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Experimental Therapeutics, Molecular Targets, and Chemical Biology |
1 Department of Bioengineering and McGowan Institute for Regenerative Medicine, University of Pittsburgh; 2 Department of Medicine and 3 Cardiovascular Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; 4 Roswell Park Cancer Institute, Buffalo, New York; and 5 Cardiovascular Imaging Center, University of Virginia, Charlottesville, Virginia
Requests for reprints: Flordeliza Villanueva, Cardiovascular Institute, University of Pittsburgh, S-568 Scaife Hall, 200 Lothrop Street, Pittsburgh, PA 15213. Phone: 412-647-5840; Fax: 412-647-4227; E-mail: villanuevafs{at}msx.upmc.edu.
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Abstract |
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Key Words: Targeting • Tumor • Angiogenesis • Molecular Imaging • Ultrasound
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Introduction |
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vß3, which is selectively expressed on angiogenic endothelium (4), has been targeted for the purposes of imaging and drug delivery using magnetic liposomes (5) or echogenic microbubbles (MB; ref. 6) and particles loaded with therapeutic agent (7). Numerous small peptides have been identified that specifically interact in vivo with various types of endothelial cells (EC), including angiogenic endothelium (8, 9). Tumor vasculature in particular has been targeted using angiogenesis-specific peptides containing asparagine-glycine-arginine (NGR), arginine-glycine-aspartate (RGD), or histidine-tryptophan-glycine-phenylalanine (HWGF; refs. 10–12). Using a peptide display library, Brown et al. identified various tumor vasculature–specific binding sequences, including the tripeptide arginine-arginine-leucine (RRL; ref. 13).
Contrast-enhanced ultrasound is a recently developed imaging technique that utilizes i.v. injected, gas-filled, acoustically active MBs that transit unimpeded through the microcirculation, acting as RBC tracers (14, 15) during ultrasound imaging. Recently, these MBs have been modified to adhere to specific endothelial surface epitopes, allowing for the ultrasonic detection of these molecular epitopes. For example, our group has showed that MBs targeted to the endothelial inflammatory marker intercellular adhesion molecule 1 (ICAM-1) selectively bind to cultured endothelial cells overexpressing that marker (16), and that these MBs can be used to echocardiographically detect acute cardiac allograft rejection in vivo (17).
Based on the above considerations, in the current study we hypothesized that ultrasound contrast MBs targeted to tumor vasculature via conjugation with the tumor-binding peptide RRL would preferentially adhere in vitro to tumor-derived versus normal endothelium, and that this selective binding phenomenon would result in increased contrast enhancement during in vivo ultrasound imaging of various solid tumors as compared with normal control myocardium in a murine model.
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Materials and Methods |
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Custom-synthesized 9-mer cyclic peptides contained either the RRL sequence or a glycine control sequence (Genemed Synthesis, South San Francisco, CA). The RRL-containing cyclic peptide comprised the RRL sequence bracketed by glycines and terminated on both ends with cysteine residues (CGGRRLGGC). The control peptide was identical except with the substitution of glycines for the RRL sequence (CGGGGGGGC). Disulfide bonds between the terminal cysteines on each peptide maintain the cyclic structure. Both cyclic peptides were biotinylated at the amino-terminal cysteine.
Microbubble Preparation
Phospholipid-based ultrasound contrast MBs were prepared as previously described (18, 19). Briefly, perfluorobutane gas was dispersed by sonication in a solution of phosphatidyl choline, polyethylene glycol stearate, and biotinylated phosphatidylethanolamine. The resulting product comprised a perfluorobutane gas bubble encapsulated by a biotin-containing phospholipid shell.
The MBs were conjugated to the cyclic peptides via avidin/biotin bridging chemistry. Biotinylated MBs were incubated in a saturating solution of streptavidin (Molecular Probes, Eugene, OR). Unbound streptavidin was removed by washing and centrifugation. The MBs were then incubated in a saturating solution of biotinylated cyclic peptide (100 µg/mL; either RRL or control) and washed again.
MBs linked to the RRL-containing peptide were designated MBRRL, and those linked to the glycine control peptide were designated MBControl. Microbubble diameter as measured by electrozone sensing (Multisizer-II, Beckman-Coulter, Fullerton, CA) was 3.2 ± 1.0 µm. The MB synthesis protocol used excess quantities of RRL peptide that ensured saturation of the MB surface with a maximum amount of RRL peptide, which, based on prior experience with MBs, is on the order of 60,000 peptides per MB (17).
The MBs show no significant toxicity, and animals are routinely recovered without difficulty following contrast ultrasound imaging (17, 20, 21). The agent does not initiate inflammation or microvascular obstruction. Biodistribution of the MBs follows the distribution of blood flow in vivo (14).
In vitro Studies
Cell Culture. Tumor-derived ECs selectively isolated by collagenase digestion from s.c. SCC-VII squamous cell carcinomas grown in C3H/HeJ mice (22) (7th to 8th passage) and human coronary artery ECs (3rd to 5th passage; Cambrex Corp., East Rutherford, NJ) were grown to confluence on glass coverslips in endothelial basal medium (Cambrex Corp.) supplemented with 5% fetal bovine serum. The tumor-derived cells were chosen because they had been used for the original identification of RRL (13) and thus had known binding affinity with RRL. Human coronary artery ECs were chosen to represent normal endothelium; our laboratory has experience manipulating their inflammatory status and investigating targeted MB adhesion to these cells (16, 17, 19).
In vitro Perfusion Protocol. A previously described rectangular parallel plate perfusion chamber (19, 23) was used to examine adhesion of MBs to cultured tumor–derived and normal ECs. Coverslips of ECs (n = 6-8 per condition) were briefly incubated with quinacrine dihydrochloride (50 µmol/L) as a fluorescent label, mounted in the chamber, fastened with a vacuum seal, and primed with culture medium at 37°C. A suspension of either MBRRL or MBControl (3.33 x 106 MBs/mL) in culture medium was perfused through the system at a flow rate corresponding to a wall shear rate of 100 s–1 for 3 minutes, followed by a 3-minute MB-free wash at 100 s–1. Immediately after each perfusion, the chamber was mounted on an epifluorescent microscope (Zeiss, Thornwood, NY), 20 randomly selected microscopic fields were imaged at x1,000, and the number of cells and adhered MBs was quantified.
In vivo Experiments
Mouse Tumor Model. Experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee and complied with the NIH policies on laboratory animal research.
To create solid tumors, 2 x 106 PC3 or Clone C tumor cells were injected s.c. into the flanks of athymic nude mice and allowed to expand for 6 weeks until the tumors were 
0.5 cm3. PC3 cells are derived from metastatic human prostate carcinoma (CRL-1435, American Type Culture Collection, Rockville, MD; ref. 24). Clone C cells are originally derived from murine NIH3T3 stable transfectants, engineered to secrete high levels of fibroblast growth factor 1 (25).
Histologic Confirmation of In vivo RRL Binding. Previous in vitro pilot studies suggested that RRL becomes bound and internalized from the endothelial surface (data not shown). We wanted to extend these studies to show in vivo tumor localization of i.v. injected RRL, prior to in vivo MBRRL imaging. Accordingly, NH2 terminus fluoresceinated peptides containing either RRL or the control sequence GGG (CGGRRLGGC and CGGGGGGGC; Sigma-Genosys, The Woodlands, TX) were injected via the tail vein (1µg in 0.2 mL saline) at hourly intervals for a total of five injections into athymic mice bearing PC3 or Clone C tumors. Fifteen minutes after the last injection, the mice were sacrificed and their vasculature flushed with saline. The heart, lung, kidney, liver, spleen, intestine, and tumors were harvested and frozen in ornithine carbamyl transferase. Unstained cryosections were observed for fluorescein localization. Tumor blood vessels were identified as luminal structures containing blood cells visualized under phase contrast/differential interference contrast microscopy. Digital images were acquired and rendered under identical settings and conditions.
Contrast-Enhanced Ultrasound. Ultrasound imaging used a HDI-5000 ultrasound system and broadband L12-5 transducer (Philips Medical, Bothell, WA) in pulse inversion mode. Images were acquired with a mechanical index (a measure of acoustic output energy) of 0.6, which induces the MB destruction that creates a measurable backscatter signal (26). A small plastic bag of water was positioned as an acoustic interface between the ultrasound probe and the mouse flank. Probe position, gain settings, and midfield focus were initially optimized and maintained throughout each experiment. Time-triggered images (up to four frames) of the tumor or myocardium were acquired at baseline (preinjection), 120 seconds, and 135 seconds after injection of MBs. Images were acquired within the linear range of the ultrasound system and using a linear postprocessing map, such that image videointensity (brightness) was proportional to the number of MBs in the ultrasound field. Images were recorded digitally and analyzed offline, and average pixel intensity (videointensity) was measured in regions of interest encompassing either the tumor or the left ventricle. Background-subtracted images were color-coded using a map in shades of red, progressing to orange, yellow, then white, representing increasing videointensity change (greater MB adhesion).
The goal of the ultrasound image protocol and analysis was to differentiate between the acoustic signal due to adhered MBs and the signal due to MBs still freely circulating in the bloodstream. A previously described method was used to isolate the acoustic signal due to adhered MBs (17, 20, 26). In pharmacokinetic studies of MB contrast agents using intravital microscopy of rat cremaster muscle microvasculature,6 we have consistently observed that by 120 seconds after i.v. MB injection, <2% of the original contrast remains in the circulation. In addition, in pilot imaging studies of mice receiving equivalent doses of a standard, nontargeted contrast agent (Optison, Amersham Health, Princeton, NJ), tumor videointensity was no longer detectable by 120 seconds after injection, suggesting that minimal MBs remained freely circulating after this period. Because MB destruction is required to detect MB presence (acoustic signal), imaging was suspended for 120 seconds after injection to allow for accumulation of MB binding while awaiting washout of unbound MBs. The contrast enhancement in the ultrasound image at 120 seconds should thus derive predominantly from adherent MBs, whereas any contrast at 135 seconds should be attributable to any persistently circulating MBs replenishing the beam in the intervening 15 seconds. Thus, the "videointensity difference" between the 120-second frame (adhered + circulating MBs) and the 135-second frame (predominantly circulating MBs) was defined as a conservative measure of the signal attributable to MB adhesion only (17, 20).
An additional index, VITargeted, calculated as the mean videointensity difference for MBRRL injections minus the videointensity difference for MBControl injections in the same mouse, resulted in a measure that reflected only the acoustic signal due to specific adhesion of the RRL-MBs (17).
Targeted Ultrasound Imaging of Mice. Eleven mice bearing tumors (5 with Clone C and 6 with PC3 tumors) were ultrasonically imaged. To prove the tumor specificity of MBRRL binding, it was necessary to show absence of persistent contrast enhancement of nonneoplastic microvasculature after MBRRL injection. We thus also imaged normal myocardium in non–tumor-bearing mice (n = 10).
Animals were anesthetized with sodium pentobarbitol (i.p. loading dose, 25 mg/kg; i.v. maintenance doses, 25 mg/kg every 15 min), intubated, and ventilated (Harvard rodent ventilator, 0.1 mL, 150 Hz). The right jugular vein was catheterized for administration of saline and MB solutions.
Mice were given an initial bolus injection of 5 x 106 Optison MBs in 0.05 mL saline during time-triggered pulse inversion ultrasound imaging to optimize image settings and to verify adequate probe position and perfusion of the tumor tissue. Mice with tumors that could not be adequately visualized (poor contrast opacification) were excluded from the study. Complete washout of the Optison was verified in selected animals by demonstrating the lack of detectable intravascular contrast material at 2 minutes. Five minutes after complete Optison washout, an i.v. injection of 5x 106 MBRRL or MBControl in 0.05 mL saline was given as a bolus followed by a 0.2-mL saline flush. Ultrasound images were acquired as discussed above. Each mouse received paired injections of MBRRL and MBControl in random order to minimize any sequence bias.
Tumor Histology. Following imaging, several tumors were harvested and sectioned for immunohistochemistry to visualize blood vessel distribution using antibodies against endothelial factor VIII.
Statistical Analysis
Results are expressed as mean ± SD. In vitro adhesion data were analyzed with two-way ANOVA. Acoustic intensities of ultrasound images were analyzed using two-tailed Student's t tests. Statistical significance was defined as P < 0.05.
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Results |
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In vivo Targeted Ultrasound Imaging. Of the 21 animals studied, 4 tumor-bearing mice were excluded from the study due to inadequate tumor perfusion as qualitatively visualized during the initial Optison imaging.
Figure 4 shows representative background-subtracted, ultrasound images of two different tumors in two mice. The topmost panels show images of a Clone C tumor obtained 120 seconds after i.v. injection of MBRRL (Fig. 4A) or MBControl (Fig. 4B). Corresponding images from a PC3 tumor in another mouse are shown for MBRRL (Fig. 4C) and MBControl (Fig. 4D). There was intense contrast enhancement (acoustic signal) in both tumors after injection with MBRRL (Fig. 4A and C). The corresponding images for MBControl (Fig. 4B and D) showed only mild contrast enhancement.
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Representative ultrasound images of normal myocardial tissue in a third mouse are also shown in Fig. 4H and I. There was little difference in videointensity resulting from the two types of MBs, suggesting that there was only minimal adhesion of the RRL-bubble to normal microvasculature. For all image sets, there was no significant tissue opacification 15 seconds later in any of the injections (images not shown), confirming that there were few remaining circulating MBs, and indicating that the acoustic signal at 120 seconds was due predominantly to adhered MBs.
Quantitative videointensity data are shown in Fig. 5. Each line represents a single mouse, with the first point representing the videointensity value (indicative of the number of adhered bubbles) from the MBControl injection, and the second point representing the MBRRL injection. There was little difference in myocardial signal intensity between MBControl and MBRRL. There was a substantial increase in signal in all but one of the tumor-bearing mice when using MBRRL versus MBControl, indicating specific retention of the tumor-targeted agent in the tumor microvasculature.
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Discussion |
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Targeted MB Adhesion to Tumor Endothelium. The in vitro experiments used a parallel plate perfusion chamber, which has been widely utilized to study cellular adhesive events under controlled shear conditions (27, 28). MBs targeted via RRL selectively bound to cultured tumor–derived but not normal ECs under physiologically relevant shear conditions. In comparison, MBs conjugated to a control glycine peptide (MBControl) showed minimal adherence to both cell types, suggesting specific binding interactions between the RRL sequence and its ligand on the endothelial surface. MBControl binding was nonetheless greater than zero, which may indicate a degree of nonspecific adhesion by the cyclic peptide. The magnitude of this effect, however, was small compared with the specific RRL adhesive interaction, suggesting that neither the lipid components of the MB shell nor presence of avidin and biotin moieties contributed significantly to the observed adhesion.
In vivo Localization of RRL. The fluorescent RRL studies showed for the first time that this peptide preferentially adheres to tumor vasculature of both Clone C and PC3 tumors in vivo. Furthermore, tagged RRL localization was not associated with native organs, except for some binding to renal tubules and glomeruli. These histology data suggest that the target of RRL, albeit as yet unidentified, is not only tumor specific but also endothelial in location. This is key because the endoluminal location of the target is a requirement for the binding of targeted MBs, which remain exclusively within the intravascular space. In addition, the target of RRL is accessible by injecting the peptide through a peripheral vein, as would be the route of MB injection, and binds to its target under the physiologic shear conditions to which MBRRL would be exposed. These findings, together with the in vitro data, offered a basis for interpreting the ultrasound imaging data in terms of specific microbubble-endothelial interactions.
Ultrasound Imaging of Tumor Angiogenesis. MBs targeted via RRL generated enhanced ultrasound opacification of tumors relative to normal myocardial tissue, which parallels our in vitro data showing greater adhesion of MBRRL to tumor endothelium than to normal endothelium. There was slight contrast enhancement of MBControl to both tissue types (Fig. 4B, D, and I), which is consistent with our in vitro observation that some MBControl adhere to endothelial cells, possibly due to nonspecific interactions between the control peptide and the endothelium, as discussed above. Alternatively, attachment of MBControl may represent MB binding to activated, endothelium-adherent leukocytes, as has previously been shown in models of acute inflammation (21). Regardless of the mechanism, the magnitude of contrast enhancement resulting from nonspecific attachment was significantly lower than that generated by MBRRL attachment.
As seen in Fig. 4, the pattern of tumor opacification often showed higher contrast enhancement in the tumor periphery than the core. This spatial distribution could indicate that the tumor periphery is more angiogenic, expressing a higher density of the endothelial tumor-specific ligand for RRL than the tumor core. However, because the initial Optison images delineated a similar pattern of perfusion, we believe that the opacification is matching the anatomic pattern of the tumor vasculature, with a highly vascularized tumor periphery and a necrotic tumor core. This hypothesis was validated by histologic examination of harvested tumor specimens (Fig. 4E-G), which showed large vessels predominantly in the tumor periphery, with necrosis and poor vascularity in the tumor center.
Comparison to Previous Studies. Previously reported techniques for the noninvasive imaging of tumor angiogenesis rely on anatomic measurements of tumor size, blood flow, or vascular permeability (29–31), whereas others have used molecular imaging techniques to assess tumor endothelial-specific molecular markers (5, 32, 33) . These functional imaging techniques most often utilize ligands (usually peptides) that bind to tumor- or angiogenesis-specific endothelial markers that are visualized by radiolabeling or conjugation to a contrast agent. Examples include magnetic resonance imaging using anti–vß3 antibody-bearing contrast agents (5), positron emission tomography using RGD-bearing 18F-labeled glycopeptides (32), and 99mtechnetium-labeled RGD peptide (33).
Contrast-enhanced ultrasound has some advantages over other imaging modalities for the functional assessment of vascular beds in vivo. Magnetic resonance imaging generally offers good spatial resolution but currently has a relatively poor signal-to-noise ratio, translating to low sensitivity (34). Scintigraphic methods generally yield high sensitivity but suboptimal spatial resolution (35). Both magnetic resonance imaging and positron emission tomography are hindered by their requirement for instrumentation typically limited to tertiary care centers. Conventional, noncontrast ultrasound can image only the larger vessels and has limited utility for assessing microvascular tumor angiogenesis (35). Conversely, contrast-enhanced ultrasound has good spatial resolution, is portable, widely available, offers real-time imaging, and uses contrast agents that remain exclusively intravascular, minimizing nonspecific signals from extravasated contrast.
Targeted contrast ultrasound has previously been utilized to detect vascular disease, including thrombus using fibrinogen-targeted MBs (36), inflammation using MBs targeted to P-selectin (37) or ICAM-1 (16), and acute cardiac transplant rejection by targeting ICAM-1 (17).
Contrast-enhanced ultrasound has only recently been investigated as a tool for imaging angiogenesis. Leong-Poi et al. reported that MBs bearing anti-integrin antibodies adhered to fibroblast growth factor–stimulated vessels overexpressing v integrins in vivo (6). We advance upon this work, first, by using short cyclic peptide rather than animal-derived antibodies, which are costly and immunogenic, and second, by investigating tumor neovasculature rather than nontumor laboratory models of generalized angiogenesis. Our study is thus unique in that we targeted ultrasound contrast MBs to tumor vasculature using a clinically relevant in vivo model of neoplasia and a targeting moiety (RRL) raised specifically for tumor endothelium. Furthermore, we were able to show specific adherence of the targeted agent to tumor versus normal vasculature.
Limitations. Limitations of the parallel plate studies are those inherent to in vitro experiments in general. Cultured ECs are an imperfect model for in situ endothelium. Perfusate flow was nonpulsatile and included no blood components. Tumor-derived and normal ECs were of different species (murine versus human); however, we have observed that RRL does species cross-react with some human tumors, including sarcomas and prostate tumors such as PC3.7 RRL may show differential binding interactions between different tumor types (as seen in Fig. 4), and the RRL peptide may internalize beyond the endothelial lining, as the extravascular fluorescence in Fig. 3 may indicate. The molecular target for the tumor-binding peptide RRL is as yet unknown; the identification of the specific target is beyond the scope of this proof-of-principle study. Preliminary investigations into this issue indicate that RRL is likely not a standard endothelial inflammatory marker, as its binding to ECs in vitro does not increase with inflammatory stimulation (data not shown).
Summary. Our data show the ability of targeted contrast-enhanced ultrasound to noninvasively detect tumor angiogenesis in vivo. Contrast MBs targeted to tumor vasculature via conjugation with the tumor-binding peptide RRL were shown to preferentially bind in vitro to tumor-derived cultured endothelium. Furthermore, we demonstrates the noninvasive, in vivo ultrasonic detection of angiogenic tumor vasculature in a tumor-bearing mouse model and showed that this technique could distinguish between normal tissue and tumor tissue.
Such ultrasonic molecular imaging of tumor angiogenesis may permit the functional assessment of tumor vasculature in the clinical setting and could thus offer noninvasive identification of tumors, assessment of malignant potential, and improved monitoring of tumor response to antiangiogenic therapies.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank David Fischer and Melissa Csikari for their valuable technical assistance.
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Footnotes |
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7 G.E.R. Weller, M.K.K. Wong, R.A. Modzelewski, E. Lu, A.L. Klibanov, W.R. Wagner, and F.S. Villanueva, unpublished data.
Received 7/29/04. Revised 9/21/04. Accepted 11/ 8/04.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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vß3 for angiogenesis. Science 1994;264:569–71.vß3-targeted magnetic resonance imaging. Nat Med 1998;4:623–6.[CrossRef][Medline]
v integrins. Circulation 2003;107:455–60.vß3 integrin expression using 18F-labeled RGD-containing glycopeptide and positron emission tomography. Cancer Res 2001;61:1781–5.vß3 integrin for tumor imaging. Bioconjug Chem 2002;13:561–70.[CrossRef][Medline]
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