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Probing the structural and molecular diversity of tumor vasculature Renata Pasqualini, Wadih Arap and Donald M. McDonald The molecular diversity of the vasculature provides a rational basis for developing targeted diagnostics and therapeutics for cancer. Targeted imaging agents would offer better localization of primary tumors and metastases, and targeted therapies would improve efficacy and reduce side effects. The development of targeted pharmaceuticals requires the identification of specific ligand–receptor pairs, and knowledge of their cellular distribution and accessibility. Using in vivo phage display, a technique by which we can identify organ-specific and disease-specific proteins expressed on the endothelial surface, it is now possible to decipher the molecular signature of blood vessels in normal and diseased tissues. These studies have already led to the identification of peptides that target the normal vasculature of the brain, kidney, pancreas, lung and skin, as well as the abnormal vasculature of tumors, arthritis and atherosclerosis. Membrane dipeptidase in the lungs, interleukin-11 receptor in the prostate, and aminopeptidase N in tumors are examples of molecular targets on blood vessels. Corresponding confocal-microscopic imaging and ultrastructural studies are providing a more complete understanding of the cellular abnormalities of tumor blood vessels, and the distribution and accessibility of potential targets. The combined approach offers a strategy for creating a ligand–receptor map of the human vasculature, and forms a foundation for the development and application of targeted therapies in cancer and other diseases.

Renata Pasqualini Wadih Arap Dept of Genitourinary Medical Oncology, The University of Texas, M.D. Anderson Cancer Center, Houston, TX 77030, USA. Donald M. McDonald* Cardiovascular Research Institute, Comprehensive Cancer Center and Dept of Anatomy, University of California, San Francisco, CA 94143, USA. *e-mail: dmcd@ itsa.ucsf.edu

The targeting of therapeutics and imaging agents to blood vessels, using probes that bind to specific molecular addresses in the vasculature, is a major research topic. These studies have led to the development of a technique by which small peptides that target receptors on endothelial cells can be identified [1]. This approach, using large RANDOM PEPTIDE LIBRARIES (see Glossary) displayed on the surface of BACTERIOPHAGE, has made it possible to find receptors that are expressed on blood vessels in an organ-specific or disease-related manner. In particular, molecular addresses have been identified on the blood vessels of tumors. The identification of ligand–receptor pairs on tumor vessels is leading to a better understanding of the vasculature of tumors, and how it changes during tumor progression and metastasis. This technique, termed ‘in vivo PHAGE DISPLAY’, also makes it possible to localize novel vascular markers in other diseases where angiogenesis and VASCULAR REMODELING occur, such as arthritis, atherosclerosis and diabetic retinopathy. Molecular targets on blood vessels can be identified using an in vivo selection system. A random peptide phage library is injected intravenously, and peptides that home selectively to specific organs or tumors are then recovered. This approach is uncovering a vascular address system that can be used http://tmm.trends.com

for organ-specific targeting of normal blood vessels and angiogenesis-related targeting of blood vessels in tumors. Methods are also being developed for determining the in vivo distribution of probes targeted to the vasculature [2,3], and their organ-specificity, vessel-specificity and cellular targets. The use of labeled probes, which can be visualized by fluorescence microscopy, confocal microscopy, scanning electron microscopy and transmission electron microscopy, is helping to define organ-, vessel- and cell-specific differences in the vasculature of normal organs, and of tumors and other disease sites. Understanding the spatial and cellular diversity of normal organs and diseased tissues has lagged behind the advances made by the Human Genome Project. Many key genes are expressed in functionally important and accessible regions of the body, but sites of expression can have a focal distribution [4]. For example, the vasculature of primary or metastatic tumors exhibits abnormalities that are not found elsewhere. However, tumor vessels are heterogeneous; certain abnormalities can develop in some tumor vessels and not in others. Potential targets for therapeutic intervention could be overlooked in high-throughput sequencing or gene-array approaches that do not take into account the cellular and molecular specificity and heterogeneity of the tissue. In vivo phage display is being used to analyze differences between the normal and diseased microvasculature. The key objectives are to identify and localize novel vascular markers in cancer, and to characterize changes that occur during tumor progression. Similar information could be obtained about the vasculature in chronic inflammatory diseases, such as rheumatoid arthritis, chronic bowel disease and asthma. Although the initial focus has been on organ-specific targeting, the next step will be to target particular regions of organs, such as pancreatic islets or the glomeruli of the kidney, and eventually to target functionally specific vessels within these regions. Similarly, the initial approach of targeting primary tumors could readily be expanded to targeting metastases. Putative human homologs have been identified from ligands and receptors isolated by in vivo phage display in mouse models. However, mouse-derived probes might not always be useful for targeting drug delivery to receptors on human blood vessels, owing to differences in the expression patterns of proteins in

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Glossary Bacteriophage: A virus that infects and propagates in bacteria. Often shortened to ‘phage’. Biopanning: Screening of a phage-display peptide library against one or more targets. Cationic liposomes: Positively charged lipid vesicles used as drug delivery vehicles. Continuous capillaries: Capillaries with endothelial cells that lack holes or discontinuities and have comparatively low solute flux. Discontinuous capillaries: Capillaries with endothelial cells that have transcellular openings not covered by a diaphragm, and permit the extravasation of macromolecules. Fenestrated capillaries: Capillaries having endothelial cells with a variable number of tiny holes, typically covered by a diaphragm, which permit high water and solute flux. Pericytes: Cells that are located within the vascular basement membrane and envelop endothelial cells of capillaries and small venules. Phage display: Screening with phage random peptide libraries to identify molecular targets to which specific phage bind. Pilus-positive bacteria: Bacteria that have surface pili (hairlike protein tubes originating from the cell membrane that allow bacteria to attach to other cells or environmental surfaces). Random peptide libraries: Large (~109) random collections of peptides, displayed as recombinant molecules on the surface of bacteriophage. Vascular basement membrane: A layer of specialized extracellular matrix, made by endothelial cells and pericytes on the outer surface of blood vessels, and consisting mainly of type IV collagen, laminin, fibronectin, other proteins and glycosaminoglycans. Vascular map: A ligand–receptor-based molecular map of blood vessels in the body. Vascular heterogeneity: The organ-, tissue- and vessel-specific differences in the vasculature associated with functional differences in normal blood vessels, and environmental differences of blood vessels in diseased tissues. Vascular remodeling: Structural changes in existing vessels, without the growth of new vessels. Vascular zip codes: Molecular addresses on blood vessels.

various tissues. Thus, the construction of a human molecular VASCULAR MAP is required for the development of effective targeted therapies for human disease. In this review, we examine conventional views of the structural and functional diversity of blood vessels, and go on to describe studies showing that the microvasculature of different organs is even more heterogeneous than originally appreciated. Additional diversity is evident in diseased states, and we summarize here the structural abnormalities of tumor vessels that are conspicuous manifestations of altered gene expression profiles. We then describe how in vivo phage display can be used to identify distinctive molecular features of tumor vessels that can be used as targets for drug delivery. Structural heterogeneity of the vasculature Diversity of normal blood vessels

The concept of structural heterogeneity within the vasculature developed in tandem with advances in the understanding of the functions of different organs. An early indication that capillaries are not all alike came from the observation of organ-related differences in vascular permeability [5,6]. Another key indicator emerged from the examination of blood vessels in different organs by transmission electron microscopy in the 1950s. These studies identified three general types of capillaries: CONTINUOUS, FENESTRATED and DISCONTINUOUS [7–10]. Capillaries in skeletal muscle, heart, lung and brain have a http://tmm.trends.com

continuous endothelium; capillaries in many endocrine glands, exocrine glands, choroid plexus, intestinal villi, gall bladder and several other organs have endothelial fenestrations covered by diaphragms; capillaries of renal glomeruli have endothelial fenestrations without diaphragms; and capillaries (sinusoids) in the liver, spleen and bone marrow have a discontinuous endothelium [7–10]. Although fenestrated capillaries are often classified as two types (those with diaphragms and those without), and more elaborate classifications have been proposed [7], the three types of capillaries that were originally proposed are still widely recognized today in textbooks of histology, pathology and circulatory physiology [10–13]. Differences in the ultrastructural features of capillary endothelial cells in different organs are indicators of functional specializations. In the 1960s, the blood–brain barrier was recognized as an example of specialized capillary endothelial cells joined by tight junctions [14,15]. Similar capillaries were found to be responsible for the blood–testis barrier [16,17]. By contrast, selectively permeable capillaries in renal glomeruli are crucial for normal glomerular filtration [18,19], and endothelial cells of high endothelial venules are specialized in mediating lymphocyte traffic from the blood into lymphoid tissue [20,21]. Distinctive structural features of sinusoids in the liver, spleen and bone marrow have also been associated with the unique functions of these organs [8,22,23]. The emerging structural diversity of the microvasculature led Guido Majno, in 1965, to observe that ‘electron microscopy has disclosed that there are almost as many varieties of capillaries as there are organs and tissues’ [8]. He went on to suggest that ‘it may well develop that each organ or tissue has its own distinctive variety of capillaries’ [8]. Based on morphological evidence alone, it was reasonable for Maya and Nicolae Simionescu to conclude nineteen years later that ‘each organ and tissue and each segment of their microvascular beds has its own characteristic endothelium’ [10]. These morphological differences are consistent with the obvious functional diversity of the vasculature across different organs, giving rise, for example, to differences in vascular permeability. At one end of the permeability spectrum is the brain, and at the other are kidney, liver, spleen and bone marrow [5,6]. Brain capillaries are made tight by junctional proteins and these capillaries express transporters that move essential amino acids, sugars and nucleotides across the endothelial barrier [24]. Pulmonary capillaries express large amounts of the water channel aquaporin-1 [25–27], a distinct form of angiotensin-converting enzyme [28,29], and have an uptake mechanism for removing 5-hydroxytryptamine from the bloodstream [30–32]. Endothelial cells of capillaries in skeletal muscle, adipose tissue and heart express low-density-lipoprotein (LDL) receptors, but those of liver sinusoids do not [33]. Liver sinusoids

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express a variety of specialized receptors, including transferrin receptors [34], Fc receptors (which bind the Fc portion of immunoglobulin complexes) [35,36] and scavenger receptors that bind and internalize modified LDL and related ligands [37,38]. Many additional levels of morphological specialization of the vasculature have been identified, and these greatly expand upon the structural diversity afforded by the three general capillary classifications. In particular, the specific features of each of the three types vary from organ to organ. For example, capillaries of the liver, spleen and bone marrow exhibit conspicuous structural and functional differences from one another, even though all are designated ‘discontinuous’ [8,10]. Similarly, fenestrated capillaries of renal glomeruli are markedly different from those of the choroid plexus or thyroid gland. Additional heterogeneity is evident in the hierarchical segments of the microvasculature; the earliest microscopic studies showed that resistance vessels (arterioles), exchange vessels (capillaries) and mediator-responsive outflow vessels (venules) were different from one another [39]. These differences have been further elucidated as technology has improved [8–10]. Unlike arterioles or capillaries, postcapillary venules (the smallest vessels found just downstream of capillaries) express receptors for histamine, bradykinin, substance P and other inflammatory mediators that increase endothelial permeability and lead to leukocyte adherence and migration [8,10,40]. The endothelial cells of venules also differ from those of capillaries by not having glycoconjugates that bind wheat-germ agglutinin [41], and by exhibiting inducible expression of P-selectin [42–44]. There are many additional manifestations of endothelial heterogeneity [45]; even individual endothelial cells have heterogeneous surface features, as illustrated by ultrastructurally distinct microdomains [10]. Studies of the distribution of binding and internalization of fluorescent CATIONIC LIPOSOMES by endothelial cells suggest that additional levels of VASCULAR HETEROGENEITY are superimposed on the standard morphological classifications [46]. After intravenous injection, cationic liposomes initially coat much of the endothelial surface, but after approximately one hour they are internalized into endosomes and lysosomes of endothelial cells in a distinctive organ- and vessel-specific fashion [46]. Uptake by capillary endothelial cells is greatest in lung, ovary and anterior pituitary, less in muscle and heart, and nearly absent in brain and pancreatic islets. In lymph nodes and intestinal Peyer’s patches, the uptake is sparse in capillaries but abundant in high endothelial venules. In the liver, spleen and bone marrow, sinusoidal endothelial cells take up the liposomes. Unlike cationic liposomes, anionic, neutral and sterically-stabilized polyethylene-glycol-coated (stealth) neutral liposomes are not taken up by endothelial cells in any of these organs. http://tmm.trends.com

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Importantly, the pattern of binding and uptake of cationic liposomes does not match the distribution of the three types of capillaries or other previously identified heterogeneities of the vasculature. For example, both anterior pituitary and pancreatic islets have fenestrated capillaries, but uptake of cationic liposomes is much greater in the pituitary. The uptake pattern is consistent with the presence of heterogeneously distributed endothelial-cell receptors for which cationic liposomes (or complexes of liposomes and plasma proteins) are ligands. In vivo phage display could be used to examine the molecular addresses responsible for this heterogeneity. Abnormalities of tumor blood vessels

Tumor blood vessels differ from those in normal organs and from newly formed blood vessels in healing wounds, chronically inflamed tissues and other sites of angiogenesis. Most tumor vessels have an irregular diameter and random branching, and lack the defining structural features of arterioles, capillaries or venules [47]. The heterogeneity, structural irregularity and defective walls of tumor vessels break the rules of normal blood-vessel construction. The thickness of tumor blood-vessel walls does not correlate with the size of the vessel, most wide-caliber tumor vessels having thin walls like those of capillaries. Despite the large size of some tumor vessels, blood flow is typically poor and can even stop or change directions in individual vessels [48]. One consistent abnormality of tumor vessels is their leakiness to macromolecules [48,49]. This might aid metastasis by facilitating the movement of tumor cells into the bloodstream, and might also lead to the accumulation of fibrin (and other changes) in the extracellular matrix thereby favoring angiogenesis [50]. The degree of blood-vessel leakiness increases with the histological grade and malignant potential of tumors [51], a property of tumor vessels that aids the delivery of chemotherapeutic drugs, particularly macromolecular agents, antibodies, liposomes and viral vectors. There is surprisingly little information about how, why, when and where tumor vessels leak; widespread variability exists in endothelial permeability among different tumors, regions of the same tumor and even different vessels of a tumor, and depending upon host environment and hormonal status [48–50,52]. The absence of endothelial cells would produce an extreme form of blood-vessel leakiness by directly exposing tumor cells to the bloodstream. It has been reported that in uveal melanomas, blood flows through channels lined by cancer cells rather than by endothelial cells, a phenomenon termed ‘vasculogenic mimicry’ [53]. Although a similar occurrence has been described in certain other tumors [54,55], re-examination of uveal melanomas by different investigators failed to detect any blood-filled spaces that were not lined by endothelial cells [56]. The presence of extravascular blood lakes in

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Fig. 1. Scanning-electronmicroscopic images of the luminal surface of blood vessels in normal mouse mammary gland and in MCa-IV mammary carcinoma. (a) In a normal mammary-gland venule, endothelial cells form a monolayer on the luminal surface. The cells are relatively uniform in size and shape, flat and tightly apposed to one another. (b) In a tumor blood vessel, endothelial cells on the luminal surface are severely deformed, separated by wide intercellular spaces, overlap one another and have cellular projections of up to 50 µm in length. Scale bars represent 10 µm. Reproduced, with permission, from Ref. [60].

some tumors complicates the matter [47,57], and tests that determine whether potential vasculogenic-mimicry channels are actually connected to the bloodstream are difficult to apply to human tumors. The term ‘vasculogenic mimicry’ is now used in different ways by different investigators; although it was originally applied to tumors in vivo, the term is also used for blood-vessel-like behavior of tumor cells grown in culture, and for the expression of endothelial-cellrelated genes by tumor cells [58]. The use of the term to describe in vitro phenomena or gene expression patterns of tumor cells has not evoked the controversy that it has for tumors in humans. A different arrangement observed in some tumors is the presence of blood vessels lined by a mosaic of cells that differ in their immunohistochemical characteristics [59]. Based on confocal-microscopic studies, the lining of these ‘mosaic’ vessels was initially thought to be formed by a mixture of endothelial and tumor cells [59]. However, ongoing ultrastructural analyses suggest that the mosaic is actually the result of heterogeneous patterns of expression of immunohistochemical markers on endothelial cells and not the presence of tumor cells. What properties of tumor vessels give rise to their functional abnormalities? The endothelial cells, PERICYTES (mural cells) and VASCULAR BASEMENT MEMBRANE of tumor vessels are all defective. Experiments using implanted and spontaneous tumors in mice have identified multiple abnormalities in the endothelial-cell monolayer, pericyte coat and basement membrane that could explain their leakiness and unusual functional properties [60,61]. For example, the endothelial cells that line blood vessels in Lewis lung carcinomas and MCa-IV breast http://tmm.trends.com

carcinomas are disorganized, irregularly shaped, loosely interconnected and in some cases multilayered [60,61]. Furthermore, in MCa-IV breast carcinomas, endothelial cells are separated by intercellular openings with an average width of nearly 2 µm, and 14% of the vessel surface is lined by irregular, overlapping endothelial cells with cytoplasmic projections of up to 50 µm in length (Fig. 1). In addition, many tumor vessels have abluminal endothelial sprouts that penetrate deep into the perivascular tumor tissue [60,61]. These abnormalities are not restricted to implanted tumors, because similar features are present in the vasculature of spontaneous pancreatic tumors in RIP-Tag transgenic mice. These mice develop multifocal islet β-cell carcinomas as a result of expression of the SV40 large T-antigen under the control of the insulin-gene regulatory region. More than 97% of blood vessels in RIP-Tag-mouse pancreatic tumors, MCa-IV mammary tumors and Lewis lung tumors have pericytes with immunoreactivity to desmin or to α-smooth-muscle actin [61]. Despite their abundance, pericytes on tumor vessels are disorganized, have an abnormally loose association with endothelial cells, and can have cytoplasmic processes that extend deep into the tumor tissue (Fig. 2). As RIP-Tag tumors develop, α-smoothmuscle-actin-immunoreactive pericytes replace the desmin-immunoreactive pericytes that are normally present on capillaries of pancreatic islets. The vascular basement membrane, as visualized by immunoreactivity with laminin, type-IV collagen, fibronectin or nidogen, covers more than 99% of the vessel surface in RIP-Tag, MCa-IV and Lewis lung tumors. However, in these tumors it has a loose association with endothelial cells and pericytes,

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Fig. 2. Confocalmicroscopic images of pericytes on a normal venule and those on a tumor vessel in MCa-IV mouse mammary carcinoma. Pericytes are made visible by α-smooth-muscle-actin immunoreactivity (red), and endothelial cells are shown by CD31 immunoreactivity (green). (a) In a normal mouse, pericytes on the venule (larger vessel) are closely associated with the endothelium, irregularly shaped and have multiple cytoplasmic processes that incompletely cover the vessel wall. On the small arteriole (small vessel), smooth-muscle cells tightly surround the endothelium and are regularly arranged around the vessel. (b) Pericytes on the tumor blood vessel are disorganized, loosely associated with the endothelium, and some project away from the endothelium into the tumor parenchyma. Other pericytes overlap one another. Scale bars represent 20 µm. Reproduced, with permission, from Ref. [61].

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consists of multiple layers, and has extensions that penetrate deep into the tumor parenchyma. Redundant layers of basement membrane serve as a historical record of tumor vessels undergoing continuous growth, regression and remodeling [62]. CD31-immunoreactive sprouts of up to 100 µm in length radiate from the abluminal surface of many tumor vessels [60,61], but are rarely found on normal vessels except during embryonic development. The tip of such sprouts consists of solid endothelial-cell cords with no detectable lumen. Sleeves of pericytes and their enveloping basement membrane are closely associated with sprouts and extend well beyond their tips. Thus, pericytes might guide the growth of endothelial sprouts during tumor angiogenesis. Another peculiarity of tumor vessels is their avid binding and uptake of cationic liposomes from the bloodstream. This property was first identified in the vasculature of pancreatic-islet β-cell carcinomas in RIP-Tag transgenic mice [63]. The uptake of fluorescently labeled cationic liposomes by endothelial cells of tumor vessels is much greater than that of corresponding normal vessels [63]. Liposomes taken up by endothelial cells enter endosomes and multivesicular bodies [63]. The uptake of cationic liposomes by tumor vessels indicates the potential for selective targeting of these vessels. By contrast, anionic, neutral and stealth liposomes leak out of tumor blood vessels but are not taken up by endothelial cells [52,63]. In summary, most tumor blood vessels show structural and functional abnormalities in all components of the blood-vessel wall. Some of these http://tmm.trends.com

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abnormalities, which are the product of soluble and matrix-associated factors from tumor and stromal cells, in combination with the hypoxic environment, might provide novel therapeutic targets. Changes in gene expression underlying the structural abnormalities lead to the presence of distinctive proteins on the endothelial surface, which can be detected by in vivo phage display. In addition, openings between endothelial cells contribute to vessel leakiness and could allow phage to gain access to pericytes, vascular basement membrane and beyond. Hence, pericytes and basement membrane exposed by openings in the endothelium of tumor vessels might be accessible to phage peptide libraries, facilitating the identification of molecular targets. Probing the molecular heterogeneity of the vasculature

Molecules that could be used as receptors for targeted therapies can be identified by probing the molecular diversity of cell surfaces. This approach has several advantages over the identification of proteins by gene profiling or biochemical purification of isolated cells. First, proteins naturally positioned in cell membranes are more likely to maintain their functional conformation, as compared with isolated receptors that can be lost upon purification and immobilization outside the context of intact cells. Second, many cell-surface receptors require the cell-membrane environment to function, so that homo- or hetero-dimeric interactions can occur. For example, integrins are heterodimeric cell-adhesion molecules that require the association of α- and β-subunits to function. Effective targeting of integrins

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Fig. 3. Mapping the vasculature of humans by in vivo phage display. A random peptide library displayed on bacteriophage, depicted as differently shaped proteins on the surface of cigar-shaped phage particles, is the source of ligands that home to receptors selectively expressed on endothelial cells of different vascular beds. After intravenous administration in a patient (left), some phage expressing random peptides home to corresponding receptors on endothelial cells of the vasculature, in normal organs such as prostate or skin, or in pathological tissues such as tumors. These phage are recovered and the targeting peptides identified. The receptors targeted by the phage, shown here as c-, v-, square or oval shapes on endothelial cells, are also identified. This yields ligand–receptor pairs that can be used to target specific types of blood vessels.

is better achieved in the context of whole cells because their affinities and activation states depend on molecules that associate with their cytoplasmic domain. Third, probing cell surfaces allows the selection of membrane receptors in an unbiased functional assay, without preconceived ideas about the nature of the receptor repertoire, making it possible to identify unknown receptors as targets. However, despite these advantages it is often difficult to identify useful targets on isolated cells, owing to the complexity of multiple targets expressed simultaneously on a given cell population. In vivo phage display

The filamentous bacteriophage used for in vivo phage display are genetically engineered to express on their surface small peptides encoded by a piece of DNA inserted into their genome [64–66]. In this way, libraries of phage expressing random peptides can be produced. These random phage peptide libraries were first created in the 1980s, and were used to map the epitope-binding sites of immunoglobulins [64,65]. Recently, random phage libraries have been injected intravenously and used to identify phage peptides that interact with molecules specifically expressed on the blood vessels of a particular organ or tumor (Fig. 3). After removal of whole organs, tumors, or tissue biopsies, bound phage are eluted and amplified by growth in host bacteria. This amplification process is highly efficient because the phage infect and propagate in PILUS-POSITIVE BACTERIA that are not lysed by the phage but instead secrete multiple copies. Amplification is followed by several http://tmm.trends.com

rounds of BIOPANNING – repeated injection of phage, vascular perfusion, tissue removal, and phage isolation and amplification – until a population of phage that selectively bind to blood vessels in the organ of interest is obtained. The DNA corresponding to the insert is then sequenced within the phage genome, allowing the insert to be reproduced as a synthetic peptide outside the context of the phage particle [64,65,67–71]. Once the target receptor of a particular peptide is identified, it can be isolated, purified and cloned using standard biochemical methods. Many peptide ligands and their receptors on blood vessels have been identified in normal organs and in tumors using this approach [1,70,72]. For example, the receptor for a peptide that bound specifically in the lung was identified as a membrane dipeptidase expressed mainly in the lung vasculature. Similarly, the interleukin-11 receptor is a vascular address that can be selectively targeted in the prostate gland. Addresses that have been identified in tumor vessels include receptors that belong to families of proteins involved in angiogenesis. For example, αv integrins are targets in tumor vessels and are also expressed in blood vessels in arthritis and retinal neovascularization. Other examples are aminopeptidase N (CD13), the proteoglycan NG2 and matrix metalloproteinase-2 and -9. These findings suggest that it might be possible to exploit the distinctive expression patterns of vascular receptors in the development of novel therapeutic delivery strategies [1,70,72]. The range of potential applications of in vivo phage display is broad, and the past decade has seen considerable progress in the development of screening methods using random phage libraries to isolate peptide ligands. The use of peptide libraries has made it possible to characterize interaction sites and receptor–ligand binding motifs, such as those of antibodies involved in inflammatory reactions, and of integrins that mediate cellular adherence. This method has also been used to identify novel peptide ligands that might lead to the development of peptidomimetic drugs or imaging agents [1]. In addition to peptides, larger protein domains, such as single-chain antibodies, can be displayed on the surface of phage particles [73,74], and sequences that bind most efficiently can be isolated by biopanning [75–77]. Vascular addresses of blood vessels

Vascular targeting exploits molecular differences that exist in blood vessels of different organs and tissues, as well as differences between normal blood vessels and angiogenic or remodeled blood vessels. Differences in plasma-membrane proteins (‘VASCULAR ZIP CODES’) can be used to target therapeutic or imaging agents directly to a particular organ or tumor. For the treatment of cancer, this approach might reduce or eliminate some of the problems associated with conventional therapy, such as poor

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Acknowledgements We thank the members of our respective laboratories and their collaborators for valuable insights and for generating much of the data featured in this review. Our work was supported by grants from the National Institutes of Health (CA-90270 and CA-8297601 to R.P., CA-90270 and CA-9081001 to W.A., and HL-24136 and HL-59157 to D.M.M.), grants from University of California Biotechnology Strategic Targets for Alliances in Research (BioSTAR Project 00–10106) and MBT Munich Biotechnology AG (to D.M.M.), awards from CaP CURE, the Gilson–Longenbaugh Foundation, the Susan G. Komen Breast Cancer Foundation and the V Foundation (to R.P. and W.A.), and funding from AngelWorks Foundation and the Vascular Mapping Project (to R.P., W.A. and D.M.M.).

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tissue penetration and development of drug resistance. In addition, targeting therapeutic agents to tumor vessels makes it possible to combine blood vessel destruction with the conventional antitumor actions of drugs. Indeed, in mice bearing human cancer xenografts, this approach has proved more efficacious and less toxic than conventional therapy [71,78,79]. In mouse models, peptides that home to specific blood vessels have been used to guide the delivery of cytotoxic drugs (such as doxorubicin), pro-apoptotic peptides, metalloprotease inhibitors, cytokines (such as tumor necrosis factor), fluorophores and gene-therapy vectors. In general, coupling to homing peptides yields targeted compounds that are more effective and less toxic than the untargeted parental compounds. Many of these targeted therapies have been tested in animal models of cancer [1]. It might eventually be possible to determine the molecular profiles of blood vessels in clinical conditions. The systematic infusion of phage libraries before surgery or biopsies could make it possible to identify novel vascular targets. Translation of high-throughput in vivo phage-display technology might also provide a functional link between genomics and proteomics. Based on the promise of peptide- or peptidomimetic-targeting entities, therapeutic applications will eventually be developed. Receptor–ligand pairs identified during patient screenings should identify the best tools for the development of targeted therapies that can be tested in clinical trials [1]. Vascular targeting can also be used to prevent metastasis. The fact that cancer cells preferentially target to particular metastatic sites suggests that the vasculature of these sites has unique receptors, accessible to and recognizable by circulating tumor cells. An example of this is the homing of prostate cancer cells to the axial bone marrow. Although physicians have been aware of the non-random nature of metastasis for more than a century, the mechanistic basis of site-specificity remains largely unknown. Because no molecular tools were available until recently to study this phenomenon systematically in vivo, the identification and isolation of recognition molecules has been slow. Furthermore, endothelial cells of tumor vessels undergo phenotypic changes when grown in culture, thereby limiting the usefulness of in vitro studies [80,81]. However, candidate receptors on blood vessels at sites of

References 1 Kolonin, M. et al. (2001) Molecular addresses in blood vessels as targets for therapy. Curr. Opin. Chem. Biol. 5, 308–313 2 Trepel, M. et al. (2002) In vivo phage display and vascular heterogeneity: implications for targeted medicine. Curr. Opin. Chem. Biol. 6, 399–404 3 Arap, W. et al. (2002) Steps toward mapping the human vasculature by phage display. Nat. Med. 8, 121–127 4 Pasqualini, R. and Arap, W. (2002) Translation of vascular proteomics into individualized http://tmm.trends.com

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metastasis are now being identified by in vivo phage display. Such receptors are potential targets for therapeutics that block the binding of cancer cells responsible for metastasis. In vivo phage display is being used in the development of therapies targeted specifically to the vasculature and these make it possible to characterize the architecture and cellular composition of the vasculature of normal organs and during the progression of cancer. Many studies have now documented the specialization of the vasculature in tumors, both with respect to structural abnormalities and to potential molecular targets (reviewed in [1,2]). In vivo phage display is leading to the identification of peptides that home to tumor vessels, and such tumor-homing peptides can target therapies to tumors [68,79,82]. Conclusions

With increasing understanding of the structural abnormalities of tumor blood vessels, and development of the in vivo phage-display technique to probe the molecular diversity of the vasculature, the identification of ligand–receptor pairs for vascular targeting can be translated into real clinical applications. By guiding anticancer agents selectively to tumor vessels, new and improved forms of targeted anticancer therapy can be developed. In addition to the promise of peptide-guided therapy, progress in this field is providing a road map of the molecular anatomy of blood vessels in normal organs and in cancer. Hence, the targeting of the vasculature of normal and diseased organs could be the basis of a new pharmacology for the treatment of malignancies and inflammatory conditions, by taking advantage of drugs that target blood vessels located specifically at sites of disease. This should improve efficacy and reduce side effects. The heterogeneity of endothelial cells has long been recognized, but the extent, diversity, significance and potential usefulness of this heterogeneity are now being more fully appreciated and exploited. The recognition that distinctive disease-specific changes occur in the vasculature – where remodeling goes hand-in-hand with angiogenesis – adds another level of diversity. The in vivo phage-display approach provides a strategy for preparing a ligand-based map of the human vasculature, and forms a foundation for the development and application of targeted therapies.

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The relationship between the roles of BRCA genes in DNA repair and cancer predisposition Andrew Tutt and Alan Ashworth The proteins encoded by the breast-cancer-susceptibility genes, BRCA1 and BRCA2, have recently been implicated in DNA-repair processes, thereby improving our understanding of how the loss of these genes contributes to cancer initiation and progression. It appears that the role of BRCA1 in DNA repair, which could involve the integration of several pathways, is broader than that of BRCA2. BRCA1 functions in the signalling of DNA damage and its repair by homologous recombination, nucleotide-excision repair and possibly non-homologous end-joining. BRCA2 has a more specific role in DNA repair, regulating the activity of RAD51, which is required for homologous recombination. An improved understanding of the interactions of BRCA1 and BRCA2 with other proteins in large macromolecular complexes is helping to reveal their exact role in DNA repair.

Andrew Tutt Alan Ashworth* The Breakthrough Breast Cancer Research Centre, The Institute Of Cancer Research, Fulham Road, London, UK SW3 6JB. *e-mail: [email protected]

Women who inherit loss-of-function mutations in one of the breast-cancer-susceptibility genes, BRCA1 or BRCA2, have a risk of up to 85% of developing breast cancer by the age of 70 [1]. These two genes are thought to act as tumour suppressors because the wild-type allele is frequently lost in tumours of heterozygous carriers. As well as breast cancer, carriers of mutations in these genes have an elevated risk of cancers of the ovary, prostate and pancreas. BRCA1 and BRCA2 are large proteins, with only a small number of structural features, providing few clues to their normal functions. Many proteins associate with BRCA1 and/or BRCA2 but, in most cases, little is known about the functional significance of these interactions [2,3]. However, there are exceptions. For example, the RING domain of BRCA1 has been implicated in ubiquitin-mediated protein degradation, and the BRC repeats in BRCA2 bind the important DNA-repair protein, RAD51 [2,3]. A wide variety of http://tmm.trends.com

functions have been proposed for the BRCA proteins, both in transcriptional regulation, and in DNA repair and recombination and cell-cycle checkpoint control [2,3]. BRCA1 and the response to DNA damage

The effective repair of DNA damage requires damage-sensing mechanisms, and then transduction of damage signals to downstream effectors that arrest the cell cycle at checkpoints and repair the damage (Fig. 1) [4,5]. The related kinases, ATM (ataxia telangiectasia mutated) and ATR (ataxia telangiectasia and Rad3 related), are central to the DNA-damage response. Loss of ATM abolishes the checkpoints at the G1–S transition, in S phase and at the G2–M boundary [6]. ATM controls the phosphorylation of several proteins involved in DNA-damage-related checkpoints and repair, such as p53, MDM2, NBS1 and the downstream kinase, CHK2 (cell-cycle-checkpoint kinase 2). ATM also phosphorylates BRCA1 after treatment with ionizing radiation (IR), in a manner that is required for the BRCA1-mediated response to DNA damage [7]. Like ATM, ATR phosphorylates BRCA1 [8], but whereas BRCA1 phosphorylation after IR is largely ATM-dependent [7], phosphorylation in response to hydroxyurea-induced replication arrest or UV damage requires ATR [8]. Furthermore, ATR colocalizes with BRCA1 in response to replication arrest. This suggests that the pathways by which ATM and ATR activate BRCA1 are distinct, and that they respond to different classes of DNA damage.

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