Osteosarcoma is the most common primary bone tumor in dogs, accounting for 85% of all bone cancers.1 In dogs, it is highly malignant because it is locally aggressive and has a high probability of metastasis. Radiotherapy is a commonly used local treatment and can be used with palliative or curative intent protocols as part of a multimodal therapeutic approach.
Conventional fractioned radiation therapy for local control of bone tumors has generally been unsuccessful because of the large total radiation dose required for tumor control and adverse radiation effects on noncancerous, healthy tissues.2,3 Stereotactic radiotherapy is a novel method of local osteosarcoma control in dogs and potentially in humans as well. The technique uniquely allows precise and accurate delivery of ablative doses of radiation directly to the tumor with relative sparing of the adjacent healthy tissues and organs.4,5
Excellent local tumor control has been achieved with SRT in dogs with naturally developing osteosarcoma of the extremities.5,6 However, in our experience and as indicated in other reports,5 the likelihood of treated dogs developing pathological fractures as a result of treatment is high with existing SRT protocols. More studies are needed to evaluate various dose and fractionation protocols as well as potential adjunctive treatments to achieve adequate local tumor control with lower total radiation doses than are presently administered and preservation of bone remodeling capability. To facilitate such studies, a reliable model of canine osteosarcoma needs to be developed in rodents that can be used to evaluate and refine the use of SRT and improve existing treatments for osteosarcoma.
Models of canine osteosarcoma have been developed in rats and mice to help characterize the disease and evaluate the efficacy of new treatments. Many of these models are heterotopic models involving IM or SC implantation of osteosarcoma cells.7–9 Heterotopic models have inherent limitations because osteosarcoma cells are not exposed to the natural tumor microenvironment. Consequently the biological behavior of osteosarcoma and response to radiation therapy in a heterotopic model may not reflect the behavior in the natural disease process.7–9 Specifically the effect of SRT on tumor-affected and adjacent healthy bone and loss of bone remodeling capability and development of pathological fractures cannot be evaluated. Orthotopic models, on the other hand, may more reliably be used to predict treatment outcomes, particularly local treatments such as SRT. The purpose of the study reported here was to develop a reproducible, temporally predictable orthotopic model of canine osteosarcoma in rats and subsequently evaluate the feasibility of delivering SRT through use of a clinically relevant fractionation scheme.
Materials and Methods
Animals—Immunocompromised athymic nude rats (RH-Foxn1rnu) that were 6 to 8 weeks of age were obtained from the National Institutes of Health and housed at a laboratory animal resources facility. Each was identified by an ear tag. Rats rather than mice were chosen for the model because their larger size allows for the use of image-guided SRT with clinically relevant total dose and fractionation schedules, which would not be feasible in mice.
Osteosarcoma cells—Cells from the Abrams osteosarcoma cell line transfected with luciferase were used.a The cells were grown at 37°C with 5% CO2 in minimum essential mediumb supplemented with 10% fetal calf serum,c 7.5% sodium bicarbonate,b minimum essential media essential amino acids,b 10mM nonessential amino acids,b l-glutamine,b and antibiotic-antimycotic solution.b Upon confluency, cell cultures were split approximately every 3 days. Luciferase activity was confirmed by treating the cells for 5 minutes with 3.57μM luciferin and viewing them with a microscope imaging systemd at a 30-second exposure with medium binning. The cultured osteosarcoma cells were validated to be of canine origin and free of contamination through multiplex PCR assay of the mitochondrial DNA.10
Experimental design—Three sequential experiments were conducted to develop and characterize the canine osteosarcoma model and evaluate the feasibility of the model for assessing clinically relevant SRT doses. In the first experiment, 12 rats were assigned to transcortical injection of osteosarcoma cells into the proximal tibial metaphysis of the left hind limb. Tumor engraftment and progression were to be monitored weekly via radiography, bioluminescence imaging, and measurement of urine pyridinoline concentration, beginning immediately after cell injections (baseline). Three rats were to be selected each week by random drawing of ear tag identification numbers for euthanasia at 2, 3, 4, and 5 weeks after osteosarcoma cell injection to obtain samples for histologic evaluation.
In the second experiment, 6 rats were assigned to undergo osteosarcoma cell injection in the distal aspect of the left femur. Tumor engraftment and progression were to be monitored weekly via radiography, bioluminescence imaging, and measurement of urine pyridinoline concentration, beginning immediately after cell injections. Urine samples (approx 500 μL) were to be collected via manual expression of the bladder or cystocentesis once a week, then stored in individually labeled Eppendorf tubes at −20°C until pyridinoline analysis. All 6 rats were to be euthanized 6 weeks after cell injection, and histologic evaluation of the treated femurs was to be performed.
In the final experiment, 8 rats were assigned to undergo osteosarcoma cell injection in the distal aspect of the left femur, followed 2 weeks later by SRT consisting of 36 Gy spread over 3 days at a dose of 12 Gy/d. Tumor engraftment and progression were to be monitored weekly via radiography, bioluminescence imaging, and measurement of urine pyridinoline concentration, beginning immediately after cell injections. Urine samples were to be obtained on a weekly basis for measurement of pyridinoline as described for the second experiment. Rats were to be euthanized 6 weeks after SRT began, and histologic evaluation of the treated femurs was to be performed.
Osteosarcoma cell injection—Rats were anesthetized by gaseous chamber induction with isoflurane (2% to 4%) and oxygen. Rats were transferred to a heated surgery table and anesthesia maintained via isoflurane (1% to 3%) mixed with 100% oxygen administered via facemask and a nonrebreathing anesthesia circuit. For the tibial injections, a 22-gauge hypodermic needle was inserted transcortically into the proximal metaphysis of each rat and 1 × 106 osteosarcoma cells in 50 μL of PBS level solution was injected. For the femoral injections, a 22-gauge needle was inserted into the femur at the level of the trochanteric fossa, advanced distally within the medullary canal with a rotating motion to the distal metaphysis, and then withdrawn. A second needle attached to a 1-mL syringe was then advanced to the distal metaphyseal region, and 1 × 106 osteosarcoma cells in 50 μL of PBS solution were injected.
Bioluminescence imaging—Bioluminescence imaging was performed weekly, beginning immediately after cell injections. Five minutes prior to imaging, each rat was placed in an anesthetic chamber and anesthesia was induced with isoflurane (2% to 4% in oxygen). Anesthesia was subsequently maintained with an admixture of isoflurane (1% to 3%) and 100% oxygen administered via face mask. While anesthetized, rats were injected intraperitoneally with 150 mg of luciferinb/kg. They were subsequently positioned in left lateral recumbency, and images of the left tibia or femur were obtained at 1- and 3-minute intervals at medium binning.d Maximum fluorescence was recorded as an estimate of the degree of luciferase expression for each rat at each evaluation as an indicator of tumor cell viability.
Radiography—While rats were anesthetized, digital radiography of the tibias or femurs was performed to monitor the onset of tumor-associated osteolysis and tumor progression. Rats were positioned in left lateral recumbency with the affected limb down and the contralateral limb positioned away from the affected limb. Lateral and anteroposterior radiographic views were obtained. Images were acquirede with settings of 40 kV and 3.72 mA at 0.14 seconds, and stored electronically for comparison and analysis.
Pyridinium cross-links ELISA—Pyridinoline has been used as a marker of tumor-associated osteolysis to monitor tumor development and response to treatment in rats with metastatic bone cancer.11 Samples were diluted 1:20 in PBS solution, and pyridinoline analysis was performed with a commercial ELISAf per the manufacturer's instructions. Urine creatinine concentration was measured with a commercial kitg so that pyridinoline values could be corrected for differences in urine creatinine concentration. This correction was performed by dividing the pyridinoline values by the creatinine values. Mean ± SD corrected pyridinoline values were plotted at each weekly assessment point.
SRT—Stereotactic radiotherapy was initiated 2 weeks after osteosarcoma cell injection and after confirmation of tumor cell engraftment via radiography. In preparation, rats assigned to receive SRT were individually anesthetized via the same protocols as previously described. Each rat was subsequently covered in a pliable plastic air bubble packing material to help maintain normothermia. Breathing was observed via closed-circuit video monitors when personnel were required to be outside the radiation suite. Noninvasive immobilization was achieved by positioning each rat in left lateral recumbency (affected femur down) within a customized bolush and cushioni indexed to the couch of the linear acceleratorj via the baseplate. Any space between the rat and bolus was ablated with petroleum jelly.
Transaxial CT images of the affected femur were captured via onboard cone-beam CT.k Cone-beam CT images were reconstructed with a slice thickness of 1 mm and imported into the computerized treatment planning system. Contouring consisted of identifying the GTV, noncancerous bone, and skin. The GTV was defined as the entire femur, including any radiographically detectable tumor that extended beyond the cortical margins. For all rats, the CTV was identical to the GTV. The PTV was the result of a symmetric 2-mm expansion beyond the GTV or CTV. A SRT plan consisting of 7 isocentrically placed fields was created. A static multileaf collimator (5-mm leaf width at isocenter) was used to increase the dose conformality achieved within the tumor volumes, while preferentially sparing surrounding unaffected tissues outside the PTV. Each plan was normalized to achieve a minimum of 99% of the desired dose within the GTV and a minimum of 95% of the desired dose within the PTV as determined through evaluation of a dose-volume histogram.
Prior to delivery of each SRT fraction, an orthogonal pair of kilovoltage radiographs of the affected femur was obtained with the onboard imaging system. These radiographs were matched to the digitally reconstructed radiographs derived from the original cone-beam CT. Any changes in the couch position, based on the matching process, were made to ensure precision and accuracy of SRT delivery. Each field of the SRT plan was then delivered individually.
Rats were anesthetized daily and underwent on 12-Gy fraction of SRT daily for 3 days for a total treatment dose of 36 Gy. When the multiple fractions of SRT were administered, a symmetric 2-mm PTV expansion assured inclusion of the GTV through the digital matching of an orthogonal pair of kilovoltage radiographs to the digitally reconstructed radiographs. On completion of SRT each day, rats were allowed to recover from anesthesia and returned to their cages. Rats were monitored serially for their response to SRT.
Quality-control testing of the linear accelerator was performed daily with a phantom and dosimetry filml to ensure the planned radiation dose was indeed delivered through the relatively small treatment field.12 The measured dose difference relative to the calculated dose was 3.2%.
Assessment of radiation effects—Rats that underwent SRT were monitored daily by direct observation for radiation-induced skin changes after treatment. The severity of any skin effects was graded with the Veterinary Radiation Therapy Oncology Group grading scheme13 each day until the apparent effects resolved as follows: 0, no pathological change; 1, mild skin effects such as erythema, dry desquamation, alopecia, or depilation; 2, moderate skin effects such as patchy moist desquamation without edema; and 3, severe skin effects such as confluent moist desquamation with edema or ulceration, necrosis, or hemorrhage. Digital photographic images of the limb that underwent SRT were obtained daily after SRT administration to document any adverse effects until any effects had resolved.
Histologic evaluation—All rats from all experiments were euthanized at the previously specified points, soft tissues were removed from the limbs in which osteosarcoma had been induced, and bones were placed in neutral-buffered 10% formalin. In preparation for evaluation, tibias and femurs were removed from the formalin and decalcified in 10% formic acid for 5 days. Bones were sectioned in the midsaggital craniocaudal plane and stained with H&E. Preparation and analysis were performed at the Veterinary Diagnostic Laboratory of Colorado State University. Histologic slides were interpreted by a board-certified veterinary pathologist with experience with osteosarcoma assessment (BEP).
Statistical analysis—All data are reported as mean ± SD. Corrected pyridinoline and luciferase data were evaluated for differences over time via repeated-measures ANOVA with statistical software.m The pairwise t test was used to identify differences in mean pyridinoline and luciferase values between experimental groups. Differences were considered significant at P < 0.05.
Results
Animals—Ten of 12 rats in the experiment involving tibial injection with canine osteosarcoma cells and all 14 rats that received a femoral injection (6 that received no treatment and 8 that underwent SRT) had successful tumor engraftment.
Osteosarcoma cell injection technique—Penetration of the needle through the cortical bone of the proximal aspect of the tibia was not reliable and repeatable. Frequently, more than 1 attempt was required to penetrate the cortical surface because of the triangular cross section of the bone. In some rats, a 0.5- to 1.0-cm skin incision was made to expose, directly see, and stabilize the proximal portion of the tibia to facilitate transcortical needle passage. This additional step resulted in an increase in the duration of anesthesia and increased the probability of surgical complications. The presence of a transcortical defect perpendicular to the long axis of the bone potentially allowed osteosarcoma cells to escape into the surrounding soft tissues. Soft tissue swelling and radiographic mineralization of soft tissues were observed in 1 of 12 rats following osteosarcoma cell injection, consistent with extraosseous osteosarcoma development. Introduction of the needle through the cortical bone of the trochanteric fossa of the femur and advancement into the distal metaphyseal region resulted in a more repeatable injection technique than with the transcortical tibial technique.
Radiography—In both the tibial and femoral injection experiments, rats had radiographic evidence of tumor 2 weeks after osteosarcoma cell injection. The observed radiographic changes were similar to those commonly seen in dogs with naturally developing osteosarcoma. At the 2-week assessment point, no evidence of fracture was identified in any rat.
Temporal radiographic changes in the limbs of rats in the tibial injection experiment consisted of osteolytic and osteoproliferative (mixed lytic) regions within the affected bone of the 10 rats that had successful tumor engraftment (Figure 1). Tumors progressed in these rats such that, by the fifth week after osteosarcoma cell injection, each tumor had completely engulfed the proximal end of the tibia, resulting in severe bone destruction. Also by week 5, fracture had occurred in 2 of 12 rats. At the same point in the femoral injection experiment, bone fracture was evident in most rats and the experiment concluded.
Representative lateral radiographic views of the tibia (A) and femur (B) of rats in which canine osteosarcoma cells had been injected into the proximal or distal aspects, respectively. The images show the radiographic tumor progression over the 5 weeks that followed cell injection.
Citation: American Journal of Veterinary Research 74, 3; 10.2460/ajvr.74.3.452
Bioluminescence—In the tibial injection experiment, viability of the osteosarcoma cells was fairly consistent throughout the first 4 weeks after injection; however, by the fifth week, no luciferase expression was detected (Figure 2). In the femoral injection experiment, luciferase activity increased from the time it was first measured until the fifth week, when the activity was 5 times as high.
Representative bioluminescence images of the luciferase-expressing osteosarcoma cells in the femurs of rats in which canine osteosarcoma cells were injected, followed or not followed 2 weeks later by SRT (36 Gy administered through 3 daily sessions of 12 Gy each). In the femur that received no SRT, the amount of luciferase expression increased throughout the 5-week period following cell injection. In the SRT group, luciferase expression was undetectable by week 3 and remained undetectable through the end of the experiment.
Citation: American Journal of Veterinary Research 74, 3; 10.2460/ajvr.74.3.452
Increases in urine pyridinoline concentration, which was measured as an estimate of the degree of bone resorption, corresponded to radiographic and bioluminescence findings suggestive of tumor-associated osteolysis after tumor engraftment. The pyridinoline concentration increased during the first 2 weeks after osteosarcoma cell injection and then remained persistently high through the remainder of the experiment (P < 0.005; Figure 3).
Mean ± SD urine pyridinoline concentrations at various points for rats that underwent femoral injection of canine osteosarcoma cells (week 0) and did (n = 8; dotted line) or did not (6; solid line) undergo 3 days of SRT beginning 2 weeks after cell injection. In rats that received SRT (36 Gy administered through 3 daily sessions of 12 Gy each), concentrations increased significantly (P < 0.001) after cell injection and decreased significantly (P < 0.001) to baseline values after SRT.
Citation: American Journal of Veterinary Research 74, 3; 10.2460/ajvr.74.3.452
SRT—All 8 rats were anesthetized, positioned, and imaged; all underwent SRT and recovered without anesthetic complication. Monitoring via the closed-circuit video system allowed for the adjustment of inhaled isoflurane concentration as needed, resulting in the maintenance of a depth of anesthesia suitable for replicating and maintaining subject positioning and immobilization. Treatment planning for all 8 rats resulted in a mean GTV and CTV of 0.8 mL and median GTV and CTV of 0.9 mL (range, 0.5 to 1.0 mL). The mean and median PTV were 2.1 and 2.5 mL (range, 0.5 to 2.9 mL), respectively. For each rat, the mean and median dose to 99% GTV and CTV were 36.4 Gy, and the mean and median dose to 95% of the PTV were both 36.0 Gy. The total treatment planning time for each rat was approximately 1 hour. All rats survived the procedures and received SRT as prescribed and intended.
Stereotactic radiotherapy involving 3 daily sessions at 12 Gy (total dose, 36 Gy) achieved local tumor control. One week after the last SRT fraction was administered, a decrease in luciferase expression was apparent (Figure 4). Luciferase expression was undetectable at week 2 and remained undetectable throughout the remainder of the experiment. After SRT, further radiographic progression of the tumors was not identified (Figure 5). A mean percentage tumor necrosis of 95% was histologically evident in the femurs treated with this SRT protocol.
Mean ± SD degree of luciferase expression at various points after rats underwent injection of canine osteosarcoma cells in the proximal aspect of the tibia (n = 12; dashed line) or distal aspect of the femur (6; solid line). In the femoral injection group, the luciferase expression increased from initial values until the study ended (week 5), by which time it had increased 5-fold (P < 0.005).
Citation: American Journal of Veterinary Research 74, 3; 10.2460/ajvr.74.3.452
Representative lateral radiographic views of the femur of a rat at various points after canine osteosarcoma cells had been injected into the distal aspect and SRT at 12 Gy/d for 3 days (total dose, 36 Gy) was initiated 2 weeks after osteosarcoma cell injection. The images show lack of radiographic progression of the osteosarcoma lesion, compared with lesion appearance in the untreated osteosarcoma-affected femurs shown in Figure 1
Citation: American Journal of Veterinary Research 74, 3; 10.2460/ajvr.74.3.452
Urine pyridinoline concentration increased significantly (P < 0.001) during the 3 weeks after osteosarcoma cell injection in rats that received SRT. After SRT (week 2), this concentration significantly (P < 0.001) decreased at week 4 (Figure 3). The pyridinoline concentration remained at baseline for the duration of the experiment.
The fractionated three 12-Gy SRT protocol used in the study resulted in transient, mild radiation-induced skin effects. The skin initially appeared unaffected after SRT. The maximum grade of radiation-induced skin effects was observed between days 10 and 11 after the first SRT fraction, and the effects were mild (grade 1). The skin recovered quickly such that minimal or no abnormalities were evident 15 and 18 days after the first SRT fraction and remained lesion-free for the remainder of the experiment.
Discussion
In the present study, a reproducible, temporally predictable orthotopic model of canine osteosarcoma was successfully developed in athymic rats. The feasibility of delivering SRT to the tumor-affected bone with a clinically relevant total dose and fractionation protocol was established. Injection of osteosarcoma cells into the distal metaphysis of the femur was more repeatable than transcortical injection of the proximal aspect of the tibia and resulted in less surgical tissue disruption and shorter procedure and anesthetic time. Stereotactic radiotherapy was delivered successfully in rats with experimentally induced osteosarcoma of the distal aspect of the femur. The proportion of rats with local tumor control was high, and the short-term effects of radiation on the skin were acceptable with the 36-Gy SRT protocol used (divided over 3 days in 3 fractions of 12 Gy each). This model and the ability to deliver clinically relevant doses of SRT have promise for use in future experiments to describe the short- and long-term radiobiological effects of SRT on noncancerous bone, tumor-affected bone, and surrounding soft tissues; to evaluate other dose and fractionation protocols; and to evaluate the effect of other treatments used in combination with SRT.
The first objective of our study was to develop a reliable orthotopic model of canine osteosarcoma in rats. Canine osteosarcoma cells were injected into the proximal aspect of the tibia by inserting a needle through the tibial cortex and injecting the cells into the metaphyseal region. Evidence of cell leakage into the muscular tissues was found, suggesting an inadequate representation of the localized disease. Ten of these 12 rats had tumor engraftment, which was comparable with findings in a similar orthotopic study14 in mice.
Because of the technical difficultly and variability associated with osteosarcoma cell injection into the proximal aspect of the tibia, we chose to inject the cells into the distal aspect of the femur via a distant access site for the second experiment. The remote nature of the access point to the site of osteosarcoma cell injection reduced the potential for extraosseous tumor development. Because of the closed nature of the intramedullary cavity, osteosarcoma cell injection should be slow because rapid injection can result in death due to embolization as observed in pilot experiments conducted in our laboratory (data not shown). This technique was reliably repeatable and minimized the potential for extraosseous tumor cell leakage after injection. In addition, the distal femoral site has a larger soft tissue envelope than the proximal tibial site, allowing for better delivery of SRT and minimization of adverse skin effects.
Neoplastic lesions were confirmed in all rats via postmortem histologic evaluation. In vivo tumor progression was observed via radiography and luciferase imaging. Increases in urine pyridinoline concentration have been detected in dogs with osteosarcoma and can be useful in monitoring response to treatment.15,16 In the study rats, a significant increase in urine pyridinoline concentration was evident 2 weeks after osteosarcoma cell injection. This increase persisted throughout the remainder of the experiment and indicated an ongoing osteolytic process induced by the tumor.17,18
All rats had successful tumor engraftment in the femoral injection experiment. A 100% engraftment rate was also observed in a previous allogenic orthotopic model in rats in which rat osteosarcoma cells were injected into the distal aspect of the femur.19 In non-experimental settings, osteosarcoma most commonly develops in the metaphyseal regions of long bones (distal portion of the femur, proximal portion of the tibia, proximal portion of the humerus, and distal portion of the radius).20 The femoral injection technique allowed for growth of the tumor from the medullary canal with eventual extension through the cortical bone from the endosteal surface, which is consistent with natural tumor development.
Our second objective was to evaluate the feasibility of delivering SRT in our model of canine osteosarcoma. Although other models have been used to evaluate the efficacy of radiation therapy in mice, the use of rats in our model with bones larger than in mice allowed for the use of SRT and higher total doses of radiation than would be achievable in mice.4 We evaluated the feasibility of administering SRT in 3 fractions of 12 Gy each (total dose, 36 Gy) and the ability to achieve local tumor control by measuring percentage tumor necrosis and acute radiation-induced effects on noncancerous, healthy tissues (skin) surrounding the tumor. This is a clinically relevant radiation dose and fractionation protocol that we have used for successful local tumor control in dogs with appendicular osteosarcoma.
A previous study21 of radiation therapy for treatment of osteosarcoma in dogs showed that a mean percentage tumor necrosis of ≥ 80% correlates with excellent local tumor control and an increase in survival rate. In the present study, SRT resulted in a mean percentage tumor necrosis of 95%. In a previously reported heterotopic model of canine osteosarcoma in mice, < 25% tumor necrosis was achieved after radiation therapy.4 This is compared with a mean of 95% tumor necrosis observed in our orthotopic model in rats as well as in dogs treated with the same SRT radiation protocol.4,21 The lower percentage of tumor necrosis in the heterotopic model study4 was most probably attributable to the smaller radiation dose (10 to 15 Gy). In our orthotopic model, the tumor did not progress in size radiographically, and luciferase activity decreased after SRT, in contrast to findings in the rats with femoral tumors not treated with SRT, which had substantial local tumor progression to the point of fracture within 5 weeks after osteosarcoma cell injection. Significant decreases in urine pyridinoline concentration after SRT relative to concentrations in rats not treated with SRT was indicative of a lower amount of tumor-associated bone resorption consistent with local tumor control. Urine pyridinoline concentration and other biomarkers have been used as indicators of response to radiation treatment of osteosarcoma in other studies.17,18,22
The orthotopic model reported here more realistically represented the response to SRT of dogs with osteosarcoma than have models of canine osteosarcoma in mice. This SRT protocol achieved tumor control while minimalizing short-term adverse effects associated with radiation therapy. With the intramuscular heterotopic model in mice,4 no adverse radiation effects have been reported, but this is probably because of the lower radiation dose used, which also resulted in a lower percentage of tumor necrosis. Although the present study involved a small number of rats, it yielded important insight into the degree of tumor necrosis achievable and the short-term effects associated with a clinically relevant total dose and fractionation SRT protocol. Use of 3 fractions of 12 Gy each resulted in a high probability of local tumor control, while minimizing the adverse effects of radiation on healthy tissue. We have observed similar degrees of tumor necrosis and short-term radiation skin effects in client-owned dogs with natural osteosarcoma in our clinic with this SRT fractionation protocol. Longer follow-up after SRT is recommended in subsequent studies to observe whether pathological fracture develops as a late complication in the orthotopic rat model, which, as our experience shows, occurs in 30% to 40% of dogs with naturally developing disease after SRT. Although beyond the scope of the present study, documentation of the histologic short- and long-term effects of SRT on the tissues (muscle, peripheral nerve, cartilage, blood vessels, and skin) within and surrounding the radiation treatment field is recommended for future experiments involving the orthotopic osteosarcoma model in rats.
ABBREVIATIONS
| CTV | Clinical target volume |
| GTV | Gross tumor volume |
| PTV | Planned target volume |
| SRT | Stereotactic radiotherapy |
Provided by the Colorado State University Animal Cancer Center, Fort Collins, Colo.
Mediatech Inc, Manassas, Va.
Atlas Biologicals, Fort Collins, Co.
Xenogen IVIS 100, Caliper Life Sciences Inc, Hopkinton, Mass.
MinXray TR90, MinXray Inc, Northbrook, Ill.
MicroVue pyridinium cross-links kit, Quidel Corp, San Diego, Calif.
Creatinine Kit, Cayman Chemical Co, Ann Arbor, Mich.
Superflab Bolus, Elimpex-Medizintechnik, Moedling, Austria.
VacLoc Cushion, Civco, Kalona, Iowa.
Varian Medical Systems Inc, Palo Alto, Calif.
Gafchromic film, Ashland Inc, Covington, Ky.
Phillips Big Bore PET-CT, Phillips Healthcare, Andover, Mass.
IBM SPSS, IBM SPSS Inc, Armonk, NY.
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