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Experimental Therapeutics |
Department of Medical Oncology [J. D., R. M-D., C. W., S. F., C. U.] and Institute of Molecular Oncology [N. E., H. H., D. M.], Tumor Biology Center, 79106 Freiburg, Germany; Department of Diagnostic Radiology, Section of Medical Physics, Albert-Ludwigs-University Hospital, 79106 Freiburg, Germany [J. H.]; Department of Anatomy, Johannes Gutenberg-University, 55099 Mainz, Germany [M. A. K.]; and Oncology Therapeutic Area and Core Technology Area, Novartis Pharma AG, Basel, Switzerland [P. R. A., J. W.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Surprisingly, no changes in extravasation occurred under treatment with PTK787/ZK 222584 as compared with the control group, whereas a significant decrease in vessel permeability occurred. Furthermore, an increase in partial blood volume was found in the PTK787/ZK 222584-treated group, although vessel density was reduced as seen by histology. Using the corrosion cast technique, reduction in vessel density was significant but not very pronounced and predominantly attributable to the loss of microvessels only. This finding correlated with a shift to large vessel diameters in the primary tumors of PTK787/ZK 222584-treated animals and with reduction of blood flow velocity in the tumor feeding renal artery. From these findings, we conclude that the treatment with PTK787/ZK 222584 primarily reduces the number of tumor microvessels, accompanied by a hemodynamic dilation of the remaining vessels. This dilation could influence the result of dyMRI such that no change in extravasation or even an increase in partial tumor blood volume could be observed.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Various angiogenic factors produced by solid tumors have been identified, and their roles in the formation of new blood vessels have been elucidated. The VEGF is one of the most potent endothelial cell-specific angiogenic factors with a key role in tumor angiogenesis (4)
. VEGF is also known to induce vascular hyperpermeability. Its specificity for endothelial cells is explained by the enhanced expression of its two receptors, VEGF-R1 and VEGF-R2, on endothelial cells of tumor vessels. Therefore, VEGF-receptors are promising targets for the inhibition of angiogenesis in growing tumors. PTK787/ZK 222584 has been developed as a potent inhibitor of all three VEGF-R-tyrosine kinases (including the lymph-angiogenic VEGF-R3) in a joint effort by Novartis Pharma AG, Schering AG, and the Institute of Molecular Oncology at the Tumor Biology Center, Freiburg. Table 1
gives the IC50 of all affected tyrosine kinases. The compound has already entered clinical trials (5)
. Its properties have been described in detail elsewhere (6, 7, 8)
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The aim of our study was to evaluate the effect of antiangiogenic therapy on vessel density as detected by immunohistochemistry, vessel architecture as analyzed by corrosion casting, vessel diameter as identified by the DNA-binding fluorescent dye H33342, and vessel blood flow velocity as measured by color Doppler imaging ultrasound. dyMRI was used to investigate the effect of the compound on vessel permeability, leakage space, and partial blood volume. GdDOTA (Dotarem) was used as contrast agent to detect extravasation, whereas intravascular iron oxide nanoparticles (Endorem) were applied to detect partial tumor blood volume.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture.
RENCA cells were originally obtained from a tumor that arose spontaneously in the kidney of BALB/c mice. Monolayers of murine RENCA cells were grown in RPMI 1640 with phenol red supplemented with 10% FCS, 2 mM L-glutamine, 100 units of penicillin/ml, and 100 µg of streptomycin/ml. RENCA cells were cultured in a humidified atmosphere of 95% air and 5% carbon dioxide at 37°C. Media were routinely changed every 3 days. Cells were released from the tissue flasks by treatment with 0.05% (Versene) trypsin/EDTA, and viability was monitored using the cell analyzer system Casy 1 from Schärfe System (Reutlingen, Germany). For the experiments, cells were collected during logarithmic growth phase.
Animal Experiments.
All experiments were carried out according to the guidelines of the Ethical Committee of the Regierungspräsidium (Freiburg, Germany) and the Eidgenössische Tierschutzverordnung approved by the Veterinary Office Basle. Female BALB/c mice were used at 6–8 weeks of age (approximate weight, 20 g). The injection of 106 RENCA cells in 0.2-ml aliquots into the subcapsular space of the left kidney was performed through a flank incision after the animals were anesthetized with 0.5–1.5 volume percent isoflurane with an oxygen flow of 1.5 l/min. The injection of 106 RENCA cells in syngeneic BALB/c mice induced progressive development of a primary tumor mass in the left kidney. One week after application, the primary tumor was macroscopically visible. At 10 days, spontaneous metastases developed in the regional lymph nodes, in the lung, the peritoneum, and the liver. The mean survival time of RENCA-bearing mice was 
32 days when 106 RENCA cells were injected.
Administration of Drug.
Therapy with PTK787/ZK 222584 or the vehicle was initiated 1 day after tumor cell inoculation. Mice received either PTK787/ZK 222584 (50 mg/kg, p.o. once daily) or vehicle (distilled water, p.o. once daily) until the animals were sacrificed. Fifteen animals were included in both groups. Oral applications were performed using a gastric tube. Animal weights were taken every second day.
Administration of Fluorescent Dye H33342.
The DNA-binding fluorescent dye H33342 was obtained from Aldrich Chemicals Ltd. (Gillingham, United Kingdom). Solutions were prepared in sterile saline immediately before use. H33342 was injected via one of the lateral tail veins at a dose of 20 mg/kg. This dose was chosen because it was well below the toxic limit for this compound and allowed easy identification of labeled cells in histological sections (12)
. Mice were killed 1 min after injection.
Evaluation of Tumors.
A previous publication (7)
reports on kinetics of tumor growth and on investigations of antitumor and antiangiogenic activity on day 14 in comparison to day 21, showing a more significant efficacy on day 21. For this reason and because of larger tumor masses on day 21 for the detection by dyMRI, day 21 was chosen for the analysis. Therefore, after 2 weeks of treatment all mice were sacrificed for determination of weight and volume of primary tumors, lung weight, and number of lung metastases and the number of metastases in the abdominal lymph nodes. Volumes of primary tumors were evaluated macroscopically by measuring their extensions in three orthogonal dimensions. The number of metastases in the lung and abdominal lymph nodes were counted using a dissection microscope. In the abdominal cave, all visible lymph nodes were assessed for detection of metastasis, knowing that in healthy animals visible lymph nodes are usually absent. Lymph nodes were inspected randomly with a microscope to confirm tumor-bearing tissue.
Immunohistochemistry.
For histological examination of the tumor vasculature, tumor tissues and lungs were frozen immediately in liquid nitrogen. Cryosections of tissue with a thickness of 5–10 µm were taken from both groups. For visualizing the blood vessels, immunohistochemical staining for CD31 (Pecam-1 and MEC13.3; Becton Dickinson GmbH) was performed, and vessels were counted microscopically using a defined magnification (x200). Furthermore, immunohistochemical staining for VEGF-receptor 2 (anti-FLK-1; PharMingen) was performed, and FLK-1-positive cells were counted using a defined magnification (x200).
Additional sections of primary tumors were air-dried and studied under UV illumination using a Zeiss microscope (Carl Zeiss, Oberkochen, Germany) equipped with an epifluorescent source (x200). Blood vessel borders were identified by the surrounding halo of fluorescent H33342-labeled cells. For detecting vessel density, all recognized vessels were counted. The lumen enclosed by the halos was measured using a square scoring system. Vessels were characterized by their lumen size and assigned to one of the following groups: group 1, no detectable lumen; group 2, lumen less than one quadrangle; group 3, lumen = one quadrangle (= 62.5 x 62.5 µm2); and group 4, lumen more than one quadrangle.
Currently, vascular density is investigated in "hot spots" only. This might lead to false high values of vessel density, e.g., in tumor tissues, because these data are not correlated to the number of "hot spots" found in the tissues. In our studies, a minimum of three slides from separate areas of each tumor were used for all analysis (CD-31 and dye H33342). Therefore, sections were representative for the whole tumor. Furthermore, the evaluation was performed by two persons who were blinded with respect to treatment of the animals.
dyMRI.
The mouse tail vein was cannulated for contrast agents bolus application before placing the animal in the magnet. MRI experiments were carried out on a Bruker DBX 47/30 spectrometer (Bruker Medical, Fällanden CH) at 4.7 T equipped with a self-shielded 20-cm bore gradient system 21 days after tumor cell application. A resonator probe with inner diameter of 40 mm has been used. During the MRI experiments, the animals were anesthetized with 1.5% isoflurane (Abbott, Cham, Switzerland) in a 1:2 mixture of O2:N2O administered with a face mask. They were positioned on a cradle in supine position inside the resonator. The exact position of the animal was assessed by a scout imaging sequence.
Gradient recalled echo imaging sequences have been used for determination of blood vessel permeability. Acquisition parameters were: repetition delay TR, 20 ms; echo delay TE, 3.1 ms; number of averages NA, 8; image matrix, 1282, field-of-view FOV, 3.5 x 3.5 cm; slice thickness, 1.5 mm. Sixty-four sequential images with a time resolution of 20 s have been recorded. After the 10th repetitive image, a bolus application of 30 µl Dotarem (Guerbet AG, Zürich, Switzerland) was given manually by the tail vein during 20 s.
Partial tumor blood volume was assessed by gradient recalled echo imaging sequence with the following measuring parameters: repetition delay TR, 10.7 ms; echo delay TE, 3.1 ms; number of averages NA, 2; image matrix, 128 x 64; field-of-view FOV, 3.5 x 3.5 cm; slice thickness, 2 mm. Forty sequential images with a time resolution of 1 s have been recorded. Sixty µl of Endorem were injected into the tail vein during acquisition of image 11 using a spectrometer-controlled infusion pump. Heart rate or respiration was not monitored, and accordingly no triggering of the imaging sequence was applied.
The theoretical background for the measurement of vascular permeability and tracer leakage space has been described by Tofts (13)
and Rudin et al. (14)
. The tracer uptake curves derived from the experimental signal enhancement were analyzed by least square fitting (SigmaPlot) using the bi-exponential model function:
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) and the final tracer uptake were computed as [d(S/S0 - 1)/dt]0 = a*ka and (S/S0 - 1)
= a + b, respectively. Tumor blood volume (TBV) was estimated as described previously (15
, 16)
according to TBV 
-ln(S/S0).
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Corrosion Casting.
BALB/c mice received either PTK787/ZK 222584 (50 mg/kg, p.o. once daily) or vehicle (distilled water) one day after inoculation of RENCA cells as described above. After 21 days of treatment, the animals were thoracotomized in deep anesthesia, an olive-tipped cannula was inserted into the ascending aorta, and up to 30 ml of body warm saline were perfused to rinse the vascular system. Vessels were fixed by a 2–5 min infusion of 25 ml of body-warm glutaraldehyde solution (2.5% phosphate-buffered glutaraldehyde, pH 7.4; 400 milliosmoles). The final casting was performed by the application of 5–15 ml of Mercox CL 2B diluted with methylmetacrylate (4:1).
After having sacrificed the animal, polymerization of casting medium took place in a water bath (40–50°C). Primary tumors were taken out, and tissue was dissolved in a 7% KOH solution for 7–9 days. Casts of the primary tumors were coated with gold for electron microscopy. Microvascular corrosion casting is an established method to investigate vascular patterns of almost all organs. In the past, evaluation of these specimens using scanning electron microscope pictures had not allowed for quantitation, because depth could not be taken into account. However, scanning electron stereo images pairs allowed for quantitative three-dimensional measurements. Therefore, three-dimensionally reconstructed scanning electron microscope images (Stereoscan 180, Cambridge instruments) were used for quantitative assessment of vessel diameters, intervessel distances, and interbranch distances. This method was described in detail elsewhere (17 , 18) .
Statistical Analysis.
All values reported were expressed as mean ± SE. Mann-Whitney t test was applied to all statistical analyses, except for corrosion cast results. Here, the Mann-Whitney rank sum test was used. P < 0.05 was considered statistically significant.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The primary tumor volumes in the left kidney of the PTK787/ZK 222584-treated group was compared with the control group. Treatment with PTK787/ZK 222584 for 21 days resulted in a 24% inhibition of primary tumor growth. The effect of PTK787/ZK 222584 on lung metastases was assessed by comparing the number of lung metastases in treated animals with those in untreated animals. Treatment resulted in 30% decrease of the number of lung metastases. Visible lymph nodes were reduced to 44.21% (data not shown).
In contrast to previous experiments, the reduction in tumor size by PTK787/ZK 222584 was not significant for all tumor sites, although vessel density detected by immunohistochemistry was dramatically reduced in all tumor tissues. This finding indicates a partial angiogenesis-independent growth of the fast-growing RENCA tumor. This hypothesis was supported by earlier findings (7) , where vessel density declined in the period between 2 and 3 weeks after inoculation without slowing down tumor growth, indicating that the proliferation rate of tumor cells was higher than that of endothelial cells. Furthermore, tumors may have the possibility to infiltrate vessels for blood supply in murine models.
Effect of PTK787/ZK 222584 on Vessel Density.
For histological examination of tumor vasculature, primary tumor tissues of all groups were stained for CD31. Initial examination of all tissue sections at low magnification showed homogeneous vessel density with lack of hot spots (Fig. 1)
. Comparison of vessel density in primary tumors of untreated animals with those of PTK787/ZK 222584-treated animals revealed a significant decrease of vessel density by 48% (P = 0.00031) under treatment. The reduction in vessel density did not lead to the heterogeneous appearance of vessel distribution.
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Effect of PTK787/ZK 222584 on Vessel Permeability and GdDOTA Extravasation.
Twenty-one days after tumor cell inoculation, primary tumors could be located by high-resolution MRI technique (Fig. 3)
in all animals (n = 30). Vessel permeability and extravasation were measured in 13 of the 15 animals of the control group and in 14 of the 15 animals in the PTK787/ZK 222584-treated group by using contrast agent Dotarem. In the 3 remaining animals, the application of contrast agent could not be performed, probably because of the small-diameter tail veins.
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shows the time course of contrast agent extravasation. Vessel permeability, which is known to correlate with the maximal slope, was significantly decreased in the PTK787/ZK 222584-treated group (48%; P = 0.033). Astonishingly, no significant difference between both groups could be detected with regard to extravasation, represented by the maximal uptake of the contrast agent.
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. In these experiments, all animals received a bolus infusion of the intravascular contrast agent Endorem during MRI. Tumor blood volume was assessed in 12 of the 15 animals of the control group and in 12 of the 15 animals in the PTK787/ZK 222584 treatment group.
The time course of Endorem uptake into the tumor is shown in Fig. 5
. Relative tumor blood volume was estimated by: TBV = -ln(S/S0) (compare methods). Comparing maximal Endorem uptake in primary tumors of untreated animals with PTK787/ZK 222584-treated animals, a significant increase in treated animals by 44% (P = 0.038) becomes apparent, reflecting an increased partial blood volume under therapy.
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Previously, we anesthetized the animals by an i.p. application of Rompun/Ketanest, whereas in this experiment, the animals were anesthetized by an isoflurane anesthesia. It is known that gas anesthesia does not influence the vessel activity as strongly as systemic anesthesia (19) . This fact may have caused a stronger variability in blood flow velocity, influencing parameters such as blood pressure and heart rate in the animals anesthetized with isoflurane.
Effect of PTK787/ZK 222584 on Vessel Diameter.
The effects of PTK787/ZK 222584 treatment on the volume of primary tumor vessels were detected by the use of H33342. Vessels could be identified in all histological sections by the surrounding halo of fluorescent H33342-labeled cells (Fig. 6)
. Tissues showed homogeneous vessel density distribution as already described by immunohistochemistry technique. Significant reduction of vessel density in PTK787/ZK 222584-treated animals by 58% (P = 5.93 x 10-6) shown by immunohistochemical technique could be confirmed (data not shown). Sections of primary tumors from treated animals exhibited a shift of vessel diameters toward larger lumen as compared with control animals (Fig. 7)
. Vascular lumen enclosed by the halos was quantified using a square scoring system. A significant decrease of the number of small vessels (diameter, <62.5 µm) by 76% (P = 1.95 x 10-5) occurred under therapy, whereas significant increases of the number of large vessels (diameter, 
62.5 µm) by 307% (P = 0.043) and by 582% (P = 0.015) were observed, respectively.
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. Primary tumors showed a low or missing vessel hierarchy, heterogeneous vascular density, and the presence of evasates and of blind-ending vessels (Fig. 8)
. Comparison of corrosion casts of the control group (Fig. 8, Ab and Bb)
and the PTK787/ZK 222584-treated group (Fig. 8, Ac and Bc)
revealed a significant difference for vessel diameters and intervessel distances (Fig. 9)
. The vessels in the PTK787/ZK 222584-treated group were found to have 26% (P = 0.0001) larger diameter than the vessels of the controls. Mean intervessel distance was increased by 20% (P = 0.0001) in the PTK787/ZK 222584-treated animals. The reduction of mean interbranch distance in treated animals was not significant (data not shown). In summary, the vessels in primary tumors of the PTK787/ZK 222584-treated group were smaller in number but larger in diameter as compared with the control group.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The use of the small molecular contrast agent GdDOTA (Dotarem), which rapidly leaks to the interstitial space, allows detection of vessel permeability and leakage space. Comparison of the rate of contrast agent uptake into tumor tissue, indicating tumor vessel permeability, between control and treated animals revealed a significant decrease of vessel permeability in the treated animals. Because, however, the rate of GdDOTA leakage is also dependent on tumor perfusion, the observed reduction in blood flow velocity might have slightly enhanced the observed vascular permeability decrease. Furthermore, changes in tumor perfusion by the treatment might influence our permeability measure. Thus, this parameter is rather an index of perfusion and permeability. However, the GdDOTA extravasation curve is to slow to be heavily influenced by perfusion changes.
Leakage space, detected by the maximum GdDOTA uptake, was not affected by PTK787/ZK 222584. Furthermore, the use of the large molecular contrast agent Endorem, which is expected to remain intravascular during the experimental time of 30 s, allows detection of total tumor blood volume. Surprisingly, the maximal uptake of Endorem, giving the partial blood volume in the primary tumors, was significantly higher for treated animals as compared with untreated animals. This result was in striking contrast to the significantly reduced vessel density and the reduction in FLK-1-positive cells as seen by histology.
The increase in tumor blood volume detected by dyMRI could be explained by the shift to larger blood vessel diameters in tumor tissues of treated animals as seen by DNA-binding fluorescent dye histology and the corrosion cast set up. The observed dilation of the remaining vessels most probably occurs to compensate for the decrease of tumor blood volume because of small vessel loss. The observed reduction of blood flow velocity in the tumor feeding renal artery in the treated animals, assessed by Color Doppler imaging sonography, supports this hypothesis. Tumor blood volume measurements by dyMRI should therefore be interpreted with care unless distribution of vessel size is assessed in parallel. In literature a method for noninvasive determination of mean vessel size index (20) is described, which, however, was not applied in the present study.
Tumor perfusion is dependent on the morphology and three-dimensional architecture of the vascular network. The latter can be studied by scanning electron microscopy of vascular corrosion casts. Qualitative comparison of primary tumors and healthy kidney of a BALB/c mouse revealed loss of the hierarchy and loss of the organ-specific vasculature in primary tumors. Quantitative analysis of casts from PTK787/ZK 222584-treated animals revealed significant differences of intervessel distance and vessel diameter as compared with control mice. Intervessel distance in primary tumors of PTK787/ZK 222584-treated animals estimated by evaluating the three-dimensionally reconstructed vascular network was significantly increased as compared with vehicle-treated mice. This is the first time that the antiangiogenic activity of PTK787/ZK 222584 is detected by three-dimensional vessel bed analysis.
Vessel diameter was found to vary between 10 and 100 µm in primary tumors of the orthotopic RENCA model, whereas the diameter of vessels with capillary wall structure range from 6 to 55 µm in human primary tumors (renal clear cell carcinoma, basalioma) and from 5 to 80 µm in xenografted tumors (sarcomas and colon carcinoma; Ref. 21 ). In primary tumors of PTK787/ZK 222584-treated animals, vessel diameters were significantly increased as compared with vehicle-treated mice, indicating primary reduction of microvessels. Studies with other VEGF-receptor tyrosine kinase inhibitors such as SU5416 and SU6668 revealed, in line with our study, reduction of microvessel density (22) . Other investigators reported, in contrast to our results, a decrease of tumor vessel diameters by using a VEGF antibody (21 , 22) . The apparently contradictory results of our study demonstrate the importance of assessing different independent vascular parameters by complementary methods to get a clear picture of the mode of action of antiangiogenic drugs.
In conclusion, our results show that selective inhibition of VEGF-receptor activity by PTK787/ZK 222584 leads to antitumor effects without obvious side effects. Treatment with PTK787/ZK 222584 results in a significant reduction of tumor vessel density detected independently by three methods. In addition, we showed that the decrease of vessel density was primarily caused by a pronounced reduction of microvessel density, whereas the remaining vessels were found to be dilated. Furthermore, dyMRI revealed significant reduction of tumor vessel permeability without change of leakage space after PTK787/ZK 222584 treatment. Color Doppler revealed reduced blood flow velocity which, in combination with vessel dilation, may be the result of a possible organ-specific compensatory effect. The very sophisticated and subtle effect of VEGF-receptor tyrosine kinase inhibitors such as PTK787/ZK 222584 on the tumor vascular bed can only be evaluated by the combination of complementary in vivo analysis techniques such as dyMRI and Doppler ultrasound imaging.
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FOOTNOTES |
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1 This work was supported by Novartis Pharma AG, Basel, Switzerland; Schering AG Berlin, Germany; Deutsche Forschungsgemeinschaft, Germany; Fördergesellschaft KTB, Freiburg, Germany; and Kirstins Weg, Neuwied, Germany. 
2 To whom requests for reprints should be addressed, at Tumor Biology Center, Department of Medical Oncology, Breisacher Strasse 117, D-79106 Freiburg, Federal Republic of Germany. Phone: 49-761-206-2178; Fax: 49-761-206-2180; E-mail: drevs{at}tumorbio.uni-freiburg.de
3 The abbreviations used are: MRI, magnetic resonance imaging; dyMRI, dynamic enhanced MRI; VEGF, vascular endothelial growth factor; H33342, Hoechst 33342; GdDOTA, gadolinium-DOTA.
Received 6/25/01. Accepted 5/ 8/02.
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Y. R. Kim, A. Yudina, J. Figueiredo, W. Reichardt, D. Hu-Lowe, A. Petrovsky, H. W. Kang, D. Torres, U. Mahmood, R. Weissleder, et al. Detection of Early Antiangiogenic Effects in Human Colon Adenocarcinoma Xenografts: In vivo Changes of Tumor Blood Volume in Response to Experimental VEGFR Tyrosine Kinase Inhibitor Cancer Res., October 15, 2005; 65(20): 9253 - 9260. [Abstract] [Full Text] [PDF]
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P. Marzola, A. Degrassi, L. Calderan, P. Farace, E. Nicolato, C. Crescimanno, M. Sandri, A. Giusti, E. Pesenti, A. Terron, et al. Early Antiangiogenic Activity of SU11248 Evaluated In vivo by Dynamic Contrast-Enhanced Magnetic Resonance Imaging in an Experimental Model of Colon Carcinoma Clin. Cancer Res., August 15, 2005; 11(16): 5827 - 5832. [Abstract] [Full Text] [PDF]
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A. L. Thomas, B. Morgan, M. A. Horsfield, A. Higginson, A. Kay, L. Lee, E. Masson, M. Puccio-Pick, D. Laurent, and W. P. Steward Phase I Study of the Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of PTK787/ZK 222584 Administered Twice Daily in Patients With Advanced Cancer J. Clin. Oncol., June 20, 2005; 23(18): 4162 - 4171. [Abstract] [Full Text] [PDF]
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J. Drevs, U. Zirrgiebel, C. I. M. Schmidt-Gersbach, K. Mross, M. Medinger, L. Lee, J. Pinheiro, J. Wood, A. L. Thomas, C. Unger, et al. Soluble markers for the assessment of biological activity with PTK787/ZK 222584 (PTK/ZK), a vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitor in patients with advanced colorectal cancer from two phase I trials Ann. Onc., April 1, 2005; 16(4): 558 - 565. [Abstract] [Full Text] [PDF]
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D. W. Davis, R. Takamori, C. P. Raut, H. Q. Xiong, R. S. Herbst, W. M. Stadler, J. V. Heymach, G. D. Demetri, A. Rashid, Y. Shen, et al. Pharmacodynamic Analysis of Target Inhibition and Endothelial Cell Death in Tumors Treated with the Vascular Endothelial Growth Factor Receptor Antagonists SU5416 or SU6668 Clin. Cancer Res., January 15, 2005; 11(2): 678 - 689. [Abstract] [Full Text] [PDF]
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R. Ansiaux, C. Baudelet, B. F. Jordan, N. Beghein, P. Sonveaux, J. De Wever, P. Martinive, V. Gregoire, O. Feron, and B. Gallez Thalidomide Radiosensitizes Tumors through Early Changes in the Tumor Microenvironment Clin. Cancer Res., January 15, 2005; 11(2): 743 - 750. [Abstract] [Full Text] [PDF]
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 V. Mastyugin, E. McWhinnie, M. Labow, and F. Buxton A Quantitative High-Throughput Endothelial Cell Migration Assay J Biomol Screen, December 1, 2004; 9(8): 712 - 718. [Abstract] [PDF]
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P. Traxler, P. R. Allegrini, R. Brandt, J. Brueggen, R. Cozens, D. Fabbro, K. Grosios, H. A. Lane, P. McSheehy, J. Mestan, et al. AEE788: A Dual Family Epidermal Growth Factor Receptor/ErbB2 and Vascular Endothelial Growth Factor Receptor Tyrosine Kinase Inhibitor with Antitumor and Antiangiogenic Activity Cancer Res., July 15, 2004; 64(14): 4931 - 4941. [Abstract] [Full Text] [PDF]
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D. W. Davis, K. Inoue, C. P. N. Dinney, D. J. Hicklin, J. L. Abbruzzese, and D. J. McConkey Regional Effects of an Antivascular Endothelial Growth Factor Receptor Monoclonal Antibody on Receptor Phosphorylation and Apoptosis in Human 253J B-V Bladder Cancer Xenografts Cancer Res., July 1, 2004; 64(13): 4601 - 4610. [Abstract] [Full Text] [PDF]
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T. Inai, M. Mancuso, H. Hashizume, F. Baffert, A. Haskell, P. Baluk, D. D. Hu-Lowe, D. R. Shalinsky, G. Thurston, G. D. Yancopoulos, et al. Inhibition of Vascular Endothelial Growth Factor (VEGF) Signaling in Cancer Causes Loss of Endothelial Fenestrations, Regression of Tumor Vessels, and Appearance of Basement Membrane Ghosts Am. J. Pathol., July 1, 2004; 165(1): 35 - 52. [Abstract] [Full Text] [PDF]
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J. L. Evelhoch, P. M. LoRusso, Z. He, Z. DelProposto, L. Polin, T. H. Corbett, P. Langmuir, C. Wheeler, A. Stone, J. Leadbetter, et al. Magnetic Resonance Imaging Measurements of the Response of Murine and Human Tumors to the Vascular-Targeting Agent ZD6126 Clin. Cancer Res., June 1, 2004; 10(11): 3650 - 3657. [Abstract] [Full Text] [PDF]
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A. M. Kamat, T. Karashima, D. W. Davis, L. Lashinger, M. Bar-Eli, R. Millikan, Y. Shen, C. P. N. Dinney, and D. J. McConkey The proteasome inhibitor bortezomib synergizes with gemcitabine to block the growth of human 253JB-V bladder tumors in vivo Mol. Cancer Ther., March 1, 2004; 3(3): 279 - 290. [Abstract] [Full Text]
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 J. Schoenberger, D. Grimm, P. Kossmehl, M. Infanger, E. Kurth, and C. Eilles Effects of PTK787/ZK222584, a Tyrosine Kinase Inhibitor, on the Growth of a Poorly Differentiated Thyroid Carcinoma: An Animal Study Endocrinology, March 1, 2004; 145(3): 1031 - 1038. [Abstract] [Full Text] [PDF]
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Y. van Hensbergen, H. J. Broxterman, S. Rana, P. J. van Diest, M. C. A. Duyndam, K. Hoekman, H. M. Pinedo, and E. Boven Reduced Growth, Increased Vascular Area, and Reduced Response to Cisplatin in CD13-Overexpressing Human Ovarian Cancer Xenografts Clin. Cancer Res., February 1, 2004; 10(3): 1180 - 1191. [Abstract] [Full Text] [PDF]
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P. Marzola, A. Degrassi, L. Calderan, P. Farace, C. Crescimanno, E. Nicolato, A. Giusti, E. Pesenti, A. Terron, A. Sbarbati, et al. In Vivo Assessment of Antiangiogenic Activity of SU6668 in an Experimental Colon Carcinoma Model Clin. Cancer Res., January 15, 2004; 10(2): 739 - 750. [Abstract] [Full Text] [PDF]
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 A R Padhani MRI for assessing antivascular cancer treatments Br. J. Radiol., December 1, 2003; 76(suppl_1): S60 - S80. [Abstract] [Full Text] [PDF]
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J. S. Beebe, J. P. Jani, E. Knauth, P. Goodwin, C. Higdon, A. M. Rossi, E. Emerson, M. Finkelstein, E. Floyd, S. Harriman, et al. Pharmacological Characterization of CP-547,632, a Novel Vascular Endothelial Growth Factor Receptor-2 Tyrosine Kinase Inhibitor for Cancer Therapy Cancer Res., November 1, 2003; 63(21): 7301 - 7309. [Abstract] [Full Text] [PDF]
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B. Ruggeri, J. Singh, D. Gingrich, T. Angeles, M. Albom, H. Chang, C. Robinson, K. Hunter, P. Dobrzanski, S. Jones-Bolin, et al. CEP-7055: A Novel, Orally Active Pan Inhibitor of Vascular Endothelial Growth Factor Receptor Tyrosine Kinases with Potent Antiangiogenic Activity and Antitumor Efficacy in Preclinical Models Cancer Res., September 15, 2003; 63(18): 5978 - 5991. [Abstract] [Full Text] [PDF]
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, avascular areas; 
, tumor vessels.