Materials and Methods

Canine mastocytoma cell line C2—The C2 cell line^a is 1 of 2 canine mast cell lines established in 1990.¹⁷ The cell line was obtained from a dog with terminal mast cell disease with recurrence of the primary tumor and distant metastasis to the liver, spleen, lymph nodes, and bone marrow. Biopsy specimens were obtained from a recurrent subcutaneous MCT at the primary site. The C2 cells were cultured in Iscove modified Dulbecco medium^b supplemented with 10% fetal calf serum^c and antimicrobials (penicillin, streptomycin, gentamicin, and amphotericin B)^c in 5% CO₂ at 37°C. They were passaged every 3 to 5 days and thawed from original stock every 6 to 8 weeks. In all experiments, cells from the third to fifteenth passage were used. Cell counts were obtained with a hemacytometer. Cell viability was determined by use of the trypan blue^d exclusion method, which evaluates the uptake of dye into nonviable cells. Cell suspensions with a viability > 95% were used in all experiments.

Growth curves—To evaluate growth curves and the population-doubling time of the C2 mastocytoma cell line, 2.5 × 10⁴ cells/well were plated into 6-well plates and counted on 10 consecutive days until plateau growth phase was reached. The same steps were performed with cells following irradiation. Duplicate samples were obtained, and all experiments were performed in triplicate.

Radiation experiments—For all radiation experiments, cells were counted and the required cell number was suspended in culture medium and transferred into 12-mL tubes with a diameter of approximately 11 mm. Tubes were completely filled with cell suspension to achieve a homogeneous dose distribution and then packed into 1.5-cm tissue equivalent bolus material to reach complete dose buildup on the tube surface. Cells were irradiated at room temperature (22°C) from a single port with 0, 2, 4, 6, and 8 Gy from a linear accelerator source^e with a dose rate of 200 monitor units/min (equal to approx 200 cGy/min). A source-surface-distance set-up technique with a source-to-bolus surface distance of 100 cm was used for monitor unit calculations. Tubes for the different groups were transported to the linear accelerator facility together to ensure that cells remained in the tubes for the same period of time and were treated under the same experimental conditions. Cells in irradiated tubes were centrifuged, resuspended, and plated onto different plate types, depending on the experiment.

Survival assays—Proliferational incorporation of (methyl-hydrogen 3 [³H]) thymidine and functional MTT assays were used as survival assays. To include all apoptotic and mitotic cell deaths, assays were performed 5 days after irradiation, which exceeds 2 normal cell cycles of C2 cells. When used this way, both assays are reported to be comparable to the clonogenic assay, which represents the gold standard for assessing cell survival.^18,19

[Methyl-³H] thymidine incorporation assay—After irradiation, cell survival rates were determined with the (methyl-³H) thymidine incorporation assay, which detects the rate of newly synthesized DNA. Following irradiation, 0.6, 1.2, and 2.5 × 10³ cells/well were plated in 96-well plates in a 100-μL volume. After a 5-day incubation period, 1 MCi of (methyl-³H) thymidine^f was added and the cells were incubated again for 18 hours. Cells were then harvested on filter membranes.^g (Methyl-³H) thymidine incorporated in newly synthesized DNA was collected on the filter membranes. Filters were air-dried, and radioactivity was counted in a β-scintillation counter.^h All samples and experiments were performed in triplicate.

MTT colorimetric assay—After irradiation, cell survival rates were additionally determined with the MTT assay, which measures changes in the function of mitochondrial dehydrogenases in viable cells.²⁰ Following irradiation, 4, 6, and 8 × 10³ cells/well were placed into 96-well, U-bottomed microtiter plates in a 100-μL volume and incubated for 5 days at 37°C in a humidified atmosphere containing 5% CO₂. Optimal concentration and incubation time with MTTⁱ were adjusted for irradiated and nonirradiated cells, as needed. On day 5, cell counts were performed, plates were centrifuged for 5 minutes at 1,525 X g, supernatants were discarded, and the cells were further incubated in the presence of MTT solution (500 μg/mL) for 4 hours at 37°C. Following centrifugation, cell pellets were solubilized in 100 μL of dimethyl sulfoxide.ⁱ Plates were placed on a rocking shaker and incubated at 22°C overnight. The following day, absorbance was measured with an ELISA reader^j and respective software. A wavelength of 570 nm and a reference wave of 690 nm were used.

To generate survival curves, absolute values (cpm for the (methyl-³H) thymidine incorporation assay and OD for the MTT assay) of nonirradiated control groups were normalized to 100% and relative proliferation of irradiated groups was determined with the following formula: cpm (OD) of irradiated cells/cpm (OD) of nonirradiated cells × 100 = relative proliferation.

Evaluation of apoptosis—Flow cytometric analysis with an annexin staining kit^k was performed to detect radiation-induced apoptosis in C2 cells. Following irradiation, cells were plated in 6-well plates and assayed at different time points (5 hours and 1, 2, 3, and 4 days). Annexin staining was performed according to the manufacturer's instructions with slight modifications. Annexin-stained samples (5 × 10⁶ cells/sample) were first analyzed to determine spectral overlap of annexin-positive cells (530/30 bandpass filter) into the emission region of PI (610/20 bandpass filter). Thereafter, PI was added to the sample and the samples were again evaluated for annexin and PI staining in combination. A set of 5 experiments was performed. For all experiments, cells were analyzed^l after exclusion of cell debris in FSC-SSC. For excitation, a 488-nm wavelength blue laser was used. For statistical reasons, results of 30,000 cells of the double-labeled probes (annexin plus PI) were recorded, and list mode data were processed later.

VEGFR-1 and VEGFR-2 expression—Flow cytometric analysis was used to evaluate the effects of radiation on VEGFR-1 and VEGFR-2 expression in the different treatment groups. Cross-reactive affinity-purified polyclonal rabbit anti-human VEGFR-1 antibodies^m were used to detect VEGFR-1 expression, and the monoclonal antibody A-3ⁿ was used to identify VEGFR-2 expression. Binding of primary antibodies was detected by use of fluorochrome-labeled secondary antibodies.^o Polyclonal rabbit IgG^p and monoclonal mouse IgG1^q served as isotype controls. Irradiated cells were plated in 24-well plates, incubated, and harvested immediately after irradiation and 3 and 6 days later. Expression of VEGFR-1 and VEGFR-2 prior to irradiation served as a baseline for comparison. Cell labeling for flow cytometric analysis was performed in a 2-step procedure. First, cells were incubated for 30 minutes on ice with the primary antibodies directed against VEGFR-1 and VEGFR-2 and their respective isotype controls. In the second step, after washing in wash solution (1% fetal calf serum in PBS solution^b [without Ca²⁺ or Mg²⁺]), cells were incubated for another 30 minutes with fluorochrome-labeled secondary antibodies. To evaluate intracellular receptor expression, cells were fixed and made permeable with saponin.ⁱ Saponin at a concentration of 0.1% was added to a fixation solution (10X PBS solution [without Ca²⁺ or Mg²⁺] with 3% formaldehyde^c in aqua bidist). Additionally, a saponin-containing wash solution was used (2% fetal calf serum with 0.1% saponin in PBS solution [without Ca²⁺ or Mg²⁺]). Following 20 minutes of incubation in fixation solution, cells were washed twice in saponin wash solution and labeling was performed as described. For statistical reasons, 20,000 cells were analyzed and list mode data were processed later. During the analysis, cells of interest were gated in FSC and SSC plots to exclude cell debris from the analysis. In all experiments in which the cells were fixed and made permeable, a parallel group of irradiated and nonirradiated cells was stained with PIⁱ to determine live-dead cell populations. Experiments were performed 3 times with conjugate and isotype controls included.

Comparison of canine and human VEGF ELISA kits—Commercially available human and canine VEGF ELISA kits^r were used according to the manufacturer's guidelines. Human and canine recombinant VEGF values were measured in human and canine assays in a crossover study. All antibodies used were part of the ELISA kit. Standard curves were generated by use of 2-fold serial dilutions to create concentrations from 2,000 to 31.25 pg/mL. All samples were collected in quadruplicate, and experiments were performed twice.

Canine VEGF ELISA—The VEGF concentrations were measured in cell-free supernatants and cell lysates by use of the canine VEGF ELISA kit^r according to the manufacturer's guidelines. Following irradiation, 5 × 10⁴ cells/well were plated into 24-well plates and incubated at 37°C. At different time points, immediately after plating (day 0) and 1, 2, and 3 days after irradiation, cells were harvested and the cell numbers were recorded for each treatment group. Plates were centrifuged for 8 minutes at 1,525 X g, and the supernatants were collected. The resulting cell pellets were resuspended in aqua bidist and incubated for 5 minutes at 22°C. Then, a 1:10 dilution of 10X PBS solution was added to the cell lysates and all samples were stored at −80°C until assayed. The OD of each well was determined by use of a microplate reader^j and the respective software. The VEGF values were normalized to the cell number of the corresponding culture dish (VEGF [pg/mL]/10⁶ cells). All standards, controls, and samples were prepared in duplicate and assayed twice. All experiments were conducted 3 times.

Statistical analysis—Data sets were analyzed by use of a commercially available software package.^s Significance was determined by use of 1-way ANOVA, as appropriate. Before calculation, data distribution was checked for normality by use of the Kolmogorov-Smirnov t test. All means were calculated from at least 3 experiments, and error bars represented SD of the mean. A value of P < 0.05 was considered significant.

Results

Radiosensitivity—Growth curves were evaluated to determine the cell-killing rate of the C2 cells after irradiation. A significantly different growth inhibition was detected on day 6 after radiation among the irradiated groups as well as between irradiated groups and the control group. Radiation dose–dependent growth curves for the different treatment groups (0, 2, 4, 6, and 8 Gy) of irradiated C2 cells were determined (Figure 1).

View Full Size

Growth curves (A) for irradiated canine mastocytoma C2 cells. Notice dose-dependent growth inhibition after irradiation of cells with single doses of 0, 2, 4, 6, and 8 Gy. Survival curves (B [methyl-³H thymidine incorporation] and C [MTT assay]) of irradiated canine mastocytoma C2 cells are also illustrated. Notice a dose-dependent decrease in surviving cells after irradiation and that the plotted graphs obtained by use of the 2 techniques are nearly identical. Values are mean ± SD.

Citation: American Journal of Veterinary Research 70, 9; 10.2460/ajvr.70.9.1141

• Download Figure
• Download figure as PowerPoint slide

Survival curves—The (methyl-³H) thymidine incorporation assay and the MTT colorimetric assay were used 5 days after irradiation to evaluate radiation sensitivity. In all irradiated groups, significantly decreased survival rates were measured in both assays, compared with nonirradiated controls (Figure 1).

Radiation-induced apoptosis in C2 cells—To examine the role of radiation-induced apoptosis in C2 cells, unlabeled cells were evaluated first for appropriate gating of annexin-positive and PI-negative cells. Radiation-induced apoptotic cell death was detected (Figure 2). Five hours after irradiation, the number of apoptotic cells was still low in the irradiated and nonirradiated groups (mean ± SD, 5.53 ± 3.1% for all groups). A dose-dependent increase in the apoptotic cell fraction was detected on day 1. In all experiments, a peak was reached 24 to 48 hours after irradiation. Mean ± SD peak values of the apoptotic fraction for the 0-, 2-, 4-, 6-, and 8-Gy groups were 6.05 ± 2.02%, 16.65 ± 2.98%, 30.43 ± 6.35%, 33.93 ± 7.59%, and 40.73 ± 4.93%, respectively. After day 2, the apoptotic fraction slowly decreased, reaching baseline values in all irradiated groups by day 4 (mean ± SD, 5.62 ± 1.6% for all groups).

View Full Size

Dot plots of radiation-induced apoptosis of C2 cells analyzed via flow cytometry (annexin-V and PI staining), with a specific gate for the apoptotic cell fraction (gate P5). The number in the lower right corner of the gate represents apoptotic cell fraction (%) determined from the total number of cells analyzed. After irradiation (0, 2, 4, 6, and 8 Gy), cells were cultured further, and the apoptotic cell fraction was quantified at 5 hours and on days 1, 2, 3, and 4. Notice a dose-dependent increase in apoptotic cells following irradiation. The percentage of apoptotic cells reached a peak on day 1 and returned to baseline values on day 4. One representative experiment is illustrated. A = Annexin-negative (⁻) and PI⁻ cells. B = Annexin-positive (⁺) and PI⁺ cells. The P5 gate is set on annexin⁺ and PI⁻ cells on the basis of dot plots of unlabeled cells.

Citation: American Journal of Veterinary Research 70, 9; 10.2460/ajvr.70.9.1141

• Download Figure
• Download figure as PowerPoint slide

Expression of VEGFR-1 after irradiation—Flow cytometric analysis was used to determine whether C2 cells expressed the VEGFR-1 receptor on their surface or in the cytoplasm before or after irradiation. Only 2.9% of nonirradiated C2 cells expressed the VEGFR-1 receptor on the cell surface (Figure 3). Radiation did not induce an upregulation of this receptor expression at any time point, regardless of the radiation dose (range of expression, 1.9% to 2.9%). After appropriate cell fixation, attainment of permeability, and labeling with anti–VEGFR-1 antibody, few cells with low fluorescence intensity expressed VEGFR-1 intracellularly.

View Full Size

Dot plots of flow cytometric detection of VEGFR-1 on the surface of C2 cells 6 days after irradiation with 0, 2, 4, 6, and 8 Gy. A small population of C2 cells expressed VEGFR-1 on the cell surface. Results of a representative experiment of 3 independent experiments is illustrated. Column A represents FSC-SSC, column B represents an isotype control, and column C represents C2 cells stained against VEGFR-1. Columns B and C both have positive cells within gate P2. The VEGFR-1⁺ cells in gate P2 from column C are back-gated into column A, thereby identifying FSC-SSC properties of VEGFR-1⁺ cells. The percentage value in the upper right corner represents the fraction of VEGFR-1⁺ cells.

Citation: American Journal of Veterinary Research 70, 9; 10.2460/ajvr.70.9.1141

• Download Figure
• Download figure as PowerPoint slide

Expression of VEGFR-2 after irradiation—Flow cytometric analysis was used to detect VEGFR-2 expression on the surface of the C2 cells. Whether C2 cells were capable of storing this receptor intracellularly was also investigated. No expression of VEGFR-2 could be detected on the cell surface. However, after fixation and attainment of permeability of the cells, VEGFR-2 receptor was found in the cell cytoplasm (Figure 4). Radiation induced dose-dependent reduction of VEGFR-2–positive cells (range, 89.4% in the 0-Gy group to 26.7% in the 8-Gy group). Parallel analysis of live-to-dead cell ratio via PI staining also revealed a strong radiation dose–dependent reduction in number of viable cells (range, 79.5% in the 0-Gy group to 45.2% in 8-Gy group). Although intracellular VEGFR-2 analysis of live cells was not possible (because of required cell permeabilization for the intracellular receptor detection), a parallel backgating of PI-negative (live cells) and VEGFR-2–positive cells into FSC-SSC dot plots indicated that live cells expressed VEGFR-2. Thus, radiation did not induce any change in the intracellular expression of VEGFR-2, regardless of dose or time point.

View Full Size

Scatter plots of flow cytometric analysis of VEGFR-2 expression in the cytoplasm of C2 cells 3 days after irradiation (0, 2, 4, 6, and 8 Gy). Expression of VEGFR-2 was observed in the cell cytoplasm of the viable cell population and did not change, regardless of the radiation dose. Columns A and B represent native cells stained with PI. Column A represents FSC-SSC. Column B represents PI⁺ cells in gate PI; PI⁺ cells are back-gated into column A, thereby identifying FSC-SSC properties of live (PI⁻) and dead (PI⁺) cells. The percentage value in the upper left corner represents the fraction of live cells. Columns C and D represent fixed and permeabilized cells, respectively, with VEGFR-2 staining. Column C represents FSC-SSC, and column D represents VEGFR-2⁺ cells in gate R2. The percentage value in the upper right corner represents the fraction of VEGFR-2⁺ cells. The VEGFR-2⁺ cells are back-gated into column C, thereby identifying FSC-SSC properties (live vs dead) of VEGFR-2⁺ cells. Results of 1 representative experiment of 3 seperate experiments are illustrated.

Citation: American Journal of Veterinary Research 70, 9; 10.2460/ajvr.70.9.1141

• Download Figure
• Download figure as PowerPoint slide

Comparison of canine and human ELISA VEGF kits—Until recently, only the human ELISA was available for detection of canine VEGF. Crossover assays were performed to determine the detection capabilities of the newly available canine assay in comparison to the human kit (Figure 5). Results indicated that the canine ELISA measured canine VEGF more adequately than the human kit, which underestimated canine VEGF concentrations. Canine assay sensitivity was 39.25 pg/mL (sensitivity is defined as 3 SDs from the signal given by the zero blank), intra-assay variance was 3.64%, and interassay variance was 3.62%.

View Full Size

Comparison of results of human (A and C) and canine (B and D) VEGF ELISAs.^r Recombinant human and canine recombinant VEGF (rVEGF) were used in a crossover assay experiment as samples and standards, respectively. All samples were collected in quadruplicate, and the experiments were performed in duplicate. Only 60% of canine recombinant VEGF was detected with the human sandwich immunoassay (C). In contrast, the canine assay detected 100% of canine recombinant VEGF and was also able to detect 100% of human recombinant VEGF (B and D).

Citation: American Journal of Veterinary Research 70, 9; 10.2460/ajvr.70.9.1141

• Download Figure
• Download figure as PowerPoint slide

VEGF protein secretion after irradiation—The canine ELISA kit was used to evaluate whether radiation induced upregulation of VEGF expression in C2 cells. Irradiated and nonirradiated C2 cells produced VEGF constitutively. The main portion of VEGF was secreted into the cell-free supernatant and accumulated over time (up to 7,000 pg/mL per 10⁶ cells on day 3; Figure 6). Only low concentrations of VEGF were detected in the cell lysates (up to 120 pg/mL per 10⁶ cells on day 3), and no accumulation of VEGF was observed in the cell cytoplasm. To account for decreasing numbers of viable and VEGF-secreting cells with higher doses of radiation, absolute VEGF values were corrected for cell number (pg/mL per 10⁶ cells). No radiation-induced upregulation of normalized mean or median VEGF concentrations was detected, regardless of the radiation dose and time point measured.

View Full Size

Box plots (median, interquartile range, range, and outliers) of results of VEGF detection in the supernatants of C2 cells. The VEGF concentrations normalized to the number of viable cells were measured immediately after (A) and at 1 (B), 2 (C), and 3 (D) days after exposure to single radiation doses of 0, 2, 4, 6, and 8 Gy, respectively. No radiation-induced increase in secreted VEGF was detected at any of the time points. Box plots of results for 3 experiments performed in duplicate are illustrated.

Citation: American Journal of Veterinary Research 70, 9; 10.2460/ajvr.70.9.1141

• Download Figure
• Download figure as PowerPoint slide

Discussion

Results of the present study indicated that C2 cells were radiosensitive in a radiation dose–dependent manner. To the authors' knowledge, no previous data are available on the radiosensitivity of the C2 cell line. However, a number of clinical studies^21,22 found good response of naturally developing MCTs to radiation therapy, which is in agreement with results of the present study. There are few reports on the radiosensitivity of mast cells in human medicine. Recent data on a human bone marrow–derived mast cell line and human-derived mast cells reveal that these cell lines are resistant to radiation-induced cytotoxicosis.²³ Discrepancy between the results of the present study and the reported radiation resistance in bone marrow–derived mast cells and human-derived mast cells could be attributed to the different detection methods used as well as different radiosensitivity of neoplastic versus normal mast cells. The present study proved the radiosensitivity of C2 cells by use of MTT and (methyl-³H) thymidine assays, which were used as survival assays, and by induction of apoptosis. Soule et al²³ evaluated radiation sensitivity only via apoptosis assays (lactate dehydrogenase and DNA fragmentation) on day 3 after irradiation. We suggest that day 3 after irradiation might be too late to detect apoptotic cell death. This is supported by results of another study²⁴ that revealed that even for cells with late apoptosis, most cells have completed their apoptotic process 48 to 72 hours after treatment. In the present study, apoptotic cell death reached its maximum 24 to 48 hours after irradiation, indicating the radiosensitivity of this cell line. The increase in the apoptotic cell fraction was followed by a slow decrease after day 2, and the apoptotic rate reached baseline values 4 days after irradiation.

Radiation has been reported to activate multiple signaling pathways within cells that can alter cell survival.^25,26 Radiation therapy enhances VEGF secretion in several tumors (eg, glioblastoma and various carcinomas) and contributes to radiation resistance by upregulating and intensifying angiogenic pathways and tumor cell proliferation.⁷ However, the role of VEGF was not known in irradiated canine mastocytoma cells. In the past, the human VEGF ELISA was the only system available for measuring canine VEGF.^27,28 In 2007, a new canine VEGF sandwich ELISA became available. However, the manufacturer did not publish data on human cross-reactivity with canine VEGF. Results of the present study indicated that only 60% of canine recombinant VEGF was detectable with the human sandwich immunoassay. In contrast, the canine sandwich immunoassay was able to detect 100% of canine and human recombinant VEGF. Scheidegger et al²⁹ detected a minimal difference (5%) between human and canine VEGF sequences. However, VEGF requires posttranslational processing at the level of glycosylation and disulfide bond formation.³⁰ Partial cross-reactivity could be attributable to the posttranslational modifications of the canine and human recombinant VEGF protein in bacterial and insect cells, respectively, or the difference in the affinity of the antibodies. Results from the present study indicated that the canine VEGF immunoassay was superior in detection of canine VEGF, compared with results for the previously available human kit. Thus, the canine kit was used for the detection of VEGF in all our experiments.

In the present study, the exposure of C2 cells to various doses of radiation did not induce increased production of VEGF. Mean and median concentrations of normalized VEGF were not significantly different between irradiated and control groups at any time point. Irradiated tumor cells synthesized VEGF in a consistent manner, resulting in accumulation of VEGF in cell-free supernatants over time. A constitutive production of VEGF by nonirradiated C2 cells has already been reported in a recently published study.² In the present study, the same basic VEGF production was observed in the nonirradiated control groups. Under normoxic conditions, C2 cells did not upregulate VEGF expression in response to radiation. Hypoxic conditions were excluded in the present study by maintaining microenvironmental conditions (5% CO₂ and 37°C) in cell culture. It is further suggested that C2 cells do not use VEGF as an autocrine growth regulator because the growth of C2 cells after irradiation was not influenced by the accumulation of high concentrations of VEGF. This conclusion was further supported by our finding that neither VEGFR-1 nor VEGFR-2 was upregulated in irradiated tumor cells, regardless of the radiation dose. Our findings are also in agreement with those in a recent study² in nonirradiated C2 cells that revealed no growth stimulation by adding VEGF to the cell culture and no growth inhibition when VEGF was blocked by monoclonal antibody.

Differences between the low amounts of VEGF found in cell lysates (≤ 120 pg/mL per 10⁶ cells) and the high concentrations in the supernatants (≤ 7,000 pg/mL per 10⁶ cells) can be explained by the fact that VEGF is a secreted protein; synthesis is followed by rapid export of the protein out of the cell, leaving the intracellular VEGF concentration quite stable.³¹ Results of the present study cannot rule out the possibility that a fraction of the extracellular VEGF is derived from the disintegration of dying cells, but even if this is the case, the influence seems to be minor. However, secreted concentrations of VEGF in vivo could have an impact on angiogenesis and the oxygen supply required for tumor growth.

In canine cells, VEGFR-1 and VEGFR-2 have been described.²⁹ Rebuzzi et al² detected VEGFR-1 in nonirradiated C2 cells on the cell surface via flow cytometry, and VEGFR-1 and VEGFR-2 were identified at the mRNA and protein level in C2 cells. In the same study,² VEGFR-2 was not found on the cell surface. The influence of radiation on this receptor's expression profile in C2 cells was unknown. In the present study, VEGFR-1 was detected on the surface of a minor population of cells (about 2%). No radiation-induced upregulation of VEGFR-1 expression was observed, regardless of radiation dose and time point. Vascular endothelial growth factor receptor-2 could not be detected on the cell surface of C2 cells, which was in agreement with results of the previous study.² However, most of the viable cells expressed VEGFR-2 in the cell cytoplasm. Again, radiation did not upregulate VEGFR-2 expression, regardless of dose and time point. Gampel et al³² found that, in endothelial cells, a substantial proportion of VEGFR-2 is held in an endosomal storage pool. Results of the present study were in agreement because the VEGFR-2 was found intracellularly in C2 cells.

It is known that solid tumors contain areas that are affected by hypoxia, nutrient deprivation, and acidity. Tumor cells in such a microenvironment are genetically unstable and metastasize frequently.^33,34 Further, hypoxic tumor cells are somewhat resistant to the killing effects of radiation and some commonly used chemotherapy drugs.^35,36 In the present study, normoxic conditions were provided, and future studies will be needed to evaluate C2 cell line survival and VEGF and VEGFR expression after radiation in hypoxic conditions.

A limitation of this in vitro MCT model is the missing opportunity to evaluate cell signaling, effector mechanisms, and influence of the microenvironment. However, an advantage of this in vitro model is the chance to study the impact of radiation selectively on tumor cells without the influence of the tumor interstitium.

The present study revealed that the C2 cell line was radiosensitive and a fraction (≤ 40%) of cells died via apoptosis in a dose-dependent manner. Further, C2 cells did not upregulate VEGF or VEGFR in response to radiation. However, the amount of secreted VEGF could promote angiogenesis in tumor tissue in an in vivo setting. Whether the use of antiangiogenic or apoptosis-modulating agents in combination with radiation therapy could improve treatment outcome in aggressive MCTs of dogs requires further investigation. Improved understanding of the molecular interplay between radiation and angiogenic factors should help in the design of new therapeutic strategies.