Established cell lines are utilized extensively to study tumor biology and preclinical therapeutic development. However, they may not accurately recapitulate the heterogeneity of their corresponding primary disease. B-cell tumor cells are especially difficult to maintain under conventional culture conditions, limiting access to samples that faithfully represent this disease for preclinical studies. Here, we used primary canine diffuse large B-cell lymphoma to establish a culture system that reliably supports the growth of these cells. CD40 ligand, either expressed by feeder cells or provided as a soluble two-trimeric form, was sufficient to support primary lymphoma cells in vitro. The tumor cells retained their original phenotype, clonality and known karyotypic abnormalities after extended expansion in culture. Finally, we illustrate the utility of the feeder cell-free culture system for comparable assessment of cytotoxicity using dog and human B-cell malignancies. We conclude that this system has broad applications for in vitro preclinical development for B-cell malignancies.
"Cryopreserved tumor cells obtained from sterile lymph node biopsy samples from dogs with diffuse large B cell lymphoma (DLBCL) were cultured with 100 ng/mL of megaCD40L (Enzo Life Science, Plymouth Meeting, PA) as previously described , . The human T-cell leukemia cell line, Jurkat, was from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in RPMI1640 medium (Gibco/BRL, Grand Island, NY) containing 10% fetal bovine serum (FBS; Atlas Biologicals, Fort Collins, CO), 2-mercaptoethanol (Gibco/BRL), HEPES, L-glutamine, sodium pyruvate (Mediatech Inc., Manassas, VA), non-essential amino acids (Sigma Aldrich, St. Louis, MO), and Primocin (Invivogen, San Diego, CA). "
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to evaluate the activity of Selective Inhibitors of Nuclear Export (SINE) compounds that inhibit the function of the nuclear export protein Exportin 1 (XPO1/CRM1) against canine tumor cell lines and perform a Phase I clinical trial of KPT-335 in dogs with spontaneous cancer to provide a preliminary assessment of biologic activity and tolerability.
Canine tumor cell lines derived from non-Hodgkin lymphoma (NHL), mast cell tumor, melanoma and osteosarcoma exhibited growth inhibition and apoptosis in response to nanomolar concentrations of SINE compounds; NHL cells were particularly sensitive with IC50 concentrations ranging from 2-42 nM. A Phase I clinical trial of KPT-335 was performed in 17 dogs with NHL (naive or relapsed), mast cell tumor or osteosarcoma. The maximum tolerated dose was 1.75 mg/kg given orally twice/week (Monday/Thursday) although biologic activity was observed at 1 mg/kg. Clinical benefit (CB) including partial response to therapy (PR, n = 2) and stable disease (SD, n = 7) was observed in 9/14 dogs with NHL with a median time to progression (TTP) for responders of 66 days (range 35-256 days). A dose expansion study was performed in 6 dogs with NHL given 1.5 mg/kg KPT-335 Monday/Wednesday/Friday; CB was observed in 4/6 dogs with a median TTP for responders of 83 days (range 35-354 days). Toxicities were primarily gastrointestinal consisting of anorexia, weight loss, vomiting and diarrhea and were manageable with supportive care, dose modulation and administration of low dose prednisone; hepatotoxicity, anorexia and weight loss were the dose limiting toxicities.
This study provides evidence that the novel orally bioavailable XPO1 inhibitor KPT-335 is safe and exhibits activity in a relevant, spontaneous large animal model of cancer. Data from this study provides critical new information that lays the groundwork for evaluation of SINE compounds in human cancer.
PLoS ONE 02/2014; 9(2):e87585. DOI:10.1371/journal.pone.0087585 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We performed genomewide gene expression analysis of 35 samples representing 6 common histologic subtypes of canine lymphoma and bioinformatics analyses to define their molecular characteristics. Three major groups were defined on the basis of gene expression profiles: (1) low-grade T-cell lymphoma, composed entirely by T-zone lymphoma; (2) high-grade T-cell lymphoma, consisting of lymphoblastic T-cell lymphoma and peripheral T-cell lymphoma not otherwise specified; and (3) B-cell lymphoma, consisting of marginal B-cell lymphoma, diffuse large B-cell lymphoma, and Burkitt lymphoma. Interspecies comparative analyses of gene expression profiles also showed that marginal B-cell lymphoma and diffuse large B-cell lymphoma in dogs and humans might represent a continuum of disease with similar drivers. The classification of these diverse tumors into 3 subgroups was prognostically significant, as the groups were directly correlated with event-free survival. Finally, we developed a benchtop diagnostic test based on expression of 4 genes that can robustly classify canine lymphomas into one of these 3 subgroups, enabling a direct clinical application for our results.
[Show abstract][Hide abstract] ABSTRACT: ABSTRACT In humans, passive immunotherapy with anti-CD20 monoclonal antibodies (mAbs) has created immeasurable improvements in outcomes of patients with B-cell malignancies. However, the lack of comparable reagents has precluded development of this approach in dogs. We developed a novel anti-canine CD20 mAb designated as 6C8. 6C8 recognized the extracellular domain of canine CD20 and showed high-affinity binding to canine CD20 in solution, as well as in its native conformation on canine B-cells. The 6C8 target was expressed invariably in B-cell lineage cells, but not in T-cells or in myeloid cells. 6C8 promoted phagocytosis of B-cell lymphoma cells by macrophages, but in its current framework, it did not induce direct cytotoxicity or complement dependent cytotoxicity. In summary, we have established a novel anti-canine CD20 mAb that is useful as a diagnostic tool to phenotype B-cells, and which could be integrated as a tool for passive immunotherapy to treat dogs with B-cell disorders.
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