Identification of Three Molecular and Functional Subtypes in Canine Hemangiosarcoma through Gene Expression Profiling and Progenitor Cell Characterization

American Journal Of Pathology (Impact Factor: 4.59). 02/2014; 184(4). DOI: 10.1016/j.ajpath.2013.12.025
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Canine hemangiosarcomas have been ascribed to an endothelial origin based on histologic appearance; however, recent findings suggest that these tumors may instead arise from hematopoietic progenitor cells. To clarify this ontogenetic dilemma, we used genome-wide expression profiling of primary hemangiosarcomas and identified three distinct tumor subtypes associated with angiogenesis (group 1), inflammation (group 2), and adipogenesis (group 3). Based on these findings, we hypothesized that a common progenitor may differentiate into the three tumor subtypes observed in our gene profiling experiment. To investigate this possibility, we cultured hemangiosarcoma cell lines under normal and sphere-forming culture conditions to enrich for tumor cell progenitors. Cells from sphere-forming cultures displayed a robust self-renewal capacity and exhibited genotypic, phenotypic, and functional properties consistent with each of the three molecular subtypes seen in primary tumors, including expression of endothelial progenitor cell (CD133 and CD34) and endothelial cell (CD105, CD146, and αvβ3 integrin) markers, expression of early hematopoietic (CD133, CD117, and CD34) and myeloid (CD115 and CD14) differentiation markers in parallel with increased phagocytic capacity, and acquisition of adipogenic potential. Collectively, these results suggest that canine hemangiosarcomas arise from multipotent progenitors that differentiate into distinct subtypes. Improved understanding of the mechanisms that determine the molecular and phenotypic differentiation of tumor cells in vivo could change paradigms regarding the origin and progression of endothelial sarcomas.

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    • "Canine hemangiosarcoma and human angiosarcoma have been classified historically as tumors of malignant endothelium due to their histology and the expression of endothelial cell surface markers [2,5]. Based on the expression of early hematopoietic and endothelial progenitor markers, other studies have challenged this idea, suggesting instead that hemangiosarcomas might arise from bone marrow-derived angioblastic progenitors [6-8]. A similar analogy of progenitor cell origin has been drawn for human angiosarcoma [9]. "
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    ABSTRACT: Background Human angiosarcoma and canine hemangiosarcoma are thought to arise from vascular tissue or vascular forming cells based upon their histological appearance. However, recent evidence indicates a hematopoietic or angioblastic cell of origin for these tumors. In support of this idea, we previously identified an endothelial-myeloid progenitor cell population with high expression of endothelial cell markers and the myeloid cell marker, colony stimulating factor 1 receptor (CSF-1R). Here, we further characterized these cells to better understand how their cellular characteristics may impact current therapeutic applications. Methods We performed cell enrichment studies from canine hemangiosarcoma and human angiosarcoma cell lines to generate cell populations with high or low CSF-1R expression. We then utilized flow cytometry, side population and cell viability assays, and fluorescence based approaches to elucidate drug resistance mechanisms and to determine the expression of hematopoietic and endothelial progenitor cell markers. Results We demonstrated that cells with high CSF-1R expression enriched from hemangiosarcoma and angiosarcoma cell lines are more drug resistant than cells with little or no CSF-1R expression. We determined that the increased drug resistance may be due to increased ABC transporter expression in hemangiosarcoma and increased drug sequestration within cellular lysosomes in both hemangiosarcoma and angiosarcoma. Conclusions We identified drug sequestration within cellular lysosomes as a shared drug resistance mechanism in human and canine vascular sarcomas marked by high CSF-1R expression. Taken together, our results demonstrate that studies in highly prevalent canine hemangiosarcoma may be especially relevant to understanding and addressing drug resistance mechanisms in both the canine and human forms of this disease.
    Vascular Cell 10/2014; 6(1):20. DOI:10.1186/2045-824X-6-20
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    ABSTRACT: Canine hemangiosarcoma is a rapidly progressive disease that is poorly responsive to conventional chemotherapy. Despite numerous attempts to advance treatment options and improve outcomes, drug resistance remains a hurdle to successful therapy. To address this problem, we used recently characterized progenitor cell populations derived from canine hemangiosarcoma cell lines and grown as non-adherent spheres to identify potential drug resistance mechanisms as well as drug-resistant cell populations. Cells from sphere-forming cultures displayed enhanced resistance to chemotherapy drugs, expansion of dye-excluding side populations and altered ATP-binding cassette (ABC) transporter expression. Invasion studies demonstrated variability between cell lines as well as between sphere and monolayer cell populations. Collectively, our results suggest that sphere cell populations contain distinct subpopulations of drug-resistant cells that utilize multiple mechanisms to evade cytotoxic drugs. Our approach represents a new tool for the study of drug resistance in hemangiosarcoma, which could alter approaches for treating this disease.
    Veterinary and Comparative Oncology 08/2014; DOI:10.1111/vco.12114 · 2.73 Impact Factor
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    ABSTRACT: Background: Epigenetic alterations including DNA methylation and histone modifications are the key factors in the differentiation of stem cells into different tissue subtypes. The generation of cancer stem cells (CSCs) in the process of carcinogenesis may also involve similar kind of epigenetic reprogramming where, in contrast, it leads to the loss of expression of genes specific to the differentiated state and regaining of stem cell-specific characteristics. The most important predicament with treatment of cancers includes the non-responsive quiescent CSC. Scope of review: The distinctive capabilities of the CSCs make cancer treatment even more difficult as this population of cells tends to remain quiescent for longer intervals and then gets reactivated leading to tumor relapse. Therefore, the current review is aimed to focus on recent advances in understanding the relation of epigenetic reprogramming to the generation, self-renewal and proliferation of CSCs. Major conclusion: CSC-targeted therapeutic approaches would improve the chances of patient survival by reducing the frequency of tumor relapse. Differentiation therapy is an emerging therapeutic approach in which the CSCs are induced to differentiate from their quiescent state to a mature differentiated form, through activation of differentiation-related signalling pathways, miRNA-mediated alteration and epigenetic differentiation therapy. Thus, understanding the origin of CSC and their epigenetic regulation is crucial to develop treatment strategy against not only for the heterogeneous population of cancer cells but also to CSCs. General significance: Characterizing the epigenetic marks of CSCs and the associated signalling cascades might help in developing therapeutic strategies against chemo-resistant cancers.
    Biochimica et Biophysica Acta (BBA) - General Subjects 09/2014; 1840(12). DOI:10.1016/j.bbagen.2014.09.017 · 4.38 Impact Factor
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