Radiolabeled cyclic RGD peptides as radiotracers for tumor imaging

The integrin family comprises 24 transmembrane receptors, each a heterodimeric combination of one of 18α and one of 8β subunits. Their main function is to integrate the cell adhesion and interaction with the extracellular microenvironment with the intracellular signaling and cytoskeletal rearrangement through transmitting signals across the cell membrane upon ligand binding. Integrin αvβ3 is a receptor for the extracellular matrix proteins containing arginine–glycine–aspartic (RGD) tripeptide sequence. The αvβ3 is generally expressed in low levels on the epithelial cells and mature endothelial cells, but it is highly expressed in many solid tumors. The αvβ3 levels correlate well with the potential for tumor metastasis and aggressiveness, which make it an important biological target for development of antiangiogenic drugs, and molecular imaging probes for early tumor diagnosis. Over the last decade, many radiolabeled cyclic RGD peptides have been evaluated as radiotracers for imaging tumors by SPECT or PET. Even though they are called “αvβ3-targeted” radiotracers, the radiolabeled cyclic RGD peptides are also able to bind αvβ5, α5β1, α6β4, α4β1, and αvβ6 integrins, which may help enhance their tumor uptake due to the “increased receptor population.” This article will use the multimeric cyclic RGD peptides as examples to illustrate basic principles for development of integrin-targeted radiotracers and focus on different approaches to maximize their tumor uptake and T/B ratios. It will also discuss important assays for pre-clinical evaluations of the integrin-targeted radiotracers, and their potential applications as molecular imaging tools for noninvasive monitoring of tumor metastasis and early detection of the tumor response to antiangiogenic therapy.


INTRODUCTION
Cancer is the second leading cause of death worldwide (Siegel et al. 2015). Most patients will survive if the cancer can be detected at the early stage. Accurate and rapid detection of rapidly growing and metastatic tumors is of great importance before they become widely spread. There are several imaging modalities available for the diagnosis of cancer, including X-ray computed tomography (CT), ultrasound (US), nuclear magnetic resonance imaging (MRI), and nuclear medicine procedures. While CT, US and MRI are better suited for anatomic analysis of solid tumors, molecular imaging with positron emission tomography (PET) and single-photon emission computed tomography (SPECT) offers significant advantages with respect to sensitivity and specificity because they are able to provide the detailed information related to biochemical changes in tumor tissues at the cellular and molecular levels (Mankoff et al. 2008;Shokeen and Anderson 2009;Tweedle 2009;Correia et al. 2011;Gaertner et al. 2012;Laverman et al. 2012b;Jamous et al. 2013). The most sensitive molecular imaging modalities are SPECT (*10 -10 mol/L) and PET (10 -10 -10 -12 mol/L) using radiotracers Gaertner et al. 2012). According to their biodistribution properties, radiotracers are classified as those whose biodistribution is determined by their chemical and physical properties, and those whose ultimate distribution is determined by their receptor or enzyme binding. The latter class is called target-specific radiotracers. Peptides are often used as targeting biomolecules (BM) for receptor binding in order to achieve high tumor specificity. Many radiotracers have been developed to target the receptors overexpressed on tumor cells and/or tumor vasculature (Mankoff et al. 2008;Shokeen and Anderson 2009;Tweedle 2009;Correia et al. 2011;Gaertner et al. 2012;Laverman et al. 2012b;Jamous et al. 2013).
A large number of radiolabeled cyclic RGD (arginine-glycine-aspartic) peptides have been evaluated as SPECT or PET radiotracers for tumor imaging Wu et al. 2005;Jia et al. 2006;Liu et al. 2006;Zhang et al. 2006;Alves et al. 2007;Dijkgraaf et al. 2007a, b;Liu et al. 2007;Wu et al. 2007;Jia et al. 2008;Li et al. 2008b;Liu et al. 2008a;Shi et al. 2008;Wang et al. 2008a, b;Liu et al. 2009a, b;Shi et al. 2009a, b, c;Kubas et al. 2010;Dumont et al. 2011;Jia et al. 2011;Shi et al. 2011a, b;Zhou et al. 2011b;Nwe et al. 2012;Pohle et al. 2012;Zhou et al. 2012;Ji et al. 2013a, b;Li et al. 2013;Simecek et al. 2013;Tsiapa et al. 2013;Maschauer et al. 2014;Yang et al. 2014;Zheng et al. 2015). Many excellent review articles have appeared to cover their nuclear medicine applications (D' Andrea et al. 2006;Liu 2006;Meyer et al. 2006;Cai and Chen 2008;Liu et al. 2008b;Liu 2009;Stollman et al. 2009;Beer and Chen 2010;Dijkgraaf and Boerman 2010;Haubner et al. 2010;Beer et al. 2011;Michalski and Chen 2011;Zhou et al. 2011a;Danhier et al. 2012;Tateishi et al. 2012. This article is not intended to be an exhaustive review of current literature on radiolabeled cyclic RGD peptides. Instead, it will use the multimeric cyclic RGD peptides to illustrate some basic principles for new radiotracer development and to address some important issues associated with integrintargeted radiotracers. It will focus on different approaches to maximize the tumor uptake and T/B ratios. Authors would apologize to those whose work has not been cited in this article.

RADIOTRACER DESIGN
Integrin-targeted radiotracer Figure 1 shows the schematic construction of an integrin-targeted radiotracer (Liu 2006(Liu , 2009. The cyclic RGD peptide serves as a ''vehicle'' to carry the isotope to integrins expressed on both tumor cells and activated endothelial cells of tumor neovasculature. BFC is a bifunctional coupling agent to attach the appropriate radionuclide to a cyclic RGD peptide (Liu and Edwards 2001;Liu 2004Liu , 2008Liu and Chakraborty 2011). The pharmacokinetic modifying (PKM) linker is often used to improve excretion kinetics of radiotracers (Liu and Edwards 2001;Liu 2004Liu , 2008. For a new radiotracer to be successful in clinics, it must show clinical indications for several of high-incidence tumor types (namely breast, lung, and prostate cancers). Renal excretion is necessary in order to maximize the tumor-to-background (T/B) ratios. The main objective of tumor imaging is to achieve the following goals: (1) to detect the presence of tumor at early stage, (2) to distinguish between benign and malignant tumors, (3) to follow the tumor growth and tumor response to a specific therapy (chemotherapy, radiation therapy, or combination thereof), (4) to predict success or failure of a specific therapeutic regimen, and (5) to access the prognosis of a particular tumor.

Radionuclide
The choice of radionuclide depends largely on the modality for tumor imaging. More than 80% of radiotracers for SPECT in nuclear medicine departments are 99m Tc compounds due to optimal nuclear properties of 99m Tc and its easy availability at low cost (Liu and Edwards 2001;Liu 2004Liu , 2008Liu and Chakraborty 2011). The 6-h half-life is long enough to allow radiopharmacists to carry out radiosynthesis and for physicians to collect clinically useful images. At the same Fig. 1 Schematic presentation of the integrin a v b 3 -targeted radiotracers. The cyclic RGD peptide is used as the targeting biomolecule (BM) to carry the isotope into the tumor tissue. BFC is used to attach the isotope to the targeting biomolecule. PKM linker is utilized to modify its pharmacokinetics time, it is short enough to permit administration of 20-30 mCi of 99m Tc without imposing a significant radiation dose to the patients. 18 F is a cyclotronproduced isotope suitable for PET. It has a half-life of 110 min. Despite its short half-life, the availability of preparative modules makes 18 F radiotracers more accessible to clinicians (Anderson et al. 2003). 64 Cu is another PET isotope to develop target-specific radiotracers. It has a half-life of 12.7 h and a b ? emission (18%, E max = 0.655 MeV). Despite poor nuclear properties, 64 Cu is a viable alternative to 18 F for research programs that wish to incorporate high sensitivity and spatial resolution of PET, but cannot afford to maintain the expensive isotope production infrastructure (Anderson et al. 2003). 68 Ga is generator-produced PET isotope with the half-life of 68 min. The 68 Ge- 68 Ga generator can be used for more than a year. 68 Ga could become as useful for PET as 99m Tc for SPECT (Maecke et al. 2005). The 68 Ga-labeled somatostatin analogs have been studied for PET imaging of somatostatin-positive tumors in both pre-clinical animal models and cancer patients (Henze et al. 2005;Koukouraki et al. 2006a, b). Gallium chemistry and related nuclear medicine applications have been reviewed recently (Maecke et al. 2005).

Integrins as molecular targets for tumor imaging
Angiogenesis is a requirement for tumor growth and metastasis (Hwang and Varner 2004;Weigelt et al. 2005). The angiogenic process depends on the vascular endothelial cell migration and invasion, and is regulated by cell adhesion receptors. Integrins are such a family of receptors that facilitate the cellular adhesion to and the migration on extracellular matrix proteins, and regulate the cellular entry and withdraw from the cell cycle (Albelda et al. 1990;Falcioni et al. 1994;Carreiras et al. 1996;Bello et al. 2001;Sengupta et al. 2001;Cooper et al. 2002;Zitzmann et al. 2002;Hwang and Varner 2004;Jin and Varner 2004;Weigelt et al. 2005;Sloan et al. 2006;Zhao et al. 2007; Hodivala-Dilke 2008; Barczyk et al. 2010;Taherian et al. 2011;Gupta et al. 2012;Sheldrake and Patterson 2014). The integrin family comprises 24 transmembrane receptors (Table 1) (Sheldrake and Patterson 2014). Their main function is to integrate the cell adhesion and interaction with the extracellular microenvironment with the intracellular signaling and cytoskeletal rearrangement through transmitting signals across the cell membrane on ligand binding. Many integrins are crucial to the tumor initiation, progression, and metastasis. Among the 24 members, the a v b 3 is studied most extensively for its role in tumor angiogenesis and metastasis (Albelda et al. 1990;Falcioni et al. 1994;Carreiras et al. 1996;Bello et al. 2001;Sengupta et al. 2001;Cooper et al. 2002;Zitzmann et al. 2002;Hwang and Varner 2004;Jin and Varner 2004;Weigelt et al. 2005;Sloan et al. 2006;Zhao et al. 2007;Hodivala-Dilke 2008;Barczyk et al. 2010;Taherian et al. 2011;Gupta et al. 2012). It is not surprising that radiolabeled cyclic RGD peptides are often called ''a v b 3 -targeted'' radiotracers in majority of the literature (D' Andrea et al. 2006;Liu 2006;Meyer et al. 2006;Cai and Chen 2008;Liu et al. 2008b;Liu 2009;Stollman et al. 2009;Beer and Chen 2010;Dijkgraaf and Boerman 2010;Haubner et al. 2010;Beer et al. 2011;Michalski and Chen 2011;Zhou et al. 2011a;Danhier et al. 2012;Tateishi et al. 2012).
The changes in the a v b 3 expression levels and activation state have been well documented during tumor growth and metastasis (Hwang and Varner 2004;Weigelt et al. 2005;Sloan et al. 2006;Zhao et al. 2007;Hodivala-Dilke 2008;Barczyk et al. 2010;Gupta et al. 2012). The a v b 3 is expressed in low levels on epithelial cells and mature endothelial cells, but it is highly expressed in many solid tumors, which include osteosarcomas, glioblastoma, melanomas, and carcinomas of lung and breast (Albelda et al. 1990;Falcioni et al. 1994;Carreiras et al. 1996;Bello et al. 2001;Sengupta et al. 2001;Cooper et al. 2002;Zitzmann et al. 2002;Hwang and Varner 2004;Jin and Varner 2004;Weigelt et al. 2005;Sloan et al. 2006;Zhao et al. 2007; Hodivala-Dilke 2008; Barczyk et al. 2010;Taherian et al. 2011;Gupta et al. 2012). Studies show that a v b 3 is overexpressed on tumor cells and activated endothelial cells of tumor neovasculature (Pilch et al. 2002;Taherian et al. 2011). It is believed that the a v b 3 expressed on endothelial cells modulate cell adhesion and migration during angiogenesis, while the a v b 3 overexpressed on carcinoma cells potentiate metastasis by facilitating invasion and movement of tumor cells across blood vessels (Sloan and Anderson 2002;Minn et al. 2005;Dittmar et al. 2008;Lorger et al. 2009;Omar et al. 2010). It has been shown that the a v b 3 expression levels correlate with the potential for metastasis and aggressiveness of tumors, including glioma, melanoma, and carcinomas of the breast and lungs (Zhao et al. 2007;Hodivala-Dilke 2008). The a v b 3 is considered as an important biological target to develop antiangiogenic drugs (Gottschalk and Kessler 2002;Kumar 2003;Jin and Varner 2004;D' Andrea et al. 2006) and molecular Fig. 2 Examples of BFCs useful for radiolabeling of cyclic RGD peptides. HYNIC and MAG 2 are useful for 99m Tc-labeling while DOTA, NOTA, and their derivatives are better suited for chelation of 64 Cu and 68 Ga. For 18 F-labeling, 4-FB, 4-FBz, 2-FP, and 2-FDG are often used as prosthetic groups. The Al(NOTA) chelate is highly efficient for radiosynthesis of 18 F radiotracers using a kit formulation Table 1 Natural integrin ligands and their corresponding recognition peptide sequences

Cyclic RGD peptides as targeting biomolecules
The a v b 3 is a receptor for the extracellular matrix proteins with the exposed RGD tripeptide sequence. Theoretically, both linear and cyclic RGD peptides can be used as targeting biomolecules. A major drawback of linear RGD peptides are their low binding affinity (IC 50 [ 100 nmol/L), lack of specificity (a v b 3 vs. a IIB b 3 ), and rapid degradation by proteases in serum. Cyclization of RGD peptides via the linkers, such as S-S disulfide, thioether, and rigid aromatic rings, leads to the increased receptor binding affinity and selectivity (Aumailley et al. 1991;Gurrath et al. 1992;Mü ller et al. 1992;Haubner et al. 1996). It seems that the a IIB b 3 is less sensitive to variations in the RGD peptide backbone and can accommodate a larger distance or spacer than a IIB b 3 and a v b 5 (Pfaff et al. 1994). Incorporation of the RGD sequence into a cyclic pentapeptide framework ( Fig. 3: c(RGDfV) and EMD121974) could significantly increase the binding affinity and selectivity of a v b 3 /a v b 5 over a IIb b 3 (Aumailley et al. 1991;Gurrath et al. 1992;Mü ller et al. 1992;Pfaff et al. 1994;Haubner et al. 1996). The addition of a rigid aromatic ring into the cyclic hexapeptide structure (Fig. 3: DMP728 and DMP757) enhances the binding affinity of a IIB b 3 Jacobson et al. 2011;Danhier et al. 2012).
The structure-activity studies indicated that the amino acid residue in position 5 has little impact on a v b 3 /a v b 5 binding affinity (Aumailley et al. 1991;Gurrath et al. 1992;Mü ller et al. 1992;Haubner et al. 1996). The valine (V) residue in c(RGDfV) can be replaced by lysine (K) or glutamic acid (E) to afford c(RGDfK) and c(RGDfE), respectively, without changing their a v b 3 / a v b 5 binding affinity. Figure 4 shows several examples of monomeric cyclic RGD peptides that have high affinity for a v b 3 and a v b 5 . Among the radiotracers evaluated in pre-clinical tumorbearing models, [ 18 F]Galacto-RGD ( Fig. 4: 2-[ 18 F]fluoropropanamide c(RGDfK(SAA); SAA = 7-amino-Lglyero-L-galacto-2,6-anhydro-7-deoxyheptanamide) was the first one under clinical investigation for visualization of a v b 3 expression in cancer patients Haubner et al. 2005). The results from imaging studies in cancer patients showed that there was sufficient a v b 3 for PET imaging. The tumor uptake of [ 18 F]Galacto-RGD correlates with the a v b 3 levels in cancer patients Beer et al. 2007. However, the radiotracers derived from monomeric cyclic RGD peptides all had low tumor uptake with T/B ratios because of their relatively low a v b 3 binding affinity.
It must be noted that cyclic RGD peptides bind not only a v b 3 but also other integrins. While the a v b 3 plays pivotal role in the tumor growth and progression, a IIB b 3 is critical for the platelet aggregation during thrombosis formation. The interaction between a v b 3 and a IIb b 3 facilitates the adhesion of tumor cells to the vasculature and often leads to metastasis (Felding-Habermann et al. 1996;Bakewell et al. 2003). The a v b 5 is very similar to a v b 3 in the ligand binding site region and has a similar expression pattern and function to those of a v b 3 . Both a v b 5 and a v b 3 are highly expressed on the activated endothelial cells and have similar roles in angiogenesis, promoting angiogenic response to different growth factors (Bakewell et al. 2003;Goodman et al. 2012). The a v b 5 has been shown to overexpress on a wide range of tumor types (Goodman et al. 2012;Boger et al. 2014). A number of tumors co-express a v b 3 and a v b 5 (Sung et al. 1998;Erdreich-Epstein et al. 2000;Graf et al. 2003;Humphries et al. 2006;Monferran et al. 2008;Bianchi-Smiraglia et al. 2013;Roth et al. 2013;Vogetseder et al. 2013;Boger et al. 2014;Navarro-Gonzalez et al. 2015), because both engage the same ECM ligands and activate complementary cell signaling pathways in order to promote tumor progression (Sung et al. 1998;Bianchi-Smiraglia et al. 2013). It was also reported that the tumor cell expression of a v b 3 , a v b 5 , a 5 b 1 , a 6 b 4 , a 4 b 1 , and a v b 6 is correlated with the progression of various tumors (Vogetseder et al. 2013;Boger et al. 2014). The structures of other RGD-binding integrins (a v b 6 , a v b 8 , a v b 1 and a 8 b 1 ) have not yet been studied in details (Sheldrake and Patterson 2014).

MAXIMIZING BINDING AFFINITY VIA MULTIMERIZATION
The multivalent concept has been used to develop radiotracers with the increased tumor-targeting capability. For example, E[c(RGDfK)] 2 (RGD 2 ) was the first cyclic RGD dimer for development of diagnostic ( 99m Tc) and therapeutic ( 90 Y and 64 Cu) radiotracers (Liu et al. 2001a;, 2006Jia et al. 2006Jia et al. , 2008. RGD tetramers RGD 4 was also used to develop SPECT and PET radiotracers (Wu et al. 2005;Liu et al. 2007Liu et al. , 2008a. Both the in vitro assays and biodistribution data showed that the radiolabeled Fig. 3 Examples of monomeric cyclic RGD peptides as targeting biomolecules for the development of a v b 3 -targeted radiotracers. EMD121974 has been under clinical investigations as an ''orphan drug'' for treatment of glioblastoma either stand-alone or in combination with radiation therapy. DMP728 and DMP757 were originally developed as anti-thrombotic agents ( 99m Tc,18 F, and 64 Cu) multimeric cyclic RGD peptides have higher a v b 3 binding affinity and better tumor uptake than their monomeric analogs Liu 2009). It is important to note that multimeric RGD peptides are not necessarily multivalent . Two factors (Fig. 5:   Fig. 5 Top: Schematic illustration of the interactions between cyclic RGD peptide dimers and a v b 3 . A The distance between two RGD motifs is not long enough for simultaneous integrin a v b 3 binding. However, the RGD concentration is ''locally enriched'' in the vicinity of neighboring integrin a v b 3 once the first RGD motif is bound. B The distance between two RGD motifs is long due to the presence of two linkers (L). As a result, the cyclic RGD dimer is able to bind integrin a v b 3 in a ''bivalent'' fashion. In both cases, the end-result would be higher integrin a v b 3 binding affinity for the multimeric cyclic RGD peptides. Bottom: Selected cyclic RGD peptide dimers and tetramers useful for development of a v b 3 -targeted radiotracers. The D 3 , G 3 , PEG 4 , and sugar linkers are used to increase the distance between two RGD motifs and to improve radiotracer excretion kinetics from non-cancerous organs Radiolabeled cyclic RGD peptides as radiotracers REVIEW bivalency and enhanced local RGD concentration) contribute to the high a v b 3 binding affinity of cyclic RGD peptides . The concentration factor exists in all multimeric RGD peptides regardless of the linker length. Given the short distance (6 bonds excluding side-arms of K-residues) between two RGD motifs in E[c(RGDfK)] 2 and E[c(RGDyK)] 2 , it is unlikely that they would bind to two adjacent a v b 3 sites simultaneously. However, the binding of one RGD motif to a v b 3 will increase the ''local concentration'' of second RGD motif in the vicinity of a v b 3 sites (Fig. 5B). The concentration factor may explain the higher tumor uptake of radiolabeled ( 99m Tc,111 In, 90 Y, 18 F, and 64 Cu) E[c(RGDfK)] 2 and E[c(RGDyK)] 2 than their monomeric derivatives Dijkgraaf and Boerman 2010;Beer et al. 2011;Michalski and Chen 2011;Zhou et al. 2011a). The key for bivalency is the distance between two cyclic RGD motifs. For example, this distance is 38 bonds in PEG 4 -E[c(RGDfK(PEG 4 ))] 2 (3P-RGD 2 : PEG 4 = 15-amino-4,7,10,13-tetraoxapentadecanoic acid), and 26 bonds G 3 -E[c(RGDfK(G 3 ))] 2 (3G-RGD 2 : G 3 = Gly-Gly-Gly), which are long enough for them to achieve the bivalency. As a result, HYNIC-3P-RGD 2 (IC 50 = 60 ± 3 nmol/L) and HYNIC-3G-RGD 2 (IC 50 = 59 ± 3 nmol/L) have much higher a v b 3 binding affinity than HYNIC-P-RGD 2 (P-RGD 2 = PEG 4 -E[c(RGDfK)] 2 : (IC 50 = 89 ± 7 nmol/L)) Wang et al. 2008b). 99m Tc-3P-RGD 2 and 99m Tc-3G-RGD 2 had higher breast tumor uptake than 99m Tc-P-RGD 2 (Fig. 6) Wang et al. 2008b). Since the tumor uptake of 99m Tc-3P-RGD 2 and 99m Tc-3P-RGD 2 is comparable to that of 99m   suggests that the contribution from ''concentration factor'' may not be as significant as that from the ''bivalency.''

MAXIMIZING RADIOTRACER UPTAKE BY TARGETING MULTIPLE RECEPTORS
Two most important factors affecting the radiotracer tumor uptake are receptor binding affinity and receptor population. The receptor binding affinity is critically important for selective tumor localization and tumor uptake of radiolabeled cyclic RGD peptides . The receptor population is equally important for the receptor-based molecular imaging. It will not be possible to image the tumor if that it has very limited or no receptor expression even if the receptor ligand has high receptor binding affinity. There are two approaches to maximize the target population. The first approach (Fig. 7A) involves the use of the same cyclic RGD peptide to target two or more integrins (such as a v b 3 , a v b 5 , a 5 b 1 , a 6 b 4 , a 4 b 1 , and a v b 6 ). Another approach (Fig. 7B) involves the use of a bifunctional peptide that is able to target two different receptors, such as a v b 3 and bombesin (BBN) receptor. By targeting two different receptors, the radiotracer will have more opportunities to localize in the tumor due to the larger populations of two receptors than that of a single receptor. The so-called ''bivalent heterodimers'' (Fig. 7) has been used to target the a v b 3 and BBN receptors (Li et al. 2008c;Liu et al. 2009c, d). The xenografted PC-3 and MDA-MB-435 tumor-bearing models were used to evaluate their tumor-targeting capability and biodistribution properties. It is well-established that the xenografted PC-3 tumors have low a v b 3 expression Ji et al. 2013c). It was also shown that the xenografted MDA-MB-435 tumor has little BBN receptor expression (Liu et al. 2009c, d). Therefore, both PC-3 and MDA-MB-435 tumor-bearing models are not appropriate to Fig. 6 Direct comparison of tumor uptake for 99m Tc-P-RGD 2 , 99m Tc-3G-RGD 2 , 99m Tc-3P-RGD 2 , and 99m Tc-RGD 4 in athymic nude mice bearing MDA-MB-435 breast cancer xenografts. The biodistribution data were adapted from Shi et al. (2008) and Wang et al. (2008b) prove the concept of ''bivalent heterodimers.'' For the bifunctional radiotracers to achieve the bivalency, the a v b 3 and BBN receptors must be co-localized and the distance between them must be short. Otherwise, it would not be advantageous even if they might be able to target both individual receptors. Unfortunately, there is lack of concrete experimental data to demonstrate if the c(RGDfK)-BBN(7-14) and c(RGDyK)-BBN(7-14) conjugates are ''bivalent'' for tumor targeting, and whether there is indeed a ''synergetic effect'' between the cyclic RGD and BBN(7-14) peptides. Another challenge associated with the ''bifunctional heterodimer concept'' is which binding unit actually contributes to the radiotracer tumor uptake.  a v b 3 , a v b 5 , a 5 b 1 , a 6 b 4 , a 4 b 1 , and a v b 6 ). B Schematic illustration of the interactions between the bifunctional peptide and two different receptors (a v b 3 and BBN receptor). By targeting two different receptors, the radiotracer will have more opportunities to localize in the tumor because of the increased receptor population. The two targeted receptors (e.g., a v b 3 /a v b 5 or a v b 3 /BBN) must be co-localized and the distance between them must be short for the bifunctional radiotracer to achieve ''simultaneous receptor binding.'' Bottom: Selected examples of bifunctional peptides containing c(RGDfK)/c(RGDyK) and Aca-BBN(7-14)NH 2 (e-aminocaproic acid-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH 2 )

Integrin specificity
Blocking experiment (Fig. 8A) has been used to demonstrate the a v b 3 specificity of radiolabeled RGD peptides with a known a v b 3 antagonist (e.g., c(RGDfK) or RGD 2 ) as the blocking agent. This experiment is often performed by biodistribution or imaging (PET or SPECT). The blocking agent is pre-or co-injected with the radiotracer. Co-injection or pre-injection of excess blocking agents (such as RGD 2 ) will result in partial or complete blockage of the radiotracer tumor uptake (Fig. 8B). It is important to note that there is also a significant reduction in radiotracer uptake in the a v b 3positive organs (e.g., eyes, intestine, kidneys, lungs, liver, muscle, and spleen). The normal organ uptake is consistent with the b 3 and CD31 staining data for the liver, kidneys, and lungs from the tumor-bearing athymic nude mice.

LINEAR RELATIONSHIP BETWEEN RADIOTRACER TUMOR UPTAKE AND A V B 3 EXPRESSION
It has been shown that the radiolabeled cyclic RGD peptides are useful for non-invasive imaging of tumors in cancer patients (Beer et al. , 2007Haubner et al. 2005). It is the total a v b 3 level that will contribute the tumor uptake of a a v b 3 -targeted radiotracer. The capability to visualize the a v b 3 expression provides new opportunities to characterize the tumor angiogenesis noninvasively, to select appropriate patients for antiangiogenic treatment, and to monitor the tumor response to antiangiogenic drugs. However, Fig. 8 A Comparison of organ uptake (%ID/g) for 99m Tc-2P-RGD 2 in athymic nude mice bearing U87MG glioma xenografts in the absence or presence of excess RGD 2 at 60 min p.i. Co-injection of excess RGD 2 resulted in significant reduction in the uptake of 99m Tc-2P-RGD 2 in the tumor and normal organs. B Comparison of the 60-min biodistribution data of 111 In-3P-RGD 2 and 111 In-3P-RGK 2 in athymic nude mice bearing U87MG glioma xenografts. The low tumor uptake for 111 In-3P-RGK 2 indicates that the radiolabeled cyclic RGD dimers are RGD-specific. The experimental data were adapted from Shi et al. (2011a) REVIEW there were only a few reports on the correlation between the a v b 3 expression levels and radiotracer tumor uptake (Beer et al. , 2007Haubner et al. 2005;Zhang et al. 2006). 99m Tc-3P-RGD 2 was studied for its capability to monitor the a v b 3 expression in five different tumorbearing animal models (U87MG, MDA-MB-435, A549, HT29, and PC-3). IHC staining was performed to determine the a v b 3 and CD31 (a biomarker for tumor vasculature) expression levels in xenografted U87MG, MDA-MB-435, A549, HT29, and PC-3 tumor tissues . It was found that the total a v b 3 expression levels on the tumor cells and tumor neovasculature follow the general ranking trend: In contrast, the CD31 expression levels follow the general ranking order of U87MG = HT29 [ MDA-MB-435 = A549 [ PC-3 (Fig. 9). More importantly, there is an excellent relationship between the tumor uptake and the a v b 3 expression levels . The linear relationship between the tumor uptake (%ID/g) and a v b 3 density suggests that 99m Tc-3P-RGD 2 is useful for non-invasive monitoring of the a v b 3 expression levels in cancer patients. 99m Tc-3P-RGD 2 has been used to monitor the tumor response to antiangiogenesis treatment with linifanib (ABT-869) (Ji et al. 2013b, d), a multi-targeted receptor tyrosine kinase inhibitor targeting vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptors (Albert et al. 2006;Shankar et al. 2007;Wong et al. 2009;Zhou et al. 2009;Hernandez-Davies et al. 2011;Jiang et al. 2011;Tannir et al. 2011;Luo et al. 2012). We found that there was a significant decrease in tumor uptake (%ID/cm 3 ) and T/M ratios of 99m Tc-3P-RGD 2 in the xenografted U87MG model, while no significant changes in tumor uptake of 99m Tc-3P-RGD 2 were seen in the PC-3 model after linifanib treatment (Ji et al. 2013d). The uptake changes in MDA-MB-435 tumors were between those observed in the U87MG and PC-3 models (Ji et al. 2013b). This is consistent with the tumor a v b 3 expression levels . Highly vascularized tumors (e.g., U87MG) with higher level of a v b 3 and CD31 have better tumor response to linifanib therapy than poorly vascularized tumors (e.g., PC-3) with low levels of a v b 3 and CD31 (Fig. 10). Thus, 99m Tc-3P-RGD 2 might be a screening tool to select appropriate patients who will benefits most antiangiogenic treatment. If the tumor has a high a v b 3 expression, as indicated by high tumor uptake of 99m Tc-3P-RGD 2 at the time of diagnosis, antiangiogenic therapy would more likely be effective. If the tumor has little a v b 3 expression (low uptake of 99m Tc-3P-RGD 2 ), antiangiogenic therapy would not be effective regardless the amount of antiangiogenic drug administered into the patient. 99m Tc-3P-RGD 2 SPECT/CT has been used as a noninvasive imaging tool to monitor the tumor growth and progression of breast cancer lung metastasis (Albert et al. 2006;Ji et al. 2013d). Figure 11 shows the SPECT/CT images of athymic nude mice (n = 8) with breast cancer lung metastasis. As expected, the SPECT/CT images showed no detectable metastatic breast tumor lesions in the lungs at week 4 ( Fig. 11:   Fig. 9 Relationship between the tumor uptake (%ID/g: radioactivity density) and relative b 3 or CD31 levels in five xenografted tumors (U87MG, MDA-MB-435, A549, HT29, and PC-3). The total b 3 expression was represented by the percentage of red area over total area in each slice of tumor tissue. Each data point was derived from at least 15 different areas of same tissue (9100 magnification). Experiments were repeated three times independently with similar results. The experimental data were adapted from Zhou et al. (2011b) top). By week 6, small breast cancer lesions started to appear in the mediastinum and lungs. At week 8, SPECT/CT images revealed many metastatic cancer lesions in both lungs (Albert et al. 2006). Figure 11 (bottom) compares the %ID (left) and %ID/cm 3 (right) uptake values of 99m Tc-3P-RGD 2 in the lungs. Even though the lung uptake of 99m Tc-3P-RGD 2 (0.41 ± 0.05 %ID) at week 4 seemed to be higher than that in the control animals (0.36 ± 0.06 %ID), this difference was not significant (p [ 0.05) within the experimental errors. At week 6, the tumor burden in the lungs became significant. The lung uptake of 99m Tc-3P-RGD 2 was much higher (0.89 ± 0.12 %ID, p \ 0.01) than that in the control group. By week 8, the uptake of 99m Tc-3P-RGD 2 in the lungs was increase to 1.40 ± 0.42 %ID. In all cases, the lung size remained relatively unchanged (1.21-1.32 cm 3 ) during the 8-week study period. Fig. 10 Linear relationship between the %ID/cm 3 tumor uptake change at days 1 (top), 4 (middle) and 11 (bottom) after linifanib therapy and the expression levels of the a v b 3 (left) and CD31 (right) in three tumor-bearing animal models. The %ID/cm 3 tumor uptake values of 99m Tc-3P-RGD 2 were calculated from SPECT/CT quantification and reported as the mean plus/minus standard error of the mean based on results from five animals (n = 5). The %ID/cm 3 tumor uptake change was calculated by deducting the %ID/cm 3 tumor uptake of 99m Tc-3P-RGD 2 on days 1, 4, and 11 from its original value on -1 day (before linifanib therapy) in the same animal. The average %ID/cm 3 tumor uptake change is used as the indicator of tumor response to linifanib treatment. The experimental data were adapted from Zheng et al. (2014) REVIEW CLINICAL EXPERIENCES WITH 99M TC-3P-RGD 2

MONITORING TUMOR METASTASIS
The excellent in vivo tumor-targeting efficacy of 99m Tc-3P-RGD 2 in animal models guaranteed its further clinical application. In a first-in-human study, 99m Tc-3P-RGD 2 was investigated for its capability to noninvasively differentiate solitary pulmonary nodules (SPNs) . Among the 21 patients with SPNs, 15 (71%) were diagnosed as malignant while 6 (29%) were benign. The sensitivities for CT interpretation and 99m Tc-3P-RGD 2 SPECT visual were 80% and 100%, respectively. All SPNs classified as indeterminate via CT can be sensitively diagnosed by 99m Tc-3P-RGD 2 scintigraphy. 99m Tc-3P-RGD 2 uptake in the malignant and benign nodules was well confirmed by ex vivo IHC staining of a v b 3 . These results demonstrated the feasibility of using 99m Tc-3P-RGD 2 scintigraphy in differentiating SPNs . A multicenter study was performed in 70 patients with suspected lung lesions (Zhu et al. 2012). The results clearly demonstrated that 99m Tc-3P-RGD 2 SPECT effectively detects lung malignancies, but with relatively low specificity. Whole-body planar scanning and chest SPECT are complementary for the evaluation of primary tumor and metastasis (Zhu et al. 2012). In a recently study, the potential of 99m Tc-3P-RGD 2 SPECT in the detection of RAIR DTC lesions was conducted ). 99m Tc-3P-RGD 2 SPECT identified all the target RAIR metastatic lesions, and there was a significant correlation between the mean tumor-to-background ratios and mean growth rates of target lesions. It is concluded that 99m Tc-3P-RGD 2 imaging can be used for the localization and growth evaluation of RAIR lesions, thus providing a promising imaging strategy to monitor the efficacy of antiangiogenic therapy ). 99m Tc-3P-RGD 2 SPECT was also evaluated and compared to 99m Tc-MIBI for the capability to assess the breast cancer lessons (Ma et al. 2014). It was found that the mean T/NT Radiolabeled cyclic RGD peptides as radiotracers REVIEW ratio of 99m Tc-3P-RGD 2 in malignant lesions was significantly higher than that in benign lesions (3.54 ± 1.51 vs. 1.83 ± 0.98, p \ 0.001). The sensitivity, specificity, and accuracy of 99m Tc-3P-RGD 2 SMM were 89.3%, 90.9%, and 89.7%, respectively, with a T/NT cut-off value of 2.40. The mean T/NT ratio of 99m Tc-MIBI in malignant lesions was also significantly higher than that in benign lesions (2.86 ± 0.99 vs. 1.51 ± 0.61, p \ 0.001). The sensitivity, specificity, and accuracy of 99m Tc-MIBI SMM were 87.5%, 72.7%, and 82.1%, respectively, with a T/NT cut-off value of 1.45. According to the ROC analysis, the area under the curve for 99m Tc-3P-RGD 2 SMM (area = 0.851) was higher than that for 99m Tc-MIBI SMM (area = 0.781), but the statistical difference was not significant.

CLINICAL EXPERIENCES WITH 18 F-ALFATIDE AND 18 F-ALFATIDE II
18 F-labeled RGD compounds suffer from multistep and time-consuming synthetic procedures, which will limit their clinic availability. To overcome this shortcoming, the Al(NOTA) chelate has been used for 18 F-labeling of P-RGD 2 (Lang et al. 2011). The application of NOTA-AlF chelation chemistry and kit formulation allows one-step 18 F-labeling. Under the optimal conditions, the radiotracer [ 18 F]AlF(NOTA-P-RGD 2 ) (denoted as 18 F-Alfatide) was prepared in relatively high yield (42.1 ± 0.02) with more than 95% radiochemical purity. The whole radiosynthesis including post-labeling chromatographic purification was accomplished within 20 min. Nine patients with a primary diagnosis of lung cancer were examined by both static and dynamic PET imaging with 18 F-alfatide, and one tuberculosis patient was investigated using both 18 F-alfatide and 18 F-FDG imaging. It was found that 18 F-alfatide PET identified all tumors, with mean standardized uptake values of 2.90 ± 0.10. Tumor-to-muscle and tumor-to-blood ratios were 5.87 ± 2.02 and 2.71 ± 0.92, respectively. It was concluded that PET scanning with 18 F-alfatide allows specific imaging of avb3 expression with good contrast in lung cancer patients.