative of the PDGFRα-positive human osteosarcoma cell line HuO9 (HuO9-H3) was implanted in the gastrocnemius of mice. Olaratumab/1E10 was administered twice weekly once tumors grew to ~ 150 mm3. When tumors reached an average of volume of 1600 mm3, tumor-bearing limbs were amputated and four separate treatment cohorts were evaluated. These cohorts were as follows: (1) continuous IgG control antibody treatment, (2) continuous olaratumab/1E10 treatment prior to and after amputation, (3) olaratumab/1E10 treatment pre-amputation followed by IgG administration post-amputation, and (4) IgG treatment pre-amputation followed by olaratumab treatment post-amputation. A statistically significant and prolonged overall survival (OS) benefit (p&amp;lt;0.001) was observed in the continuous olaratumab/1E10 treatment group only, and correlated with a reduced tumor burden in the lung as determined by histologic evaluation. An olaratumab/1E10-dependent statistically significant OS benefit (p&amp;lt;0.001) was also observed in the HuO9-H3 and PDGFRα/PDGFRβ-positive MG63.3 human osteosarcoma cell lines introduced via tail vein injection. Interestingly, histologic review shows reduced tumor burden in the lung and lung pleura/mediastinum of these models. Mouse-specific bright field in situ hybridization (BRISH) showed increased expression of PDGFRA, PDGFRB, associated ligands, and VEGFA in the murine lung stroma prior to histologically apparent metastases. These data indicate that olaratumab/1E10-mediated PDGFRα blockade significantly increases OS in preclinical mouse models of human osteosarcoma and suggest a novel role for the PDGFRα pathway in the pathogenesis of metastatic osteosarcoma lung lesions. Citation Format: Amy K. LeBlanc, Gerard J. Oakley, Caitlin D. Lowery, Arnulfo Mendoza, Ling Ren, Timothy Holzer, Kelly Credille, Cynthia Winings, Amanda Estelle, Mia Chen, Patrick Finnegan, Wayne Blosser, Andrew Schade, Symantha Melemed, Louis F. Stancato. The anti-platelet-derived growth factor receptor α antibody olaratumab (Lartruvo) increases overall survival in metastatic mouse models of human osteosarcoma [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B01.

Zoledronate Enhances the Cytotoxicity of Gamma Delta T Cell Immunotherapy in an Orthotopic Mouse Model of Osteolytic Osteosarcoma

Zoledronate Enhances the Cytotoxicity of Gamma Delta T Cell Immunotherapy in an Orthotopic Mouse Model of Osteolytic Osteosarcoma

Systemic administration of β-glucan of 200 kDa modulates melanoma microenvironment and suppresses metastatic cancer

ABSTRACTConverting an immunosuppressive melanoma microenvironment into one that favors the induction of antitumor immunity is indispensable for effective cancer immunotherapy. In the current study we demonstrate that oat-derived β-(1-3)-(1-4)-glucan of 200 kDa molecular size (BG34-200) previously shown to mediate direct interaction with macrophages could alter the immune signature within melanoma microenvironment. Systemic administration of BG34-200 resulted in reversion of tolerant melanoma microenvironment to an immunogenic one that allows M1-type activation of macrophages, the induction of pro-inflammatory cytokines/chemokines including IFN-γ, TNF-α, CXCL9, and CXCL10, and enhanced IRF1 and PD-L1 expression. In turn, BG34-200 induced a superior antitumor response against primary and lung metastatic B16F10 melanoma compared to untreated controls. The enhanced tumor destruction was accompanied with significantly increased tumor infiltration of CD4+ and CD8+ T cells as well as elevated IFN-γ in the tumor sites. Systemic administration of BG34-200 also provoked systemic activation of tumor draining lymph node T cells that recognize antigens naturally expressing in melanoma (gp100/PMEL). Mechanistic studies using CD11b-knockout (KO), CD11 c-DTR transgenic mice and nude mice revealed that macrophages, DCs, T cells and NK cells were all required for the BG34-200-induced therapeutic benefit. Studies using IFN-γ-KO transgenic mice showed that IFN-γ was essential for the BG34-200-elicited antitumor response. Beyond melanoma, the therapeutic efficacy of BG34-200 and its immune stimulating activity were demonstrated in a mouse model of osteosarcoma. Together, BG34-200 is highly effective in modulating antitumor immunity. Our data support the potential therapeutic use of this novel immune modulator in the treatment of metastatic melanoma.

Memory T Cells Expressing an NKG2D-CAR Efficiently Target Osteosarcoma Cells.

Purpose: NKG2D ligands (NKG2DL) are expressed on various tumor types and immunosuppressive cells within tumor microenvironments, providing suitable targets for cancer therapy. Various immune cells express NKG2D receptors, including natural killer (NK) cells and CD8+ T cells. Interactions between NKG2DL and NKG2D receptors are essential for NK-cell elimination of osteosarcoma tumor-initiating cells. In this report, we used NKG2D-NKG2DL interactions to optimize an immunotherapeutic strategy against osteosarcoma. We evaluated in vitro and in vivo the safety and cytotoxic capacity against osteosarcoma cells of CD45RA- memory T cells expressing an NKG2D-4-1BB-CD3z chimeric antigen receptor (CAR).Experimental Design: CD45RA- cells from healthy donors were transduced with NKG2D CARs containing 4-1BB and CD3z signaling domains. NKG2D CAR expression was analyzed by flow cytometry. In vitro cytotoxicity of NKG2D-CAR+ CD45RA- T cells against osteosarcoma was evaluated by performing conventional 4-hour europium-TDA release assays. For the in vivo orthotopic model, 531MII YFP-luc osteosarcoma cells were used as targets in NOD-scid IL2Rgnull mice.Results: Lentiviral transduction of NKG2D-4-1BB-CD3z markedly increased NKG2D surface expression in CD45RA- cells. Genetic stability was preserved in transduced cells. In vitro, NKG2D-CAR+ memory T cells showed significantly increased cytolytic activity than untransduced cells against osteosarcoma cell lines, while preserving the integrity of healthy cells. NKG2D-CAR+ memory T cells had considerable antitumor activity in a mouse model of osteosarcoma, whereas untransduced T cells were ineffective.Conclusions: Our results demonstrate NKG2D-4-1BB-CD3z CAR-redirected memory T cells target NKG2DL-expressing osteosarcoma cells in vivo and in vitro and could be a promising immunotherapeutic approach for patients with osteosarcoma. Clin Cancer Res; 23(19); 5824-35. ©2017 AACR.

Ets2 anchors the prometastatic function of mutant p53 in osteosarcoma.

Mutations in the tumor suppressor p53 occur in a majority of human cancers. Some gain-of-function (GOF) p53 mutations endow tumor cells with increased metastatic ability, although our understanding of the underlying mechanism remains incomplete. In this issue of Genes & Development, Pourebrahim and colleagues (pp. 1847-1857) develop a new mouse model of osteosarcoma in which a GOF mutant p53 allele is expressed specifically in osteoblasts, while the tumor microenvironment remains wild type for p53, allowing for the study of cell-autonomous functions. In this model, the role of GOF mutant p53 in promoting lung metastasis is shown to be critically dependent on the transcription factor Ets2 and is accompanied by the elevated expression of a cluster of small nucleolar RNAs (snoRNAs).

Integrative genome analysis of somatic p53 mutant osteosarcomas identifies Ets2-dependent regulation of small nucleolar RNAs by mutant p53 protein.

TP53 is the most frequently mutated gene in human cancer. Many mutant p53 proteins exert oncogenic gain-of-function (GOF) properties that contribute to metastasis, but the mechanisms mediating these functions remain poorly defined in vivo. To elucidate how mutant p53 GOF drives metastasis, we developed a traceable somatic osteosarcoma mouse model that is initiated with either a single p53 mutation (p53R172H) or p53 loss in osteoblasts. Our study confirmed that p53 mutant mice developed osteosarcomas with increased metastasis as compared with p53-null mice. Comprehensive transcriptome RNA sequencing (RNA-seq) analysis of 16 tumors identified a cluster of small nucleolar RNAs (snoRNAs) that are highly up-regulated in p53 mutant tumors. Regulatory element analysis of these deregulated snoRNA genes identified strong enrichment of a common Ets2 transcription factor-binding site. Homozygous deletion of Ets2 in p53 mutant mice resulted in strong down-regulation of snoRNAs and reversed the prometastatic phenotype of mutant p53 but had no effect on osteosarcoma development, which remained 100% penetrant. In summary, our studies identify Ets2 inhibition as a potential therapeutic vulnerability in p53 mutant osteosarcomas.

Integration of genomic, transcriptomic and functional profiles of aggressive osteosarcomas across multiple species

In complex, highly unstable genomes such as in osteosarcoma, targeting aberrant checkpoint processes (metabolic, cell cycle or immune) may prove more successful than targeting specific kinase or growth factor signaling pathways. Here, we establish a comparative oncology approach characterizing the most lethal osteosarcomas identified in a biorepository of tumors from three different species: human, mouse and canine. We describe the development of a genetically-engineered mouse model of osteosarcoma, establishment of primary cell cultures from fatal human tumors, and a biorepository of osteosarcoma surgical specimens from pet dogs. We analyzed the DNA mutations, differential RNA expression and in vitro drug sensitivity from two phenotypically-distinct cohorts: tumors with a highly aggressive biology resulting in death from rapidly progressive, refractory metastatic disease, and tumors with a non-aggressive, curable phenotype. We identified ARK5 (AMPK-Related Protein Kinase 5, also referred to as NUAK Family Kinase 1) as a novel metabolic target present in all species, and independent analyses confirmed glucose metabolism as the most significantly aberrant cellular signaling pathway in a model system for highly metastatic tumors. Pathway integration analysis identified Polo Like Kinase 1 (PLK1)-mediated checkpoint adaptation as critical to the survival of a distinctly aggressive osteosarcoma. The tumor-associated macrophage cytokine CCL18 (C-C Motif Chemokine Ligand 18) was significantly over-expressed in aggressive human osteosarcomas, and a clustering of mutations in the BAGE (B Melanoma Antigen) tumor antigen gene family was found. The theme of these features of high risk osteosarcoma is checkpoint adaptations, which may prove both prognostic and targetable.