Preclinical Molecular Imaging of Tumor Angiogenesis

Abstract

Angiogenesis, a course that new blood vessels grow from the existing vasculature, plays important roles both physiologically and pathologically. Angiogenesis can be switched on by growth factors secreted by tumor cells, and in turn supplies more oxygen and nutrition to the tumor. More and more preclinical studies and clinical trials have shown that inhibition of angiogenesis is an effective way to inhibit tumor growth, substantiating the development of anti-angiogenesis therapeutics. Imaging technologies accelerate the translation of preclinical research to the clinic. In oncology, various imaging modalities are widely applied to drug development, tumor early detection and therapy response monitoring. So far, several angiogenesis related imaging agents are promising in cancer diagnosis. However, more effective imaging agents with less side-effect still need to be pursued to visualize angiogenesis process non-invasively. The main purpose of this review is to summarize the recent progresses in preclinical molecular imaging of angiogenesis and to discuss the potential of the current preclinical probes specific to various angiogenesis targets including vascular endothelial growth factor and its receptors (VEGF/VEGFRs), integrin α_vβ₃ and matrix metalloproteinases (MMPs). It is predicable that related investigations in the field will benefit cancer research and quicken the anti-angiogenic drug development.

INTRODUCTION OF TUMOR ANGIOGENESIS

Angiogenesis is critical in both physiological development and pathological processes such as tumor progression, wound healing, cardiovascular, inflammatory, ischemic, and infectious diseases ^1–3. For multicellular tumor clones to grow beyond 100–200 μm, they must recruit new blood vessels by angiogenesis and vasculogenesis ^4–6. It is now widely accepted that both mutations of oncogenes and tumor suppressor genes lead to the switch into an angiogenic tumor, i.e., the endogenous balance between pro-angiogenic and anti-angiogenic molecules is tipped in favor of angiogenesis ^7–9. For example, tumor tissues produce and release angiogenic growth factors such as vasculoendothelial growth factor (VEGF), the acidic and basic fibroblast growth factors (aFGF, bFGF), and the platelet-derived endothelial cell growth factor (PD-ECGF) ¹⁰. When these angiogenic growth factors bind to their corresponding specific receptors located on the endothelial cells of pre-existing blood vessels, various signal transduction pathways are activated to promote the activation of endothelial cells ^11,12. Subsequently, the original vessels undergo characteristic morphological changes, including enlargement of the diameter, basement membrane degradation, a thinned endothelial cell lining, increased endothelial number, decreased number of pericytes and detachment of pericytes ¹³. Tumor angiogenesis also involves an intricate interplay between the tumor and surrounding or supportive cells, including vascular endothelial cells, pericytes, smooth muscle cells, fibroblasts and tumor-associated macrophages ¹⁴. Tumor vessels can grow by several different patterns including sprouting, intussusception or incorporation of bone marrow-derived endothelial precursors. In addition, tumor cells can co-opt existing vessels ¹⁵. Sprouting angiogenesis is the most important mechanism for tumor vascularization, which involves several steps from the growth of endothelial sprouts from preexisting post-capillary venules to the growth and remodeling process of the primitive network into a complex network ^2,16–18.

At the sprouting tips of growing vessels, endothelial cells secrete matrix metalloproteinases (MMPs) to facilitate the degradation of extracellular matrix and cell invasion ¹⁹. Cell surface adhesion molecules such as integrins also play an important role in endothelial cell migration and in contact with the extracellular tumor matrix, facilitating cell survival ^20,21. Next, a lumen within an endothelial cell tubule has to be formed, which requires interactions between the extracellular matrix and cell-associated surface proteins, such as galectin-2, PECAM-1, and VE-cadherin ²². Finally, newly formed vessels are stabilized through the recruitment of smooth muscle cells and pericytes. Unlike blood vessels in healthy tissues, the tumor vasculatures appear as disorganized tubular structures, which are often interconnected, tortuous, highly leaky, resembling premature sinusoidal vasculatures ^23,24. These abnormal vessels usually lack a clear separation between arterioles and venules and the recruitment of pericytes and vascular smooth muscle cells ²⁵.

IMAGING OF TUMOR ANGIOGENSIS

Anti-angiogenesis therapy and tumor response assessment

In as early as 1971, Folkman proposed that anti-angiogenesis might be an effective anticancer strategy ⁶ based on the observation that tumor growth was associated with marked vascularity ²⁶. Recognition of the VEGF pathway as a key regulator of angiogenesis has led to the development of several VEGF-targeted agents, including agents that prevent VEGF-A from binding to its receptors ²⁷, antibodies that directly block VEGFR-2 ^28,29, and small molecules that inhibit the kinase activity of VEGFR-2 thereby blocking growth factor signaling ^30–32. Many studies using VEGF-targeted therapies in murine models demonstrated that inhibition of VEGF signaling could lead to tumor endothelial cell apoptosis, although rarely did such therapies lead to regression of established tumors ^33,34.

So far, hundreds of molecules with anti-angiogenic activity in preclinical models have been reported, and many of them have entered clinical testing in oncology. Several of them have been approved for human use in solid tumors. For example, Bevacizumab, a monoclonal antibody against VEGF-A, is the first clinically available angiogenesis inhibitor in US ³⁵. Another category of VEGF/VEGFR targeted therapeutics is tyrosine kinase inhibitors. These agents compete with ATP for binding within the intracellular domain of various wild-type and/or mutated receptor tyrosine kinases ³⁶. Unlike Bevacizumab, which targets extracellular VEGF, tyrosine kinase inhibitors target the intracellular signaling pathways of VEGFRs ³⁷.

The most commonly used end-point for assessing anti-angiogenic treatment in clinical studies is microvessel density (MVD), measured from biopsies taken before and at one or more times after treatment, using a variety of immunohistochemical vascular markers such as CD34, CD31, CD105 and von Willebrand factor (vWF) to identify the vessels ³⁸. However, measurement of MVD is problematic for assessing the vascular efficacy of antiangiogenic agents ³⁹ since blocking angiogenesis may be accompanied by a proportional reduction in tumor growth that would not result in a net change in MVD. Besides, vessel counts and/or density may remain unchanged even in the face of effective therapy ⁴⁰. Therefore, although a reduction in MVD following treatment is indicative of an antiangiogenic effect, it does not mean that no change in MVD is indicative of no antiangiogenic effect, as is commonly assumed.

Non-invasive imaging methods for measuring functional vascular volume are available and can provide a noninvasive means of detecting angiogenesis within and about the perimeter of the whole tumor and give functional information. For instance, PET studies with ¹⁵O-oxygen and related tracers can offer direct physiological measurement of circulatory parameters of regional blood flow and vascular volume ⁴¹. Ultrasound (particularly microbubble contrast enhanced ultrasound) is also a valuable imaging modality to determine the tumor microvascular blood volume and blood velocity ⁴². Especially, dynamic contrast-enhanced ultrasonography (DCE-US) allows repeated examinations and provides both morphologic and functional analyses. Several quantitative parameters considered as indicators of tumor flow such as the peak intensity (PI) or time-to-PI can be extracted from the time-intensity curves of contrast uptake ⁴³. Power Doppler ultrasonography has been used to demonstrate the presence of blood flow in small vessels and it was also found that the vascular signal correlates with histopathological quantification of the vascular density of synovial tissue ⁴⁴.

Dynamic contrast-enhanced MRI (DCE-MRI) has also been well established to investigate angiogenesis within tumors, and in particular the response to antiangiogenic therapy. The leakage of MR contrast agent through tumor vessels results in a fast “wash-in” of contrast coupled with the rapid “wash-out,” and allows a functional analysis of the tumor microcirculation ⁴⁵. DCE-MRI has been the most utilized pharmacodynamic imaging modality in early phase clinical trials of angiogenic inhibitors. This functional imaging technique is non-invasive and can be used to serially assess tumor vasculature in vivo ⁴⁶. However, like MVD measurements, a negative effect on vascular volume indicated by non-invasive imaging cannot be interpreted as absence of antiangiogenic effect, either ⁴⁷. Indeed, a study in a xenograft model of human breast cancer showed a poor correlation between MVD and fractional blood volume estimates as measured by functional MRI and macromolecular contrast agents ³⁴. Tumor blood flow rate is also an accessible end-point for clinical studies. A decrease in tumor blood flow rate is expected if MVD is decreased and its measurement would provide additional functional information linked to oxygen availability and tumor growth. However, some pre-clinical studies have demonstrated an increase in tumor blood flow rate following antiangiogenic therapy. For example, Teicher et al. ⁴⁸ showed that tumor blood flow and oxygenation significantly was increased in the first weeks of treatment with TNP-470, a synthetic analogue of fumagillon. Following antiangiogenic therapy, blood flow rate within individual vessels may be improved, which has been termed as “normalizing tumor vasculature” ⁴⁹. The mechanisms may lie in that the most immature and inefficient tumor blood vessels are “pruned” from the tumor vascular network by antiangiogenic therapy, leaving a more efficient system ⁴⁹. In addition, many pro-angiogenic growth factors are associated with high vascular permeability and their withdrawal can reverse this effect ⁵⁰. It is possible that a decrease in vascular permeability to macromolecules could improve blood flow rate by reducing tumor interstitial fluid pressure. Thus, measurement of vascular permeability or interstitial fluid pressure could provide alternative end-points for assessing tumor vascular effects of antiangiogenic agents ⁴⁷.

Molecular imaging of tumor angiogenesis

Compared with traditional method, molecular imaging usually exploits specific molecular probes as well as intrinsic tissue characteristics as the source of imaging contrast, and provides the potential for understanding the integrative biology, earlier detection and characterization of disease, and evaluation of treatment ⁵¹. Imaging probes with high affinity and specificity would be the key to successful molecular imaging. Currently, several important angiogenesis related targets including VEGF/VEGFRs, integrins, and MMPS are being intensively investigated to evaluate both tumor angiogenesis and tumor response to various anti-angiogenesis drugs.

Imaging VEGF/VEGFRs

In view of the critical role of VEGF/VEGFR in cancer progression, development of VEGF- or VEGFR-targeted molecular imaging probes could serve as a new paradigm for the assessment of anti-angiogenic therapeutics, and for better understanding the role and expression profile of VEGF/VEGFR in many angiogenesis-related diseases. Due to the soluble and more dynamic nature of VEGF, imaging VEGF expression and explanation of the imaging results can be difficult, although single photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging of VEGF has been performed with radiolabeled anti-VEGF antibodies ⁵². VG76e, an IgG₁ monoclonal antibody that binds to human VEGF, was labeled with ¹²⁴I for PET imaging of solid tumor xenografts in immune-deficient mice ⁵³. Whole-animal PET imaging studies revealed a high tumor-to-background contrast. Although VEGF specificity in vivo was demonstrated in this report, the poor immunoreactivity (< 35%) of the radiolabeled antibody limits the potential use of this tracer. HuMV833, the humanized version of a mouse monoclonal anti-VEGF antibody MV833, was also labeled with ¹²⁴I and the distribution and biological effects of HuMV833 in patients in a phase I clinical trial were investigated ⁵⁴. Patients with progressive solid tumors were treated with various doses of HuMV833 and PET imaging using ¹²⁴I-HuMV833 was carried out to measure the antibody distribution in and clearance from tissues. It was found that antibody distribution and clearance were quite heterogeneous not only between and within patients but also between and within individual tumors. Bevacizumab, a humanized monoclonal antibody against VEGF, has been labeled with ¹¹¹In to image VEGF-A expression in nude mice model or patients with colorectal liver metastases ⁵⁵. Although enhanced uptake of ¹¹¹In-bevacizumab in the liver metastases was observed in 9 of the 12 patients, there was no correlation between the level of ¹¹¹In-antibody accumulation and the level of VEGF-A expression in the tissue as determined by in situ hybridization and ELISA ⁵⁵. Bevacizumab has also been labeled with the PET isotope ⁸⁹Zr for noninvasive in vivo VEGF visualization and quantification. On small-animal PET images, radiolabeled bevacizumab showed higher uptake compared with radiolabeled human IgG in a human SKOV-3 ovarian tumor xenograft. Tracer uptake in other organs was seen primarily in the liver and spleen (Figure 1) ⁵². However, the slow distribution and clearance of antibodies within tumors also make it not an optimal tracer to monitor therapy response of tumors to anti-angiogenic treatment.

Figure 1.

Noninvasive imaging of VEGF expression in vivo by ⁸⁹Zr-labeled VEGF antibody Bevacizumab. A. Coronal CT image (left) with clear subcutaneous localization of SKOV-3 tumor (arrow) and fusion of microPET and CT images (right) (168 h after injection) (arrows indicate SKOV- 3 tumors). B. Coronal planes of microPET images at different time points after ⁸⁹Zr-Bevacizumab injection. Reproduced from reference ⁵² with permission.

The more rational design is to use radiolabeled VEGF isoforms for SPECT or PET imaging of VEGFR expression. With SPECT imaging, recombinant human VEGF₁₂₁ was labeled with ¹¹¹In for the identification of ischemic tissue in a rabbit model, where unilateral hind-limb ischemia was created by femoral artery excision ¹⁸. VEGF₁₂₁ has also been labeled with ^99mTc through an “Adapter/Docking” strategy and the tracer was tested in a murine mammary carcinoma ⁵⁶. Cai et al. have labeled VEGF₁₂₁ with ⁶⁴Cu for PET imaging of tumor angiogenesis and VEGFR expression ⁵⁷. MicroPET imaging revealed the dynamic nature of VEGFR expression during tumor progression in that even for the same tumor model, VEGFR expression level can be dramatically different at different stages. Indeed, the uptake of ⁶⁴Cu-DOTA-VEGF₁₂₁ in the tumor peaked when the tumor size was about 100–250 mm³. Both small and large tumors had lower tracer uptake indicating a narrow range of tumor size with high VEGFR-2 expression. The tumor uptake value obtained from PET imaging had good linear correlation with the relative tumor tissue VEGFR-2 expression as measured by Western blot ⁵⁸.

Random radiolabeling or bioconjugation may impair VEGF conformation and result in high uptake in major organs, such as liver and kidney ⁵⁹. Modification of the protein at a specific site or using a linker can avoid impairing the structure of VEGF. Blankenberg et al. constructed recombinant VEGF containing a cysteine containing peptide tag (C-tag) that allows for site-specific modification of C-tag-containing fusion proteins with a bifunctional chelator, HYNIC (hydrazine nicotinamide)-maleimide ⁶⁰. With ^99mTc labeled HYNIC-VEGF, 4T1 murine breast tumors showed a decreased contrast agent uptake after treatment. In the following study, a new single-chain Cys-tagged VEGF (scVEGF) was developed by fusion of two fragments (amino acids 3–112) of human VEGF₁₂₁ ⁵⁶. The scVEGF showed a high binding affinity to VEGFR-2 and receptor mediated internalization into PAE/KDR cells. After being conjugated with Cy dye molecule, ^99mTc and ⁶⁴Cu, in vivo imaging indicated that all the tumors showed enhanced contrast agent uptake by the optical, SPECT and PET imaging. We developed an alternative site-specific modification of VEGF with an Avi-tag fused to the C-terminus of VEGF₁₂₁ to allow site-specific biotinylation without disruption of VEGF₁₂₁ function ⁶¹. The biotinylated VEGF₁₂₁-Avi (VEGF-Avib) was able to form a stable complex with streptavidin-IRDy800 (SA800). VEGF-Avib/SA800 had significantly higher tumor signal intensity in a 67NR murine breast cancer xenograft model. Tumor-to-background ratio was higher than those of randomly labeled control VEGF₁₂₁ probes at all different times up to 66 hours, confirming the superiority of site-specific labeling strategy (Figure 2).

Figure 2.

In vivo optical imaging with site-specifically labeled VEGF₁₂₁. A. Optical image of 67NR tumors by VEGF₁₂₁-Avi-IRDye800 (VEGF-Avi/800) at different time point after tracer injection. B. Optical image of 67NR tumors by VEGF₁₂₁-Avi-biotin/streptavidin-IRDye800. C. Optical image of 67NR tumors by VEGF_mutant-Avi-biotin/streptavidin-IRDye800 (VEGFm-Avib/SA800). D. Optical image of 67NR tumors by streptavidin-IRDye800 (SA800). Reproduced from reference ⁶¹ with permission.

It is well known that all VEGF-A isoforms bind to both VEGFR-1 and VEGFR-2. A VEGFR-2-specific PET tracer has been developed using the D63AE64AE67A mutant of VEGF₁₂₁ (VEGF_DEE) generated by recombinant DNA technology. The renal uptake of ⁶⁴Cu-DOTA-VEGF_DEE was significantly lower than that of ⁶⁴Cu-DOTA-VEGF₁₂₁ as rodent kidneys expressed high levels of VEGFR-1 based on immunofluorescence staining ⁶². With the development of new tracers with better targeting efficacy and desirable pharmacokinetics, clinical translation will be critical for the maximum benefit of VEGF-based imaging agents. Peptidic VEGFR antagonists can be labeled with short-lived isotopes such as ¹⁸F and they may allow for higher throughput than antibody- or protein-based radiotracers, as one hour post-injection is usually sufficient for a peptide-based tracer to clear from the non-targeted organs and give high contrast images ⁶³. One peptide (QKRKRKKSRYKS) encoded by VEGF-A₁₈₉ exon 6 was reported ⁶⁴ and labeled with ¹⁸⁸Re for SPECT imaging of VEGFR in tumor-bearing nude mice. Planar imaging with SPECT demonstrated significant radioactivity accumulation in tumor 1 h after injection of the labeled peptide and disappearance of radioactivity 3 h later, facilitating repetitive imaging with the peptide for therapy response monitoring ⁶⁵.

Ultrasonography (US) is by far one of the most commonly used clinical imaging modalities because it is safe and cost effective. Ultrasonic contrast agents such as microbubbles have been the subject of active research, especially in recent years, with added interest in developing site-directed ultrasonic contrast agents ⁵¹. With at least several micrometers in diameter, microbubbles are too large to extravasate so only the tumor endothelium can be targeted ⁶⁶. Moreover, acoustic destruction of “payload-bearing” microbubbles can be used to deliver drugs or to augment gene transfection ⁶⁷. Angiogenesis-targeted microbubbles may also have applications in site-specific therapy for ischemic tissues or tumors. Thus, VEGFR-2 is an excellent candidate for targeted untrasound imaging since it is almost exclusively expressed on activated endothelial cells ⁶⁸. In a mouse model of pancreatic adenocarcinoma, anti-VEGFR2 or anti VEGF-VEGFR complex antibodies conjugated microbubbles were used to image and quantify vascular effects of two different anti-tumor therapies in both subcutaneous and orthotopic pancreatic tumors ⁶⁶. Significant signal enhancement of tumor vasculature was observed when compared with untargeted or control IgG-targeted microbubbles. Video intensity from targeted microbubbles also correlated with the expression level of the target (VEGFR-2 or the VEGF-VEGFR complex) and with MVD in tumors under therapy. In another report, Willmann et al. have imaged VEGFR-2 expression in two murine tumor models using anti-VEGFR2 monoclonal antibody conjugated microbubbles ^69,70. Contrast-enhanced ultrasound imaging using targeted microbubbles showed significantly higher average video intensity compared with control microbubbles in both tumor models. These studies support that targeted microbubbles can be used for non-invasive, vascularture-targeted molecular imaging of tumor angiogenesis and for in vivo monitoring of vascular effects after therapy. Recently, BR55, an ultrasound contrast agent was evaluated in vitro and in vivo for the molecular imaging of tumoral angiogenesis ⁷¹. BR55 was based on a heterodimer peptide targeting the VEGFR2, and incorporation of a biospecific lipopeptide into the microbubble membrane. BR55 showed a similar accumulation in tumor as microbubble bearing the specific antibody. In the meantime, Pillai et al. attached a heterodimeric peptide to a pegylated phospholipid and showed that the resulting construct retained nanomolar affinity for its target, VEGFR-2. They demonstrated that the phospholipid-PEG2000-peptide is smoothly incorporated into gas-filled microbubbles and provides imaging of angiogenesis in a rat tumor model ⁷².

Although optical imaging may not be widely used in clinical settings, near infrared (NIR) (700–900 nm) approaches provide opportunities for rapid and cost-effective preclinical evaluation in small animal models. Optical imaging has been used to study gene expression ⁷³, tumor angiogenesis, physiological function of tumors, and tumor metastasis ⁷⁴. In a transgenic mouse model where a VEGF promoter was chosen to drive a GFP reporter gene, VEGF expression during wound healing and possible impairment of wound healing due to collateral tissue damage was imaged in vivo ⁷³. Human VEGF has also been conjugated to a self-assembled “dock and lock” system and retained its functional activities ⁷⁵. After incorporating an additional cysteine residue for site-specific modification, a NIR fluorescent dye Cy5.5 (maximum emission 696 nm) was conjugated and the resulting Cy5.5-VEGF was used for in vivo imaging. Although tumor contrast was observed after administration of the probe, no information was reported about the whole body distribution of Cy5.5-VEGF ^75,76. To develop a dual-function PET/NIRF probe, our group conjugated VEGF protein and DOTA chelator on an amine-functionalized QD (DOTA-QD-VEGF) for VEGFR-targeted PET/NIRF imaging. The DOTA-QD-VEGF exhibited VEGFR-specific binding in both cell-binding assay and cell staining experiment. Both NIR fluorescence imaging and microPET showed VEGFR-specific delivery of conjugated DOTA-QD-VEGF nanoparticle and prominent reticuloendothelial system uptake. Moreover, good correlation was also observed between the results measured by ex vivo PET and NIRF organ imaging ⁷⁷. Another component of optical imaging is bioluminescence imaging (BLI), which can be used to detect very low levels of signal because the emitted light is virtually background free ⁷⁸. Non-invasive indirect imaging of VEGF expression with BLI in living transgenic mice has also been reported, where a two-step transcriptional amplification approach was used to augment the transcriptional activity of the relatively weak VEGF promoter ⁷⁹.

Imaging of integrins

Integrins are a family of receptors comprised of a family of heterodimeric glycoproteins, which are involved in the formation of new blood vessels in tumors ⁸⁰. Integrins expressed on endothelial cells are related to cell survival and migration during angiogenesis, while integrins expressed on carcinoma cells modulate metastasis by facilitating invasion and movement across the vessels ⁸¹. Each integrin member consists of an α and a β subunit, 18 different α and 8 different β subunits are known in mammals, which can combine to 24 different integrin receptors ^82,83. Among them, the α_vβ₃ integrin, which binds to arginine-glycine-aspartic acid (RGD)-containing components of the interstitial matrix such as vitronectin, fibronectin and thrombospondin ^84,85, is expressed in a number of tumor types such as melanoma, late stage glioblastoma, ovarian, breast, and prostate cancer ^63,86,87. Integrin α_vβ₃ promotes angiogenesis and endothelial cell survival and that antagonism of this integrin suppresses angiogenesis by inducing endothelial cell apoptosis in vitro and in vivo ^80,88,89. Apart from α_vβ₃, integrin α_vβ₁, α_vβ₅, α₅β₁, and α₄β₁ also play important roles in regulating angiogenesis.

Integrins are ideal pharmacological targets based on both the key role they played in angiogenesis, leukocytes function and tumor development and easy accessibility as cell surface receptors interacting with extracellular ligands ⁹⁰. So far, the integrin superfamily represents the best opportunity of targeting both antibodies and small-molecule antagonists for both therapeutic and diagnostic utility in various key diseases ⁹¹. Preclinical studies and clinical trials showed that quite a few integrin targeting antibodies were effective in blocking tumor growth and metastasis ^80,92,93. Small molecular antagonists, mainly based on RGD containing peptides and RGD peptidomimetics ^94–96, also showed potent inhibition of angiogenesis. More detailed information can be found in several recently published review articles ^90,97.

First in vivo application of radioiodinated RGD peptides revealed the receptor-specific tumor uptake but also predominantly hepatobiliary elimination, resulting in high activity concentration in the liver and small intestines ⁹⁸. Consequently, several strategies to improve the pharmacokinetics of radiohalogenated peptides have been studied including conjugation with sugar moieties, hydrophilic amino acids and polyethylene glycol (PEG) ^99–102. Besides radiohalogenated RGD peptides, a variety of radiometalated tracers have been developed as well, including peptides labeled with ¹¹¹In, ^99mTc, ⁶⁴Cu, ⁹⁰Y, ¹⁸⁸Re and ⁶⁸Ga ^103–106. Most of them are based on the cyclic pentapeptide c(RGDfK) or c(RGDyK) and are conjugated via the γ-amino function of a lysine with different chelator systems, like diethylene triamine pentaacetic acid (DTPA), the tetrapeptide sequence H-Asp-Lys-Cys-Lys-OH, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA). While all these compounds have shown high receptor affinity and selectivity and specific tumor accumulation, the pharmacokinetics of most of them still need to be improved ¹⁰⁷. NC100692 is a cyclic synthetic RGD containing ligand with disulfide bond. The compound ^99mTc-NC100692 by GE Healthcare has been used for SPECT imaging in preclinical and clinical studies ¹⁰⁸. Another radiotracer, ¹⁸F-AH111585 has the core sequence of ACDCRGDCFCG and was also applied to detect tumors in metastatic breast cancerpatients using PET imaging ¹⁰⁹.

In a human melanoma M21 model, ¹⁸F-Galacto-RGD showed a tumor uptake of 1.5 %ID/g at 120 min postinjection (p.i.) ^110,111. A correlation between integrin expression and tracer accumulation was observed in imaging studies with mice bearing melanoma tumors with increasing amounts of α_vβ₃-positive cells ¹¹². ¹⁸F-Galacto-RGD has also been applied to patients and successfully imaged α_vβ₃ expression in human tumors with good tumor/background ratios ¹¹³. Rapid clearance of ¹⁸F-Galacto-RGD from the blood pool and primarily renal excretion was confirmed by following biodistribution and dosimetry studies. Background activity in lung and muscle tissue was low and the calculated effective dose is very similar to an ¹⁸F-FDG scan ¹¹⁴. Standard uptake values (SUVs) and tumor/blood ratios from static emission scans at ~ 60 min. p.i. of ¹⁸F-Galacto-RGD were found to correlate with the intensity of immunohistochemical staining of α_vβ₃ expression as well as with the microvessel density ¹¹⁵. There was no obvious correlation between the tracer uptake of ¹⁸F-FDG and ¹⁸F-Galacto-RGD in patients with various tumors, indicating that α_vβ₃ expression and glucose metabolism are not closely correlated in tumor lesions and that consequently ¹⁸F-FDG cannot provide similar information as ¹⁸F-Galacto-RGD ¹¹⁶.

Within physiological ¹⁸F-Galacto-RGD uptake area, such as liver, spleen and intestine, lesion identification is still problematic ¹¹⁷. Therefore, multimeric RGD peptides have been developed in order to provide more effective antagonists with better targeting capability and higher cellular uptake through the integrin-dependent binding ¹¹⁸. The underlying rationale is that the interaction between integrin α_vβ₃ and RGD-containing ECM-proteins involves multivalent binding sites with clustering of integrins. Wester and Kessler groups have synthesized a series of monomeric, dimeric, tetrameric and octameric RGD peptides. These compounds contain different numbers of c(RGDfE) peptides connected via PEG linker and lysine moieties, which are used as branching units ^119,120. We also developed a series of multimeric RGD peptides labeled with ¹⁸F or ⁶⁴Cu for PET imaging to improve the tumor-targeting efficacy and pharmacokinetics ^{104,121–125}. ¹⁸F-FB-E[c(RGDyK)]₂ (abbreviated as ¹⁸F-FRGD2) showed predominantly renal excretion and almost twice as much tumor uptake in the same animal model compared with the monomeric tracer ¹⁸F-FB-c(RGDyK) ^121,122. Tumor uptakes quantified by microPET scans in six tumor xenograft models correlated well with integrin α_vβ₃ expression level measured by SDS-PAGE autoradiography. The tetrameric RGD peptide-based tracer, ¹⁸F-E[E[c(RGDfK)]₂]₂, showed significantly higher receptor binding affinity than the corresponding monomeric and dimeric RGD analogues and demonstrated rapid blood clearance, high metabolic stability, predominant renal excretion and significant receptor-mediated tumor uptake with good contrast in xenograft-bearing mice ¹²⁵. Therefore, ¹⁸F-E[E[c(RGDfK)]₂]₂ is a promising agent for peptide receptor radionuclide imaging as well as targeted internal radiotherapy of integrin α_vβ₃ positive tumors. Compared with tetramer, RGD octamer further increased the integrin avidityby another 3-fold. In vivo microPET imaging showed that ⁶⁴Cu-DOTA-RGD octamer had slightly higher initial tumor uptake and much longer tumor retention in U87MG tumor that express high level of integrin ¹²⁶. However, higher renal uptake of the octamer was also observed, which was attributed mainly to the integrin positivity of the kidneys. Several novel dimeric RGD also have been developed by insertion a Gly(3) or PEG(4) linkers between two RGD monomers.^127,12818F labeled PEG₄-E[PEG₄-c(RGDfK)]₂ wasnamed as ¹⁸F-FP-P-PRGD₂. MicroPET imaging with ¹⁸F-FP-P-PRGD₂ revealed high tumor contrast and low background in tumor-bearing nude mice (Figure 3). Biodistribution studies confirmed the in vivo integrin αvβ3-binding specificity of ¹⁸F-FP-P-RGD₂ ¹²⁹. Initial studies in healthy volunteers have been tested and further clinical trials of this dimeric RGD peptide tracer on currently inderway.

Figure 3.

MicroPET imaging of integrin expression using ¹⁸F labeled RGD dimer peptide with PEG₄ linkers. A. Whole-body (top coronal, bottom transaxial) microPET images of U87MG tumor-bearing mice at different time points after injection of ¹⁸F-FP-P-PRGD₂. B. MicroPET images at 60 min after injection of ¹⁸F-FP-P-PRGD₂ in U87MG and MDA-MB-435 tumor-bearing mice without/with a blocking dose of c(RGDyK). Reproduced from reference ¹²⁹ with permission.

Besides RGD peptides, in vivo imaging using Abegrin, a humanized monoclonal antibody against human integrin α_vβ₃, has been performed after DOTA conjugation and ⁶⁴Cu labeling. MicroPET studies revealed that ⁶⁴Cu-DOTA-Abegrin had a very high tumor activity accumulation in integrin α_vβ₃ positive U87MG tumors ¹³⁰. Knottins are small constrained polypeptides that share a common disulfide-bonded framework and a triple-stranded β-sheet fold ¹³¹. Using yeast surface display screening, Kimura et al. ¹³² identified RGD containing knottin peptides with high binding affinity to α_vβ₃, α_vβ₅, and α₅β₁ integrins (IC₅₀ = 10–30 nM). The integrin binding knottin peptides were labeled with ⁶⁴Cu, ¹⁸F or Cy5.5 for integrin imaging in tumor models. Compared with c(RGDfk), higher tumor uptake and lower liver uptake of integrin-binding knottins were observed in both PET and optical images ¹³³. The same group also developed a dual-labeled integrin-binding knottin peptides for PET and near-infrared fluorescence imaging of integrin expression on a U87MG tumor model (Figure 4) ¹³⁴. However, further investigation is still needed to claim the superiority of these RGD-containing knottin peptides to monoric and multimeric RGD peptides.

Figure 4.

Dual-labeled integrin-binding knottin peptide for microPET and NIRF imaging. A. MicroPET imaging of DOTA/Cy5.5-knottin peptide in mice bearing U87MG tumor. B. Mice were co-injected with an excess amount of unlabeled c(RGDyK) in addition to labeled knottin peptides as blocking experiment. C. NIRF imaging of DOTA/Cy5.5-knottin peptide in mice bearing U87MG tumor (arrows indicate U87MG tumors). D. Mice were co-injected with an excess amount of unlabeled c(RGDyK) in addition to labeled knottin peptides as blocking experiment. T, tumor; K, kidney; Bd, bladder; L, liver. Reproduced from reference ¹³⁴ with permission.

Integrin α_vβ₃ has also been investigated as an imaging target using nanoparticle-based tracers. However, the main purpose of these studies is not to evaluate receptor expression levels, but to provide guidance for integrin targeted drug delivery or therapy, which is a little different from previously described peptide- or antibody- based imaging. Cai et al. ¹³⁵ developed a quantum dot (QD)-based probe for both NIRF and PET imaging. QD surface modification with RGD peptides allows for integrin α_vβ₃ targeting and DOTA conjugation enables PET imaging after ⁶⁴Cu-labeling. Using this dual-modality probe, it was found that the majority of the probe in the tumor was within the tumor vasculature. Further observation with an intravital microscopy confirmed that RGD-QD does not extravasate. With a subcellular (approximately 0.5 μm) resolution, RGD-QD was found only bind as aggregates rather than individually ¹³⁶.

Besides evaluating the functional properties of tumor vasculature, several targeted MRI contrast agents have been reported to image integrin α_vβ₃ after conjugation of iron oxide nanoparticles with RGD peptide. To develop a bifunctional iron oxide (IO) nanoparticle probe for PET and MRI scans of tumor integrin α_vβ₃ expression, RGD peptides were coupled to the surface of polyaspartic acid (PASP)-coated IO nanoparticles (RGD-PASP-IO). Both small-animal PET and T2-weighted MRI show integrin-specific delivery of conjugated RGD-PASP-IO nanoparticles and prominent reticuloendothelial system uptake ¹³⁷. In another study, Xie et al. synthesized ultrasmall c(RGDyK) peptide-coated IONPs (<10 nm in hydrodynamic diameter) and demonstrated their in vivo tumor-specific targeting capability ¹³⁸. Recently, IONPs were coated with a PEGylated amphiphilic triblock copolymer, making them water soluble and function-extendable. These particles were then conjugated with IRDye800 and c(RGDyK) for integrin α_vβ₃ targeting. Successful tumor homing in vivo was perceived in a subcutaneous U87MG glioblastoma xenograft model by both magnetic resonance imaging (MRI) and NIRF imaging ¹³⁹.

Targeted ultrasound contrast agents significantly and selectively enhance the detection of a targeted vasculature. Integrin α_vβ₃ targeted ultrasonic contrast agents were prepared by incorporation either cyclic analogs of RGD peptide or the anti-α_vβ₃ antibody LM609 into microbubbles. Acoustic studies illustrate a backscatter amplitude increase from monolayers exposed to the targeted contrast agents of up to 13-fold (22 dB) relative to enhancement due to control bubbles. In addition, a linear dependence between the echo amplitude and bubble concentration was observed for bound agents ¹⁴⁰. Recently, the above mentioned integrin-binding knottin peptide was attached to the shell of perfluorocarbon-filled microbubbles (named as MB-Knottin_Integrin) for contrast-enhanced ultrasound imaging. In vivo ultrasound imaging signal was significantly enhanced after the administration of MB-Knottin_Integrin than after the administration of control peptides.¹⁴¹

Imaging of matrix metalloproteinases

Matrix metalloproteinases (MMPs) are a family of zinc- and calcium-dependent endopeptidases which are responsible for the enzymatic degradation of connective tissue, and thus facilitate endothelial cell migration during angiogenesis ¹⁴². In addition, MMPs process and release bioactive molecules such as growth factors, proteinase inhibitors, cytokines and chemokines ¹⁴³. Based on the different substrates specificity, MMPs family can be classified as collagenase (MMP-1, -8, -13, and -18), gelatinase (MMP-2 and -9), stromelysin (MMP-3, -10, and -11), matrilysin (MMP-7 and -26), while the other MMPs like MMP-12, -19, -20, -23, -27 and MMP-28 were not well characterized till now. Most MMPs are of secreted type while a few members (such as membrane-type (MT) MMPs) anchor to the cell membrane either with the type I transmembrane domain or a glycosylphosphatidylinositol (GPI) linkage ^144,145.

MMPs play an important role in new blood vessel formation ^146–148. Many strategies have been developed to image MMP levels for the assessment of angiogenesis ^149,150. One of them is called “smart probes” which have been developed to contain fluorescent dyes and MMP cleavable sequences ^151,152. It has been reported a MMP-2-sensitive probe was activated by MMP-2 in vitro, producing up to an 850% increase in near-infrared fluorescent signal intensity, and MMP-2-positive tumors were easily identified as high-signal-intensity regions as early as 1 hour after intravenous injection of the MMP-2 probe ¹⁵³. Since then, various strategies have been investigated to further improve the signal magnification, tumor delivery and enzymatic activation of the activatable probes. For example, Tsien and colleagues developed an activatable cell-penetrating peptides (ACPPs) system which is composed of two strands, a Cy5-conjugated polycation (Arg; CPPs-Cy5) and a polyanion (Glu). These domains were bridged by a MMP-cleavable peptide linker. Subsequent cleavage of the linker by MMPs dissociated the inhibitory polyanions, leaving the CPPs-Cy5 free to be internalized. In vivo, these ACPPs probes successfully identified HT-1080 tumors overexpressing MMP-2 and -9 ¹⁵⁴. In addition, ACPPs can target many xenograft tumor models from different cancer sites, as well as a thoroughly studied transgenic model of spontaneous breast cancer (mouse mammary tumor virus promoter driving polyoma middle T antigen, MMTV-PyMT) with the selectivity for MMP-2 and -9. Moreover, in accord with the known local distribution of MMP activity, accumulation is strongest at the tumor-stromal interface in primary tumors and associated metastases ¹⁵⁵. To improve the absolute tumor fluorescence and tumor-to-background fluorescence contrast, ACPPs were conjugated to dendrimers (ACPPDs). ACPPDs were used for guiding surgery and resulted in fewer residual cancer cells left in the animal after surgery. Animals whose tumors were moved with ACPPD guidance had a long-term tumor-free survival (Figure 5) ¹⁵⁶. These findings implicated a novel strategy to translate these activatable probes into the clinics.

Figure 5.

ACPPs guide surgery. A. White light image of a MDA-MB-435 tumor-bearing mouse. B. Optical image of GFP-labeled tumor cells from the same mouse as in A (arrow indicate MDA-MB-435 tumor). C. Optical image of Cy5-labeled free ACPP in tumor. D. Colocalization of the Cy5 free ACPP with the GFP-labeled tumor. E. White light image of the excised tumor (large arrow) shown next to the mouse. F. Fluorescence imaging of the GFP signal in the tumor bed to confirm tumor excision successfully (*). G and H. Imaging of the Cy5 signal of tumor bed shows a residual fluorescence signal (arrowhead) in remaining tissue. I. A small piece of residual tumor (arrowhead) is excised (small arrow). J and K. Visualize of dissected tumor by GFP signal (J, arrowhead) and Cy5 free ACPP signal (K, arrowhead). L. Overlay GFP and Cy5 signal in dissected tumor. Reproduced from reference ¹⁵⁶ with permission.

Dark quenchers, dyes with no native fluorescence, offer advantages over conventional FRET-based probes because they do not occupy the emission spectra ¹⁵⁷. Black hole quencher (BHQ) dyes are commercially available dark quenchers and are able to permit efficient quenching across the visible spectrum from 480 nm into the NIR through a combination of FRET and static quenching mechanisms ¹⁵⁸. Dark-quenched, dual-labeled peptide probes have been designed by using a NIR fluorophore and a dark quencher for imaging protease activity. An MMP-13 activatable peptide probe was designed using a combination of the known MMP-13 substrate GPLGMRGLGK and Cy5.5 and BHQ-3 ¹⁵². BHQ-3 has maximal absorption in the 620- to 730-nm range, which can efficiently quench NIR fluorophores such as Cy5.5. The MMP-13 probe Cy5.5-GPLGMRGLGK(BHQ-3) showed a 32-fold increase in enhancement of NIR fluorescence signals after incubation with MMP-13 in vitro, and the fluorescent recovery of the probe was strongly inhibited in the presence of an MMP-13 inhibitor. To further improve the in vivo pharmacokinetics of the activatable probes, Recently, Lee et al. developed a self-assembled polymeric nanoparticle based MMP activatable probe ¹⁵⁹. The strongly quenched MMP specific NIR peptides were used as a carrier on the surface of self-assembled chitosan tumor-homing polymeric nanoparticles. The probes produced significantly lower fluorescence in the absence of MMPs or in the present of MMP inhibitor while yielded an extremely heightened fluorescence signal when cleaved by MMP-2. MMP-positive SCC7 xenograft tumor models showed high uptake of nanosensor with minimal background signals in vivo ¹⁵⁹.

In another study, Zhang et al. ¹⁶⁰ designed QD conjugates which were coated with 5–10 copies of streptavidin, and conjugated with biotinylated peptide ligands via the strong biotin and streptavidin binding. The biotinylated peptide ligands consisted of a transporting group (transporter) that could transport QDs into cells, a blocking group (blocker) that could diminish the cellular uptake of QDs, and a sensing group, sandwiched between the transporter and blocker, that could be cleaved by MMP-2 and -7. After the MMP treatment, the QD-peptide conjugates could be efficiently taken up into cells ¹⁶⁰. The same group also developed a protease sensing nanoplatform based on QDs and bioluminescence resonance energy transfer (QD-BRET). These nanosensors consist of bioluminescent proteins as the BRET donor, quantum dots as the BRET acceptor, and MMPs substrates sandwiched between the two as a sensing group. These nanosensors can detect the MMP activity in buffers and in mouse serum with the sensitivity to a few nanograms per milliliter and secreted proteases by tumor cells ¹⁶¹. However, the in vivo application potential of these constructs needs to be further confirmed.

Via phage display techniques, the MMP specific decapeptide H-Cys-Thr-Thr-His-Trp-Gly-Phe-Thr-leu-Cys-OH (CTT) was found and could be labeled with ¹²⁵I and ^99mTc. However, this tracer has unfavourable characteristics for in vivo imaging because the metabolic stability of the compound is low and lipophilicity is high ¹⁶². Another group labeled this peptide with ¹¹¹In after conjugating it with a highly hydrophilic and negatively charged chelator DTPA. A significant correlation was observed between the accumulation in the tumor as well as tumor-to-blood ratio of ¹¹¹In-DTPA-CTT and gelatinase activity. Moreover, ¹¹¹In-DTPA-CTT showed low levels of radioactivity in the liver and kidneys ¹⁶³. CTT peptide was also labeled with ⁶⁴Cu after DOTA conjugation for PET imaging of MMP. ⁶⁴Cu-DOTA-CTT inhibited hMMP-2 and mMMP-9 with similar affinity to CTT. MicroPET imaging studies showed that ⁶⁴Cu-DOTA-CTT was taken up by MMP-2/9-positive B16F10 murine melanoma tumors, however, the low affinity for MMP-2 and MMP-9 and in vivo instability of CTT-based imaging probes need to be overcome for further applications ¹⁶⁴.

Another approach is to label small molecule MMP inhibitors (MMPIs), which are typically used as antiangiogenic drugs. In general, MMPIs possess a zinc binding group (ZBG) complexing the zinc ion of the active site and are classified into several groups owing to their lead structures ¹⁴². Different ¹⁸F and ¹¹C labeled MMPIs have been synthesized and evaluated preclinically with mixed results ^165,166. Fluorinated MMPIs based on lead structures of the broad-spectrum inhibitors N-hydroxy-2(R)-[[(4-methoxyphenyl)sulfonyl](benzyl)-amino]-3-methyl-butanamide (CGS 25966) and N-hydroxy-2(R)-[[(4-methoxyphenyl)sulfonyl](3-picolyl)-amino]-3-methyl-butanamide (CGS 27023A) have been synthesized and showed high in vitro MMP inhibition potencies for MMP-2, MMP-8, MMP-9, and MMP-13 ¹⁶⁷. However, in vivo microPET study with ¹¹C-CGS 25966 failed to demarcate MMP positive tumors ¹⁶⁸. A ¹¹C-labeled MMPI (2R)-2-[[4-(6-fluorohex-1-ynyl)phenyl]sulfonylamino]-3-methylbutyric acid ¹¹C-methyl ester (¹¹C-FMAME), has also been synthesized and applied to two animal models of breast cancer, MCF-7 xenograft transfected with IL-1 and MDA-MB-435 xenograft in athymic mice. Again, low tumor-to-blood and tumor-to muscle ratios of these tracers do not allow visualization of the tumors in microPET studies ^165,169. However, biodistribution study with ¹⁸F-labeled similar compound, (2R)-2-[4-(6-¹⁸F-Fluorohex-1-ynyl)-benzenesulfonylamino]-3-methylbutyric acid (¹⁸F-SAV03), showed higher tumor uptake of the tracer than normal organs ¹⁶⁶. Other MMPIs have also been synthesized and labeled with ¹¹¹In and ¹⁸F ^167,170. Nevertheless, significant improvements in tumor MMP targeting and in vivo pharmacokinetics are necessary before the use of MMP radiotracer imaging can be translated into the clinic. Another hindrance for MMPs imaging is the high homology between each MMP member and it is difficult to identify specific substrates to a particular MMP ¹⁷¹. Moreover, most of MMPs are expressed as soluble form, which complicate both the imaging and data analysis. It is also worth of note that there is difference between pharmacokinetic requirement for therapeutic drugs and imaging tracers. When developing a therapeutic drug, the particular tumor uptake of the drug is not emphasized if the drug showed tumor therapeutic efficacy and no side-effect to normal organs. However, for imaging tracer development, higher tumor accumulation than normal tissues is a prerequisite for clear tumor visualization.

Other angiogenesis imaging targets

Apart from VEGF/VEGFs, integrins and MMPs, more tumor angiogenes related proteins have been identified as potential imaging targets ¹⁷². For example, fibronectin, a large glycoprotein, can be found physiologically in plasma and tissues. Extra-domain B of fibronectin (EDB), consisting of 91 amino acids, is not present in the fibronectin molecule under normal conditions, but expressed in the endometrium in the proliferative phase and some vessels of the ovaries. It is an angiogenesis marker in a variety of solid tumors. By phage display, a human antibody fragment scFv (L19) was identified and has been shown to efficiently localize on neovasculature in vivo. The L19 small immunoprotein (SIP) was labeled by ⁷⁶Br and ¹²⁴I for PET imaging ^173,174. Endoglin (CD105) is a cell membrane glycoprotein mainly expressed on endothelial cells and overexpressed on tumor vasculature. Gd-DTPA liposomes were conjugated with immunoglobulins and used to detect the expression of CD105 in tumor-bearing rats by MR imaging ¹⁷⁵. E-selectin is a cell adhesion molecule and CD antigen that expressed exclusively by activated endothelial cells. ¹¹¹In-labeled E-selectin antibody was used for imaging of inflamed human synovial vasculature ¹⁷⁶. A significant difference was observed comparing to the control group. Other angiogenesis-related biomarkers, such as angiopoietins/Tie receptors ¹⁷⁷, and CD276 ¹⁷⁸ are also potential targets for angiogenesis imaging.

Imaging therapy response of tumors

Monitoring tumor response to various therapeutics is one major requirement of molecular imaging. So far, assessment of anti-angiogenic treatment in clinical studies is achieved mainly by MVD measurement and non-invasive imaging methods for measuring functional vascular volume including PET studies with ¹⁵O-oxygen and related tracers ⁴¹, dynamic contrast-enhanced ultrasonography (DCE-US) ⁴², and dynamic contrast-enhanced MRI (DCE-MRI) ⁴⁵. For example, it has been shown that DCE-MRI can detect responses to PTK/ZK (a VEGF receptor tyrosine kinase inhibitor) therapy as early as two days after therapy with significant reductions in area under gadolinium-contrast-medium curve (AUGC) ¹⁷⁹ or permeability parameters ¹⁸⁰, which also predict subsequent response. LMCM DCE-MRI has also shown significant reductions in permeability values in patients treated with the antivascular agents AG-013736 (an inhibitor of the VEGF, PDGF, c-Kit receptor tyrosine kinases) and SU5416 (a selective inhibitor of VEGFR-2 tyrosine kinase) activity ¹⁸¹.

Molecular imaging of angiogenesis related protein targets also has been reported to monitor tumor response to treatment in preclinical models. For example, tumor uptake of ¹⁸F-AH111585, a disulfide bond cyclic RGD peptide, and microvessel density were assessed in human lung cancer bearing mice treated with either low-dose paclitaxel or ZD4190, a VEGFR-2 TKI. The results showed that paclitaxel therapy reduced the MVD in LLC tumor–bearing mice and resulted in significantly reduced ¹⁸F-AH111585 tumor uptake. ZD4190 therapy also resulted in a significant decrease in ¹⁸F-AH111585 uptake in Calu-6 tumors, compared with the vehicle control–treated Calu-6 tumors ¹⁸². Moreover, the decreased uptake of ¹⁸F-AH111585 was matched by a decrease in the tumor MVD and FDG uptake remained unchanged with low-dose paclitaxel treatment. Another application is for the Src family kinases (SFK) inhibitor dasatinib treated U87MG xenografts bearing mice. Dasatinib significantly reduced ⁶⁴Cu-DOTA-c(RGDfK) uptake by up to 59% in U87MG xenografts. In contrast, tumor FDG uptake showed no significant reduction with dasatinib therapy, indicating that ⁶⁴Cu-DOTA-c(RGDfK) may provide a sensitive means of monitoring tumor response to SFK inhibition in α_vβ₃ expressing cancers in the early course of therapy ¹⁸³. Blankenberg et al applied ^99mTc-scVEGF in mice with HT29 tumor xenografts to evaluate the effects of pazopanib, a small-molecule tyrosine kinase inhibitor selectively targeting VEGFR, PDGFR, and c-Kit. The changes in VEGFR imaging reflect a dramatic pazopanib-induced decrease in the number of VEGFR-2⁺/CD31⁺ endothelial cells (ECs) within the tumor vasculature followed by a relative increase in the number of ECs at the tumor edges ¹⁸⁴.

The above mentioned data give us promising envisions as to the application of molecular imaging for therapy response monitoring. However, there are several issues need to be further explored. For example, integrins are not only overexpressed on tumor endothelial cells but also on tumor cells. In addition, there is heterogeneity of integrin expression between different tumors or even within the same tumor. In addition, tumor uptake and accumulation of imaging tracers is not only dependent on the receptor expression. Several other factors including vascular density and volume, vascular permeability and interstitial fluid pressure also affect the distribution ^185,186. All these factors will be changed after treatment, especially with anti-angiogenesis treatment.

CONCLUSIONS AND PERSPECTIVES

A multitude of imaging techniques is available for assessing tissue vasculature on a structural, functional and molecular level. A wide variety of targeting ligands (small molecules, peptidomimetics, peptides, and antibodies) have been conjugated with various imaging labels for MRI, ultrasound, optical, SPECT, PET, and multimodality imaging of angiogenesis. All these methods have been successfully used preclinically and will hopefully aid in antiangiogenic drug evaluation in animal studies. In the future, multimodality, multiplexing imaging will allow for evaluation of the angiogenic cascade in its full complexity to acquire comprehensive information. It is predictable that the new generation clinical PET/CT and preclinical microPET/microCT, as well as PET/MRI and microPET/microMRI currently in active development ^187–189, will likely play a major role in molecular imaging of angiogenesis.

To further improve imaging of the angiogenesis process at molecular level, it is necessary to identify new angiogenesis related targets and corresponding specific ligands and to optimize currently available imaging probes. Thorough and full understanding of the physiological and pathological changes during angiogenesis will be critical for new target identification. Optimization of currently available imaging probes can be achieved by oligomerization (homo or hetero), glycosylation, PEGylation, and site-specific labeling, and so on. With all the efforts, molecular imaging techniques can bridge the gap between preclinical and clinical research to develop candidate drugs that have the optimal target specificity, pharmacokinetics, and efficacy. It is expected that in the foreseeable future, anigogenesis imaging will be routinely applied in anti-cancer clinical trials.