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Ann Thorac Surg 2001;71:1657-1665
© 2001 The Society of Thoracic Surgeons
a Department for General Surgery and Thoracic Surgery, Christian-Albrechts-University, Kiel, Germany
b Molecular Oncology Research Division, Christian-Albrechts-University, Kiel, Germany
c Department of Pathology, Christian-Albrechts-University, Kiel, Germany
Accepted for publication December 14, 2000.
Address reprint requests to Dr Boehle, Department of General Surgery and Thoracic Surgery, Christian-Albrechts-University, Arnold-Heller-Strasse 7, D-24105 Kiel, Germany
e-mail: boehle{at}surgery.uni-kiel.de
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Abstract |
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Methods. Cytostatic and cytotoxic effects of CA-4PD on selected NSCLC cells, Colo-699 and KNS-62, were studied in vitro. After subcutaneous xenotransplantation the effect of systemically administrated CA-4PD on tumor growth was investigated in vivo. A newly established orthotopic xenotransplant model was employed to estimate prolongation of survival after intrapulmonary tumor induction with secondary metastatic disease.
Results. In vitro, CA-4PD displayed a time and dose dependent antiproliferative effect on human lung cancer cells. In vivo, CA-4PD significantly delayed growth of subcutaneously induced lung cancer. This growth delay was translated into a prolongation of survival in the metastasizing orthotopic xenotransplant model.
Conclusions. In vitro CA-4PD inhibits proliferation of NSCLC cells, most likely by disruption of microtubule assembly. In vivo, systemic treatment inhibits growth of subcutaneously xenotransplanted tumors by an antivascular effect. In the case of metastasizing human lung cancer this translated into a prolongation of survival.
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Introduction |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Growth of a primary malignancy or metastatic lesion above an average volume of 1 mm3 inevitably demands the generation of new blood vessels, as nutritive supply can no longer be provided by means of diffusion. Tumors can persist for months and years as an avascular lesion, where tumor cell proliferation and apoptosis reaches equilibrium. Once a subgroup of tumor cells switches to an angiogenic phenotype the tumor becomes vascularized and may start to grow exponentially [2].
The principle of angiogenesis is valid for numerous solid malignant tumors, which makes antiangiogenic strategies become a unifying concept in cancer therapy. Advantages of antiangiogenic treatment are (1) ease of access of drugs to the endothelial cell compartment, (2) targeting of a genetically stable cell population (endothelial vs tumor cells), thereby lessening the chance of drug resistance, and (3) amplification achieved because one endothelial cell supports the growth of 50 to 100 tumor cells [3]. Combretastatin A-4 was originally isolated from Combretum caffrum, a Southern African shrub [4]. It has a high affinity for tubulin at or near the colchicine binding site, causing destabilization of the tubulin polymers of the cytoskeleton [5]. Solubility in aqueous solution was improved by conversion to disodium combretastatin A4 3-O-phosphate (combretastatin A-4 prodrug, CA-4PD) from which the phosphate group is cleaved by endogenous nonspecific phosphatases under physiological conditions [6], thereby transforming the prodrug into its biologically active form. In contrast to colchicine and other tubulin-binding agents that have been investigated as agents for disrupting tumor vasculature and for which dosage is limited by toxicity, combretastatin A-4 (CA-4) is active at one-tenth of the maximum tolerated dose, offering a wide therapeutic window [7].
Combretastatin A-4 was characterized to act as an antivascular agent, causing vascular shutdown in pre-existing tumor vessels by a preferential effect on tumor vascular endothelium, leading to extensive secondary tumor cell death [8]. CA-4PD was demonstrated to have potent antivascular effects in various rodent experimental tumor models as the P22 rat carcinosarcoma [9], murine liver metastases [10], the KHT sarcoma in mice [11], the C3H mammary tumor in mice [12], and the murine colon 26 adenocarcinoma [13]. The aim of this study was to investigate whether antivascular treatment of human NSCLC is effective in a newly established preclinical murine xenotransplantation model [14].
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Material and methods |
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Cells were cultured as monolayers in 75-cm2 flasks using RPMI 1640 supplemented with 10% fetal calf serum, 1% sodium-pyruvate, and 1% L-glutamine (Life Technologies, Paisley, Scotland). Cells were maintained at 37°C in a humidified incubator gased with 5% CO2 and 95% air. When cells grew to approximately 80% confluency, they were subcultured or harvested using trypsin EDTA (Life Technologies).
Cells to be xenotransplanted were washed in PBS buffer (Life Technologies), resuspended in serum-free culture medium, counted in a hemocytometer, and equilibrated at a density of 2 x 106 cells in 50 µL injection volume.
Cell viability was tested by trypan blue staining. All cell cultures were proved to be free of mycoplasma infection by RT-PCR of supernatants from densely growing cells following the instructions of the manufacturer (Takara Shuzo, Japan).
H3-thymidine incorporation assay
For determination of a cytostatic effect of CA-4PD in vitro H3-thymidine incorporation assays were performed; then 1 x 104 cells were seeded in 100-µL culture medium in 96-well microtiter plates. Cells were allowed to attach overnight. Cells were exposed to CA-4PD in concentrations ranging from 10-8 to 10-4 mol/L for 3 hours and 24 hours, respectively; then 0.02 µCi tritiated thymidine (43.0 Ci/mmol, Amershampharmaciabiotech, Braunschweig, Germany) were added to each well 3 hours before cell harvest. After washing with PBS buffer three times, cells were lysed by incubation with trypsin EDTA. The lysate was filtered through a fiberglass filtermat (Wallac Oy, Turku, Finland) by a 96-well cell harvester (Inotech, Fairfax, VA). The filtermat was dried in a microwave oven and placed in a MicroBeta sample bag; then 4.5 mL scintillation fluid was added and the bag heat-sealed. Counting was performed in a 1450 MicroBeta scintillation counter (Wallac Oy, Turku, Finland). Each sample was prepared sixfold.
The recovery of incorporated H3-thymidine in the DNA of proliferating cells was estimated for determination of inhibition of proliferation. Each measurement was performed sixfold.
Cell viability assay
To determine CA-4PD–induced cytotoxicity, cell viability assays were performed, following the manufacturer’s instructions (EZ4U, Biomedica, Vienna, Austria). First, 5 x 103 cells in 200-µL culture medium per well were seeded in 96-well microtiter plates. Cells were allowed to attach overnight. Cells were exposed to CA-4PD in concentrations ranging from 10-8 to 10-4 mol/L for 3 hours and 24 hours, respectively. Cell viability was estimated photometrically. Each measurement was performed sixfold.
Experimental animals
All animal experiments were approved by the Review Board of the Ministry of Schleswig-Holstein. All animal have received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (1996).
Pathogen-free female SCID bg mice (Harlan Winkelmann, Borchen, Germany) were maintained in sterile polycarbonate microisolator cages under pathogen-free conditions, fed autoclaved food and water ad libitum, and handled under stringent sterile conditions in a laminar flow hood. For induction of subcutaneous tumors, 2 x 106 tumor cells were injected subcutaneously into the right flank. Animals were divided into two treatment groups and two control groups (n = 6 each) and were monitored daily for tumor growth. Tumors were gauged percutaneously in two diameters. Tumor volume was calculated by V = L x W2 x 0.52 (V = volume, L = length, W = width). At a tumor volume of 100 mm3 systemic treatment was started by daily intraperitoneal injection of 50 mg/kg body weight of CA-4PD. Control groups were injected by sterile saline intraperitoneally in corresponding volumes. After treatment was stopped animals were sacrificed by CO2 inhalation and tumors removed for histological examination.
For induction of intrapulmonary tumor growth, mice were anesthetized by intraperitoneal injection of Avertin (Sigma-Aldrich, Steinheim, Germany) 240 mg/kg and 2 x 106 tumor cells introduced below the visceral pleura of the left lung [14]. Animals were divided into two treatment groups of 6 animals each and control groups of 9 animals each. Cells were allowed to settle at the site of implantation and systemic treatment was started on day 8 postoperatively by daily intraperitoneal injection of 50 mg/kg body weight of CA-4PD for 3 weeks. Control animals were injected by sterile saline in corresponding volume. All animals were monitored daily for signs of respiratory distress or physical discomfort. When animals presented with respiratory insufficiency they were sacrificed by CO2 inhalation and tumors were removed for histological examination.
Histological examination
Tissues were fixed in formalin and stained with hematoxylin and eosin using standard procedures. Areas of tumor necrosis were estimated visually. For immunohistochemistry fresh tumor tissue was snap-frozen in liquid nitrogen.
Microvessel density
For immunohistochemical staining of intratumoral vascular endothelium, cryopreserved sections were stained by CD31 antibody (Pharmingen, Hamburg, Germany) following the manufacturer’s instructions for detection by employing APAAP reaction. Counterstaining was by hemalaun. According to the method of Weidner [15], regions with elevated vascular density were identified and the number of microvessel entities per optical field (x200 magnification) subsequently counted within these spots.
Angiogenic cytokines
Cryopreserved sections were stained for human VEGF (antibody sc-152, Santa Cruz Biotechnology, Santa Cruz, CA) and bFGF (antibody sc-79, Santa Cruz Biotechnology). Detection with Peroxidase Vectastatin Elite ABC Kit (Vector, Burlingame, CA). As a semiquantitative measure a staining index according to Mattern [16] was employed. Per 1000 tumor cells the percentage of positively stained cells was graded as: 0 = no staining, 1 = less than 25% positive cells, 2 = 25% to 50% positive cells, 3 = more than 50% positive cells. Staining intensity was graded as: 0 = no staining, 1 = weak, 2 = mean intensity, 3 = intense staining. Grades for percentage of stained cells and staining intensity were added as staining index, ranging from 0 to 6.
Apoptosis and proliferation
Cryopreserved tumor sections were stained for markers of apoptosis and proliferation. Antibody MIB1 (anti-Ki 67, Dianova, Hamburg, Germany) served as a marker of tumor cell proliferation as detected by an animal research kit (DAKO Diagnostika GmbH, Hamburg, Germany). Fragmented DNA indicative for apoptosis was detected using the Tacs TdT kit, (R&D Systems, Nordenstadt, Germany) following the manufacturer’s instructions; 1000 cells per section were counted and the percentage of positively stained tumor cells estimated.
Immunocompetence assay
When animals were sacrificed, blood was withdrawn and serum was isolated. Cryopreserved histologic sections of the corresponding tumor were incubated at 4°C overnight with 50 µL murine serum. Sections were washed in PBS three times and incubated for 1 hour with a peroxidase coupled rabbit antimouse IgG and IgM antibody in 1:1000 dilution (Dianova, Hamburg, Germany). After careful rinsing with PBS, staining was performed by adding peroxidase-H2O2-diaminobenzidine (DAB) substrate solution for 10 minutes. Nuclei were counterstained by hematoxylin. Animals displaying specific binding of murine antibodies against the human tumor tissue were rated as immunocompetent and excluded from the study.
Statistical analyses
Statistical analyses of the effects of CA-4PD on cell proliferation and viability, microvessel density (MVD), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF) indices, cell proliferation and apoptosis, and median survival of animals compared to controls were carried out using the Levene test followed by the two-tailed t test for two samples of unequal variance. Tumor growth curves compared by analysis of variance with two factors repeated measures. Final tumor volumes were compared by the Mann-Whitney U test. All calculations were performed with the statistical package SPSS for PC (SPSS, Chicago, IL).
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Results |
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Effect of CA-4PD on subcutaneously xenotransplanted human lung cancer
For determination of growth inhibition of human lung cancer by CA-4PD in vivo, tumors were subcutaneously xenotransplanted. The implantation rate was 100%. There were no procedure-related deaths or complications. Tumors grew as solitary, solid tumors without local or systemic distribution of metastasis. Tumors induced by subcutaneous injection of Colo-699 reached a volume of 100 mm3 after 23 days on average, when systemic treatment was started. During the 3-week treatment period there were no clinical signs of drug-related toxicity. Untreated tumors continued an exponential growth up to a mean volume (± SD) of 6036 mm3 (±1,655) on day 42. In comparison, tumors treated by systemical CA-4PD injection demonstrated a significant growth inhibition. Average tumor volume on day 42 was 313 mm3 (±166). These differences were of statistical significance (p < 0.01; Fig 3).
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Microvessel density
Microvessel counting did not result in significant differences between treated tumors and untreated controls. Microvessel density in Colo-699 tumors after treatment was 85 vessels (±18.6) per optical field compared with 84.6 (±19) in controls. In KNS-62, MVD was 86.4 (±26.8) in the treatment group compared with 67.8 (±12.6) in untreated controls (Fig 5).
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Previous studies demonstrated Combretastatin A-4 to display in vitro a selective toxicity toward proliferating human umbilical vein endothelial cells [7]. This effect was mediated by the induction of apoptosis [18]. In the employed xenotransplant tumor model malignant cells were of human origin, but the endothelial lining of the newly formed tumor vessels was derived from the murine endothelium by angiogenesis. In previous studies we demonstrated that CA-4PD inhibits proliferation of immortalized murine endothelial cells in vitro [19], thereby displaying a certain selectivity compared to proliferating murine fibroblasts.
In addition to the antiproliferative effect against endothelial cells, CA-4PD displayed a cytostatic effect on proliferating tumor cells Colo-699 and KNS-62 in vitro. The antiproliferative effect of CA-4PD was demonstrated to be both time and dose dependent. Both cell lines were of high mitotic activity with a doubling time of 38 hours and 35 hours, respectively. This high mitotic activity probably sensitized the cells for the cytostatic effect in vitro by disruption of microtubule assembly during mitosis. As cytotoxicity of CA-4PD was described for human umbilical vein endothelial cells by Iyer and colleagues [18], KNS-62 and COLO-699 cells were exposed to CA-4PD in a cell viability assay to estimate a potentially cytotoxic effect; 3 hours of exposure at a 10-4 mol/L concentration reduced cell viability by 31% in both cell lines, compared to a 37% and 69% inhibition of proliferation. Prolongation of drug exposure to 24 hours reduced cell viability by 46% and 55%, respectively, compared to an almost complete stop in proliferation. These findings suggest that approximately 50% of tumor cells undergo in vitro cell death after disruption of mitosis by microtubule assembly. In summary, CA-4PD displays in vitro a significant inhibitory effect on the proliferation of murine endothelial cells and a cytostatic and attributable cytotoxic effect on the evaluated proliferating human lung cancer cells. Similar observations have been reported by Zhang and colleagues [20] and el-Zayat and associates [21].
A murine xenotransplant model was used to investigate whether the antiproliferative effect of CA-4PD in vitro could be reproduced by a growth inhibition of solid tumors in vivo. So far, in vivo studies have used rodent tumors [9–13]. This is, to our knowledge, the first study of antivascular treatment in a human lung cancer xenotransplantation model.
A significant reduction of tumor perfusion up to 90% with marked subsequent cell death after administration of a single dose of CA-4PD has been described by several authors [7–12]; but Chaplin and coworkers [22] reported that, despite the induction of extensive central tumor necrosis, this was not translated into any significant effect on the growth of solid tumors. The continued growth was supposed to be attributed to an actively proliferating population of cells at the periphery of the tumor, which obtained nutritive supply by diffusion from the surrounding tissue, apparently independent from tumor vasculature. Therefore, a regimen of daily repeated intraperitoneal injections of CA-4PD for 21 days was chosen to augment the antitumoral effect of single-dose administration with respect to the short plasma half-life of 15 minutes and possible recovery of intratumoral vessels [9]. This regimen resulted in significant growth delay of subcutaneously induced human adenocarcinoma and squamous cell carcinoma of the lung. After 21 days of systemic treatment squamous cell carcinomas reached only 31% of the volume of the untreated control tumors. In adenocarcinoma the difference was even more marked; treated tumors grew only up to a volume of 5% of the untreated controls. Treated tumors displayed, on histologic examination, large central necrosis with disruption of tumor vasculature, but there was a rim of 6 to 10 viable cell layers in the tumor periphery. Tumor growth delay was of statistical significance. However, in contrast to the observed complete inhibition of proliferation in vitro, tumors exposed to CA-4PD continued to grow in vivo; as the adenocarcinoma tripled its volume within 21 days of therapy, the squamous cell carcinoma increased its volume even fivefold. Differences in the efficacy of CA-4PD in vitro and in vivo may be explained by reduced bioavailabity of CA-4PD in solid tumor tissue compared with an in vitro cell culture, resulting in a reduced exposure of both tumor and endothelial cells toward the drug. The plasma half-life of CA-4PD in female CBA mice was estimated to last only 15 minutes; binding to serum albumin may cause a certain loss of antitumor efficacy for CA-4, as demonstrated by physiologic serum concentrations in vitro [23]. The transformation of CA-4PD by endogenous phosphatases into its pharmacologically active form makes the endothelial cell compartment in vivo likely as the major site of action, as endothelial cells have previously been demonstrated to be especially rich in phosphatases [24]. This was supported by Tozer and coworkers [9], who demonstrated in an isolated perfusion model that CA-4 is accumulated in its activated form in tumor tissue in an eightfold higher concentration compared to skeletal muscle, where concentrations were equal to concentration in the perfusate. The capacity for dephosphorylation of CA-4PD in tumor homogenate was six times higher compared to that in skeletal muscle, most likely to be derived from the tumor vasculature.
Systemic treatment of xenotransplanted animals by CA-4PD did not induce a significant change in intratumoral microvessel density. This suggests a constant ratio between intracapillary endothelia and tumor cells. A reduction in microvessels by antivascular treatment subsequently induces a reduction in tumor cells due to reduced nutritive supply, resulting in a reduction in absolute tumor size, still displaying a constant ratio between proliferating tumor cells and endothelial cells. Furthermore, this finding implies that the observed inhibition of tumor growth in vivo is predominantly induced by an antivascular effect, as a mainly antitumorcell effect might have resulted in an increase in the MVD, as tumor cells were destroyed whereas endothelial cells remained.
Systemic treatment of xenotransplanted animals by CA-4PD resulted in an increase in angiogenic cytokines VEGF and bFGF. This observation illustrates the predominantly antivascular effect of CA-4PD in vivo, as CA-4PD significantly reduces in vivo intratumoral blood flow [8], subsequently inducing intratumoral hypoxia, which is known to be a strong stimulus for secretion of angiogenic cytokines [25]. Immunohistochemical staining demonstrated a marked reduction in proliferating tumor cells of 41% (Colo-699) and 24% (KNS-62) by systemic CA-4PD application. Whether this effect is delivered subsequent to reduced intratumoral blood flow or as a direct cytostatic effect remains unanswered. The observation that there was no increase in intratumoral apoptosis may be interpreted as a sign of reduced nutritive supply, preventing cells from further proliferation, and less as a sign of direct tumor cell killing by CA-4PD.
To determine whether growth delay of a subcutaneously induced carcinoma translates into prolonged survival of animals bearing metastasizing intrapulmonary tumors, the effect of systemic intraperitoneal administration of CA-4PD was investigated in a newly established orthotopic xenotransplant model of human lung carcinoma in SCID bg mice. In contrast to the nonmetastasizing model of subcutaneous tumor induction, this model is lethal because of secondary metastatic disease.
Systemic treatment with CA-4PD increased animal survival by 29% and 35%, respectively. This prolongation of survival was of statistical significance. In comparison, the primary intrapulmonary tumors of the animals in the treatment group appeared to be somewhat smaller, but at the time that respiratory insufficiency was observed, the metastatic tumor burden appeared to be equal to that of the animals serving as controls. Although it has been speculated that antivascular therapy might reduce the occurrence of metastases, CA-4PD failed to demonstrate this in our model. This finding might be explained by the macroscopic and histologic observation that the metastases grew preferentially as avascular lesions with a miliar distribution in the mediastinum and both pleural cavities measuring less than 1 mm in diameter. Immunohistochemical staining of the vascular endothelium by the antibody CD 31 in the metastatic lesion revealed in most cases the absence of neovascularization, indicating that the nutritive supply of the metastatic lesions is achieved by means of diffusion, thereby neutralizing the antivascular effects of CA-4PD. The prolongation of survival might therefore be preferentially mediated by a antiproliferative effects on the tumor cells, delaying the growth of metastases. For further investigation it might be favorable to make use of a larger-scale animal model, as the biological relevance of a 1-mm metastatic lesion might be less in a larger organism, thereby resulting in a more pronounced effect on animal survival.
In summary, CA-4PD was demonstrated to have a significant antiproliferative effect on human NSCLC cells in vitro. In vivo, proliferating endothelial cells in newly generated tumor vessels appear to be the major target of CA-4PD. The antivascular action of CA-4PD results in inhibition of subcutaneous tumor growth and prolongation of survival in animals with metastasizing human NSCLC, although metastatic spread was not inhibited.
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Acknowledgments |
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