Objective—To determine the effect of pamidronate
disodium on the in vitro viability of osteosarcoma
cells and non-neoplastic cells from dogs.
Sample Population—3 osteosarcoma and 1 fibroblast
cell lines derived from dogs.
Procedure—Cell counts and cell viability assays were
performed in cultures of osteosarcoma cells (POS,
HMPOS, and COS31 cell lines) and fibroblasts after
24, 48, and 72 hours of incubation with pamidronate
at concentrations of 0.001 to 1,000µM or with no
drug (control treatment). Percentage viability was
determined in cell samples for each concentration of
pamidronate and each incubation time. A DNA fragmentation
analysis was performed to assess bisphosphonate-
Results—Osteosarcoma cell viability decreased significantly
in a concentration- and time-dependent
manner at pamidronate concentrations ranging from
100 to 1,000µM, most consistently after 48 and 72
hours' exposure. In treated osteosarcoma cells, the
lowest percentage cell viability was 34% (detected
after 72 hours' exposure to 1,000µM pamidronate).
Conversely, 72 hours' exposure to 1,000µM
pamidronate did not significantly reduce fibroblast viability
(the lowest percentage viability was 76%). After
72 hours of exposure, pamidronate did not cause
DNA fragmentation in POS or HMPOS cells.
Conclusions and Clinical Relevance—Results indicate
that pamidronate may have the potential to inhibit
osteosarcoma growth in dogs, possibly through a
nonapoptotic mechanism. The clinical relevance of
these in vitro findings remains to be determined, but
administration of pamidronate may potentially be indicated
as an adjuvant treatment in chemotherapeutic
protocols used in dogs. (Am J Vet Res 2005;66:
Objective—To determine whether exposure of
canine osteosarcoma cells to deracoxib or piroxicam
results in decreased viability, whether the cytotoxic
effects of deracoxib and piroxicam involve induction
of apoptosis, and whether deracoxib is a more
potent inhibitor of osteosarcoma cell growth than
Sample Population—1 fibroblast and 3 osteosarcoma
Procedure—Cell counts and viability assays were
performed using osteosarcoma cells (POS, highly
metastatic POS, and canine osteosarcoma cell 31)
and fibroblasts after 72 hours of incubation with deracoxib
at concentrations of 0.5µM to 500µM or piroxicam
at concentrations of 1µM to 1,000µM.
Percentage viability was determined for each concentration.
A DNA fragmentation analysis was performed
to assess drug-induced apoptosis.
Results—Concentration of deracoxib required for
50% inhibition of cell viability (IC50) was reached in all
3 osteosarcoma cell lines and ranged from 70 to
150µM, whereas the IC50 for piroxicam was only
reached in the POS cell line at 500µM. Neither deracoxib
nor piroxicam induced sufficient toxicity in
fibroblasts to reach an IC50. Exposure of osteosarcoma
cells to cytotoxic concentrations of deracoxib and
piroxicam did not result in DNA fragmentation.
Conclusions and Clinical Relevance—Intermediate
and high concentrations of deracoxib and high concentrations
of piroxicam were cytotoxic to osteosarcoma
cells; neither drug inhibited cell viability at typical
plasma concentrations in dogs. Deracoxib inhibited
viability of cells at concentrations that did not
affect fibroblast viability. There was no evidence of
apoptosis induction for either drug; however, only 1
cell line was evaluated for apoptosis induction and
only for a limited selection of drug concentrations.
(Am J Vet Res 2005;66:1961–1967)
Objective—To characterize the radiosensitivity and capacity for sublethal damage repair (SLDR) of radiation-induced injury in 4 canine osteosarcoma cell lines.
Sample Population—4 canine osteosarcoma cell lines (HMPOS, POS, COS 31, and D17).
Procedures—A clonogenic colony-forming assay was used to evaluate the cell lines' intrinsic radiosensitivities and SLDR capacities. Dose-response curves for the cell lines were generated by fitting the surviving fractions after radiation doses of 0 (control cells), 1, 2, 3, 6, and 9 Gy to a linear quadratic model. To evaluate SLDR, cell lines were exposed to 2 doses of 3 Gy (split-dose experiments) at an interval of 0 (single 6-Gy dose), 2, 4, 6, or 24 hours, after which the surviving fractions were assessed.
Results—Mean surviving fraction did not differ significantly among the 4 cell lines at the radiation doses tested. Mean surviving fraction at 2 Gy was high (0.62), and the α/β ratios (predictor of tissue sensitivity to radiation therapy) for the cell lines were low (mean ratio, 3.47). The split-dose experiments revealed a 2.8- to 3.9-fold increase in cell survival when the radiation doses were applied at an interval of 24 hours, compared with cell survival after radiation doses were applied consecutively (0-hour interval).
Conclusions and Clinical Relevance—Results indicated that these canine osteosarcoma cell lines are fairly radioresistant; α/β ratios were similar to those of nonneoplastic, lateresponding tissues. Future clinical investigations should involve increasing the fraction size in a manner that maximizes tumor killing without adverse effects on the nonneoplastic surrounding tissues.
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 × 105 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.
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.