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Browsing by Subject "Osteosarcoma"

Now showing 1 - 6 of 6
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    Cancer gene discovery using somatic transposon mutagenesis in the mouse and systems for validation of identified candidate cancer genes and pathways.
    (2012-07) Moriarity, Branden Scott
    The conditional Sleeping Beauty (SB) transposon mutagenesis system has proven to be a successful method for cancer gene identification in solid tumors. Using tissue specific Cre recombinases (TSP-Cre) to selectively activate SB mediated mutagenesis in tissues of interest has yielded numerous novel candidate cancer genes in leukemia/lymphoma, colorectal, liver, and pancreatic cancers. We implemented the SB system for the identification of genes involved in osteosarcoma (OS). OS is the most common cancer of the bone and third most common cancer in children and adolescents. The 5-year survival rate of OS patients is 60-70%, though with metastatic disease it drops to less than 20%-30%. Identification of genes responsible for OS development and metastasis has been difficult due to its genomic instability and subsequent complexity. Consequently, there is a pressing need to identify the genes and pathways governing OS development and metastasis. To this end, we performed a forward genetic screen utilizing the conditional Sleeping Beauty (SB) transposase and mutagenic T2/Onc transposon system in Osx-Cre mice on a predisposing Trp53LSL-R270H/+ or wild type background to induce random somatic mutations that induce OS development and metastasis. One hundred and nineteen OSs were isolated from 96 predisposed and 20 wild type mice undergoing SB mutagenesis, with a subset of animals harboring metastases to the liver and/or lungs. Over 100 candidate cancer genes, both known and novel, were identified by analysis of recurrent T2/Onc insertion sites. In order to validate the candidate cancer genes identified in our screen, we developed a recombinase-based system for efficient assembly of vectors to over express or knock down 1-6 genes in mammalian cells. Further, these vectors were constructed in Piggybac transposons to allow for stable and reversible integration of the vector via transposition. In addition to cDNA over expression or shRNA knock down, we also developed methods for generation and selection of candidate cancer gene knock out cell lines using TAL effector nucleases (TALENs).
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    Comparative and molecular approaches to improve identification, classification, and therapeutic approaches to cancer
    (2013-01) Frantz, Aric M.
    A major area of contemporary research in cancer is focused on improving tumor classification into clinically relevant subgroups of disease. To achieve this, it is important to understand the molecular events that driver tumor heterogeneity both at the cellular level and at the tissue level. I initially tested the hypothesis that canine lymphoma is composed of a group of molecularly distinct entities with prognostic significance. The results show that canine lymphoma can be stratified into molecular subgroups that have prognostic value and can assist to guide therapy. Next, I tested the hypothesis that canine hemangiosarcoma (HSA) is organized hierarchically with a cancer stem cell (CSC)-like population of cells at the apex. The data show that variable numbers of CSC-like cells are invariably present in HSA. These CSC-like cells retain the capacity to differentiate into vascular, inflammatory, or adipogenic tissue, suggesting that their multipotency is a contributing factor to the observed heterogeneity in this disease. Finally, I tested the hypothesis that CSCs, or CSC-like cells from three histologically distinct types of canine cancer (HSA, osteosarcoma, and glioblastoma) share molecular and functional properties. Using a system that allowed me to eliminate tumor-specific culture conditions, I showed that despite extensive heterogeneity in CSC-like cells from these tumors, they all showed reduced activity of pathways associated with proliferation and development. In summary, my results confirm that cellular heterogeneity exists both within and among tumors. A better understanding of the mechanisms that drive this will improve patient stratification and guide efforts to develop rational, more effective therapies.
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    Developing a Mouse Model of Metastatic Osteosarcoma for Immunotherapy
    (2022-12) Sonam
    Osteosarcoma is a primary bone malignancy that primarily affects children and young adults. The 5-year survival rate for patients with localized osteosarcoma is 60-70% and only 20% for patients who present metastasis at the diagnosis. The standard of care treatment has not improved the prognosis in over 30 years and new approaches addressing tumor resistance and immune evasion are required. The long-term goals of this project are to address two objectives: i) To study the potential benefits and risks of immunotherapeutic drugs eBAT and ONIx on syngeneic mouse models of metastatic osteosarcoma, and ii) To study their mechanism of action. K7M2 is a well-characterized model of spontaneous pulmonary metastasis, but intravenous injection of these cells did not result in lung colonization, at any dosage, in the mice in our study. We inferred from the data that the tumor cells were rejected by the immune-competent host. To study the mechanism of action of ONIx, we have made progress in optimizing the protocol for the phagocytosis assay, and further analysis with a different target cell line is planned. Assays to verify the mechanism of action of eBAT are being planned. Overall, this project provided significant learnings on objective planning and designing of the experiments and provided opportunities to acquire expertise in several new methodologies.
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    Targeted Biological and Cellular Immunotherapies for Osteosarcoma
    (2023-12) Robbins, Gabrielle
    Osteosarcoma (OSA) is the most common cancer of the bone and third most common cancer diagnosed in children and adolescent. The five-year survival rate for OSA patients approaches 70%; however, this rate drops to less than 30% with metastatic disease. There have been no changes in treatment options or patient outcome in over four decades. Consequently, there is a pressing need to identify actionable targets and develop novel immunotherapies for the treatment of OSA. Immunotherapies have proven to be successful for the treatment of hematological malignancies as well as some solid tumors, including cancers of the lung, kidney, and bladder. To date, OSA remains impervious to treatment with immunotherapies. We hypothesize that treating OSA using targeted immunotherapies will decrease proliferation in vitro and decrease tumor growth and invasion/migration while improving survival in vivo. To this end, we evaluated the efficacy of two different, targeted immunotherapies for the treatment of OSA. First, we tested a monoclonal antibody (mAb) targeting SEMA4D, a candidate gene previously identified in a forward genetic screen of OSA. Anti-SEMA4D treatment decreased OSA cell proliferation and MET production in vitro. In vivo, we saw anti-SEMA4D treatment slow tumor growth, decrease collagen deposition, decrease the incidence of metastasis, and modulate the immune cell infiltrate. OSA is traditionally an immunologically ‘cold’ tumor with poor immune cell infiltration and activity. As such, we also utilized a cell-based approach to target OSA using natural killer (NK) cells. NK cells are lymphocytes of the innate immune system involved in killing infected or malignantly transformed cells. Previous work has shown that unmanipulated NK cells are minimally effective in the treatment of cancer, likely due to poor persistence and suppression within the tumor microenvironment. To circumvent these challenges, we delivered a CD70-specific chimeric antigen receptor (CAR), soluble interleukin 15 (IL-15), and a transforming growth factor beta (TGF-β) dominant negative mutant receptor. These armored CD70-CAR NK cells show remarkable in vitro killing and pro-inflammatory cytokine activity against OSA. In vivo, we see armored CD70-NK cells home to the tumor as well as sustained persistence and strong anti-tumor activity. The goal of this work was to develop novel and effective therapies to treat OSA for use in the clinical setting. Future work will focus on developing multi-targeted approaches for these hard to treat and genetically diverse tumors.
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    Uncovering Mechanisms of Osteosarcoma Metastasis
    (2016-08) Marko, Tracy
    Osteosarcoma (OS) is the most common primary malignant bone tumor, with metastatic disease responsible for most treatment failure and patient death. A better understanding of the metastatic disease state is needed to improve patient survival. The studies presented here explore a variety of questions surrounding metastatic OS. A literature search of the PubMed database was conducted to compare the prevalence of metastatic OS at diagnosis across countries. The average prevalence of metastasis at diagnosis increased as Human Development Index score (HDI) decreased, with an 18% global average. In countries with medium/low HDI, where more barriers to accessing healthcare exist, the higher prevalence of metastasis may result from treatment delay or an artificially inflated prevalence due to patients with less severe symptoms not presenting to clinic. Canine OS is a naturally occurring, spontaneous disease with more rapid disease progression and greater incidence than human OS. An understanding and utilization of studies on metastatic OS in dogs could help identify new treatment strategies to improve patient outcomes in humans and dogs. We evaluated the similarities of metastatic OS between the species by comparing risk factors for having metastatic OS at diagnosis between pet dogs from our veterinary clinic and pediatric patients in the Surveillance, Epidemiology, and End Results database. Here we build on current knowledge of canine OS by showing that primary tumors in similar anatomical locations metastasize at comparable rates in both species. A Sleeping Beauty mutagenesis screen previously conducted in our laboratory identified Slit-Robo GTPase- Activating Protein 2 (SRGAP2) as a potential suppressor of OS metastasis. SRGAP2 controls phenotypes in neurons supporting the hypothesis that it may suppress migration in the context of cancer. Although the effects of SRGAP2 in OS were not consistent across all cell lines, they tended to support this hypothesis. Additionally, expression levels of other genes in the Slit-Robo pathway were significantly altered in a subset of mouse and human OS, and SRGAP2 protein expression was lost in a subset of primary tumor samples. SRGAP2 and other axon guidance proteins likely play a role in OS metastasis, with loss of SRGAP2 contributing to a more aggressive phenotype.
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    Utilization of A 3D Culture System of Collagen-Mimic Peptide Gfoger-Based Hydrogel to Model Osteosarcoma from Engineered Ipsc
    (2021-05) Thueson, Hanna
    Modeling the early stages of human osteosarcoma development remains a significant challenge. Most existing human models are derived from patient tumor tissue which is used to establish tumor cell lines or xenograft models in immunodeficient mice. These models are largely derived from late or end stage disease and do not allow the study of the early events of transformation. Further, 2D cell lines are largely homogenous and do not replicate the heterogeneity of primary tumors. Xenografted models more closely replicate the primary tumor but can have low engraftment rates and are logistically challenging to maintain. The immunocompromised nature of xenografted mice limits the potential for immunotherapy studies. The work presented here establishes a 3D culture system to model early-stage osteosarcoma development from engineered human iPSC. When cultured as aggregates in a GFOGER (integrin-specific glycine-phenylalanine-hydroxyproline-glycine-glutamate-arginine) based hydrogel known to promote osteoblastic differentiation, osteoblasts engineered with osteosarcoma-associated mutations readily form 3D organoids. Histological analysis supports that 3D culture of iPSC-derived osteoblasts promotes a more tissue-like phenotype with increased mineralization and ECM development within the tissue construct. In addition, preliminary functional studies suggest that 3D culture promotes transformative properties and an osteosarcoma phenotype. This novel approach has potential for future applications in disease modeling, in vivo studies, and drug discovery.