Radiation Response Factors Suppresses Tumor ...

Simultaneous Inhibition of the Receptor Kinase Activity of Vascular Endothelial, Fibroblast, and Platelet-derived Growth Factors Suppresses Tumor Growth and Enhances Tumor Radiation Response Robert J. Griffin, Brent W. Williams, Robert Wild, et al. Cancer Res 2002;62:1702-1706.

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[CANCER RESEARCH 62, 1702–1706, March 15, 2002]

Simultaneous Inhibition of the Receptor Kinase Activity of Vascular Endothelial, Fibroblast, and Platelet-derived Growth Factors Suppresses Tumor Growth and Enhances Tumor Radiation Response1 Robert J. Griffin,2 Brent W. Williams, Robert Wild, Julie M. Cherrington, Heonjoo Park, and Chang W. Song Department of Therapeutic Radiology-Radiation Oncology, University of Minnesota Medical School, Minneapolis, Minnesota 55455 [R. J. G., B. W. W., H. P., C. W. S.], and Sugen, Inc, South San Francisco, California 94080 [R. W., J. M. C.]

ABSTRACT We have investigated the effect of simultaneous inhibition of multiple angiogenic growth factor signaling pathways on tumor growth, tumor blood perfusion, and radiation-induced tumor-growth delay using SU6668, an inhibitor of the receptor-tyrosine kinase activity of vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF). The SCK mammary carcinoma, FSaII fibrosarcoma, and CFPAC human pancreatic carcinoma were grown s.c. in the hind leg of A/J mice, C3H mice, and Balb/cAnNCrl-nuBr nude mice, respectively. Daily i.p. injection of 100 mg/kg of SU6668 markedly suppressed the growth of these three tumor types. SU6668 also markedly prolonged the survival time of host mice bearing SCK tumors, which appeared to be caused by a reduction of metastatic tumor growth in the lung. There was little or no change in normal tissue blood perfusion, whereas in SCK tumors the perfusion decreased by 50% at 1 h after a single i.p. injection of SU6668, slightly recovered at 4 h, and completely recovered by 8 h. Interestingly, the tumor blood flow was significantly increased above the baseline level 24 h after SU6668 injection. After extended daily i.p. injections of SU6668, the tumor blood flow in all of the three tumor types studied was markedly decreased compared with control. The observed effects of this drug on tumor blood perfusion may partially explain the effectiveness of the compound in suppressing tumor growth and extending survival of tumor-bearing mice. We also observed that daily SU6668 administration and a single dose of 15 Gy of X-irradiation was significantly more effective than either treatment alone in suppressing tumor growth. Our results suggest that SU6668 increased the radiosensitivity of tumor blood vessels. We conclude that SU6668 is a potent therapeutic agent potentially useful to suppress tumor growth and enhance the response of tumors to radiotherapy.

INTRODUCTION Among several dozen angiogenic growth factors thus far identified, VEGF,3 FGF, and PDGF are perhaps the most studied (1). The stimulation of endothelial cell proliferation by VEGF is mediated by interaction between VEGF and high-affinity receptor tyrosine kinases, VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1/KDR), which are expressed on the surface of endothelial cells (2– 6). FGF is a potent pleiotropic heparin-binding mitogen for vascular endothelial cells and tumor cells, and it synergistically acts with VEGF in stimulating new vessel growth (7–10). PDGF stimulates angiogenesis by up-regulating VEGF production and modulating the proliferation of pericytes (11, 12) and fibroblast-like cells surrounding the endothelium (13). It has been repeatedly demonstrated that antiangiogenic treatment is potentially useful to halt tumor growth. Among a number of emerging strategies that aim at nullifying tumor angiogenesis, blocking the

interaction of growth factors with their receptors has been demonstrated to be potentially useful (5, 6, 14). For example, significant antitumor effects have been observed with the use of antibodies raised against VEGFRs (5, 14). Recent studies have also demonstrated that the inhibition of the kinase activity of VEGFRs suppresses tumor growth by inhibiting angiogenesis. SU5416, 3-[(2,4-dimethylpyrrol5-yl) methylidenyl]-indolin-2-one, is a synthetic small molecule designed to inhibit VEGFR-2 (Flk-1/KDR) tyrosine kinase activity, and it causes marked growth inhibition in a variety of experimental tumors (15–20). Phase III clinical trials to evaluate this drug are presently in progress (21). In light of the important role of VEGF, FGF, and PDGF and their receptors in tumor angiogenesis, it is reasonable to expect that simultaneously antagonizing the VEGF, FGF, and PDGF signaling pathways may be more effective than antagonizing VEGF signal transduction alone. SU6668, (Z)-3-[2,4-dimethyl-5-(2-oxo-1,2-dihydro-3-ylidenemethyl)-1H-pyrrol-3-yl]-propionic acid, is a multipurpose small synthetic molecule designed to inhibit the kinase activity of FGF and PDGF receptors as well as VEGFRs (22). The antiangiogenic and antitumor effects of SU6668 have recently been demonstrated in a variety of experimental tumors (20, 22–23). Importantly, various antiangiogenic agents have been demonstrated to enhance the response of murine tumors to chemotherapy or radiotherapy. Although the mechanism is not yet completely understood, these studies may represent what is becoming a paradigm shift in the type of adjuvant treatment to be used with traditional chemotherapy or radiotherapy (14, 24 –28). In this connection, it has been reported that the inhibition of VEGFR tyrosine kinase activity using SU5416 markedly enhanced the radioresponse of experimental tumors (19). We have investigated the effect of SU6668, which inhibits VEGF, FGF, and PDGF receptor kinases, on the response of tumors to radiotherapy in the present study. To our knowledge this is one of the first studies to elucidate the effects of simultaneous inhibition of multiple angiogenic growth factors on the response of tumors to ionizing radiation. In addition, we have for the first time quantified the effect of SU6668 on tumor blood perfusion to better understand the antitumor effects of this intriguing compound alone and as an adjuvant therapy.

MATERIALS AND METHODS Tumors

SCK Tumor. This mammary carcinoma spontaneously arose in a female A/J mouse (Jackson Laboratories) in our laboratory. Cells from an early generation are stored in liquid nitrogen, and new cultures are established every 2–3 months. Received 10/23/01; accepted 1/16/02. FSaII Tumor. This fibrosarcoma of C3H mice (Jackson Laboratories) was The costs of publication of this article were defrayed in part by the payment of page originally obtained from the laboratory of Dr. Herman Suit (Massachusetts charges. This article must therefore be hereby marked advertisement in accordance with General Hospital, Boston, MA). Stock cells are stored in liquid nitrogen and 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by NIH Grant CA13353. new cultures are established every 2–3 months. The SCK and FSaII tumor cells 2 To whom requests for reprints should be addressed, at Department of Therapeutic grow well in RPMI 1640 supplemented with 10% calf serum. Radiology-Radiation Oncology, 420 Delaware Street SE, MMC 494, University of CFPAC Human Pancreatic Carcinoma. This human pancreatic carciMinnesota Medical School, Minneapolis, MN 55455. 3 noma (American Type Culture Collection, Manassas, VA) is grown in Balb/ The abbreviations used are: VEGF, vascular endothelial growth factor; FGF, fibrocAnNCrl-nuBr nude mice (Charles River Labs, Wilmington, MA). Cells stored blast growth factor; PDGF, platelet-derived growth factor; VEGFR, VEGF receptor. 1702


SU6668 AS AN ADJUVANT TO TUMOR RADIOTHERAPY

in liquid nitrogen are grown in Iscove’s modified Dulbecco’s medium with 10% FCS (Life Technologies, Inc.). Tumor Induction The SCK and FSaII tumor cells in exponential growth phase in culture were harvested using 0.25% trypsin and were washed and counted. About 2 ⫻ 105 cells in 0.05 ml of serum-free medium were injected s.c. into the hind thigh of male A/J mice for SCK tumors and female C3H mice for FSaII tumors. The SCK tumors and FSaII tumors grow to 250 mm3 in about 8 days and 12 days, respectively. The CFPAC tumor cells were harvested using a 0.05% trypsinEDTA mixture, washed, suspended in Dulbecco’s PBS, and counted. About 1 ⫻ 106 cells in 0.05 ml of PBS were injected s.c. into the hind thigh of female nude mice. The CFPAC tumors grew to 250 mm3 in ⬃21 days. The tumor size was determined using a caliper, and the tumor volume was calculated using the formula a2b/2, where a and b are the shorter and longer diameters of the tumor, respectively. Drug Treatments Starting on the day when the tumors had grown to the desired volume, depending on the purpose of the experiment, the tumor-bearing mice received i.p. once-a-day injections of 100 mg/kg SU6668 (Sugen Inc., South San Francisco, CA) dissolved in 0.05 ml of DMSO. Control animals received injections of the same volume of DMSO. Irradiation of Tumors The A/J mice bearing SCK tumors were treated with 100 mg/kg SU6668 once a day for two days. Two h after the second SU6668 treatment, the host mice were anesthetized with an i.p. injection of a mixture of 100 mg/kg ketamine and 10 mg/kg xylazine. About 15 min later, the mice were covered with a 4 mm thick lead shield and the tumor-bearing legs were gently extended into the radiation field and exposed to X-rays at a dose rate of 1.4 Gy/min. A Philips 250 kV orthovoltage machine (Philips Medical Systems, Brookfield, WI) was used for the irradiation. Blood Perfusion Determination The blood perfusion in tumors and several normal tissues was measured with the 86RbCl uptake method (29). The mice were anesthetized as described above, 5 ␮Ci of 86RbCl in 0.1 ml of PBS was injected through the lateral tail vein, and the mice were killed 60 s later by cervical dislocation. The tissues were removed, weighed, and the radioactivity was counted with a well-type gamma counter (1282 Compugamma; Pharmacia LKB Wallac, Turku, Finland). From the radioactivity in the tissue sample and that in the reference, the percentage of injected 86RbCl per gram of tissue was calculated. Statistical Analysis Data sets were analyzed using a commercially available software package (InStat 2.03, Graphpad Software, Inc.). A two-tailed Student’s t test was used to determine the validity of the differences between control and treatment data sets. A P of 0.05 or less was considered significant.

RESULTS Tumor Growth and Animal Survival. Fig. 1 shows that daily i.p. injection of SU6668 at 100 mg/kg significantly suppressed the growth of CFPAC tumors, FSaII tumors, and SCK tumors. The treatment of CFPAC tumors with SU6668 was begun when the tumor volume reached 250 –300 mm3. The volume doubling time of control CFPAC tumors was 8.9 ⫾ 0.9 days, whereas daily treatment with SU6668 increased the tumor volume doubling time to 18.8 ⫾ 0.7 days (P ⬍ 0.0001). Control FSaII tumors that averaged 250 mm3 grew to 1000 mm3, a 4-fold increase, in 9.4 ⫾ 0.7 days, whereas the volume of tumors treated with SU6668 increased four times in 13.4 ⫾ 0.8 days (P ⬍ 0.006). The growth of SCK tumors was also significantly suppressed by SU6668 treatment. To reveal whether the efficacy of SU6668 on tumor growth depends on the tumor size at the start of

Fig. 1. Growth of CFPAC human pancreatic carcinomas, FSaII murine fibrosarcomas, and SCK murine mammary carcinomas in response to daily i.p. treatment with 50 ␮l of DMSO (Control) or 100 mg/kg/day SU6668 dissolved in 50 ␮l of DMSO. Each group contained 7–10 mice. Error bars are ⫾SE. The dotted lines, the day after which one or more mice in the treatment group had died.

treatment, we began treating SCK-bearing host mice with SU6668 when the tumor volume reached either 125 mm3 or 250 mm3. SU6668 treatment was effective in suppressing the growth of both 125 mm3 and 250 mm3 tumors. The large and small control tumors grew 4-fold in volume in 6.4 ⫾ 0.3 and 5.0 ⫾ 0.8 days, respectively, whereas large and small tumors treated with SU6668 grew 4-fold in volume in 9.5 ⫾ 0.3 days and 7.9 ⫾ 1.5 days, respectively (P ⱕ 0.007). The difference between the growth delay of large (3.1 days) and small (2.9 days) tumors caused by SU6668 treatment was not statistically significant. Fig. 2 shows the effect of daily treatment of mice bearing SCK tumors with SU6668 or vehicle (DMSO) on the survival time of the host mice. The treatment was started when the tumors reached 125 mm3 (4 –5 days after tumor cell inoculation) and the day of death represents the number of days after treatments were started. The control mice treated with only the vehicle started to die on day 4, and all of the control animals died by day 12, when the primary tumor volume had reached an average of 1575 ⫾ 197 mm3. On the other hand, the mice treated with SU6668 began to die on day 10, and some tumor-bearing mice survived until day 18, when the average primary tumor volume was 1878 ⫾ 105 mm3. The median survival time of host mice treated with only the vehicle was 8 days, whereas that of mice treated with SU6668 was 14 days after the start of treatment.

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Fig. 2. Survival of SCK-tumor-bearing A/J mice treated with daily i.p. injections of either 50 ␮l DMSO or 100 mg/kg/day SU6668 dissolved in 50 ␮l of DMSO starting on day 0, when the tumor volume was ⬃125 mm3. Each group contained 10 mice.

again declined to 75% of the control value (P ⫽ 0.05). The perfusion in the skin, muscle, and kidney of A/J mice was measured concomitantly with the tumor blood perfusion. In contrast to the effects of SU6668 on tumor perfusion, there was no significant difference in normal tissue perfusion found between control and drug-treated mice except for the kidney perfusion at 1 h after SU6668 injection, which decreased to 60% of control level and recovered completely by 4 h (data not shown). Fig. 3B shows the effect of daily administrations of SU6668 on tumor blood perfusion determined by measuring 86Rb uptake in the FSaII and CFPAC tumors that were used to study the effect of SU6668 on tumor growth (shown in Fig. 1). The 86Rb uptake in the control CFPAC and FSaII tumors was 2.8 ⫾ 0.3%/g and 1.3 ⫾ 0.3%/g, respectively. At 24 h after the last of 17 daily injections with SU6668, the 86Rb uptake in CFPAC and FSaII tumors was reduced to 1.9 ⫾ 0.3%/g and 0.5 ⫾ 0.1%/g, respectively (P ⱕ 0.05). The 86Rb uptake in control SCK tumors was 2.1 ⫾ 0.2%/g, as stated above, but 24 h after 7 daily injections of 100 mg/kg SU6668, the 86 Rb uptake was reduced to 1.4 ⫾ 0.3%/g (P ⫽ 0.01). Enhancement of Tumor Radiation Response. After SCK tumors grew to about 125 mm3, we began daily treatment of the host mice with 100 mg/kg of SU6668. Tumors were irradiated with 15 Gy of X-rays 2 h after the second drug treatment, and daily treatment of host mice with SU6668 was continued after tumor irradiation until the end of the experiment. Fig. 4 shows that the suppression of tumor growth caused by daily 100 mg/kg SU6668 was slightly greater than that caused by 15 Gy of radiation, and the combination of SU6668 treatment and radiation was far more effective than either treatment alone. In Table 1 the number of days required for the tumors to grow four times in volume after various treatments are compared. The tumor-growth delay caused by daily 100 mg/kg SU6668 treatment in combination with 15-Gy irradiation was 15.3 ⫾ 1.6 days which was roughly triple that caused by either of these treatments alone

Fig. 3. A, changes in blood perfusion in SCK tumors over a 24-h time period after a single i.p. injection and 2 h after a second injection of 100 mg/kg SU6668. Each data point represents average of 8 –10 tumors ⫾1 SE. The data were normalized to the corresponding value from control tumors of mice injected with DMSO and measured at each time point. B, tumor blood perfusion in three different tumor types (average of 8 –10 tumors ⫾1 SE) after chronic daily treatment with SU6668. The CFPAC and FSaII tumors were treated with 17 daily i.p. injections of 100 mg/kg of SU6668 dissolved in 50 ␮l of DMSO, and the blood perfusion was measured 1 day after the last drug treatment. The blood flow in SCK tumors was determined 1 day after 7 daily treatments with SU6668. The control data for each group was measured in size-matched tumors of mice treated with DMSO daily, starting when the tumor volume was ⬃250 mm3.

Blood Perfusion. The effect of single or multiple injections of SU6668 on tumor blood perfusion was determined with 86Rb uptake as shown in Fig. 3. The 86Rb uptake in control SCK tumors was 2.1 ⫾ 0.2% of the total injected radioactivity per gram. As shown in Fig. 3A, when host mice received an injection of a single dose of 100 mg/kg of SU6668, the 86Rb uptake in SCK tumors decreased to about one-half of that in control mice within the 1st hour after drug administration (P ⬍ 0.008). The perfusion recovered to control level by 8 h and increased to about 40% above the corresponding control value by 24 h after injection (P ⫽ 0.03). At 2 h after a second dose of 100 mg/kg SU6668, given 24 h after the first dose, the blood perfusion

Fig. 4. Radiation-induced growth delay of SCK tumors. The mice were treated with either 50 ␮l of DMSO or 100 mg/kg SU6668 for 2 days; 2 h after the second drug treatment, the tumors were irradiated with 15 Gy. The tumor-bearing host mice were continuously treated with daily injections of drug or vehicle until the end of the experiment. Each group contained 7–10 mice; error bars, ⫾1 SE.

Table 1 Response of SCK tumors to SU6668 treatment and local irradiation

Treatment

Days required for tumors to grow four times the original volume (⫾SE)

Growth delay (days)

Control 15 Gy SU6668 SU6668 ⫹ 15 Gy

5.0 ⫾ 0.8 10.3 ⫾ 0.8 9.3 ⫾ 0.6 20.3 ⫾ 1.6

5.3 4.3 15.3

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(P ⱕ 0.0003). There was also a synergistic effect of SU6668 and tumor irradiation on animal survival. The mice in the X-irradiationalone group and SU6668-treatment-alone group began to die on day 6 and 10, respectively, whereas none of the host mice that received the combined treatments died before day 26 (data not shown). DISCUSSION Our results with two different murine tumor models and a human pancreatic carcinoma xenograft model confirm previous reports that the inhibition of angiogenesis-related receptor tyrosine kinase activity suppresses tumor growth (15–20, 22–23). The suppression of tumor growth caused by daily treatment with SU6668 (Fig. 1) was apparently related to the marked decline in tumor blood perfusion caused by SU6668 (Fig. 3). Microscopic observation of vascular density in histological preparations of tumors and changes in vascular patterns in small tumors grown in window chambers are commonly used as a measure of antiangiogenesis. Although these methods are useful to investigate morphological and structural changes in blood vessels, we believe that measuring actual tumor blood perfusion, an important and novel part of the present study, is an invaluable indicator of the action of antiangiogenic agents on existing tumor vasculature. A question that remains is what amount of the reduction of tumor blood perfusion after multiple daily treatments with SU6668 (Fig. 3B) was attributable to the inhibition of new blood vessel formation as opposed to damage to existing tumor blood vessels. It has been reported that VEGF, FGF, and PDGF may be required not only for angiogenesis but also for survival of existing endothelial cells (20). Therefore, it is probable that the marked reduction of tumor blood flow caused by extended treatment with SU6668 in the present study was caused, in part, by damage of existing tumor vasculature. In this connection, the early response of tumor microvasculature to SU6668 treatment has been recently reported (30). Here, SU6668 induced apoptosis in tumor vessels within 6 –12 h after drug administration. Moreover, SU6668 treatment resulted in a decrease in tumor mitotic index and dramatically reduced the functional microvessel density in the tumor. These data suggest that the antitumor action of SU6668 is, at least in part, dependent on a significant degree of damage to existing vessels. Interestingly, although the tumor blood perfusion decreased in the 1st hour after a single i.p. injection of SU6668, it increased by 24 h after the drug administration (Fig. 3A). This observation may be related to the findings of Laird et al. that when the tumor endothelium was undergoing breakdown several hours after SU6668 treatment, VEGF transcript levels in RNA isolated from the tumor were elevated 2- to 3-fold and then returned to near normal levels by 24 h (30). One interpretation of these data are that the rapid vessel perturbation and the accompanying reduction in tumor perfusion caused by SU6668 resulted in hypoxic stress in the tumor tissue leading to induction of VEGF transcription. VEGF is known to induce nitric oxide production leading to increases in vascular permeability and vasodilation (31), which may explain why blood perfusion was transiently elevated after the single dose of SU6668. Ultimately, repeated administration of SU6668 may render many vessels nonfunctional because of a massive loss of endothelial cells. Clearly, the complete mechanism of the dynamic changes in tumor perfusion after single or multiple injections of SU6668 needs further elucidation. The ability of SU6668 to significantly prolong the survival time of tumor-bearing host mice (Fig. 2) is potentially important and echoes the results of other recent studies (20, 23). In our studies, we treated A/J mice bearing SCK tumors, which normally metastasize to the lung, with SU6668 at 100 mg/kg/day beginning when the tumors were about 125 mm3 in size. An example of lung preparations of tumor-

Fig. 5. Typical examples of lung metastases in SCK-bearing A/J mice with and without SU6668 treatment, beginning when the tumor was 125 mm3. Left, an animal treated with seven daily injections of 50 ␮l of DMSO; right, an animal treated with seven daily injections of 100 mg/kg SU6668. The animals were killed, and the lungs were removed and processed for histology at 15 days posttumor cell-inoculation in the rear leg.

bearing A/J mice treated for 7 days with either DMSO or 100 mg/kg SU6668 is shown in Fig. 5. In a preliminary histological study, we observed that in lung sections from DMSO-treated tumor-bearing mice, there were 9.8 ⫾ 3.7 metastatic loci/section, whereas only 2.3 ⫾ 0.3 metastatic loci/section were observed in the lungs of tumor-bearing mice treated daily with SU6668. In addition, the size of the metastases in the lungs of mice treated with SU6668 was noticeably smaller than that in DMSO-treated control mice. The significant decline in the number and size of metastatic nodules in the lung seems to reflect the prolonged survival time caused by SU6668. Our future studies aim at determining the mechanism of the survival benefit afforded to tumor-bearing mice by SU6668 treatment. Our observation that SU6668 significantly enhances radiationinduced tumor-growth delay supports the growing body of data indicating that antiangiogenic agents may be used to improve the efficacy of radiotherapy or chemotherapy. The list of antiangiogenic agents that have been demonstrated to enhance the antitumor effect of radiation includes angiostatin (26), anti-VEGF or VEGFR antibodies (14, 27, 28), isocoumarin (24), TNP-470 (25), and VEGF-toxin constructs.4 The mechanisms underlying the enhancement of radiation response caused by antiangiogenic agents is unclear. To date. a time-dependent increase in tumor pO2 after treatment with antiangiogenic agents (14, 28) or an inhibition of VEGF-induced protection against, and/or repair of, radiation damage in endothelial cells (27, 32) have been suggested. In our study of the combination of SU6668 and radiation, tumors were irradiated 2 h after the second daily administration of the drug when tumor blood perfusion was at 75% of control level (Fig. 3A). Therefore, the increase in radiation response of the tumors by exposure to SU6668 did not appear to have resulted from an increase in tumor oxygenation via an increase in tumor blood perfusion. We cannot rule out, however, the possibility that SU6668 alters the oxygen consumption rate in the tumor endothelium, the tumor cells, or both. Although significant, our results lean toward an additive rather than an interactive effect of the combination of SU6668 and radiation. We are currently working to understand the optimal combined treatment scheme to produce a greater-than-additive antitumor response. A recent study has demonstrated that SU5416, an inhibitor of VEGF receptor kinase, increased the radiation-induced apoptosis in endothelial cells (19). It has also been reported that SU6668 caused apoptosis in endothelial cells in vivo (23). Taken together, these data suggest that enhancement of tumor response to radiation by SU6668 may occur most readily by increasing the radiosensitivity of tumor blood vessels. In conclusion, this study indicates that the multipotent angiogenesis inhibitor SU6668 is effective in perturbing existing tumor vascular function and further uncovers the mechanism(s) by which growth4

R. Wild et al., unpublished observations.

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factor inhibition alters tumor growth. Our data shows that SU6668, currently in Phase I clinical trials (21), has potential in combating tumor progression and may be particularly effective as an adjuvant to radiotherapy of human tumors.

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ACKNOWLEDGMENTS We thank Dr. Kathryn Dusenberry for continued support and encouragement.

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