Tumor Vasculature Cells Research Articles

Engineered Annexin A5 Variants Have Impaired Cell Entry for Molecular Imaging of Apoptosis Using Pretargeting Strategies

Phosphatidylserine (PS) on apoptotic cells is a target for diagnosis and therapy using annexin A5 (anxA5). Pretargeting is a strategy developed to improve signal to background ratio for molecular imaging and to minimize undesired side effects of pharmacological and radiotherapy. Pretargeting relies on accessibility of the target finder on the surface of the target cell. anxA5 binds PS and crystallizes in a two-dimensional network covering the PS-expressing cell surface. Two-dimensional crystallization is the driving force for anxA5 internalization by PS-expressing cells. Here, we report structure/function analysis of anxA5 internalization. Guided by structural bioinformatics including protein-protein docking, we revealed that the amino acids Arg(63), Lys(70), Lys(101), Glu(138), Asp(139), and Asn(160) engage in intermolecular salt bridges within the anxA5 trimer, which is the basic building block of the two-dimensional network. Disruption of the salt bridges by site-directed mutagenesis does not affect PS binding but inhibits trimer formation and cell entry of surface-bound anxA5. The anxA5 variants with impaired internalization are superior molecular imaging agents in pretargeting strategies as compared with wild-type anxA5.

Melanoma cell‐secreted soluble factor that stimulates ubiquitination and degradation of the interferon alpha receptor and attenuates its signaling

Melanoma cell‐secreted soluble factor that stimulates ubiquitination and degradation of the interferon alpha receptor and attenuates its signaling

Solid‐tumor radionuclide therapy dosimetry: New paradigms in view of tumor microenvironment and angiogenesis

The objective of this study is to evaluate requirements for radionuclide-based solid tumor therapy by assessing the radial dose distribution of beta-particle-emitting and alpha-particle-emitting molecules localized either solely within endothelial cells of tumor vasculature or diffusing from the vasculature throughout the adjacent viable tumor cells. Tumor blood vessels were modeled as a group of microcylindrical layers comprising endothelial cells (one-cell thick, 10 microm diameter), viable tumor cells (25-cell thick, 250 microm radius), and necrotic tumor region (> 250 microm from any blood vessel). Sources of radioactivity were assumed to distribute uniformly in either endothelial cells or in concentric cylindrical 10 microm shells within the viable tumor-cell region. The EGSnrc Monte Carlo simulation code system was used for beta particle dosimetry and a dose-point kernel method for alpha particle dosimetry. The radioactive decays required to deposit cytocidal doses (> or = 100 Gy) in the vascular endothelial cells (endothelial cell mean dose) or, alternatively, at the tumor edge [tumor-edge mean dose (TEMD)] of adjacent viable tumor cells were then determined for six beta (32P, 33P, 67Cu, 90Y 131I, and 1188Re) and two alpha (211At and 213Bi) particle emitters. Contrary to previous modeling in targeted radionuclide therapy dosimetry of solid tumors, the present work restricts the region of tumor viability to 250 microm around tumor blood vessels for consistency with biological observations. For delivering > or = 100 Gy at the viable tumor edge (TEMD) rather than throughout a solid tumor, energetic beta emitters 90Y, 32P, and 188Re can be effective even when the radionuclide is confined to the blood vessel (i.e., no diffusion into the tumor). Furthermore, the increase in tumor-edge dose consequent to beta emitter diffusion is dependent on the energy of the emitted beta particles, being much greater for lower-energy emitters 131I, 67Cu, and 33P relative to higher-energy emitters 90Y, 32P, and 188Re. Compared to alpha particle emitters, a approximately 150-400 times higher number of beta-particle-emitting radioactive atoms is required to deposit the same dose in tumor neovasculature. However, for the alpha particle emitters 211At and 213Bi to be effective in irradiating viable tumor-cell regions in addition to the vasculature the carrier molecules must diffuse substantially from the vasculature into the viable tumor. The presented data enable comparison of radionuclides used for antiangiogenic therapy on the basis of their radioactive decay properties, tumor neovasculature geometry, and tumor-cell viability. For alpha particle emitters or low-energy beta particle emitters, the targeting carrier molecule should be chosen to permit the radiopharmaceutical to diffuse from the endothelial wall of the blood vessel, while for long-range energetic beta particle emitters that target neovasculature, a radiopharmaceutical that binds to newly formed endothelial cells and does not diffuse is preferable. The work is a first approximation to modeling of tumor neovasculature that ignores factors such as pharmacokinetics and targeting capability of carrier molecules. The calculations quantify the interplay between irradiation of neovasculature, the surrounding viable tumor cells, and the physical properties of commonly used radionuclides and can be used to assist estimation of radioactivity to be administered for neovasculature-targeted tumor therapy.

Bevacizumab enhances chemosensitivity of hepatocellular carcinoma to adriamycin related to inhibition of survivin expression

In recent years, anti-angiogenesis drugs have shown promising clinical effects against many tumors, particularly in combination with chemotherapy. Although the combination has become a standard of care for many tumors, the mechanisms of the chemosensitizing activity of anti-angiogenic drugs are not fully understood. Here, we sought to determine if anti-angiogenesis drug bevacizumab could enhance the chemosensitivity of HCC by inhibition of survivin. After treatment of human umbilical vein endothelial cells (HUVECs) and hepatocellular carcinoma (HCC) cell line PLC/PRF/5 (PLC) with bevacizumab or/and adriamycin, the direct effects were examined by survival assays, and the expression of Akt, Phospho-Akt and survivin were evaluated by western blot. Tumor growth was observed in a human HCC xenograft nude mouse model treated with different drugs, and the expression of PCNA, CD31 and survivin in tumor tissues were evaluated by means of immunohistochemistry. Bevacizumab enhanced the chemosensitivity of HCC by inhibiting the VEGF-PI3K/Akt-survivin signaling cascade in endothelial cells. The combination of bevacizumab with adriamycin therapy resulted in better outcomes compared with monotherapy in hepatocellular carcinoma xenografts; bevacizumab significantly inhibited tumor angiogenesis and growth. In addition, bevacizumab reduced survivin expression in tumor tissues, including tumor vascular endothelial cells in vivo, although it did not inhibit survivin expression in tumor cells in vitro. These results implicate the bevacizumab-increased efficacy of adriamycin via an inhibition of survivin expression in malignant cells as well as tumor vasculature cells, which provides other insights into the mechanism of enhanced efficacy by combination of VEGF blocker and chemotherapeutic agents.

Abstract 384: Effects of endostar on cell functions and biological characteristics in tumor-derived endothelial cells

Abstract Tumor microvessels are essential to tumor formation, growth, and metastasis. Interventions targeting the microvessels would lead to avascular necrosis of tumor cells. Therefore, specifically targeting endothelial cells in tumor vasculature had become one of the most important strategies for cancer therapy. Endostar, a novel recombinant human endostatin with an additional nine-amino acid sequence, is a novel anti-angiogenesis drug. This present study is to determine how endostar affects the behavior of endothelial cells and tumor-derived endothelial cells both in vitro and in vivo. Human umbilical vein endothelial cells (HUVEC) were cultured and differentiated to tumor-derived endothelial cells (Td-ECs) after co-culturing with conditioned medium from A549 cells, a lung cancer cell line. The anti-angiogenesis effects of endostar on Td-ECs were examined by MTT assay, cell migration assay, flow cytometry, and TUNEL assay. Endostar exhibited a significant inhibitory effect both time and dose dependently in Td-EC cells. In addition, Endostar significantly inhibited cell migration and induced G0/G1 and S phase cell cycle arrest and apoptosis. However, under the same concentrations, Endostar has little impact on HUVEC. To study the in vivo effects, a xenograft model in nude mice was established. Endostar was injected intratumorally as localized administration, and tumor growth was closely monitored during the following days. As a result, endostar showed significant anti-angiogenesis effect and tumor growth inhibition. The concentration of Ca2+ in the treatment groups measured by fluorescence spectrometry was elevated significantly compared to that in the control group. This study revealed a novel activity of endostar in inhibiting angiogenesis, more potently in tumor-derived endothelial cells than in normal endothelial cells. This finding suggests the potential use of endostar as cancer therapeutic to be tested in clinical trials. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 384.

Abstract 132: Cytoprotective induction of nitric oxide synthase in a cellular model of 5-aminolevulinic acid-based photodynamic therapy

Abstract Photodynamic therapy (PDT) employs a photosensitizing agent, molecular oxygen, and visible light to produce reactive oxygen species that kill tumor and tumor vasculature cells. Nitric oxide (NO) produced by these cells could be pro-carcinogenic by inhibiting apoptosis or promoting angiogenesis and tumor growth. Our previous studies showed that NO from a chemical donor made COH-BR1 breast tumor cells hyperresistant to apoptotic photokilling induced by photoactivation of 5-aminolevulinic acid (ALA)-generated protoporphyrin IX localized in mitochondria. Hyperresistance was associated with inhibition of proapoptotic JNK and p38α MAP kinase phosphorylation-activation. The purpose of this study was to determine whether tumor cells upregulate NOS-generated NO as a cytoprotective measure during PDT.COH-BR1 cells sensitized in mitochondria with ALA-derived protoporphyrin IX died apoptotically following irradiation as measured by Hoechst staining. Western analysis revealed that inducible nitric oxide synthase (iNOS) was upregulated >3-fold within 4 h after ALA/light treatment, while other NOS isoforms were unaffected. Exposing cells to the iNOS inhibitor, 1400W during photochallenge enhanced caspase-3/7 activation and apoptotic killing up to 2-3-fold while substantially reducing chemiluminescence-assessed NO production, suggesting that this NO was cytoprotective. Consistently, the NO scavenger cPTIO enhanced ALA/light-induced caspase-3/7 activation and apoptotic kill by >2.5-fold. Of added significance, cells could be rescued from 1400W-exacerbated apoptosis by an exogenous NO donor, spermine-NONOate. Using Hoechst and TUNEL staining, we found that short hairpin RNA (shRNA)-induced knockdown of iNOS enhanced the apoptotic kill of ALA/light treated COH-BR1 cells. ALA/light-treated COH-BR1cells exhibited a transient post-irradiation activation of JNK and p38α as measured by Western blot analysis. Consistently, both effects were intensified and prolonged by 1400W. The survival MAP kinase ERK1/2 was deactivated more rapidly when 1400W was present during a PpIX/light challenge. Similar effects on MAP kinase activation/deactivation were observed for iNOS knockdown cells under photostress cinfirming iNOS's protective role. As demonstrated for non-photodynamic stress systems, NO could have interfered with apoptosis by inactivating participating MAP kinases and/or caspases. This is the first reported evidence for increased tumor cell resistance due to iNOS upregulation in a PDT model. Our findings indicate that stress-elicited NO in PDT-treated tumors could compromise therapeutic efficacy, and suggest NOS-based pharmacologic interventions for preventing this. (Supported by NIH Grant CA70823) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 132.

Cytoprotective induction of nitric oxide synthase in a cellular model of 5-aminolevulinic acid-based photodynamic therapy

Photodynamic therapy (PDT) employs a photosensitizing agent, molecular oxygen, and visible light to generate reactive species that kill tumor and tumor vasculature cells. Nitric oxide produced by these cells could be procarcinogenic by inhibiting apoptosis or promoting angiogenesis and tumor growth. The purpose of this study was to determine whether tumor cells upregulate NO as a cytoprotective measure during PDT. Breast tumor COH-BR1 cells sensitized in their mitochondria with 5-aminolevulinic acid (ALA)-derived protoporphyrin IX died apoptotically after irradiation, ALA- and light-only controls showing no effect. Western analysis revealed that inducible nitric oxide synthase (iNOS) was upregulated > 3-fold within 4 h after ALA/light treatment, whereas other NOS isoforms were unaffected. Exposing cells to a NOS inhibitor (L-NAME or 1400W) during photochallenge enhanced caspase-3/7 activation and apoptotic killing up to 2- to 3-fold while substantially reducing chemiluminescence-assessed NO production, suggesting that this NO was cytoprotective. Consistently, the NO scavenger cPTIO enhanced ALA/light-induced caspase-3/7 activation and apoptotic kill by > 2.5-fold. Of added significance, cells could be rescued from 1400W-exacerbated apoptosis by an exogenous NO donor, spermine-NONOate. This is the first reported evidence for increased tumor cell resistance due to iNOS upregulation in a PDT model. Our findings indicate that stress-elicited NO in PDT-treated tumors could compromise therapeutic efficacy and suggest NOS-based pharmacologic interventions for preventing this.

Signaling events in apoptotic photokilling of 5-aminolevulinic acid-treated tumor cells: Inhibitory effects of nitric oxide

Antitumor photodynamic therapy (PDT) employs a photosensitizing agent, molecular oxygen, and visible light to produce reactive oxygen species that can destroy tumor and tumor vasculature cells. NO produced by these cells could be procarcinogenic by inhibiting apoptosis and promoting angiogenesis and tumor growth. We recently showed that NO from a chemical donor or activated macrophages makes COH-BR1 breast tumor cells more resistant to photokilling sensitized by 5-aminolevulinic acid (ALA)-generated protoporphyrin IX (PpIX). Signaling events associated with this hyperresistance have now been examined. ALA-treated COH-BR1 cells containing mitochondria-localized PpIX died mainly by apoptosis after being irradiated. Underlying redox signaling associated with MAP kinase (ERK1/2, p38, JUN) phosphorylation–activation, and heme oxygenase-1 (HO-1) upregulation was studied using immunoprecipitation and Western blot methodology. ALA/light treatment resulted in activation of proapoptotic JNK and p38α, and deactivation of prosurvival p38β and ERK1/2. Involvement of both JNK and p38 in apoptosis was established by using a specific inhibitor for each. Spermine NONOate-derived NO, introduced immediately before irradiation, provided substantial protection against apoptosis. This was accompanied by greater HO-1 induction and a strong inhibition of each MAP kinase effect seen in the absence of NO. Downstream of JNK and p38α activation, a marked upregulation/activation of proapoptotic Bax and Bid was observed along with down-regulation of antiapoptotic Bcl-xL, each response being reversed by NO. These findings provide new insights into signaling activity associated with the intrinsic apoptotic pathway in ALA-PDT and how this activity can be modulated by NO.

Targeting of chemotherapy to Tie-2-receptor positive angiogenic endothelial cells of tumor vasculature.

Abstract Abstract #902 Background: There are at least three types of cells in tumor tissue which express the Tie2 receptor (Tie2R): dividing vascular endothelial cells (TVECs), Tie2R-expressing monocytes (TEMs) and Tie2R-expressing mesenchymal progenitor cells (TMPCs).
 
 We tested if the targeting of chemotherapy to these classes of Tie2R positive cells could suppress tumor cell growth.
 Material and Methods: We constructed replication incompetent adenoviral vectors carrying the cytosine deaminase (CD) suicide gene driven by the mouse Tie2R transcriptional promoter and enhancer sequences. In these viruses, we inserted an RGD-4C peptide into the HI loop of the fiber knob domain of the vector to increase its transduction efficiency in TVECs. At the same time, we added two mutations (S408E and P409A) in the AB loop of the fiber, which ablated the CAR-binding ability of the vector. The resulting vector was named AdTie2RCD(MRGD).
 Results: In vitro viral infection assays involving the human umbilical vein endothelial cells (HUVECs) showed that the RGD-modified vectors have a higher transduction efficiency as compared with their adenoviral vector counterparts which have a wild type fiber protein. Mice carrying the rat Her-2-Neu positive N202 mouse breast cancer cells and the mouse B16 melanoma cells were treated with intravenous injections of Hetastarch and the AdTie2RCD(MRGD) vector followed by intraperitoneal injections of 5-Fluorocytosine (5-FC). The Hetastarch was given before the intraveinous infusion of the adenoviral vector in order to reduce the uptake of the adenoviral vector by the reticuloendothelial cell system. The Tie2R targeted chemotherapy sensitization vectors induced greater degrees of suppression of tumor cell growth than did the control group, achieved the similar treatment effect to its CMV promoter counterpart, but with less side effect.
 
 Histological analysis showed that these vectors specifically targeted the TVECs, TEMs and TMPCs through which they may have exerted cytotoxic effects in the presence of 5-FC on the tumor cells.
 Discussion: Our results showed that N202 breast cancer cell line is more responsive to the Tie2R-targeting therapy compared to B16 mouse melanoma cell line and the Tie2R-directed anti-angiogenic therapy deserves further study, in combination with other types of therapy to pave the way for future clinic trails. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 902.

Liposomally-encapsulated cisplatin (Lipoplatin) plus gemcitabine in NSCLC: Preliminary results of a phase II trial and its antiangiogenesis potential

19024 Background: Liposomally-encapsulated cisplatin (Lipoplatin) nanoparticles evade immune surveillance, target tumor cells in mouse xenografts, primary human tumors and metastases in patients as well as endothelial cells of tumor vasculature. Thus, Lipoplatin may enjoy the cytotoxic properties of cisplatin while additionally exerting antiangiogenic effects. Lipoplatin-gemcitabine is less toxic yet at least as efficacious as cisplatin-gemcitabine in Phase III trials. Lipoplatin has an orphan drug status for pancreatic cancer in the EU. This is the first report of a multicenter, phase II Lipoplatin study in NSCLC. Methods: Eligibility criteria included confirmed diagnosis of inoperable/metastatic NSCLC, no previous chemotherapy, WHO PS 0–1, adequate end-organ function. Patients received Lipoplatin 120 mg/m2 D1,8,15 combined with gemcitabine 1 g/m2 D1,8, in 3-week cycles, with disease evaluation after 3 and 6 cycles. Primary endpoints were Response Rates and Toxicity; OS PFS and QOL are also being evaluat...

Integrin affinity modulation in angiogenesis

Integrins, transmembrane glycoprotein receptors, play vital roles in pathological angiogenesis, but their precise regulatory functions are not completely understood and remain controversial. This study aims to assess the regulatory functions of individual beta subunits of endothelial integrins in angiogenic responses induced by vascular endothelial growth factor (VEGF). Inhibition of expression of β1, β3, or β5 integrins in endothelial cells resulted in down regulation of EC adhesion and migration on the primary ligand for the corresponding integrin receptor, while no effects on the recognition of other ligands were detected. Although inhibition of expression of each subunit substantially affected capillary growth stimulated by VEGF, the loss of β3 integrin was the most inhibitory. EC stimulation by VEGF induced formation of the high affinity (activated) state of αVβ3 in a monolayer and activated αVβ3 was co-localized with VEGF receptor-2 (VEGFR-2). Inhibition of expression of β1, β3, or β5 did not affect expression levels of VEGFR-2 in EC. However, inhibition of β3, but not β1 or β5, resulted in substantial inhibition of VEGFR-2 phosphorylation stimulated by VEGF. Exogenous stimulation of αVβ3 integrin with activating antibodies augmented VEGF-dependent phosphorylation of VEGFR-2, whereas integrin blockade suppressed this response. Most importantly, activated αVβ3 was detected on endothelial cells of tumor vasculature. Activation of αVβ3 was substantially increased in highly-vascularized tumors as compared to normal tissues. Moreover, activated αVβ3 was co-localized with VEGFR-2 on endothelial cells of proliferating blood vessels. Together, these results show the unique role of αVβ3 integrin in cross-talk with VEGFR-2 in the context of pathological angiogenesis.

Peptidase in Regulatory Loop Affecting Angiogenesis

Prostate-specific membrane antigen (PSMA) is so-named because its expression is enhanced in advanced prostate carcinomas, where its increased presence correlates with a poor prognosis. The protein is also called glutamate carboxypeptidase II and is a transmembrane protein with peptidase activity. PSMA has been found in endothelial cells in tumor vasculature. Given roles of other peptidases in angiogenesis, Conway et al. explored the possibility of such a role for PSMA. They used an in vivo angiogenesis assay in knockout mice lacking PSMA to show that loss of the PSMA protein inhibited formation of new blood vessels. Proteolysis contributes to remodeling of the extracellular matrix that is necessary for angiogenesis, but further studies by the authors suggest that PSMA may instead be part of a complex regulatory loop that controls integrin signaling and activation of the p21-activated kinase 1 (PAK1). In vitro cell invasion studies with PSMA-null cells or with inhibitors of the enzyme showed that PSMA has an important role in cell invasion and in signaling from β 1 integrins to focal adhesion kinase (FAK) and PAK1. The authors confirmed that PSMA interacts with the actin-binding protein filamin A. Disruption of this interaction with a peptide designed to compete with PMSA for binding to filamin A decreased the peptidase activity of PMSA and decreased phosphorylation of PAK1 in cultured cells. PAK1 also interacts with filamin A, and the authors propose that it may compete with PMSA for binding to filamin A. The interaction of PMSA and the cytoskeletal protein filamin A may allow a feedback signal from integrin β 1 and PAK to keep PMSA activity in check. Inhibition of PAK by expression of a peptide corresponding to its autoinhibitory domain enhanced association of PMSA with filamin A, increasing its peptidase activity. Further understanding of PMSA's roles in control of angiogenesis may allow new strategies to inhibit angiogenesis in cancers and other diseases to which it contributes. R. E. Conway, N. Petrovic, Z. Li, W. Heston, D. Wu, L. H. Shapiro, Prostate-specific membrane antigen regulates angiogenesis by modulating integrin signal transduction. Mol. Cell. Biol. 26 , 5310-5324 (2006). [Abstract] [Full Text]

Utilization of bone marrow-derived endothelial cell precursors in spontaneous prostate tumors varies with tumor grade.

Id1 and Id3 genes are required for vascularization, growth, and metastasis of xenograft tumors. In Id-deficient mice, tumor transplantation and proangiogenic factors fail to mobilize and recruit circulating endothelial precursor cells (CEPs) and hematopoietic cells, leading to defective tumor angiogenesis in various models. To investigate the requirement of Id genes and bone marrow incorporation in spontaneous prostate tumors, we crossbred Id mutant mice with the transgenic adenocarcinoma of the mouse prostate (TRAMP) mice. Id1-/- Id3+/- TRAMP mice display delayed tumor growth at 24 weeks compared with wild-type TRAMP mice. Id1 and Id3 were strongly expressed in the endothelial cells of poorly differentiated prostate adenocarcinoma but not in the vasculature of well-differentiated tumors, a finding that is corroborated in human prostate tumor samples. In Id-deficient TRAMP mice, the poorly differentiated tumors show extensive hemorrhage, whereas well-differentiated tumors exhibit none. Transplantation with Id wild-type bone marrow significantly reduced the hemorrhage in poorly differentiated prostate adenocarcinomas with bone marrow-derived endothelial cells contributing to 14% of the tumor blood vessels. However, in well-differentiated prostate adenocarcinomas, there was little evidence of bone marrow-derived endothelial cell incorporation. These differences in the expression of Id genes, the effects of Id loss, and the recruitment of bone marrow-derived endothelial precursor cells in tumor vasculature between well-differentiated and poorly differentiated prostate adenocarcinoma suggest that tumor angiogenesis varies depending on the tumor grade.

Assessment of metabolism, excretion and pharmacokinetics of a single dose of [14C]-ZD6126 in patients with solid malignant tumors

3083 Background: ZD6126 is a novel vascular-targeting agent that causes disruption of the microtubular network responsible for maintaining the shape of immature, proliferating endothelial cells in tumor vasculature. This leads to selective vessel occlusion and extensive tumor necrosis. Methods: A Phase I excretion balance study of ZD6126 has been conducted in 6 patients (47–56 yrs) with solid tumors refractory to standard therapies, a WHO performance status of 0–2 and a life expectancy of ≥12 weeks. Those with significant cardiac, hematopoietic, hepatic or renal dysfunction were excluded. Patients received a single 10 min infusion of 40 mg/m2 [14C]-ZD6126 (70 μCi; 2.6 MBq) on day 1 of a 28-day cycle. Urine and feces were collected continuously for the first 8 days and blood samples were collected on days 1 and 2. Subsequent treatment comprised a single i.v. infusion of ZD6126 (56 mg/m2). Results: Pharmacokinetic analysis showed that Cmax for plasma ZD6126 was observed 5 min into the 10 min infusion. Conce...

Evaluation of the role of body-surface-area dose adjustment of ZD6126, a novel vascular-targeting agent

3065 Background: Dosing of anticancer agents using body-surface-area (BSA) normalization has been standard clinical practice for most i.v. drugs and continues to be practiced without clear biological or physiological support. A review of data from Phase I trials of 33 cancer agents has shown that dosing on a mg/m2 basis reduced variability in exposure in only 5 of the drug candidates examined (Baker et al. J Natl Cancer Inst, 2002 94:1883–8). The authors of this review recommend that BSA dosing should not be adopted unless sufficient clinical information had been generated to support such a dosing strategy. The novel vascular-targeting agent, ZD6126, causes disruption of the microtubular network responsible for maintaining the shape of immature, proliferating endothelial cells in tumor vasculature. This leads to selective vessel occlusion and extensive tumor necrosis. Toxicokinetic data in animals showed male/female differences following exposure to ZD6126 phenol (the active metabolite) and a potential for cytotoxicity at doses 10x greater than anticipated maximum clinical doses. Therefore, dosing normalized to BSA was chosen for initial clinical studies. Methods: During the Phase I program, the safety and pharmacokinetics of ZD6126 were explored at doses of 1–112 mg/m2 administered according to one of 3 schedules. Prior to Phase II evaluation, pharmacokinetic data were available from 71 patients. A high degree of variability was observed in exposure [Cmax and AUC] across the entire dose range, although Cmax and AUC generally increased with dose in a linear fashion. The relationship between BSA and clearance (CL = [Dose/AUC]) of ZD6126 phenol was explored. Results: No clear correlation between CL and BSA was seen. Statistical evaluation of the variability, as assessed by the coefficient of variation, showed that the exposure achieved with unit (mg) dosing would be comparable with that achieved when dosed on a mg/m2basis. Conclusions: These findings indicated that dosing ZD6126 on a unit dose basis would not increase variability in patient exposure to ZD6126 phenol. A unit dosing strategy has therefore been adopted for the Phase II clinical program. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration AstraZeneca

Combination docetaxel and platelet-derived growth factor receptor inhibition with imatinib mesylate in prostate cancer

The burden of metastatic disease in prostate cancer is largely distributed to bone in the form of osteoblastic metastases. Interactions between malignant epithelial cells of prostate cancer and the bone microenvironment are implicated in the progression of prostate cancer. Because prostate cancer cells in bone metastases express the platelet-derived growth factor receptor (PDGFR), it is possible that inhibition of PDGFR can be an effective means of altering the clinical course of prostate cancer. Preclinical data suggest that preferential expression of PDGFRs in endothelial cells of tumor vasculature in experimental prostate cancer bone metastases is an important target for combination therapy incorporating the PDGFR inhibitor imatinib mesylate. Clinical trials combining imatinib and docetaxel are under way in the metastatic and neoadjuvant settings. Clinical and translational data from these studies are likely to provide additional insights into the role of imatinib in combination therapy for prostate cancer.

Highlights on endoglin (CD105): from basic findings towards clinical applications in human cancer

Antibody targeting of tumor-associated vasculature is a promising therapeutic approach in human cancer; however, a specific cell membrane marker for endothelial cells of tumor vasculature has not been discovered yet. Endoglin (CD105) is a cell-surface glycoprotein most recently identified as an optimal indicator of proliferation of human endothelial cells. The finding that CD105 is over-expressed on vascular endothelium in angiogenetic tissues has prompted several pre-clinical studies designed to get a deeper understanding on the role of CD105 in angiogenesis, and to evaluate the most appropriate clinical setting(s) to utilize CD105 as a therapeutic target. In this review, the foreseeable clinical applications of CD105 in human cancer are discussed.

Sickle red blood cells accumulate in tumor.

The preferential accumulation of sickle blood cells in tumor vasculature is demonstrated noninvasively using MRI and sickle red blood cells loaded with Gd-DTPA and invasively by two other techniques. The distribution of red blood cells in rat brain tumors relative to normal brains were measured using three separate techniques: MRI of Gd-DTPA loaded cells, fluorescent microscopy detection of Oregon Green 488 fluorescence conjugated to a streptavidin-biotin complex that binds to red blood cell surface proteins, and autoradiography using a technetium (99m)Tc-labeling kit. Labeled red cells were infused intravenously in rats with brain tumors. Sickle cells preferentially accumulated in tumor relative to normal brain, with highest concentrations near the tumor / normal tissue boundary, whereas control normal red cells did not preferentially aggregate at the tumor periphery. This demonstrates the potential of sickle red blood cells to accumulate in the abnormal tumor vessel network, and the ability to detect their aggregation noninvasively and at high spatial resolution using MRI. The application of the noninvasive measurement of sickle cells for imaging tumor neovasculature, or as a delivery tool for therapy, requires further study.

Identification of tumor vascular antigens by monoclonal antibodies prepared from rat-tumor-derived endothelial cells.

We have reported the isolation and specific in vitro properties of tumor-derived endothelial cells (TEC) from rat KMT-17 fibrosarcomas transplanted into rats. To develop antibody-based tumor vascular targeting therapy for solid tumors, we have generated monoclonal antibodies (MAbs) using passive immunization of outside-out membrane vesicles of rat epididymal-fat-pad-derived capillary endothelial cells (FCEC) followed by active immunization of those of rat TEC. The MAbs produced were screened against TEC and FCEC. Of all cultured hybridomas, 75 (3.3%) of the secreted MAbs preferentially recognized TEC. We selected a total of 7 MAbs which detected antigens highly abundant in TEC, although 5 of the 7 MAbs were weakly positive for FCEC in cell-ELISA and FACS analyses. The antigens recognized by these MAbs, with the exception of MAb TES-7, were present on endothelial cells of tumor blood vessels in KMT-17 fibrosarcoma tissues, as shown by immunohistochemical analysis. Antigens of 40- and 80-kDa were recognized by MAbs TES-1, 7, 17, 21 and 26 and by MAbs TES-23 and 27 respectively. Although the function of these antigens, which are preferentially expressed on rat tumor-derived endothelial cells, is still unknown, we believe that future studies of such antigens will help elucidate the role of endothelial cells in tumor vasculature. Our results indicate that MAbs may provide a novel tool for the development of antibody-based therapy targeting tumor vasculature.

Targeting the tumor vasculature: inhibition of tumor growth by a vascular endothelial growth factor-toxin conjugate.

Tumor-derived vascular endothelial growth factor (VEGF)/ vascular permeability factor (VPF) plays an important role in neovascularization and the development of tumor stroma. Furthermore, VEGF receptors are over-expressed in the endothelial cells of tumor vasculature and almost non-detectable in the vascular endothelium of adjoining normal tissues. The differential expression of receptor offers a selective advantage for targeting cytotoxic toxin polypeptides. We have prepared a vascular targeting reagent by chemically linking recombinant VEGF to a truncated form of diphtheria toxin. The VEGF-toxin conjugate was selectively toxic to endothelial cell lines and inhibited experimental neovascularization of the chick chorioallantoic membrane. In the present study, we examined the effects of VEGF-toxin conjugate on solid tumor growth. Athymic nude mice with established subcutaneous tumors were treated with daily intraperitoneal injections of the VEGF-toxin conjugate or free toxin. When compared with control animals treated with the toxin polypeptide alone, the conjugate-treated animals displayed a significant inhibition of tumor growth. Histological analysis of tumors from conjugate-treated animals revealed hemorrhagic necrosis consistent with a vascular-mediated injury. In contrast, highly vascularized normal tissues from conjugate-treated animals demonstrated no evidence of hemorrhage or tissue injury. The conjugate was well tolerated without apparent toxicities. Our results illustrate the anti-tumor activity of a VEGF-toxin conjugate selectively targeting the tumor neovasculature.