Marc E. Salute

In vitro effects of the tyrosine kinase inhibitor, masitinib mesylate, on canine hemangiosarcoma cell lines

This study evaluated the in vitro activity of masitinib mesylate against canine hemangiosarcoma (HSA) cell lines after treatment with increasing concentrations of masitinib mesylate (0.01-100 µM) for 24, 48 and 72 h. Results indicated that masitinib mesylate caused a dose- and time-dependent decrease in HSA cell proliferation. The 50% inhibitory concentration (IC(50) ) at 72 h for three HSA cell lines (DEN, Fitz and SB) was found to be 8.56, 9.41 and 10.65 µM, respectively. Further investigation demonstrated that masitinib mesylate induced apoptosis in all HSA cell lines, including activation of caspase-3/7. Measurement of VEGF levels in cell supernatant found a statistically significant increased VEGF in close proximity to the IC(50) of each cell line followed by a decline back towards baseline. These findings indicate that masitinib mesylate causes dose-dependent HSA cell death in vitro and supports future clinical trials of masitinib for canine HSA.

Effect of bevacizumab on angiogenesis and growth of canine osteosarcoma cells xenografted in athymic mice

Objective-To investigate the effects of bevacizumab, a human monoclonal antibody against vascular endothelial growth factor, on the angiogenesis and growth of canine osteosarcoma cells xenografted in mice. Animals-27 athymic nude mice. Procedures-To each mouse, highly metastasizing parent osteosarcoma cells of canine origin were injected into the left gastrocnemius muscle. Each mouse was then randomly allocated to 1 of 3 treatment groups: high-dose bevacizumab (4 mg/kg, IP), low-dose bevacizumab (2 mg/kg, IP), or control (no treatment). Tumor growth (the number of days required for the tumor to grow from 8 to 13 mm), vasculature, histomorphology, necrosis, and pulmonary metastasis were evaluated. Results-Mice in the high-dose bevacizumab group had significantly delayed tumor growth (mean ± SD, 13.4 ± 3.8 days; range, 9 to 21 days), compared with that for mice in the low-dose bevacizumab group (mean ± SD, 9.4 ± 1.5 days; range, 7 to 11 days) or control group (mean ± SD, 7. 2 ± 1.5 days; range, 4 to 9 days). Mice in the low-dose bevacizumab group also had significantly delayed tumor growth, compared with that for mice in the control group. Conclusions and Clinical Relevance-Results indicated that bevacizumab inhibited growth of canine osteosarcoma cells xenografted in mice, which suggested that vascular endothelial growth factor inhibitors may be clinically useful for the treatment of osteosarcoma in dogs. Impact for Human Medicine-Canine osteosarcoma is used as a research model for human osteosarcoma; therefore, bevacizumab may be clinically beneficial for the treatment of osteosarcoma in humans.

Apoptotic effects of the tyrosine kinase inhibitor, masitinib mesylate, on canine osteosarcoma cells.

Osteosarcoma (OSA) is the most common primary bone tumor in dogs and the guarded prognosis highlights the necessity to find new treatments. Masitinib mesylate is a highly selective tyrosine kinase inhibitor that predominantly targets c-Kit and PDGFR-&agr;/&bgr;. This study evaluated the in-vitro activity of masitinib against three canine OSA cell lines after treatment with increasing concentrations of masitinib (0.1–50 µmol/l) at 24, 48, and 72 h. The IC50 values at 72 h for the three OSA cell lines (POS, HMPOS, and COS31) were determined to be 11.04, 7.09, and 9.74 µmol/l, respectively. In addition, increases in caspase-3/7 activity and transferase dUTP nick end labeling-positive cells indicated apoptotic cell death. Because increased levels of vascular endothelial growth factor are found in dogs with OSA, vascular endothelial growth factor in the supernatant was quantified. Overall, the study found that masitinib causes dose-time dependent OSA cell death in vitro through initiation of caspase-mediated apoptosis, which supports future OSA clinical trials.

Radiosensitivity and capacity for radiation-induced sublethal damage repair of canine transitional cell carcinoma (TCC) cell lines

Understanding the inherent radiosensitivity and repair capacity of canine transitional cell carcinoma (TCC) can aid in optimizing radiation protocols to treat this disease. The objective of this study was to evaluate the parameters surviving fraction at 2 Gy (SF(2) ), α/β ratio and capacity for sublethal damage repair (SLDR) in response to radiation. Dose-response and split-dose studies were performed using the clonogenic assay. The mean SF(2) for three established TCC cell lines was high at 0.61. All the three cell lines exhibited a low to moderate α/β ratio, with the mean being 3.27. Two cell lines exhibited statistically increased survival at 4 and 24 h in the dose-response assay. Overall, our results indicate that the cell lines are moderately radioresistant, have a high repair capacity and behave similarly to a late-responding normal tissue. These findings indicate that the radiation protocols utilizing higher doses with less fractionation may be more effective for treating TCC.

In vitro effects of Yunnan Baiyao on canine hemangiosarcoma cell lines

Yunnan Baiyao is a Chinese herbal medicine that has been utilized for its anti-inflammatory, haemostatic, wound healing and pain relieving properties in people. It has been utilized in the veterinary profession to control bleeding in dogs with hemangiosarcoma (HSA) and has been anecdotally reported to prolong survival times in dogs with this neoplasm. This study evaluated the in vitro activity of Yunnan Baiyao against three canine HSA cell lines after treatment with increasing concentrations of Yunnan Baiyao (50, 100, 200, 400, 600 and 800 µg mL(-1) ) at 24, 48 and 72 h. Mean half maximum inhibitory concentration (IC50 ) at 72 h for DEN, Fitz, SB was 369.9, 275.9 and 325.3 µg mL(-1) , respectively. Caspase-3/7 activity increased in correlation with the IC50 in each cell line which was confirmed by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL, APO-BRDU Kit; BD Biosciences, San Jose, CA, USA) assay. VEGF in cell supernatant was also quantified. Overall, the study found that Yunnan Baiyao causes dose and time dependent HSA cell death through initiation of caspase-mediated apoptosis, which supports future studies involving Yunnan Baiyao.

Development of an intramuscular xenograft model of canine osteosarcoma in mice for evaluation of the effects of radiation therapy

OBJECTIVE To develop an IM xenograft model of canine osteosarcoma in mice for the purpose of evaluating effects of radiation therapy on tumors. ANIMALS 27 athymic nude mice. PROCEDURES Mice were randomly assigned to 1 of 3 groups of 9 mice each: no treatment (control group), radiation at 10 Gy, or radiation at 15 Gy. Each mouse received 5 x 10(5) highly metastasizing parent osteosarcoma cells injected into the left gastrocnemius muscle. Maximum tumor diameter was determined with a metric circles template to generate a tumor growth curve. Conscious mice were restrained in customized plastic jigs allowing local tumor irradiation. The behavior and development of the tumor xenograft were assessed via evaluations of the interval required for tumor-bearing limbs to reach diameters of 8 and 13 mm, extent of tumor vasculature, histomorphology of tumors, degree of tumor necrosis, and existence of pulmonary metastasis and clinical disease in affected mice. RESULTS Tumor-bearing limbs grew to a diameter of 8 mm (0.2-g tumor mass) in a mean +/- SEM interval of 7.0 +/- 0.2 days in all mice. Interval to grow from 8 to 13 mm was significantly prolonged for both radiation therapy groups, compared with that of the control group. Histologic evaluation revealed the induced tumors were highly vascular and had characteristics consistent with those of osteosarcoma. Pulmonary metastasis was not detected, and there was no significant difference in percentage of tumor necrosis between groups. CONCLUSIONS AND CLINICAL RELEVANCE A reliable, repeatable, and easily produced IM xenograft model was developed for in vivo assessment of canine osteosarcoma.

Effects of aurothiomalate treatment on canine osteosarcoma in a murine xenograft model.

Osteosarcoma is a highly fatal cancer, with most patients ultimately succumbing to metastatic disease. The purpose of this study was to evaluate the effects of the antirheumatoid drug aurothiomalate on canine and human osteosarcoma cells and on canine osteosarcoma growth and metastasis in a mouse xenograft model. We hypothesized that aurothiomalate would decrease osteosarcoma cell survival, tumor cellular proliferation, tumor growth, and metastasis. After performing clonogenic assays, aurothiomalate or a placebo was administered to 54 mice inoculated with canine osteosarcoma. Survival, tumor growth, embolization, metastasis, histopathology, cell proliferation marker Ki67, and apoptosis marker caspase-3 were compared between groups. Statistical analysis was carried out using the Kaplan–Meier method with the log-rank test and one-way analysis of variance with the Tukey’s test or Dunn’s method. Aurothiomalate caused dose-dependent inhibition of osteosarcoma cell survival (P<0.001) and decreased tumor growth (P<0.001). Pulmonary macrometastasis and Ki67 labeling were reduced with low-dose aurothiomalate (P=0.033 and 0.005, respectively), and tumor emboli and pulmonary micrometastases were decreased with high-dose aurothiomalate (P=0.010 and 0.011, respectively). There was no difference in survival, tumor development, ulceration, mitotic indices, tumor necrosis, nonpulmonary metastases, and caspase-3 labeling. Aurothiomalate treatment inhibited osteosarcoma cell survival and reduced tumor cell proliferation, growth, embolization, and pulmonary metastasis. Given aurothiomalate’s established utility in canine and human medicine, our results suggest that this compound may hold promise as an adjunctive therapy for osteosarcoma. Further translational research is warranted to better characterize the dose response of canine and human osteosarcoma to aurothiomalate.

Development and Assessment of a Novel Canine Ex Vivo Corneal Model

ABSTRACT Purpose: To develop a novel ex vivo extended culture model of canine corneal epithelial cell wound healing. Materials and Methods: Canine corneoscleral rims (CSR) were obtained and, after preparation for culture, were placed on a nutating scaffold and incubated in physiological conditions. In experiment 1, eight CSR in a serum-containing antimicrobial-fortified medium were monitored for epithelial integrity and bacterial infection up to 28 days in culture. CSR were assessed histologically at the end of the culture period end points 0, 7, 14, and 28 days with accompanying scanning electron microscopic (SEM) and transmission electron microscopic (TEM) evaluation. Samples for microbial culture were obtained at days 0, 3, 7, 14, and 28. In experiment 2, uniform 8-mm-diameter superficial corneal epithelial wounds were created and monitored for re-epithelialization in the same culture conditions or in a serum-free protein equivalent medium, with four CSR per group. Standardized digital images were obtained with cobalt filter at the time of fluorescein staining and media change every six hours. Image J imaging software was used to measure the area of fluorescein retention. Re-epithelialization rates were calculated and CSR then fixed for immunohistochemistry (IHC). Results: All corneas survived to end points as described in experiment 1 with no evidence of contamination or compromised epithelial integrity. Histologically, a multilayered epithelium was maintained and corneal edema was not appreciated until day 14. SEM examination revealed epithelial cell layer confluence and migrating epithelial cells of normal cellular morphology with normal cell–cell interactions on TEM. In experiment 2, all eight corneas healed with a healing rate of 0.702 ± 0.130 mm2/h (1.25 mm/day epithelial cell migration rate) and were positive in IHC evaluation for markers of corneal fibrosis. Conclusion: This ex vivo canine corneal wound healing model is an appropriate and clinically relevant tool for assessment and modulation of epithelial wound healing.

Abstract A29: Differential expression of the gangliosides GD3 and GD2 in canine and human osteosarcoma cell lines: An immunotherapy target

Introduction: The disialyl gangliosides GD2/GD3 have been implicated in the enhancement of malignancy in a number human and animal caners (Milner et al Vet Immunology 2006). Moreover, pediatric research trials targeting GD2 osteosarcoma (OSA) surface antigens using humanized mouse antibodies is currently underway. While the coexpression of GD3 and GD2 have been documented in human OSA it has been not reported in canine OSA. Canine OSA is an accepted phenotypic and genotypic large animal model for the study of human OSA. We report on the co-expression of GD3/GD2 in three canine and one human OSA cell lines. Material and Methods: Four canine OSA cell lines (POS, HMPOS, COS31, and Doug) and one human OSA cell line (MG63) were trypsinized from T75 flasks and counted using trypan blue exclusion dye. Live cells (1x10 6 ) were spun down at 250 x gravity. Supernatant was removed and cell pellet mixed with 1ml PBS wash solution. Samples were then centrifuged, supernatant removed, and cells were washed two more times. Primary antibodies GD2 antibody (clone 14G2a, Santa Cruz) and GD3 Mel-1 (clone R24, Covance) and isotype controls were added to samples which were then incubated on ice in the dark for 30 minutes. Cells were again washed in PBS wash solution. Samples were blocked using normal goat serum, washed and then incubated with detection antibody goat anti-mouse IgG F(ab9)2 conjugated to Alexa Flour 488® or Alexa Fluor 647 Goat anti-mouse IgG2a [Molecular Probes, Eugene, OR USA] ) for 30 minutes on ice in the dark. Cells were then washed in wash buffer and re-suspended in 1ml of PBS wash solution. Samples were read at 10,000 events/tube on FACScan (BD, Franklin Lakes, NJ USA). Immunofluorescence was documented using confocal microscopy was used to establish the co-expression of GD3 and GD2 on the cell surface. Results: All cell lines, expressed varying levels of GD3 and GD2 on the cell surface. The canine OSA cell line appeared to express more GD3 (12.54%) on the surface compared to the human MG63 cell line (0.02%). Remarkably, POS, the primary tumor cell line of HMPOS, expressed very low levels of GD3 (4.65%) and GD2 (0.24%), whereas the metastatic line (HMPOS) expressed higher GD3 (25.4%). All cell lines had cells that expressed either GD2 or GD3 but not both. The human MG63 cell line expressed the highest levels of GD2 alone (68.4%) compared to canine cells (average 0.58%). The average co-expression of GD2/GD3 was 48.1% for all cell lines. Discussion: This is the first report of the expression of both GD3 and GD2 in canine OSA cells. The related cell lines POS and HMPOS showed significant variation in expression, with HMPOS (the metastatic line) showing increased expression of GD3. The differential expression of GD3/GD2 on these two cell lines may indicate a phenotype change associate with metastatic potential. The highest co-expression of GD2/GD3 was found in MG63 and COS31, but was also found in HMPOS and Doug. Current investigation includes expression of GD2 & GD3 in osteosarcoma stem cell populations and invasive potential. Conclusion: Results from this study will provide data to investigate the expression profiles of GD2/GD3 and its role in metastasis as well as targeting OSA tumor antigens. Citation Format: Rowan J. Milner, Noaki Chimura, Kristina D. Bowles, Marc Salute. Differential expression of the gangliosides GD3 and GD2 in canine and human osteosarcoma cell lines: An immunotherapy target. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A29.