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.

Establishment of a Patient-Derived Xenograft of Canine Enteropathy-Associated T-Cell Lymphoma, Large Cell Type.

The pathogenesis of canine T-cell lymphoma remains incompletely understood, partly because there are no well-established in-vivo models to study these malignancies. For this study, we generated a patient-derived tumour xenograft (PDTX) from a 10-year-old neutered male golden retriever dog with enteropathy-associated intestinal T-cell lymphoma, large cell type. One of two female, 15-week-old beige/nude/XID mice developed a visible tumour 7 weeks after sections of tumour material from the spleen were surgically implanted. The histological appearance, immunophenotype and clonal antigen receptor rearrangements of the tumour from the recipient mouse showed that it was derived from the primary canine tumour. Our results indicate that immunodeficient mice are receptive hosts to develop in-vivo PDTX models to study the pathogenesis and management of canine T-cell lymphomas.

Interactions between CXCR4 and CXCL12 promote cell migration and invasion of canine hemangiosarcoma.

The CXCR4/CXCL12 axis plays an important role in cell locomotion and metastasis in many cancers. In this study, we hypothesized that the CXCR4/CXCL12 axis promotes migration and invasion of canine hemangiosarcoma (HSA) cells. Transcriptomic analysis across 12 HSA cell lines and 58 HSA whole tumour tissues identified heterogeneous expression of CXCR4 and CXCL12, which was associated with cell movement. In vitro, CXCL12 promoted calcium mobilization, cell migration and invasion that were directly proportional to surface expression of CXCR4; furthermore, these responses proved sensitive to the CXCR4 antagonist, AMD3100, in HSA cell lines. These results indicate that CXCL12 potentiates migration and invasion of canine HSA cells through CXCR4 signalling. The direct relationship between these responses in HSA cells suggests that the CXCR4/CXCL12 axis contributes to HSA progression.

Association of Sphingosine-1-phosphate (S1P)/S1P Receptor-1 Pathway with Cell Proliferation and Survival in Canine Hemangiosarcoma.

BACKGROUND: Sphingosine-1-phosphate (S1P) is a key biolipid signaling molecule that regulates cell growth and survival, but it has not been studied in tumors from dogs. HYPOTHESIS/OBJECTIVES: S1P/S1P1 signaling will contribute to the progression of hemangiosarcoma (HSA). ANIMALS: Thirteen spontaneous HSA tissues, 9 HSA cell lines, 8 nonmalignant tissues, including 6 splenic hematomas and 2 livers with vacuolar degeneration, and 1 endothelial cell line derived from a dog with splenic hematoma were used. METHODS: This was a retrospective case series and in vitro study. Samples were obtained as part of medically necessary diagnostic procedures. Microarray, qRT-PCR, immunohistochemistry, and immunoblotting were performed to examine S1P1 expression. S1P concentrations were measured by high-performance liquid chromatography/mass spectrometry. S1P signaling was evaluated by intracellular Ca(2+) mobilization; proliferation and survival were evaluated using the MTS assay and Annexin V staining. RESULTS: Canine HSA cells expressed higher levels of S1P1 mRNA than nonmalignant endothelial cells. S1P1 protein was present in HSA tissues and cell lines. HSA cells appeared to produce low levels of S1P, but they selectively consumed S1P from the culture media. Exogenous S1P induced an increase in intracellular calcium as well as increased proliferation and viability of HSA cells. Prolonged treatment with FTY720, an inhibitor of S1P1 , decreased S1P1 protein expression and induced apoptosis of HSA cells. CONCLUSIONS AND CLINICAL IMPORTANCE: S1P/S1P1 signaling pathway functions to maintain HSA cell viability and proliferation. The data suggest that S1P1 or the S1P pathway in general could be targets for therapeutic intervention for dogs with HSA.

Canine osteosarcoma cells exhibit resistance to aurora kinase inhibitors.

We evaluated the effect of Aurora kinase inhibitors AZD1152 and VX680 on canine osteosarcoma cells. Cytotoxicity was seen in all four cell lines; however, half-maximal inhibitory concentrations were significantly higher than in human leukaemia and canine lymphoma cells. AZD1152 reduced Aurora kinase B phosphorylation, indicating resistance was not because of failure of target recognition. Efflux mediated by ABCB1 and ABCG2 transporters is one known mechanism of resistance against these drugs and verapamil enhanced AZD1152-induced apoptosis; however, these transporters were only expressed by a small percentage of cells in each line and the effects of verapamil were modest, suggesting other mechanisms contribute to resistance. Our results indicate that canine osteosarcoma cells are resistant to Aurora kinase inhibitors and suggest that these compounds are unlikely to be useful as single agents for this disease. Further investigation of these resistance mechanisms and the potential utility of Aurora kinase inhibitors in multi-agent protocols is warranted.

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.

A tumor-related lymphoid progenitor population supports hierarchical tumor organization in canine B-cell lymphoma.

BACKGROUND: Tumors have heterogeneous properties, which could be explained by the existence of hierarchically and biologically distinct tumor cells such as tumor-initiating cells (TICs). This model is clinically important, as TICs are promising targets for cancer therapies. However, TICs in spontaneous B-cell lymphoma have not been conclusively identified. HYPOTHESIS/OBJECTIVES: Tumor cells with a progenitor phenotype exist in B-cell lymphoma, reflecting a hierarchical organization. ANIMALS: Twenty-eight client-owned dogs with previously untreated B-cell lymphoma and 6 healthy dogs. METHODS: This was a prospective study. Flow cytometry was used to identify lymphoid progenitor cells (LPCs) that coexpressed hematopoietic progenitor antigens CD34, CD117, and CD133, with lymphoid differentiation markers CD21 and/or CD22 in B-cell lymphoma. The polymerase chain reaction for antigen receptor rearrangements was used to analyze clonality and relatedness of tumor populations. A xenograft model with NOD/SCID/IL-2Rγ(-/-) mice was adapted to expand and serially transplant primary canine B-cell lymphoma. RESULTS: LPCs were expanded in lymph nodes from 28 dogs with B-cell lymphoma compared with 6 healthy dogs (P= .0022). LPCs contained a clonal antigen receptor gene rearrangement identical to that of the bulk of tumor cells. Canine B-cell lymphoma xenografts in recipient mice that maintained LPCs in the tumors were recurrently observed. CONCLUSIONS AND CLINICAL IMPORTANCE: These results suggest the presence of a hierarchy of tumor cells in B-cell lymphoma as has been demonstrated in other cancers. These findings have the potential to impact not only the understanding of lymphoma pathogenesis but also the development of lymphoma therapies by providing novel targets for therapy.

Immunotherapy with autologous tumour antigen-coated microbeads (large multivalent immunogen), IL-2 and GM-CSF in dogs with spontaneous B-cell lymphoma.

Cytotoxic T-lymphocyte responses to subcellular antigens are enhanced when antigens are presented on cell-sized silica microbeads called large multivalent immunogens (LMIs). LMIs prepared with tumour cell membrane fragments have induced partial remissions in humans with melanoma and renal cell carcinoma. The purpose of this phase I study was to evaluate the safety of LMIs, prepared with autologous lymphoma cell membranes, along with subcutaneous interleukin 2 (IL-2) and granulocyte-macrophage colony stimulating factor (GM-CSF) in dogs with untreated B-cell lymphoma. After lymph node excision and induction chemotherapy, five dogs were vaccinated with three weekly doses of LMI alone; five with LMI and subcutaneous IL-2 and five with LMI, IL-2 and GM-CSF. No significant toxicity was noted, treatment did not adversely affect disease-free interval and half of the dogs showed measurable delayed-type hypersensitivity reactions to intradermal challenge with LMI, suggesting specific cell-mediated immunity.

Inactivation of the p16 cyclin-dependent kinase inhibitor in high-grade canine non-Hodgkin's T-cell lymphoma.

The significance of p16/Rb tumor suppressor pathway inactivation in T-cell non-Hodgkin's lymphoma (NHL) remains incompletely understood. We used naturally occurring canine NHL to test the hypothesis that p16 inactivation has specific pathologic correlates. Forty-eight samples (22 T-cell NHL and 26 B-cell NHL) were included. As applicable, metaphase- or array-based comparative genomic hybridization, Southern blotting, promoter methylation, and Rb phosphorylation were used to determine the presence, expression, and activity of p16. Fisher's exact test was used to test for significance. Deletion of p16 (or loss of dog chromosome 11) was restricted to high-grade T-cell NHL (lymphoblastic T-cell lymphoma and peripheral T-cell lymphoma, not otherwise specified). These were characterized by a concomitant increase of tumor cells with Rb phosphorylation at canonical CDK4 sites. Rb phosphorylation also was seen in high-grade B-cell NHL (diffuse large B-cell lymphoma and Burkitt-type lymphoma), but in those cases, it appeared to be associated with c-Myc overexpression. The data show that p16 deletion or inactivation occurs almost exclusively in high-grade T-cell NHL; however, alternative pathways can generate functional phenotypes of Rb deficiency in low-grade T-cell NHL and in high-grade B-cell NHL. Both morphologic classification according to World Health Organization criteria and assessment of Rb phosphorylation are prognostically valuable parameters for canine NHL.

Mutations of phosphatase and tensin homolog deleted from chromosome 10 in canine hemangiosarcoma.

We examined the presence of phosphatase and tensin homolog deleted from chromosome 10 (PTEN) abnormalities that could contribute to the origin or progression of naturally occurring canine endothelial tumors (hemangiosarcoma). Our results document somatic point mutations or deletions encompassing the PTEN C-terminal domain in canine hemangiosarcoma that might provide cells a survival advantage within their microenvironment. This represents the first characterization of a naturally occurring, highly metastatic tumor with biologically significant mutations of PTEN in the C-terminal domain.

CD20 expression in normal canine B cells and in canine non-Hodgkin lymphoma.

We examined the expression of CD20 in normal canine peripheral blood mononuclear cells, normal canine spleen, and canine non-Hodgkin lymphoma (NHL) to determine the feasibility of using this antigen as a diagnostic aid and as a possible target for therapy. An antibody generated against a C-terminal (intracytoplasmic) epitope of human CD20 recognized proteins of 32-36 kd in normal and malignant canine lymphocytes. This antibody showed restricted membrane binding in a subset of lymphocytes in peripheral blood, in the B-cell regions from a normal canine spleen and lymph node, and in malignant cells from 19 dogs with B-cell NHL, but not from 15 dogs with T-cell NHL. The patterns of CD20 reactivity in these samples overlapped those seen using an antibody that recognizes canine CD79a. This anti-CD20 antibody is therefore suitable as an aid to phenotype canine NHL. In contrast, normal canine B cells were not recognized by any of 28 antibodies directed against the extracellular domains of human CD20 (including the chimeric mouse-human antibody Rituximab) or by any of 12 antibodies directed against the extracellular domains of mouse CD20. Thus, the use of CD20 as a therapeutic target will require the generation of specific antibodies against the extracellular domains of canine CD20.

Diagnosis of canine lymphoid neoplasia using clonal rearrangements of antigen receptor genes.

Although the diagnosis of canine leukemia and lymphoma in advanced stages is usually uncomplicated, some presentations of the disease can be a diagnostic challenge. In certain situations, lymphoma and leukemia can be difficult to distinguish from a benign reactive proliferation of lymphocytes. Because clonality is the hallmark of malignancy, we have developed an assay that uses the polymerase chain reaction to amplify the variable regions of immunoglobulin genes and T-cell receptor genes to detect the presence of a clonal lymphocyte population. The assay detected clonally rearranged antigen receptor genes in 91% of the 77 dogs with lymphoid malignancy. Of the 24 dogs tested, that were either healthy or had clearly defined conditions not related to lymphoid malignancy, a clonally rearranged antigen receptor gene was found in one (a dog with Ehrlichia canis infection). Gene rearrangement was appropriate for the immunophenotype (immunoglobulin gene rearrangement in B-cell leukemias and T-cell receptor gene rearrangement in T-cell leukemias). Dilution analysis showed that the clonal rearrangement could be detected when 0.1-10% of the DNA was derived from neoplastic cells, depending on the source tissue. Potential applications of this assay include the diagnosis of lymphoma or leukemia in biopsy samples, cavity fluids, fine needle aspirates, bone marrow and peripheral blood; the determination of lineage (B or T cell); staging of lymphoma; and detection of residual disease after chemotherapy.

Expression and significance of p53, rb, p21/waf-1, p16/ink-4a, and PTEN tumor suppressors in canine melanoma.

The role of tumor suppressor genes in the pathogenesis of canine melanoma is incompletely understood. The genes encoding the tumor suppressors p53, Rb, p21 (waf-1), p16 (ink-4a), and PTEN have been postulated to contribute to the pathogenesis of melanoma in humans and experimental animal models. To assess whether inactivation of these genes similarly contributes to the origin and progression of canine melanoma, we examined their expression in seven distinct canine melanoma cell lines and in 31 retrospective samples (representing 29 dogs) of spontaneous canine melanoma. Various patterns suggestive of loss of tumor suppressor function emerged in these cell lines. The most frequently observed abnormality was loss or significant reduction of p16 expression in six of seven cell lines and in 21 of 26 tumor samples. Loss or significant reduction of PTEN expression was seen in four of seven cell lines and in 13 of 27 tumor samples. Although p53 was detectable in all the cell lines and in 24 of 30 tumors, exclusion of p53 from the nuclear compartment was observed in each of the cell lines and in 18 of 25 tumor samples. These results indicate that loss of function of these tumor suppressor proteins is a common occurrence that may contribute to the origin of canine melanoma. In our sample population, abnormalities in the expression or localization of one or more tumor suppressor proteins occurred with similar frequency in malignant and benign tumors; thus, additional work is necessary to determine how these proteins may impact disease progression and response to therapy.

Expression of S100a, vimentin, NSE, and melan A/MART-1 in seven canine melanoma cells lines and twenty-nine retrospective cases of canine melanoma.

We evaluated the expression of vimentin, S100a, and Melan A/MART-1 (melanoma antigen recognized by T cells 1) in seven cell lines established independently from dogs with canine melanoma. We also compared routine immunostaining of 29 clinical specimens from melanoma cases using vimentin, S100a, and neuron-specific enolase (NSE) with staining for Melan A/MART-1 as part of a diagnostic panel. All the cell lines were positive for expression of vimentin and S-100a. MelanA/MART-1 expression was seen consistently in only two of the seven cell lines. Staining for Melan A/MART-1 was most intense near areas of heavy melanin pigmentation. All except one of the clinical specimens were positive for vimentin. S 100a was expressed in the majority of both pigmented (15/20, 75%) and amelanotic (8/9, 88.8%) tumors. Seventeen of 29 (58.6%) tumors were positive for NSE. Melan A/MART-1 was expressed in 18/29 (62%) tumors, including 90% of pigmented tumors, but in no amelanotic tumors. Intensity of Melan A/MART-1 staining correlated positively with biologic behavior, with seven malignant tumors showing negative to weak staining and 10 benign tumors showing moderate to strong staining. Three malignant tumors showed moderate to intense staining for Melan A/ MART-1. Our results suggest that expression of Melan A/MART-1 may be unstable in cultured cell lines. Assessment of both S100a and Melan A/MART-1 expression is useful to confirm a diagnosis of canine melanoma, and Melan A/MART-1 may be especially informative regarding the biologic behavior of these tumors.

CDK4 expression and activity are required for cytokine responsiveness in T cells.

Stimulation of lymphocytes through the Ag receptor can lead to cytokine responsiveness or unresponsiveness. We examined the importance of cyclin-dependent kinase (CDK)4 to establish and maintain IL-2 responsiveness in human T cells. Our results show that a herbimycin A- and staurosporine-sensitive phase of CDK4 expression and activity preceded the acquisition of IL-2-responsiveness in mitogen-stimulated peripheral blood T cells. Intriguingly, CDK4 expression and activity were demonstrable in purified unstimulated peripheral blood T cells from approximately 30% (5/16) of healthy individuals examined for this study. These T cells proliferated in response to IL-2 without additional mitogens, and both the expression and activity of CDK4 and the ability to respond to cytokines were resistant to herbimycin A and staurosporine. The pattern of CDK4 expression and response to IL-2 in this subset of individuals resembled that seen in the human IL-2-dependent Kit-225 T cell line. However, in contrast to normal T cells, Kit-225 cells were rendered unresponsive to IL-2 by stimulation through the Ag receptor. In these cells, PHA, anti-CD3, or PMA induced marked reductions of CDK4 expression and activity that paralleled IL-2 unresponsiveness, and these effects were not reversible by IL-2. Furthermore, IL-2-dependent proliferation could be similarly inhibited in Kit-225 cells by overexpression of the CDK inhibitors p16/Ink4-a or p21/Waf-1a or by overexpression of a kinase-inactive CDK4 mutant. The data indicate that CDK4 expression and activity are necessary to induce and maintain cytokine responsiveness in T cells, suggesting that CDK4 is important to link T cell signaling pathways to the machinery that controls cell cycle progression.

Sustained nuclear localization of p21/WAF-1 upon growth arrest induced by contact inhibition.

We assessed the expression and distribution of p21/Waf-1 in TLM1 melanoma cells that exhibit contact inhibition and require serum for growth. The growth stage of cells stimulated to enter the mitotic cell cycle synchronously and grow to confluence was characterized by distinct, yet consistent levels and patterns of distribution of p21/Waf-1. Significantly, sustained accumulation of p21/Waf-1 in the nuclear compartment was seen only after 4 days in culture when cell-to-cell contacts were established, leading to a diminished rate of cell growth. Overexpression of wild-type waf-1 in melanoma cells reduced growth of subconfluent cells, decreased Cdk4 activity with a concomitant increase in hypophosphorylated Rb, and promoted cell death by apoptosis. The data support the premise that cell-to-cell contacts provide signals that mediate sustained nuclear localization of p21/Waf-1 leading to cell growth arrest; furthermore, an elevation in the activity of this protein can lead to apoptosis.

Quantitative and qualitative signals determine T-cell cycle entry and progression.

Cell growth and proliferation as well as cell cycle arrest and apoptosis all play integral roles in the cellular immune response. The signals that lead to cytokine production by antigen- or mitogen-stimulated T cells have been studied in detail. However, it is not fully understood how these signals promote cell cycle entry and progression to DNA synthesis in T lymphocytes, especially in primary cells. We used a model distinguishing between competence and progression phases to examine quantitative and qualitative differences in signal transduction that resulted in cell cycle entry and G1 phase arrest or led to DNA synthesis in human T cells. Resting peripheral blood T cells were rendered competent by stimulation with submitogenic concentrations of phytohemagglutinin (PHA) or they were stimulated to proliferate using mitogenic concentrations of PHA. The competent state (that is, the capacity to proliferate in response to exogenous IL-2) was characterized by calcium mobilization, a protein kinase C-dependent internalization of CD3, increased mitogen-activated protein kinase (MAPK) activity, transient translocation of AP-1 transcription factors to the nucleus, expression of immediate early genes, activation of G1-phase cyclin-dependent kinases, and increased CD25 (IL-2Ralpha) expression. However, all of these events were of lesser magnitude in T cells rendered competent than in T cells stimulated to proliferate. Furthermore, the mitogenic stimulus induced a different pattern of MAPK activation and sustained translocation of AP-1 to the nucleus with concomitant IL-2 production. The data indicate that quantitative and qualitative differences in early signaling events distinguish the acquisition of the competent state or the induction of cytokine production with a commitment to T-cell proliferation.

The molecular basis of canine melanoma: pathogenesis and trends in diagnosis and therapy.

Melanoma is a common neoplastic disease of dogs with variable presentation and biological behavior. Canine malignant melanoma is a rapidly metastatic disease that generally is incurable. The loss of function of cellular safeguards built into the genetic program and of immune surveillance systems that cooperate to prevent tumor formation and progression appear to be important underlying causes of canine malignant melanoma. In effect, many existing cancer treatments restore the function of 1 or the other of these mechanisms. For example, chemotherapy and radiotherapy often kill tumor cells by initiating a genetic suicide mechanism (apoptosis), and immunotherapy initiates or enhances a response by the body's immune cells to identify and destroy cancer cells by mechanisms that rely on direct cytotoxicity or apoptotic cell death. Nevertheless, standard therapeutic approaches have not proved effective in treatment of canine malignant melanoma, with only marginal improvement in the outcome of dogs with this disease. The advantages of an improved understanding of the molecular basis of canine cancer are underscored by recent promising advances in diagnosis and in immunologic and genetic therapies that may help reduce the mortality of dogs affected with malignant melanoma.