(Chest. 2005;128:596S-600S.)
© 2005
American College of Chest Physicians
Phenotypic Diversity of the Lung Vasculature in Experimental Models of Metastases*
Yun Oh;
Imran Mohiuddin;
Yan Sun;
Joseph B. Putnam, Jr;
Waun Ki Hong;
Wadih Arap, MD, PhD and
Renata Pasqualini, PhD
* From the Departments of Thoracic/Head & Neck Medical Oncology (Drs. Oh and Hong), Thoracic Surgery (Drs. Mohiuddin and Putnam), and Genitourinary Medical Oncology (Drs. Sun, Arap, and Pasqualini), The University of Texas M.D. Anderson Cancer Center, Houston, TX.
Correspondence to: Wadih Arap, MD, PhD, or Renata Pasqualini, PhD, M.D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030; e-mail: warap{at}mdanderson.org or rpasqual{at}mdanderson.org
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Abstract
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In vivo phage display is a screening method in which peptides homing to specific vascular beds are selected after IV administration of a random peptide library. This strategy has revealed a vascular address system that allows tissue-specific targeting of normal blood vessels and angiogenesis-related targeting of tumor blood vessels by selected peptides. Many vascular receptors or "addresses" targeted by homing peptides have been identified. One such vascular receptor of normal lung endothelium is membrane dipeptidase (MDP), which was found by in vivo phage display to bind the tripeptide motif gly-phe-glu (GFE). Our studies with GFE peptide and lung vasculature suggest that MDP mediates cancer cell adhesion to lung vasculature and the development of lung metastases, but that MDP is not present in the vasculature of lung metastases. MDP appears to occupy a vascular distribution that is similar to the pulmonary artery circulation. These results demonstrate the promise of defining critical functional and anatomic characteristics of endothelial cells in lung and other organs by in vivo phage display.
Key Words: lung endothelium • membrane dipeptidase • metastasis • phage display • random peptide library • vascular targeting
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Introduction
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Efforts to target therapeutics and imaging agents to blood vessels in an organ- specific or disease-specific manner has led to the development of a technique by which small peptides that target receptors on endothelial cells can be identified.123 This approach, using large, random peptide libraries displayed on the surface of the bacteriophage, has been termed in vivo phage display. A random peptide phage library is injected IV, and peptides that home selectively to specific organs or tumors are then recovered. Putative human homologs have been identified from ligands and receptors isolated by this approach in mouse models, suggesting that at least some ligand peptide motifs and their corresponding receptors may be evolutionarily conserved and pertinent in humans.4 In vivo phage display is uncovering a vascular address system that can be used for organ-specific targeting of normal blood vessels and angiogenesis-related targeting of blood vessels in tumors.567891011 Methods are also being developed for determining the in vivo distribution of probes targeted to the vasculature,23 and their organ-specificity, vessel-specificity, and cellular targets. As the complexity of vascular diversity is further revealed, in vivo phage display will target particular regions of organs, such as pancreatic islets or the glomeruli of the kidney,12 and eventually will identify receptor ligand pairs for functionally distinct vessels within these regions.
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Membrane Dipeptidase and Its Ligand Gly-Phe-Glu in Lung Vasculature
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Membrane dipeptidase (MDP) was biochemically identified as a vascular receptor for lung-homing peptides containing the tripeptide motif gly-phe-glu (GFE) selected by in vivo phage display,213 and appears to be a vascular receptor that binds to and mediates GFE homing to normal lung endothelium. MDP is a member of the zinc-metalloprotease family and a GP-anchored cell surface protein expressed primarily in the lung and kidney.14 In the rat and mouse, levels of MDP activity are highest in the lung.1415 MDP is involved in the metabolism of glutathione and cysteinyl leukotrienes,1416 and it is the only known mammalian enzyme that is capable of degrading ß-lactam antibiotics.14 The work presented here describes the expression and functional role of MDP in the dual circulation of the lung in the context of the vasculature of pulmonary metastases.
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Role of MDP in Experimental Melanoma Lung Metastases
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To determine whether the lung vascular receptor MDP mediates the homing of tumor cells and the subsequent development of experimental pulmonary metastases in mice, we used the C8161 human melanoma cell model of experimental lung metastases.1718 C8161 cells were coadministered IV with either the GFE-containing peptide (a ligand targeting MDP213) or a negative control peptide (Fig 1
). The median weight of lungs from mice that received GFE peptide was only 12% greater than that of normal lungs, whereas the median weight of lungs from mice that received control peptide or no peptide (vehicle alone) was 88% greater than that of normal lungs (p < 0.01 [t test]). Consistent with these findings, histologic examination showed many fewer and smaller metastatic foci in mice treated with GFE. Moreover, GFE did not affect cell viability, as demonstrated by Trypan Blue exclusion and MTT assays on aliquots of melanoma cells prepared for injection into mice; cell viability was > 95% (results not shown). These results suggest that the tripeptide GFE binds to MDP and blocks the vascular homing of metastatic melanoma cells via this receptor.
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Figure 1. The GFE lung homing peptide inhibits the experimental metastasis of IV administered C8161 melanoma. Tumor cells were preincubated for 10 min with GFE or a control peptide, as indicated, and then were injected into the tail vein (106 cells per mouse, 10 mice per group). Lung weights are shown for the three experimental groups. The average normal lung weight (0.175 g) is marked with a line. Relative to the vehicle (DMEM) and control peptide groups, p values were < 0.01. Shown is one of five experiments in which similar results were obtained.
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Bronchial vs Pulmonary Artery Perfusion of Metastases
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Photomicroscopy of lung metastases in the C8161 model demonstrated that the earliest visible lung metastases grew preferentially in close proximity to bronchioles (Fig 2
). These observations led to the hypothesis that these metastatic tumors were preferentially recruiting angiogenic blood vessels from the bronchial vs pulmonary artery circulations. To test this hypothesis, we used a rat fibrosarcoma lung metastasis model19 and isolated rat lung perfusion model.20 After surgically isolating the left pulmonary artery circulation of rats under general anesthesia, Evans blue (with red fluorescence) was perfused; the remaining systemic circulation was perfused with fluorescein isothiocyanate-dextran via left ventricular injection. Rats were sacrificed after a 2-min circulation time, and lung tissue was processed according to the method of Li et al.21 Indeed, we found that microscopic lung metastases of rat fibrosarcoma cells recruited tumor blood vessels preferentially from the bronchial vasculature, at least in early stages of growth (Fig 3
). This was a surprising result since IV inoculated cancer cells pass through the pulmonary artery circulation first and would have a greater chance to deposit in the lung near pulmonary arteriolar capillaries than around the bronchial circulation.
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Figure 2. Lung tumor foci develop preferentially around the bronchia. Top, A: trichrome-stained sections of mouse lungs with C8161 metastases demonstrate that early tumors are found centered around bronchioles (arrows indicate lumens with blue-green borders, indicating collagen within the bronchial walls). Tumors secondarily also envelop pulmonary arteries that accompany the bronchioles. Bottom, B: normal lung is shown for comparison (trichrome, magnification).
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Figure 3. Tumor vasculature is preferentially recruited from the bronchial circulation. Top left, A: fluorescein isothiocyanate (FITC)-dextran was perfused into the left ventricle and Evans blue into the isolated left pulmonary artery of anesthetized rats. Top right, B: a photograph of the operative field demonstrates left lung (LL), right lung (RL), pulmonary artery catheter (PA), clamps placed on both pulmonary veins (PV), the left ventricle of the heart (H), and the ascending aorta (AA). Bottom, C: high-power views of the earliest detectable microscopic tumor nodules, the largest of which at far right, appears to have central necrosis with only punctate peripheral perfusion.
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Distribution of MDP in C8161 Metastases to the Lung
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Finally, we analyzed the distribution of GFE binding to lung vasculature to evaluate whether MDP is expressed in the angiogenic vasculature of lung metastases because the expression of MDP in the lung endothelium closely recapitulates the distribution of a GFE-displaying phage.213 Either a GFE-displaying phage or a control insertless phage were injected IV into mice bearing C8161 lung metastases and were examined for their ability to home to tumor vasculature. Strong phage immunoreactivity was observed in normal lung tissue, but the lung metastases were clearly negative (Fig 4
). The failure of GFE-displaying phage to accumulate in tumor blood vessels indicates that its homing receptor, MDP, is not expressed in these blood vessels.
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Figure 4. GFE does not bind to the vasculature within lung tumors. Lungs were harvested from mice bearing C8161 metastases after the animals had been injected with phage displaying the GFE peptide. Tissue sections were immunostained with an anti-M13 antibody to detect phage particles. Top left, A: brown peroxidase staining indicates phages are distributed throughout the lung vasculature (arrows). Phage staining, however, is absent in tumor (T) vasculature. As a negative control for the staining, a serial tissue section obtained from the GFE-injected mouse lung was processed with the secondary antibody alone (top right, B), and control tissues are shown for skin (bottom left, C) and brain (bottom right, D).
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Conclusions
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The molecular differences based on the heterogeneous expression of MDP in the lung and lung tumors could be explained by tumors differentially recruiting the dual blood circulations of the lung. Based on our perfusion experiments, we concluded that early lung metastases derive their blood supply primarily from the bronchial artery circulation. In contrast, based on the pattern of GFE-displaying phage binding in alveolar capillaries and pulmonary arterioles, it appears that MDP is selectively expressed in the pulmonary artery vasculature.
The patterns and function of MDP based on the results presented here imply that normal organ vascular receptors are still not necessarily valid targets for imaging or treating tumors in those organs. Previous studies have identified vascular receptors that can be used to deliver therapeutic agents to cancers originating in host organs567891011 or to cancers originating in normal organs.222324 MDP likely cannot be utilized in this manner since it appears to be absent in the vasculature of lung metastases. Even if MDP were present in lung tumor vasculature, it would not be an optimal target for delivering drugs to lung tumors. Unlike the prostate and breast, the lung is an essential organ for survival, and the risk of collateral damage to normal lung vasculature may be unacceptable. On the other hand, the delivery of radioprotective agents to the lungs would remain an open possibility.
Taken together, these data illustrate the diversity and functional relevance of a vascular receptor in the lung vasculature. Novel antimetastatic strategies may emerge from interfering with tumor cell attachment to MDP and/or mechanisms underlying angiogenesis in lung cancer.
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Acknowledgements
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We thank Dr. Wolfgang Kuschinsky for references and advice on vascular imaging techniques.
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Footnotes
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Abbreviations: GFE = gly-phe-glu; MDP = membrane dipeptidase
This work was funded in part by an award from the Gillson-Longenbaugh Foundation (Drs. Arap and Pasqualini) and the Biology, Education, Screening, Chemoprevention and Treatment (BESCT) Lung Cancer Program (contract No. DAMD17–01-1–0689; Dr. Oh).
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Abstract
Introduction
