Biological and molecular characterization of a canine hemangiosarcoma-derived cell line.

Canine hemangiosarcoma (HSA) is a devastating disease. Investigation of novel therapies has been limited by the limited availability of canine HSA-derived cell lines. We report the development of a canine HSA-derived cell line, DEN-HSA, which recapitulates features of angiogenic endothelium. DEN-HSA cells were derived from a spontaneous HSA arising in the kidney of a dog. DEN-HSA displayed surface molecules distinctive of endothelial histogenesis, including factor VIII-related antigen, ICAM-1 and alpha(v)beta3 integrin. In vitro, DEN-HSA formed microvascular tube-like structures on Matrigel, and proliferated in response to a variety of angiogenic growth factors. The cells expressed mRNA and protein specific for bFGF and its receptors, and VEGF and its receptors, among others. DEN-HSA conditioned medium evoked a marked angiogenic response in a murine corneal pocket assay, indicating potent proangiogenic activity of substances secreted by this cell line. This research confirms the DEN-HSA cell line as endothelial in origin, suggests the presence of angiogenic growth factor autocrine loops, and offers the potential to utilize DEN-HSA cells for the study of novel therapies that modulate endothelial proliferation.

Systemic administration of an attenuated, tumor-targeting Salmonella typhimurium to dogs with spontaneous neoplasia: phase I evaluation.

PURPOSE: Genetically modified bacteria are a potentially powerful anticancer therapy due to their tumor targeting capacity, inherent antitumor activity, and ability to serve as efficient vectors for gene delivery. This study sought to characterize the acute and short-term toxicities and tumor colonization rates of a genetically modified Salmonella typhimurium (VNP20009) in dogs with spontaneous tumors, in the context of a phase I dose escalation trial. EXPERIMENTAL DESIGN: Forty-one pet dogs with a variety of malignant tumors received weekly or biweekly i.v. infusions of VNP20009, at doses ranging from 1.5 x 10(5) to 1 x 10(8) cfu/kg. Vital signs and clinicopathologic variables were monitored regularly. Incisional biopsies were obtained before and 1 week following the first infusion for histopathology and bacterial culture. RESULTS: The nominal maximum tolerated dose was 3 x 10(7) cfu/kg, with refractory fever and vomiting being the dose-limiting toxicities. One treatment-related acute death occurred. Bacteria were cultured from tumor tissue in 42% of cases. Thirty-five patients were evaluable for antitumor response. Major antitumor responses were seen in 15% (4 complete response and 2 partial response), and disease stabilization for at least 6 weeks in 10%. CONCLUSIONS: Administration of VNP20009 at doses with acceptable toxicity results in detectable bacterial colonization of tumor tissue and significant antitumor activity in tumor-bearing dogs.

IGF-1 receptor contributes to the malignant phenotype in human and canine osteosarcoma.

To further define the role of insulin-like growth factor-1 (IGF-1) and its receptor (IGF-1R) in osteosarcoma (OS), human OS cell lines with low (SAOS-2) and high (SAOS-LM2) metastatic potential and three canine OS-derived cell lines were studied. Cell lines were evaluated for: IGF-1R expression; expression of IGF binding proteins (IGFBPs); effect of IGF-1 on tumor cell growth, invasion, expression of urokinase plasminogen activator (uPA), and soluble uPA receptor (suPAR), and; ectopic and orthotopic tumorigenicity of the canine OS cells in athymic mice. All cell lines exhibited steady-state mRNA expression of IGF-1R. The SAOS-2 and SAOS-LM2 cells expressed 9,138 and 10,234 cell-associated binding sites, respectively. Canine OS cells expressed from 1,728 to 3,883 binding sites. Two IGF-1-treated cell lines displayed enhanced proliferation. Two cell lines formed colonies in semisolid media, and IGF-1 increased colony number. Matrigel invasion was enhanced in one cell line following IGF-1 treatment. uPA and suPAR were unchanged in SAOS-2 and SAOS-LM2 cells following IGF-1 treatment, but the highly metastatic OS line SAOS-LM2 expressed five times more suPAR and displayed enhanced invasion compared to the parental, low metastatic SAOS-2. IGFBP-5 was detected in four of five cell lines, and IGFBP-3 was detected in two canine OS cell lines. Two canine OS lines were tumorigenic, and one metastasized spontaneously. In conclusion, OS cells express IGF-1R, which can contribute to their growth and invasion. There is suggestive evidence that increasing receptor number may contribute to in vivo tumorigenesis. Additional studies are needed to determine how IGF-1/IGF-1R interactions contribute to the malignant phenotype of OS.

c-Met tyrosine kinase receptor expression and function in human and canine osteosarcoma cells.

To further characterize the role of hepatocyte growth factor-scatter factor (HGF-SF) and its receptor (c-Met) in osteosarcoma (OS), human OS cell lines with low (SAOS-2) and high (SAOS-LM2) metastatic potential, and cell lines derived from spontaneous canine OS were studied. All cell lines were evaluated for c-Met and HGF-SF expression and receptor activation using Northern, RT-PCR, and Western blot analyses, respectively. Functional activity of receptor-ligand interaction was measured using c-Met phosphorylation status, proliferation assays (anchorage-dependent and -independent), Matrigel invasion, modulation of urokinase plasminogen activator (uPA) expression, and cell dispersion (scattering). All cell lines exhibited steady-state mRNA expression of c-Met. The canine OS cell lines also expressed HGF-SF mRNA as determined by RT-PCR analysis. Western analysis showed c-Met protein expression and HGF-stimulated (human) or constitutive (canine) receptor autophosphorylation. Treatment with recombinant human HGF resulted in enhanced proliferation in 3 of 5 OS cell lines and enhanced colony formation in 2 of 5 OS cell lines. Matrigel invasion was significantly enhanced in 3 of the cell lines and uPA levels were significantly increased in the SAOS-2 cells following HGF treatment. Scattering was enhanced in both the SAOS-2 and SAOS-LM2 cells. These data support the involvement of c-Met and HGF-SF in the growth and progression of human and canine OS, and may offer new targets for the development of therapeutic strategies for OS.

Intralesional lipid-complexed cytokine/superantigen immunogene therapy for spontaneous canine tumors.

These studies sought to determine the gene expression and short-term effects of intralesional lipid-complexed immunogene therapy with constructs encoding Staphylococcus aureus enterotoxin A and canine interleukin-2 (L-SEA/cIL-2) in dogs with tumors of various histotypes, and then to assess the safety and efficacy of repeated L-SEA/cIL-2 injections in dogs with spontaneous soft tissue sarcomas (STS). In the first study, pet dogs with a variety of tumors received a single intralesional injection of L-SEA/cIL-2, and surgical excision was performed 48 h later. In the second study, dogs with histologically confirmed STS were treated weekly for a maximum of 12 weeks with escalating doses of L-SEA/cIL-2. Tumors were then surgically excised and assessed histologically and immunohistochemically. Overall, treatments were well tolerated, with no dose-limiting toxicities encountered. At 48 h, in the single injection study, plasmid DNA was detected in 14 of 16 tumor samples, and plasmid-specific mRNA was detected in 3 of 14. In the multiple injection study, the overall response rate in dogs with STS was 25%, consisting of 3 complete responses (CR) and 1 partial response (PR). Diffuse lymphoplasmacytic inflammation was observed in all tumors from patients experiencing CR or PR, whereas these changes were not evident in tumors from nonresponders. The infiltrate was composed primarily of CD3(+) cells at 48 h from the single-injection study, and was composed of both CD3(+) and CD79a(+) cells at 12 weeks in responding dogs from the multiple-injection study. In conclusion, these studies suggests that intralesional L-SEA/cIL-2 immunotherapy is well tolerated, results in detectable transgene expression in canine tumors, and has antitumor activity in dogs with spontaneous STS.

Preclinical study of dolastatin-10 in dogs with spontaneous neoplasia.

PURPOSE: To evaluate the short-term adverse effects of administration of dolastatin-10 (Dol-10) to dogs with spontaneously occurring malignant tumors. METHODS: A total of 34 tumor-bearing dogs were given Dol-10 as a rapid intravenous bolus every 14 days at starting dosages ranging from 200 to 350 microg/m(2). Acute and short-term adverse effects, antitumor response, and duration of response were characterized. RESULTS: The maximum tolerated dose varied greatly from patient to patient, but a reasonable starting dose for further studies was established at 300 microg/m(2). The median number of treatments per dog was 2 (range 1 to 17). Granulocytopenia was the dose-limiting toxicity. The overall response rate was 3%, consisting of a complete and durable (30 months) response in a dog with high-grade malignant lymphoma that was refractory to standard therapy. Two minor or transient responses were observed, and two dogs experienced disease stabilization for 8 and 16 weeks. CONCLUSIONS: Dol-10 appears to be well tolerated in tumor-bearing dogs at doses approaching those tolerated by humans. The clinical activity observed in dogs with non-Hodgkin's lymphoma warrants further investigation.