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 αvβ3 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.

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 × 105 to 1 × 108 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 × 107 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.

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. © 2004 Wiley‐Liss, Inc.

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.

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.

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 µg/m2. 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 µg/m2. 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.

Tumor and normal tissue response was assessed in 21 dogs with malignant nasal tumors given 42 Gy cobalt radiation in 9 or 10 fractions over 11 to 13 days. Local tumor/clinical relapse recurred in 68% of dogs, with a median relapse free interval (RFI) of 270 days. Median survival was 428 days. One year survival for all dogs was 60%. RFI and survival times are better than, or similar to, previous reports of dogs treated with radiotherapy only. Acute radiation effects were severe in one dog. Late effects were severe in six of 15 dogs (40%) with durable tumor control. Late effects included bilateral blindness (3), osteoradionecrosis (3), and seizures (1). These six dogs had a median survival of 705 days. Loss of vision occurred in at least one eye in nine dogs (47%). Tumor staging based on CT findings was predictive for survival duration. Tumor histology was not predictive of outcome. Labrador Retrievers were significantly over-represented. Despite comparable or improved tumor control and survival times provided by this accelerated protocol, relative to other radiotherapy reports, local failure remains the major cause of death, and late radiation effects can be severe in dogs with durable tumor control.

Primitive neuroectodermal tumors are composed of primitive neuroepithelial cells and include tumors of the central and peripheral nervous system. Neuroblastoma, medulloblastoma and retinoblastoma are examples of these rare malignant tumors that usually occur in young patients. This report describes a peripheral neuroblastoma in a 2 year old Boxer that presented with signs of renal disease and a palpable abdominal mass. The purpose of this paper is to describe the clinical presentation, imaging and immunohistological studies of this abdominal tumor in a young dog and to review the literature.

PURPOSE:To prospectively evaluate the short-term toxicoses associated with pegylated-liposomal doxorubicin (Doxil) administered to dogs with measurable tumors of various histologic types and sites. Preliminary information regarding efficacy was also generated.METHODS:A group of 51 dogs with histologically confirmed malignancies received a total of 103 Doxil treatments given i.v. every 3 weeks at dosages ranging from 0.75 to 1.1 mg/kg in the context of a phase I dose-escalation trial. Acute and short-term toxicities as well as tumor response and duration of response were characterized.RESULTS:The maximally tolerated dose in tumor-bearing dogs was established as 1.0 mg/kg i.v. every 3 weeks. The dose-limiting toxicity was a cutaneous toxicity clinically resembling palmar-plantar erythrodysesthesia (PPES). An overall response rate of 25.5% was observed with five complete responders and eight partial responders.CONCLUSIONS:Doxil appeared to be well tolerated at dosages similar to those tolerated for free doxorubicin in tumor-bearing dogs. PPES was the dose-limiting toxicity encountered, rather than myelosuppresion as is the case with free doxorubicin in dogs. Doxil as a single agent may have a broad spectrum of activity and deserves further evaluation.

Forty-eight dogs with histologically confirmed appendicular osteosarcoma (OSA) entered a prospective clinical trial evaluating treatment with amputation and up to 4 doses of carboplatin given every 21 days. The median disease-free interval (DFI) was 257 days, with 31.2% of the dogs disease-free at 1 year. The median survival time was 321 days, with 35.4% of the dogs alive at 1 year. Dogs with proximal humeral OSA had shorter DFI (P = .016) and survival (P = .037) times than dogs with OSA at other locations. Dogs with lower body weights ( < 40 kg) had longer DFI (P = .0056) and survival (P = .007) times than larger dogs. Survival times for dogs that received carboplatin were statistically longer than those previously reported for amputation alone (P < .001). DFI and survival times are similar to those previously reported for 2 to 4 doses of cisplatin. Carboplatin appears to be a well-tolerated chemotherapeutic drug that can be given safely every 21 days at a dose of 300 mg/m2. Neutropenia was the dose-limiting toxicity in this study.

Canine osteosarcoma is a highly metastatic cancer commonly seen in large breed dogs. At the time of diagnosis, approximately 90% to 95% of the dogs have established micrometastases. Dogs undergoing amputation alone have a median survival time of 3 to 4 months. Amputation followed by cisplatin chemotherapy increases median survival times to 9 to 11 months. When dogs are treated with amputation and cisplatin, followed by immunotherapy (with liposome-encapsulated muramyl tripeptide phosphatidylethanolamine), median survival times increase to 14.4 months, the longest reported median survival time for dogs with osteosarcoma treated by amputation and any form of adjuvant therapy.

Journal of Veterinary Internal MedicineVolume 9, Issue 6 p. 425-428 Open Access Response to Liposome-Encapsulated Doxorubicin (TLC D-99) in a Dog With Myeloma William C. Kisseberth, William C. Kisseberth Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WISearch for more papers by this authorE. Gregory MacEwen, Corresponding Author E. Gregory MacEwen Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WIVMD, Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin, 2015 Linden Dr W, Madison, WI 53706.Search for more papers by this authorStuart C. Helfand, Stuart C. Helfand Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WISearch for more papers by this authorDavid M. Vail, David M. Vail Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WISearch for more papers by this authorCheryl L. London, Cheryl L. London Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WISearch for more papers by this authorEvan Keller, Evan Keller Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WISearch for more papers by this author William C. Kisseberth, William C. Kisseberth Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WISearch for more papers by this authorE. Gregory MacEwen, Corresponding Author E. Gregory MacEwen Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WIVMD, Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin, 2015 Linden Dr W, Madison, WI 53706.Search for more papers by this authorStuart C. Helfand, Stuart C. Helfand Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WISearch for more papers by this authorDavid M. Vail, David M. Vail Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WISearch for more papers by this authorCheryl L. London, Cheryl L. London Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WISearch for more papers by this authorEvan Keller, Evan Keller Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WISearch for more papers by this author First published: November 1995 https://doi.org/10.1111/j.1939-1676.1995.tb03304.xCitations: 12 Used in this study was the liposome preparation TLC-99, kindly provided by The Liposome Company, Inc, Princeton, NJ. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume9, Issue6November 1995Pages 425-428 ReferencesRelatedInformation

Forty cats with previously untreated, histologically confirmed mammary gland adenocarcinoma received a radical mastectomy of the affected mammary chain(s). All cats were stratified according to clinical stage and randomized to receive either weekly intravenous liposome-encapsulated muramyl tripeptide phosphatidylethanolamine or placebo (lipid-equivalent) for eight consecutive weeks in this double-blind study. Thirty-six of the 40 cats were available for follow-up. No significant differences in either disease-free interval or survival were found as a result of treatment. Cats with clinical stage II disease had a statistically significantly longer disease-free interval (p<0.02) and overall survival (p<0.005) when compared with clinical stage III cats.

Fifty-five dogs with lymphoma were treated using a doxorubicin-based sequential combination chemotherapy protocol. Complete response, partial response, and no response were seen in 46, 4, and 5 dogs, respectively. The overall median remission duration and survival times were 36 and 51 weeks, respectively. Age, sex, weight, World Health Organization stage, World Health Organization substage (i.e., a = not ill, b = ill), serum calcium concentration, blood urea nitrogen concentration, breed and protocol alteration secondary to toxicity were evaluated for prognostic significance. Univariate analysis of prognostic factors identified sex, World Health Organization substage, and serum calcium as statistically significant (P < or = .05) variables for both survival and remission duration. Upon multivariate analysis, only substage (P = .036) was a significant prognostic factor for remission duration, whereas, both substage (P = .006) and sex (P = .005) were significant prognostic factors for survival.

Tumor necrosis factor (TNF) is a potent mediator of tumor cell killing by activated monocytes and macrophages. We measured TNF activity induced by muramyl peptides and lipopolysaccharide (LPS) in normal dogs. Canine adherent mononuclear cells were isolated and cultured in either medium alone or medium containing muramyl dipeptide (MDP) or MDP plus LPS. After 18 h, culture supernatants were collected and assayed for TNF activity. Sera from dogs injected with liposome-encapsulated muramyl tripeptide-phosphatidylethanolamine (L-MTP-PE) were also evaluated for TNF activity. TNF activity both in supernatants and in sera was detected in a 18 h WEHI-164 cell cytotoxicity assay and was confirmed by a monoclonal antibody directed against recombinant human TNF-alpha. Results showed a significant increase in TNF activity from mononuclear cells exposed to MDP or MDP plus LPS of 20% and 88%, respectively; P<0.0005. Serum TNF activity rapidly increased within 2-3 h post L-MTP-PE injection and subsequently declined to pretreatment level at 4 h post administration. This study demonstrates that MDP +/- LPS can stimulate canine adherent mononuclear cells to release TNF and intravenous injection of L-MTP-PE is capable of rendering the in vivo release of TNF in normal dogs.

The protective effect of methimazole, a commonly used antithyroid drug, on cisplatin-induced nephrotoxicity was studied. Eight dogs received 80 mg/m2 cisplatin i.v. without saline prehydration. Dogs were randomized into two groups of four dogs each: one group received 40 mg/kg methimazole i.p. at 30 min prior to and 4 h after cisplatin delivery, and the other group received saline placebo i.p. Methimazole protected dogs against the in vivo nephrotoxicity elicited by cisplatin as evidenced by clinicopathologic and histopathologic indices. Protection was not complete, as methimazole-treated animals developed mild histopathologic renal changes. Measures of renal oxidative stress did not differ between the two groups at day 5 following cisplating treatment. No difference was noted for serum thyroxine concentrations before or after therapy in either group; however, serum levels of 3,5,3′-triiodothyronine were significantly higher on day 5 in both groups of dogs receiving cisplatin, regardless of whether they received methimazole or not. Methimazole as used in this study was found to be well tolerated in dogs over the short term, with no significant clinical or clinicopathologic toxicity being observed. The results of this study support the additional evaluation of methimazole as a protectant against cisplatin-induced nephrotoxicity using the dog as a model.

Journal of Immunotherapy with Emphasis on Tumor Immunology: January 1993 - Volume 13 - Issue 1 - p 65