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Experimental Therapeutics |
Vanderbilt University School of Medicine, Nashville, Tennessee 37232 [A. J. S., E. H.]; Departments of Radiation Oncology [L. G., J. T., D. E. H.], Radiology [E. D.], and Cancer Biology [D. E. H.], Vanderbilt University School of Medicine, Nashville, Tennessee 37232-5671; and Sugen, Inc., South San Francisco, California 94080 [D. M., G. M.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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SU11248 is a p.o. available indolinone-based synthetic molecule, which was identified as a low nM-selective inhibitor of the angiogenic RTKs Flk-1/KDR and PDGFR in both biochemical and cellular assays (9) . SU11248 was also found to inhibit cellular signaling via Kit and fetal liver tyrosine kinase 3. SU11248 exhibited broad and potent antitumor activity in mice, regressing A431 human epidermoid and Colo205 human colon tumors, arresting the growth of H460 human lung, MDA-MB-435 human breast, and PC3-human prostate tumors, and substantially delaying the growth of C6 rat and SF763T human glioma xenografts (9) . In Colo205 tumors, SU11248 treatment induced a dose- and time-dependent decrease in tumor microvessel density and tumor cell proliferation and an associated increase in tumor cell apoptosis, culminating in tumor regression.
SU11248 is currently in Phase I clinical trials in patients with advanced cancer. Pharmacokinetic/pharmacodynamic studies in mice have shown that SU11248 inhibited PDGFRß and Flk-1/KDR phosphorylation in a time- and dose-dependent fashion with target plasma concentrations of 50–100 ng/ml (9)
. Sustained inhibition of Flk-1/KDR and PDGFRß phosphorylation was not required for maximum efficacy, as indicated by the demonstration that target receptor phosphorylation was suppressed for 
12 h at efficacious doses with daily administration. In this study, SU11248 was therefore administered twice daily to tumor-bearing mice when a constant efficacious dose was required. The selectivity of SU11248 demonstrated against other kinases in vitro is recapitulated in vivo by the fact that SU11248 does not inhibit epidermal growth factor receptor phosphorylation in vivo, even at high plasma concentrations (9)
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RTK antagonists have been shown to enhance the cytotoxic effects of cancer therapy through the inhibition of the phosphatidylinositol 3'-kinase/Akt viability pathway in tumor vascular endothelium. In this study, this broad spectrum RTK antagonist was administered to vascular endothelium to determine whether SU11248 enhances radiation-induced endothelial cytotoxicity. In separate experiments, SU11248 was administered in a maintenance schedule to determine whether tumor control can be maintained for prolonged time periods after irradiation. These studies show that SU11248 enhanced radiation-induced tumor vascular endothelial destruction and improved tumor control after the discontinuation of fractionated radiotherapy.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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An Eldorado 8 Teletherapy 60Co Unit (Atomic Energy of Canada Limited) was used to irradiate endothelial cell cultures at a dose rate of 0.84 Gy/min. Delivered dose was verified by use of thermoluminescence detectors.
Viability Assays.
The number of cells undergoing apoptosis was quantified by microscopic analysis of apoptotic nuclei. Cells were then fixed and stained with H&E at 24 h after treatment with 6 Gy radiation and/or 100 nM SU11248. Cells were then examined by light microscopy. For each treatment group, five high power fields (x40 objective) were examined, and the number of apoptotic and total cells was determined. From these numbers, the percentage of apoptotic cells for each group was determined.
Clonogenic survival analysis was performed as we have described previously (2) . Briefly, HUVEC culture plates were treated at each radiation dose level with/without 1 µM SU11248 for 1 h before irradiation. After treatment with radiation and/or antagonist, cells were trypsinized, counted by hemocytometer, and subcultured into fresh medium. After 14 days, the cells were fixed with cold methanol and stained with 1% methylene blue. Colonies with >50 cells were counted, and the surviving fraction was determined.
Western Immunoblots.
HUVECs were treated with 100 nM SU11248 for 1 h and/or irradiated with 6 Gy. HUVECs were washed twice with PBS and lysis buffer (20 nM Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2.5 mM Na PP, 1 mM phenylmethylsulfonyl fluoride, and 1 µg/ml leupeptin) added. Protein concentration was quantified by the Bio-Rad method. Twenty µg of total protein were loaded into each well and separated by 8 or 12% SDS-PAGE gel, depending on the size of the target protein being investigated. The proteins were transferred onto nitrocellulose membranes (Hybond ECL; Amersham, Arlington Heights, IL) and probed with antibodies to caspase 3 and caspase 3-cleaved fragment (Cell Signaling).
Tumor Vascular Window Model.
Life Technologies, Inc., penicillin-streptomycin solution (200 µl) was injected into the hind limb of the mouse before the procedure. The dorsal skin-fold window is a 3-g plastic frame applied to the skin of the mouse and remains attached for the duration of the study. The chamber was screwed together, whereas the epidermis was incised and remained open with a plastic covering. The midline was found along the back, and a clip was placed to hold the skin in position. A template, equivalent to the outer diameter of the chamber, was traced, producing the outline of the incision. A circular cut was made tracing the perimeter (7-mm diameter) of the outline followed by a crisscross cut, thus producing four skin flaps. The epidermis of the four flaps was then removed using a scalpel with an effort to follow the hypodermis superior to the fascia. The area was then trimmed with fine forceps and iris scissors. The template was removed, and the top piece of the chamber was fixed with screws. During surgery, the area was kept moist by applying moist drops of PBS with 1% penicillin/streptomycin solution. The bottom portion of the chamber was put in place, and the top was carefully positioned on the cut side so that the window and the circular incision were fitted. Antibiotic ointment was applied at this time. The three screws that held the chamber together were then positioned into the chamber holes and tightened so that the skin was not pinched, thus avoiding diminished circulation. Tumor blood vessels developed in the window within 1 week.
We studied the time- and dose-dependent response of tumor blood vessels to radiation using the window model. Vascular windows were treated with 3 Gy of superficial X-rays using 80 kVp (Pantak X-ray Generator). Five mice were studied in each of the treatment groups. SU11248 (40 mg/kg) was injected i.p. 15 min before irradiation. The window frame was marked with coordinates, which were used to photograph the same microscopic field each day. Vascular windows were photographed using x4 objective to obtain a x40 total magnification. Color photographs were used to catalogue the appearance of blood vessels on days 0–7. Photographs were scanned into Photoshop software, and vascular center lines were positioned by ImagePro Software and verified by an observer blinded to the treatment groups. Tumor blood vessels were quantified by the use of ImagePro software, which quantifies the vascular length density of blood vessel within the microscopic field. Center lines were verified before summation of the vascular length density. The mean and 95% confidence intervals of vascular length density for each treatment group were calculated, and variance was analyzed by the general linear models and Bonferroni t test.
Tumor Growth Delay Assays.
C57BL/6 mice received s.c. injections in the right thigh with 106 viable cells of a murine glioblastoma (GL261) or lung carcinoma (LLC) suspended in 0.2 ml of a 0.6% solution of agarose. Each set of six mice was stratified into four groups on day 1 (SU11248 vehicle control, radiation, SU11248, and SU11248 + radiation) to control for mean tumor volume. An equal number of large- and intermediate-sized tumors were present in each group. Mouse tumors were stratified into groups so that the mean tumor volume of each group was comparable. Mean tumor volumes for LLC were 425 ± 40, 420 ± 40, and 432 ± 45 mm3 and for GL261 were 530 ± 50, 510 ± 35, and 535 ± 55 mm3 for groups of tumors treated with radiation alone, SU11248 alone, or SU11248 and radiation, respectively.
Irradiated mice were immobilized in Lucite chambers, and the entire body was shielded with lead except for the tumor-bearing hind limb. Radiation was administered within 30 min of SU11248 (40 mg/kg) i.p. injection. A total dose of 21 Gy was administered to the appropriate mice in seven fractionated doses of 3 Gy on days 1, 2, 3, 4, 5, 7, and 8, the first dose being administered on day 1. Tumor volumes were measured three times weekly using skin calipers as described previously (12, 13, 14)
. The volumes were calculated from a formula (a x b x c / 2) that was derived from the formula for an ellipsoid (
d3 / 6). Data were calculated as the percentage of original (day 1) tumor volume and graphed as fractional tumor volume ± SE for each treatment group.
For the maintenance study of SU11248, C57BL/6 mice bearing GL261 hind limb tumors were stratified as described above into six groups. Mean tumor volumes were 320 ± 28, 310 ± 23, and 330 ± 29 mm3 in groups treated with radiation alone, SU11248 alone, or SU11248 and radiation, respectively. Radiation was administered within 30 min of SU11248 (40 mg/kg) i.p. injection. A total dose of 18 Gy was given to the appropriate groups in six fractionated doses of 3 Gy on days 1, 2, 3, 4, 5, and 8. After day 8, three groups received twice daily SU11248 injections (20 mg/kg, 8 h apart) for 7 days (days 9, 10, 11, 12, 15, 16, and 17), and after a 4-day interruption in therapy (days 18–21), they were subsequently maintained for the remainder of the experiment. Tumor volumes were assessed as above.
Amplitude-modulated Doppler Blood Flow Analysis.
Blood flow within these tumors was quantified by Power Doppler after the third fraction of irradiation. Tumor blood flow was imaged with a 10-5 MHz linear Entos probe attached to a HDI 5000 (probe and HDI 5000 from ATL/Philips, Bothell, WA) as we have described previously (15)
. Power Doppler sonography images were obtained with the power gain set to 82%. A 20-frame cineloop sweep of the entire tumor was obtained with the probe perpendicular to the long axis of the lower extremity along the entire length of the tumor. Color area was quantified using HDI lab software (ATL/Philips). This software allows direct evaluation of Power Doppler cineloop raw. The color area was recorded for the entire tumor. Five mice were entered into each treatment group. Values for color area were averaged for each tumor set, and treated groups were compared with controls with the unpaired Student t test.
Microscopy of Tumor Sections.
C57BL6 mice bearing LLC tumors were treated with 3 Gy alone, SU11248 alone, or SU11248 and radiation. Twenty-four h after treatment, tumors were fixed, sectioned, and stained. Phosphorylation of the molecular target for SU11248, PDGFRß, was studied by use of the phospho-specific antibody (Cell Signaling). TUNEL staining was used to identify apoptotic cells (DeadEnd Colorimetric TUNEL System, Promega no. G7130). Sections were counterstained with antibody to the endothelial-specific antigen von Willebrand factor using rabbit anti-VWF, IgG (Dako, Carpinteria, CA). Secondary antibody: peroxidase-labeled goat antirabbit IgG, Dako stained with Vector Nova Red Substrate kit (Vector, Burlingame, CA) Sections were imaged by light microscopy using x200 under oil emersion.
Statistical Analysis.
We used the general linear model (logistic regression analysis) to test for associations between the numbers of apoptotic cells present in culture, clonogenic survival, tumor blood flow, and tumor volumes. We applied the Bonferroni method to adjust the overall significant level equals to 5% for the multiple comparisons in this study. All statistical tests were two-sided, and differences were considered statistically significant for P < 0.05. SAS software version 8.1 (SAS Institute, Inc., Cary, NC) was used for all statistical analyses.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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shows the percentage of cells undergoing apoptosis after treatment with SU11248 alone for cells treated with SU11248 alone, 6 Gy alone, or SU11248 followed by 6 Gy. Untreated control cells show 2% apoptotic nuclei as compared with 7 and 8% after treatment with SU11248 or radiation, respectively (P > 0.1). HUVECs treated with SU11248 followed by 6 Gy showed 21% of cells with apoptotic nuclei at 24 h, which was significantly greater than either agent alone (P < 0.02) or untreated control cells (P < 0.001). Caspase activation was studied to verify the role of apoptosis in the interaction between SU11248 and radiation. Fig. 1B
shows the Western immunoblot staining for caspase 3. This shows that caspase 3 was not cleaved in endothelial cells after treatment with either SU11248 alone or radiation alone, whereas caspase 3 was cleaved in HUVECs treated with SU11248 followed by irradiation.
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shows the surviving fraction of HUVECs treated with vehicle or 1 µM SU11248 before the indicated dose of radiation. There was no reduced plating efficiency of HUVECs treated with SU11248 alone. HUVECs treated with SU11248 before irradiation showed a significant reduction in clonogenic survival as compared with radiation alone (*, P < 0.05).
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shows the vasculature within LLC tumors implanted into the dorsal skin-fold window in C57BL6 mice. Representative photographs of tumor vasculature before and 48 h after treatment with SU11248, 3 Gy, or SU11248 followed by 3 Gy indicate that RTK inhibition increases tumor vascular destruction as compared with either agent alone. Five mice were treated in each of the treatment groups, and the vascular length density after treatment was quantified. Mean vascular length densities over 4 days are shown as a bar graph. Within 72 h, VLD in tumors was significantly reduced to 8% of that at 0 h (P < 0.01). In comparison, tumors treated with either 3 Gy or SU11248 alone showed an insignificant reduction in VLD to 75 and 84% that of 0 h, respectively. Combined SU11248 and 3 Gy achieved significantly greater reduction in VLD as compared with either agent alone (P < 0.05).
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). The mean fold increase in tumor volumes in five mice in each of the treatment groups (vehicle, SU11248, vehicle + 21 Gy, and SU11248 + 21 Gy) are shown. Time to doubling of LLC tumor size was 5, 6, 8, and 16 days for each group, respectively. Both LLC and GL261 tumors showed a significant increase in tumor growth delay when SU11248 was added before daily 3-Gy fractions as compared with either agent alone (*, P < 0.05).
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, shows immunohistochemical staining of LLC tumors with and without SU11248 administration. This shows that PDGFRß phosphorylation was detected in the stroma and endothelium of tumors before treatment. At 3 h after SU11248 administration, PDGFR phosphorylation could not be detected by immunohistochemistry (Fig. 4D)
. This indicates that SU11248 is biologically active within mouse tumor models after systemic administration.
To determine whether prolonged growth delay correlated with reduction in tumor blood flow, amplitude modulated Power Doppler was used to monitor blood flow. Fig. 5
shows representative images of signal intensity of blood flow in GL261 tumors on day 5 of treatment. Reduced blood flow in tumors treated with SU11248 and radiation correlated with the improved tumor growth delay that was found in Fig. 4
. The bar graph shows the average blood flow within GL261 tumors. Tumors treated with SU11248 and radiation approached a significant reduction in blood flow as compared with tumors treated with radiation alone (*, P < 0.053) and a trend toward significance when compared with SU11248 alone (P = 0.15).
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, arrow A), three groups, one drug alone, one radiation + vehicle, and one combined therapy were maintained with twice daily i.p. administrations of 20 mg/kg SU11248 for an additional 7 days. Maintenance treatment (main in Fig. 6
) was discontinued after day 17 (arrow B) and subsequently resumed after 4 days of interruption of SU11248 administration (arrow C). Fig. 6
shows the means of changes in tumor volumes in five mice in each of the treatment groups. Each of the maintenance groups showed significantly delay in growth to 4-fold that of day 0 tumors (P < 0.05 in all groups). Reinitiation of SU11248 at day 21 (arrow C) produced a second phase of tumor growth delay.
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shows TUNEL staining of endothelial cells costained for VWF in tumor blood vessels. Tumors treated with SU11248 alone or radiation alone developed no TUNEL staining, whereas SU11248 followed by irradiation resulted in TUNEL staining positive in endothelial cells (Fig. 6D)
. We found no tumor cells staining positive with TUNEL stain after treatment with SU11248 and radiation. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although the predominant effect of SU11248 may be at the level of tumor vascular endothelium, PDGFR antagonists could prevent tumor regrowth by direct effect on tumor cells. For example, the PDGFR inhibitor STI571 has been somewhat effective in treatment of solid tumors (17 , 18) . Several other human cancers may overexpress c-Kit or PDGFR, and clinical trials to evaluate the role of STI571 in the treatment of such cancers are currently ongoing (17) . STI571 inhibited PDGF-mediated growth and lead to apoptosis of osteosarcoma cells in vitro by selective inhibition of the PDGFR tyrosine kinase (18) . Although SU11248 could prevent tumor growth through inhibition of tumor cell proliferation, in this study, apoptosis (TUNEL staining) was limited to tumor vascular endothelial cells. This indicates that the primary site of interaction between SU11248 and radiation was at the vascular endothelium.
With regard to enhancement of cytotoxicity in tumor vascular endothelium, we found reduced clonogenic survival in irradiated endothelial cells pretreated with SU11248 as compared with radiation alone. The proposed mechanism of interaction between RTK antagonists and cytotoxic therapy is the attenuation of signal transduction through the phosphatidylinositol 3'-kinase/Akt pathway (19) . Attenuation of this pathway alters the levels of Bcl gene products, resulting in increased susceptibility to apoptosis (20) . This model of diminished cell viability is supported by this study, which shows SU11248 significantly increased radiation-induced apoptosis in vascular endothelial cells pretreated with the broad spectrum RTK antagonist SU11248. Caspase 3 was cleaved only in HUVECs treated with both SU11248 and radiation but not either agent alone. This suggests that RTK inhibition, together with cytotoxic therapy, may be a beneficial combination in cancer therapy. The enhancement in radiation-induced cytotoxicity was also measured by clonogenic assay, which shows a subtle, albeit significant reduction in surviving fraction of endothelial cells. This interaction between SU11248 and radiation was minimized by washing the kinase inhibitor off of cultures after 1-h incubation. Washing and feeding of cell cultures were required because colonies will not form if this VEGF receptor antagonist remains on cells during clonogenic assays. Taken together with positive TUNEL staining in tumor endothelium, these data support the notion that apoptosis is one mode of endothelial cell death when SU11248 is combined with radiation.
Tumors produce growth factors that could contribute to tumor resistance to cytotoxic therapy. Moreover, growth factors that activate RTKs on tumor microvascular endothelium include VEGF, PDGF, as well as others. This study addresses the contribution of RTK signaling to radioresistance. SU11248 blocks signaling from a broad spectrum of receptors, which could be advantageous (9) . Several methods have been described to study microvessel density, including fluorescent dyes (21) and vascular staining (22) . Two measures of tumor vasculature (Doppler and Vascular Window) were used to assess the effectiveness of SU11248 at enhancing radiation-induced tumor vascular destruction. Intravital tumor vascular window provided a measure of vascular length density that was significantly reduced when SU11248 was added before irradiation. This finding was supported by amplitude modulated Doppler blood flow measurement, which showed reduced microvascular blood flow when tumors were treated with SU11248 together with radiation as compared with either agent alone. Taken together, these findings suggest that RTK inhibition enhances the therapeutic effect of radiation, in part, through improved tumor vascular destruction. The contribution of tumor vascular destruction on tumor control is additionally supported by previous studies that show antiangiogenic agents enhance radiation-induced tumor control (2 , 7 , 16 , 23, 24, 25, 26, 27, 28, 29) . Mechanisms of interaction between SU11248 and radiation could involve increased cytotoxicity within vascular endothelium or within tumor cells. Histological assessment of each of these cellular components consisted of TUNEL staining, which showed only vascular endothelial cells undergoing apoptosis while tumor cells showed no response. This finding indicates that the predominant mechanism of interaction between this VEGF receptor antagonist and radiation is within the tumor blood vessels.
Diminished tumor blood flow results in hypoxia, which could contribute to radioresistance (30) . The effect of hypoxia could be overcome by the concomitant reduction in nutrients and growth factors achieved by ischemia. To evaluate whether the ischemia induced by SU11248 and radiation has an impact on tumor control, we studied growth delay in hind limb tumors. This showed that destruction of tumor vasculature correlates with improved growth delay as compared with tumors treated with radiation alone. Although hypoxia could have a lessening effect upon the interaction between these agents, there was a significant improvement in tumor growth delay.
Upon completion of treatment with SU11248 and radiation, tumors rapidly regrow. SU11248 was developed as an antiangiogenic agent (9) . The study of maintaining SU11248 after the completion of radiation indicates that tumor growth delay can be improved by continuation of this antiangiogenic agent. This maintenance schedule significantly delayed regrowth as compared with tumors receiving no maintenance SU11248. Prior studies have shown that resumption of antiangiogenic agents can again accomplished tumor control (31) . Likewise, the resumption of SU11248 additionally delayed tumor regrowth. The clinical significance of this schedule of administration is that ongoing Phase I trials show accumulation of SU11248 necessitating drug holidays. We found that resistance to antiangiogenic therapy does not develop in mice bearing GL261 tumor grafts.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported, in part, by NIH Grants R01-CA58508, R01-CA70937, R01-CA88076, R21-CA89674, CA89888, and the Vanderbilt Lung Cancer SPORE Grant P50-CA90949, Vanderbilt-Ingrim Cancer Center Grant CCSG P30-CA68485, and a grant from Sugen Inc. 
2 To whom requests for reprints should be addressed, at Department of Radiation Oncology, Vanderbilt University, 1301 22nd Avenue South, B-902 The Vanderbilt Clinic, Nashville, Tennessee 37232-5671. Phone: (615) 343-9244; Fax: (615) 343-3075, E-mail: Dennis.Hallahan{at}mcmail.vanderbilt.edu
3 The abbreviations used are: RTK, receptor tyrosine kinase; PDGF, platelet-derived growth factor; Flk-1/KDR, fetal liver kinase-1; PDGFR, PDGF receptor; PDGFRß, PDGF receptor ß; FGF, fibroblast growth factor; VEGF, vascular endothelial growth factor; LLC, Lewis lung carcinoma; HUVEC, human umbilical vein endothelial cell; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; VLD, vascular length density.
Received 12/18/02. Accepted 5/ 8/03.
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, P < 0.001. B, the induction of apoptosis was verified by studying caspase 3 cleavage. Total cellular protein was extracted at 6 h after irradiation and separated by PAGE. Shown is the Western immunoblot using antibodies to intact caspase 3 and cleaved caspase 3.
