Aric M. Frantz

Molecular Profiling Reveals Prognostically Significant Subtypes of Canine Lymphoma

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.

Genome-wide Association Study Identifies Shared Risk Loci Common to Two Malignancies in Golden Retrievers

Dogs, with their breed-determined limited genetic background, are great models of human disease including cancer. Canine B-cell lymphoma and hemangiosarcoma are both malignancies of the hematologic system that are clinically and histologically similar to human B-cell non-Hodgkin lymphoma and angiosarcoma, respectively. Golden retrievers in the US show significantly elevated lifetime risk for both B-cell lymphoma (6%) and hemangiosarcoma (20%). We conducted genome-wide association studies for hemangiosarcoma and B-cell lymphoma, identifying two shared predisposing loci. The two associated loci are located on chromosome 5, and together contribute ~20% of the risk of developing these cancers. Genome-wide p-values for the top SNP of each locus are 4.6×10-7 and 2.7×10-6, respectively. Whole genome resequencing of nine cases and controls followed by genotyping and detailed analysis identified three shared and one B-cell lymphoma specific risk haplotypes within the two loci, but no coding changes were associated with the risk haplotypes. Gene expression analysis of B-cell lymphoma tumors revealed that carrying the risk haplotypes at the first locus is associated with down-regulation of several nearby genes including the proximal gene TRPC6, a transient receptor Ca2+-channel involved in T-cell activation, among other functions. The shared risk haplotype in the second locus overlaps the vesicle transport and release gene STX8. Carrying the shared risk haplotype is associated with gene expression changes of 100 genes enriched for pathways involved in immune cell activation. Thus, the predisposing germ-line mutations in B-cell lymphoma and hemangiosarcoma appear to be regulatory, and affect pathways involved in T-cell mediated immune response in the tumor. This suggests that the interaction between the immune system and malignant cells plays a common role in the tumorigenesis of these relatively different cancers.

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

Canine hemangiosarcomas have been ascribed to an endothelial origin based on histologic appearance; however, recent findings suggest that these tumors may arise instead 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.

CD40 ligand is necessary and sufficient to support primary diffuse large B-cell lymphoma cells in culture: a tool for in vitro preclinical studies with primary B-cell malignancies

Abstract 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.

Identification of drug‐resistant subpopulations in canine hemangiosarcoma

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.

Modulation of fatty acid metabolism and immune suppression are features of in vitro tumour sphere formation in ontogenetically distinct dog cancers

Non-adherent, 3-dimensional sphere formation is used as an in vitro surrogate to evaluate cellular potential for tumour initiation and self-renewal. To determine if a shared molecular program underlies the capacity for sphere formation by cells originating from diverse tumour types, we characterized molecular and functional properties of 10 independent cell lines derived from 3 ontogenetically distinct dog cancers: hemangiosarcoma, osteosarcoma and glial brain tumours. Genome-wide gene expression profiling identified tumour-of-origin-dependent patterns of adjustment to sphere formation in a uniform culture condition. However, expression of the stem/progenitor markers CD34 and CD117, resistance to cytotoxic drugs and dye efflux (side population assays) showed no association with these gene expression profiles. Instead, primary sphere-forming capacity was inversely correlated with the ability to reform secondary spheres, regardless of tumour ontogeny. Primary sphere formation seemed to be proportional to the number of pre-existing cells with sphere-forming capacity in the cell lines. Cell lines where secondary sphere formation was more proficient than primary sphere formation showed enrichment of genes involved in fatty acid synthesis and immunosuppressive cytokines. In contrast, cell lines where secondary sphere formation was approximately equivalent to or less proficient than primary sphere formation showed upregulation of CD40 and enrichment of genes involved in fatty acid oxidation. Our data suggest that in vitro sphere formation is associated with upregulation of gene clusters involved in metabolic and immunosuppressive functions, which might be necessary for self-renewal and for tumour initiation and/or tumour propagation in vivo.

Constitutive expression and roles of interleukin-8 in canine hemangiosarcoma

Results IL-8 mRNA expression was variable among the tissue samples and both IL-8 mRNA and protein were variable among the cell lines. In contrast, IL-8 receptor mRNA and protein showed minimal variance. “IL-8 high” and “IL-8 low” groups were defined from the HSA tumor samples based on gene expression profiles. The “IL-8 high” group was associated with a “reactive microenvironment,” showing enrichment of coagulation, inflammation, and fibrosis networks. However, IL-8 added exogenously and IL-8 blockade using neutralizing antibodies had no effect on HSA cell proliferation, despite apparent response to these signals at the level of gene expression. Similarly, neither addition nor blockade of IL-8 protected cells from apoptosis. IL-8 mRNA was elevated in HSA cancer stem cells, but exogenous IL-8 attenuated self-renewal of these cells. Conclusion The results of this study suggest that IL-8 is a driver of tumor heterogeneity, steering cells away from self-renewal and towards partial differentiation. It also could act to recruit (or produce from the tumor) inflammatory and pro-angiogenic cells to the microenvironment. We are testing this hypothesis in a robust xenograft model. These experiments will establish if IL-8 plays a role in progression and metastasis of canine HSA, and allow us to define the therapeutic potential of IL-8 blockade.

Hemangiosarcoma and its cancer stem cell sub-population are effectively killed by a toxin targeted through epidermal growth factor and urokinase receptors

Background Targeted toxins have the potential to overcome intrinsic or acquired resistance of cancer cells to conventional cytotoxic agents. We hypothesized that EGFuPA-toxin, a bispecific ligand-targeted toxin consisting of a deimmunized Pseudomonas exotoxin conjugated to epidermal growth factor (EGF) and urokinase (uPA), would efficiently target and kill cells derived from canine hemangiosarcoma (HSA), a highly chemotherapy resistant tumor, as well as cultured hemangiospheres, used as a surrogate for cancer stem cells (CSC).

997. Immune-Mediated Loss of Transgene Expression in Mouse Liver Following Transposon Delivery

An advantage of plasmid-based vector systems for gene therapy over virus-based is their low immunogenicity. The Sleeping Beauty (SB) transposon system, which has the ability to integrate genes into human chromosomes, is a candidate for gene therapy of mucopolysaccharidoses (MPS), genetic disorders of lysosomal metabolism that require sustained expression of the therapeutic transgene. However, our attempts to achieve long-term expression of human alpha -L- iduronidase (hIDUA) or beta-glucuronidase (hGUSB) without immune suppression resulted in almost complete loss of transgene expression and maintenance by 4 weeks postinjection in both MPS and wild type (WT ) mice. In cyclophosphamide-immune-suppressed MPS I mice, the initial supranormal, 1-day plasma activity levels dropped approximately 150-fold by 2 weeks, but then persisted at detectable levels in some mice (see abstract by Aronovich et al). This suggested that cells that expressed the human therapeutic gene induced immune response and were cleared from the liver of some treated mice.