Nuclear Oncology, 1 Ed.



Zhao-Hui Jin • Takako Furukawa • Tsuneo Saga


Angiogenesis is a tightly regulated process involving the formation of new blood vessels from pre-existing vasculature.1 Angiogenesis is fundamental to embryogenesis, uterine maturation, placental development, corpus luteum formation, and wound healing.1,2 Angiogenesis is also characteristic of pathologic processes including neoplasm, psoriasis, endometriosis, arthritis, macular degeneration, regional ileitis, atherosclerosis, and chronic inflammatory diseases.35 In 1971, Folkman,6 an American medical scientist best known for his research on tumor angiogenesis, proposed that tumor cells were capable of stimulating endothelial cell proliferation by means of a soluble “tumor angiogenesis factor,” and emphasized the importance of angiogenesis in tumor growth, leading to extensive research to understand and control the angiogenic pathways in tumors. Angiogenesis is jointly regulated by the nature of each tumor and a complex network of several cell types, various growth factors/receptors, extracellular matrix (ECM) proteins/proteases, and signaling pathways involved in endothelial proliferation, migration, and tube formation and function.5,7,8 Tumor angiogenesis has been accepted as an important target and indicator of therapeutic outcome and prognosis, because it is a key feature of malignant solid tumors and plays a critical role in tumor growth, invasion, and metastasis.9,10 A large number of angiogenesis inhibitors have been developed, based on cells, genes, monoclonal antibodies (mAbs), proteins, peptides, or small molecules, for differentially blocking the multiple and complex steps of angiogenesis, whereby the formation of new blood vessels can be suppressed and growth or spread of tumors can be stopped or slowed.7,10,11 Several angiogenesis inhibitors such as bevacizumab (Avastin), sorafenib (Nexavar), and sunitinib (Sutent) for the treatment of certain types of cancer have been approved by the Food and Drug Administration (FDA) in the United States and many other countries; many other drugs are in active clinical trials.10,12 As clinical practice and research proceed, it is critical to identify noninvasive biomarkers for patient selection/stratification, dose and dosage optimization, prediction/monitoring of treatment response, and detecting early signs of drug resistance that may be acquired by activation or upregulation of an alternative pathway.13,14 Unlike cytotoxic chemotherapeutic drugs, angiogenesis inhibitors are typically cytostatic; that is, they slow or stop tumor growth rather than causing tumor shrinkage. Therefore, routine methods for evaluating chemotherapeutic efficiency, including changes in tumor volume or morphology detected by computed tomography (CT) and magnetic resonance imaging (MRI), may not be suitable for assessing the response to antiangiogenic treatment.15

Great advances in imaging instrumentation have made possible the noninvasive molecular imaging of tumor angiogenesis in vivo; thus, tumor angiogenesis can be assessed and evaluated indirectly or directly.1618 Indirect angiogenesis imaging is nontargeted, measuring differences in perfusion, vessel permeability, and blood volume between tumor and normal vasculature. Contrast-enhanced or dynamic contrast–enhanced CT and MRI can be used to evaluate or quantify perfusion and vascular permeability. Positron emission tomography (PET) with oxygen-15 (15O) water, carbon-11 (11C)-carbon monoxide (CO) or C15O can be used to visualize and quantify tumor blood volume. Ultrasound with contrast-enhanced microbubbles also shows promise to evaluate tumor blood volume. By contrast, direct tumor angiogenesis imaging targets specific molecules in the angiogenic pathway, also known as targeted angiogenesis imaging. Recent advances in nuclear medicine imaging technology, including PET and single photon emission computed tomography (SPECT), have enabled direct angiogenesis imaging with high sensitivity and improved spatial resolution. These techniques have become the predominant strategy for noninvasive in vivo visualization, quantitation, and monitoring of tumor angiogenesis and antiangiogenic treatment efficacy.19

This chapter introduces the most extensively studied biomarkers and development of corresponding radionuclide-based probes for nuclear medicine imaging of tumor angiogenesis (Table 37.1). The main tools for nuclear medicine imaging are PET and SPECT, each with advantages and shortcomings, and each of the radionuclides in PET or SPECT radiopharmaceuticals has a specific decay mode, physical half-life, chemical properties, and method of production. Detailed descriptions can be found in recent reviews.20,21

TABLE 37.1


αvβ3 Integrin as a Marker for Tumor Angiogenesis

Integrins, a family of transmembrane glycoproteins and cell adhesion receptors,22 mediate cell–cell and cell–matrix interactions23 and outside-in/inside-out modes of signal transduction.24,25 An integrin contains two noncovalently associated α and β subunits; in mammals, 19 α and 8 β subunits have been characterized, forming 24 distinct integrin heterodimers through different subunit combinations.26,27 The vitronectin receptor, αvβ3 integrin, is one of the most interesting and extensively studied members of the integrin family, with important roles in tumor growth, invasion, metastasis, and angiogenesis.26,28 It is highly expressed on activated endothelial cells during angiogenesis and on some types of tumor cells, such as invasive melanoma, glioblastoma, osteosarcoma, neuroblastoma, and carcinomas of different origins.26,28,29 αvβ3 Integrin is important for tumor angiogenesis imaging because of its high expression levels, but also because the activation state of αvβ3 occurs during the final stage of angiogenesis. The latter is especially helpful when using it as a biomarker for monitoring tumor response to angiogenesis inhibitors. As explained by Choyke,30 “There are multiple mechanisms by which new vessels can be generated; inhibition of one pathway can lead to compensation by another. An imaging agent that targets the ‘final common pathway’ of angiogenesis would be more widely useful than a highly specific agent that is relevant only under certain circumstances; integrins hold promise clinically, precisely because they seem to be part of this final common pathway.”

Many integrins, including αvβ3, bind to their natural ligands: A wide variety of ECM proteins such as fibronectin, vitronectin, osteopontin, fibrinogen, collagens, and laminin via the common tripeptide sequence Arg-Gly-Asp (single letter coding: RGD).31,32 However, the specificity of the receptor–ligand recognition and binding is also attributed to sequences neighboring the RGD, as well as its constitutive spatial conformation.33,34 Cyclic pentapeptides containing the RGD (cRGD) are optimized synthetic ligands possessing a high affinity and selectivity for αvβ3 integrin.35,36 Based on this lead structure, a series of cRGD-containing radiolabeled tracers have been developed for the purpose of tumor angiogenesis imaging.37,38 In addition, other RGD-based peptidomimetics or peptide sequences obtained from phage-display peptide library screening have also been developed.38,39 The most published preclinical and clinical studies on the use of radiolabeled tracers for detection of αvβ3 integrin are based on RGD peptides.

RGD-Based Radionuclides for Angiogenesis Imaging Via Targeting of αvβ3 Integrin

The first generation of RGD-based tracers was developed by Haubner et al.40 They modified the lead cyclo(-Arg-Gly-Asp-D-Phe-Val-) peptide to retain high selectivity and affinity for αvβ3 through substitution of D-Phe or Val with tyrosine (Tyr) to allow electrophilic radiohalogenation by the iodogen method. This led to two iodine-125 (125I, γ-ray emitter, half-life: 59.4 days)-labeled tracers, 125I-3-iodo-D-Tyr4-cyclo(-Arg-Gly-Asp-D-Tyr-Val-) and 125I-3-iodo-D-Tyr5-cyclo(-Arg-Gly-Asp-D-Phe-Try-). In vitro binding assays of isolated and immobilized integrin receptors revealed that the two modes of tyrosine replacement and subsequent radiolabeling had no influence on αvβ3 affinity and selectivity; however, 125I-3-iodo-D-Tyr4-cyclo(-Arg-Gly-Asp-D-Tyr-Val-) showed better αvβ3-specific tumor-targeting efficiency in vivo. Both tracers had fast elimination kinetics, resulting in a rapid decrease in tumor tracer accumulation and predominant hepatobiliary excretion, leading to high radioactivity accumulation in the liver and intestine. These promising results and serious limitations inspired many research groups to explore optimal tracers for tumor angiogenesis imaging. Some of these have already entered clinical trials, and some promising agents are undergoing preclinical studies.

RGD-Based Tracers in Clinical Trials


[18F]Galacto-RGD was developed by Haubner et al.41 at the University of Munich by introducing a sugar amino acid (SAA) (7-amino-L-glycero-L-galacto-2,6-anhydro-7-deoxyheptanoic acid) to the cyclic RGD pentapeptide to form cyclo(-Arg-Gly-Asp-D-Phe-Lys(SAA)-) to increase hydrophilicity, reducing hepatobiliary excretion, and allowing fluorine-18 (18F, commonly used positron emitter for clinical patients, half-life: 110 minutes) labeling with 4-nitrophenyl 2-18F-fluoropropionate. In vitro binding assays using the isolated immobilized integrin receptors (αIIbβ3, αvβ5, and αvβ3) demonstrated the αvβ3 integrin-binding specificity of [18F]Galacto-RGD, which exhibited 200- to 1,200-fold greater affinity for αvβ3 than for αvβ5 and αIIbβ3. The in vivo αvβ3 integrin-targeting specificity of [18F]Galacto-RGD was tested by using αvβ3-positive M21 and αvβ3-negative M21-L human melanoma xenografts. Tracer uptake in M21 is approximately four times greater than in M21-L between 60 and 120 minutes post injection (p.i.); this was confirmed by a competitive blocking assay with excess αv-selective peptide cyclo(-Arg-Gly-Asp-D-Phe-Val-).

Published in 2005,42 the first study of PET imaging with [18F]Galacto-RGD (133 to 200 MBq) was performed in 19 patients with metastatic malignant melanoma (seven patients), sarcoma (ten patients), or osseous metastases (two patients), and demonstrated rapid blood clearance and primarily renal excretion of this tracer. Dynamic scanning revealed that tumor tracer uptake peaked at ∼10 minutes p.i., followed by a plateau with no or minimal reduction in radioactivity until 60 minutes p.i. [18F]Galacto-RGD uptake in 29 tumor lesions had a mean standardized uptake value (SUV) of 3.7 ± 2.3 (ranging from 1.2 to 9) at 72 minutes p.i., indicating great inter- and intraindividual diversity of αvβ3 expression in cancer patients. The tumor-to-blood (T/B) and tumor-to-muscle (T/M) ratios increased over time, with peak ratios of 3.1 ± 2 and 7.7 ± 4.3, respectively, at 72 minutes p.i. Nearly 80% of the 29 lesions could be visualized by PET imaging.

To determine the clinical correlation of [18F]Galacto-RGD uptake with αvβ3 integrin expression (distribution pattern and levels), or microvessel density (MVD), a clinical study was performed in 19 patients with solid tumors (musculoskeletal system, ten patients; melanoma, four patients; head and neck cancer, two patients; glioblastoma, two patients; and one breast cancer patient) who were examined with [18F]Galacto-RGD PET before surgical removal of the tumor lesions.43 Twenty-six specimens were collected from representative areas with low and high SUVs and then snap frozen. Immunohistochemical staining revealed no αvβ3 expression in normal tissue and in the two lesions without tracer uptake whereas all lesions with enhanced uptake showed immunohistochemical αvβ3 expression. In benign lesions (pigmented villonodular synovitis, neurofibroma, and inflammatory tissue), αvβ3 was located only on the vasculature, whereas in malignant tumors, the patterns of αvβ3 expression varied considerably. In lymph-node metastasis and cutaneous metastasis from melanoma, αvβ3 was predominantly located on tumor cells. In squamous cell carcinoma of the head and neck (SCCHN), αvβ3 was located on the neovasculature. In the other tumor entities, αvβ3 was located on the neovasculature and on tumor cells in varying degrees. SUVs and T/B ratios significantly correlated with the intensity of immunohistochemical αvβ3 staining (Spearman’s r = 0.92 and 0.94, respectively; p < 0.0001) and with the MVD of αvβ3-positive vessels (Spearman’s r = 0.84 and 0.90, respectively; p < 0.0001).

A clinical trial investigated the efficacy of [18F]Galacto-RGD PET to image αvβ3 expression on the neovasculature in SCCHN patients.44 Eleven patients with primary SCCHN diagnoses were enrolled. Ten of twelve tumors could be detected (Fig. 37.1), and the other two tumors (<5 mm) failed for delineation. Tumor kinetics were consistent with a two-tissue compartmental model with reversible specific binding. Immunohistochemistry confirmed αvβ3expression in all tumors with αvβ3 being located on the microvessels in all specimens (see Fig. 37.1) and with only one tumor showing αvβ3 expression on both tumor cells and vasculature. This study suggests the feasibility of performing angiogenesis assessments with [18F]Galacto-RGD PET in SCCHN, which might be useful for planning and response evaluation of antiangiogenic therapies combined with chemotherapy and/or radiotherapy.

FIGURE 37.1. Patient with a squamous cell carcinoma of the head and neck in the right mandible (arrows, A and B). [18F]Galacto-RGD PET (A) and PET/MRI image fusion (B) show heterogeneous intense tracer accumulation in the lesion, whereas there is only low background uptake in parts of the oral cavity and very low uptake in the parotid glands and in muscle tissue. Immunohistochemical evaluation of αvβ3 expression at low-power magnification (C)and at high-power magnification (D) shows intense staining of the neovasculature (arrows) without staining of the tumor cell complexes. SUV, standardized uptake value. (Reprinted from Beer AJ, Grosu AL, Carlsen J, et al. [18F]galacto-RGD positron emission tomography for imaging of αvβ3 expression on the neovasculature in patients with squamous cell carcinoma of the head and neck. Clin Cancer Res. 2007;13:6610–6616, with permission from AACR.)

A breast cancer clinical trial was performed in 16 patients with invasive ductal carcinoma.45 [18F]Galacto-RGD PET performed well for primary tumor detection but did not show sufficient sensitivity for lymph-node staging. Immunohistochemical staining in primary tumors revealed αvβ3 expression predominantly on endothelial cells, but also on tumor cells. This work indicates the ability of [18F]Galacto-RGD PET to detect primary breast cancer, but also suggests that when assessing angiogenesis of breast cancer using αvβ3 as a biomarker, the results must be interpreted carefully, as part of the signal might come from the tracer binding on tumor cells themselves. Similar findings were obtained in patients with malignant glioma.46


[18F]Fluciclatide, also known as 18F-AH111585, is under development as a monomeric cyclic RGD-based radioligand for αvβ3 integrin with the core sequence ACDCRGDCFCG, originally discovered in a phage-display library.47[18F]Fluciclatide was designed by cyclization and introduction of multiple disulfide bridges to stabilize the molecule with minimal disruption of the RGD pharmacophore, and by introduction of a polyethylene glycol (PEG)-like spacer at the C terminus to stabilize the peptide against degradation by carboxypeptidases and prolonging the circulation life span, thereby increasing tumor uptake and retention.48

[18F]Fluciclatide has entered clinical trials to evaluate safety and metabolic stability and to determine the pharmacokinetic and tumor-targeting efficacy.49,50 However, the limitation of [18F]fluciclatide is the relatively high radioactivity in the liver and gastrointestinal tract because of the hepatobiliary excretion.49,50 Dynamic PET scan was performed in seven patients with metastatic breast cancer after receiving [18F]fluciclatide (198.8 to 292.1 MBq), and all 18 tumors could be visualized either as a distinct increase in radioactive accumulation in comparison with the surrounding normal tissue (Fig. 37.2A, B) or, in the case of liver metastases, as regions of deficient accumulation (Fig. 37.2C) because of the high background activity in normal liver tissue.50 Compared with the rapid uptake of [18F]Galacto-RGD in tumors, which peaked at 10 to 15 minutes p.i.,42,44 the PEGylated [18F]fluciclatide accumulated in tumor lesions gradually, reaching a plateau by 50 to 60 minutes p.i. A two-tissue reversible model was established as the best quantitative methodology for analysis of [18F]fluciclatide PET data, suggesting that the k(3)/k(4) ratio is a reasonable measure of specific binding, and for tumor/healthy tissue differentiation.51 In addition, clinical trials in patients with malignant melanoma, renal cell carcinoma (RCC), and renal oncocytoma (the most common benign solid renal tumor) are ongoing52 It is worth noting that it would be valuable if [18F]fluciclatide PET imaging can distinguish RCC from oncocytoma because in clinical practice it is not always possible to do so with ultrasonography, CT scanning, or MRI.

Another important application of [18F]fluciclatide PET is to monitor tumor response to antiangiogenic therapy as reported by Battle et al.53 In their experimental study in mice bearing αvβ3 highly expressing U87MG tumor models, sunitinib (60 mg/kg orally daily, two 5-day cycles with 2 days free between cycles), the FDA-approved multitargeted receptor tyrosine kinase (RTK) inhibitor, inhibited tumor uptake of [18F]fluciclatide and significantly reduced the tumor MVD obtained at the end of therapy, but without significant changes in tumor volumes. Earlier work done by Morrison et al.54 also showed the promise of [18F]fluciclatide PET for monitoring response to treatment with different antiangiogenesis drugs, low-dose paclitaxel (a mitotic inhibitor with antiangiogenic activity at low doses) and ZD4190 (a substituted 4-anilinoquinazoline acting as an RTK inhibitor-type angiogenesis inhibitor) as well as different tumor models in mice, mouse Lewis lung carcinoma (LLC), and human lung adenocarcinoma Calu-6, both of which have established low levels of tumor-cell αvβ3 expression.

FIGURE 37.2. 18F-AH111585 PET of metastatic lesions and corresponding CT images showing increased signal in periphery of lesions in patient with lung and pleural metastases (A), intralesion heterogeneity of uptake within pleural metastasis in PET image, which was not demonstrated as necrosis on corresponding CT section (B), and liver metastases imaged as hypointense lesions because of high background signal (C). High uptake in spleen is possibly because of blood pooling. See also color bar for PET images. (Reprinted with permission of the Society of Nuclear Medicine from Kenny LM, Coombes RC, Oulie I, et al. Phase I trial of the positron-emitting Arg-Gly-Asp (RGD) peptide radioligand 18F-AH111585 in breast cancer patients. J Nucl Med. 2008;49:879–886.)


2-((2S,5R,8S,11S)-5-benzyl-8-(4-((2S,3R,4R,5R,6S)-6-((2-(4-(3-18F-fluoropropyl)-1H-1,2,3-triazol-1-yl)acetamido)methyl)-3,4,5-trihydroxytetrahydro-2H-pyran-2-carboxamido)butyl)-11-(3-guanidinopropyl)-3,6,9,12,15-pentaoxo-1,4,7,10,13-pentaazacyclopentadecan-2-yl) acetic acid ([18F]RGD-K5), a click-chemistry– derived, RGD-based cyclic peptidomimetic tracer, was developed as an αvβ3 integrin marker for PET.38,55 It contains a metabolically stable, yet highly polar 1,2,3-antitriazole moiety that increases the tracer’s excretion via the renal pathway, thus circumventing unwanted liver uptake. Compared with [18F]Galacto-RGD, the preparation of [18F]RGD-K5 is simple and straightforward using click chemistry, consisting of a single reaction that can be readily automated.56 Although [18F]RGD-K5 was designed to target αvβ3 integrin, it may also bind other integrins because the selectivity of RGD-K5 toward αvβ3 integrin is only 2.3 to 3.4-fold higher versus related integrins.57

Whole-body PET/CT on four healthy humans receiving 583 ± 78 MBq [18F]RGD-K5 demonstrated its safety, favorable pharmacokinetic characteristics with metabolic stability, rapid blood clearance, and predominant renal excretion.55 It was also shown that the biodistribution and dosimetry of [18F]RGD-K5 in monkeys can adequately predict similar data in humans.55

The initial evaluation of [18F]RGD-K5 PET compared with 18F-FDG PET was performed in 12 patients with breast cancer.38 More than 77% of 157 FDG-positive lesions could be detected on [18F]RGD-K5 PET, and no correlation was observed with tracer uptake. Tumor uptake of [18F]RGD-K5 did not correlate with MVD, one of the histologic parameters of angiogenesis. Further study on the correlation between the intratumoral distribution of [18F]RGD-K5 and αvβ3 integrin localization, as well as the correlation of tracer uptake with integrin expression may help determine the efficacy of this tracer for tumor angiogenesis imaging.


[18F]FPPRGD2, 4-nitrophenyl 2-18F-fluoropropionate (18F-NFP)-labeled PEGylated dimeric RGD peptide, PEG3-E[c(RGDyk)2], was developed at Stanford University Medical Center and approved by the FDA as an Investigational New Drug (IND 104150). It was designed by using the dimeric cRGD peptide and PEG3 as the space linker between two cRGD motifs to enhance receptor-binding affinity.58 Compared with the monomeric peptide tracer [18F]Galacto-RGD, [18F]FPPRGD2 showed high αvβ3 affinity to human glioblastoma U87MG cells in vitro and high αvβ3-specific tumor uptake with comparable tumor contrast in U87MG tumor-bearing mice.58

Mittra et al.59 reported a pilot study of [18F]FPPRGD2 PET in five healthy volunteers, showing the safety, favorable dosimetry, and pharmacokinetics of this tracer. [18F]FPPRGD2 PET is suitable for brain, lung, and breast cancers. For reliable and routine clinical PET studies, Chin et al.60 developed an automated multistep radiosynthesis of clinical-grade [18F]FPPRGD2 with favorable biodistribution in human studies, with low background signal in the head, neck, and thorax (Fig. 37.3).

Stanford University Medical Center recruited six female patients with breast cancer61 and five patients with suspected recurrent glioblastoma multiforme (GMB).62 [18F]FPPRGD2 PET/CT and 18F-FDG PET/CT were performed within 2 weeks for each patient, and brain MRI was added for suspected recurrent GBM patients. Vital signs, electrocardiogram, and blood examination indicated the safety of [18F]FPPRGD2. There was consistent distribution in all patients with primary clearance through the kidneys and hepatobiliary system. Intense uptake of [18F]FPPRGD2 was observed in the three primary breast lesions at 45 minutes p.i., with SUVmax values ranging from 8.1 to 9.4 (average: 8.75 ± 0.9), compared to 18F-FDG uptake with SUVmax 11.9. Eleven metastatic breast cancer lesions also showed intense uptake of [18F]FPPRGD2, with SUVmax values ranging from 4.8 to 9.4 (average: 7.12 ± 1.9), compared with 18F-FDG uptake with SUVmax values ranging from 2.2 to 4.8 (average: 3.13 ± 1.4). Among the five patients suspected of developing recurrent GMB, one patient had recurrent GBM identified only on the [18F]FPPRGD2 PET. Another patient had recurrent GBM identified on [18F]FPPRGD2 PET and brain MRI but not on 18F-FDG PET. Another patient had recurrent GBM identified on all three scans. The remaining two patients had no recurrent GBM as determined by [18F]FPPRGD2 PET, 18F-FDG PET, and brain MRI. Uptake of [18F]FPPRGD2 in the three recurrent GBM lesions showed SUVmax values of 2.3 to 2.9 (average: 2.6 ± 0.3). [18F]FPPRGD2 uptake was seen in the brain ventricles and at the edge of the surgical incision (wound repair) in all patients, but was not noted in the normal brain. These findings show that [18F]FPPRGD2 PET is a promising candidate for detection of primary and metastatic breast cancers, as well as recurrent GBM lesions. Further evaluation with larger cohorts will verify these preliminary data and clarify the relationship between tracer uptake and histologic localization of αvβ3integrin to explore the potential of [18F]FPPRGD2 for angiogenesis imaging.

FIGURE 37.3. Clinical-grade [18F]FPP(RGD)2 (518 MBq/14 mCi) injected intravenously to a healthy human volunteer. Images include a coronal fusion PET/CT image (A, left side) and maximum intensity projection (MIP) image (A, right side) as well as serial MIP images (B)(A) was obtained 1 hour post injection and has the major organs of uptake labeled as follows: 1, brain ventricles; 2, lung; 3, liver; 4,gallbladder; 5, bowel; 6, urinary bladder; 7,salivary glands/oropharynx; 8, thyroid glands; 9, spleen; and 10, kidneys. The renal and hepatobiliary clearance routes of [18F]FPP(RGD)2 are evident. Above the diaphragm, there is only very limited uptake. The combined image in (B) shows the temporal stability of [18F]FPP(RGD)2 at five time-points post injection. (With kind permission from Springer Science and Business Media: Chin FT, Shen B, Liu S, et al. First experience with clinical-grade [18F]FPP(RGD)2: An automated multi-step radiosynthesis for clinical PET studies. Mol Imaging Biol. 2012;14:88–95.)

The utility of [18F]FPPRGD2 PET to monitor tumor response to antiangiogenic therapy was investigated in mice bearing αvβ3-positive human MDA-MB-435 breast tumors.63 The mice received 3 days (days 1 to 3) of treatment with ZD4190 (100 mg/kg/day, oral). Compared to the almost unchanging [18F]FPPRGD2 uptake in the control tumors between days 0 and 7, [18F]FPPRGD2 uptake in ZD4190-treated tumors decreased significantly relative to the baseline level (day 0) by 26.74% ± 8.12% on day 1 and by 41.19% ± 6.63% on day 3, then returned to baseline on day 7, at which time the tumor volume of ZD4190-treated mice was significantly lesser than that of the control mice. In parallel, ZD4190 suppressed expression of αvβ3 integrin on tumor vasculature and on tumor cells on days 1 and 3, but not day 7. Unlike ZD4190, treatment with Abraxane (an FDA-approved nanoparticle albumin-bound paclitaxel: 25 mg/kg every other day, three doses, intravenous) did not induce significant changes of αvβ3 integrin expression on the tumor cells.64 The decreased tumor uptake of [18F]FPPRGD2 in the Abraxane-treated group correlated with the decreased expression of αvβ3 integrin on vascular endothelial cells. The collapse of microvessel cavities observed in Abraxane-treated tumors further confirmed the antiangiogenic effects of Abraxane. Together, these studies illustrate that [18F]FPPRGD2 is a promising PET tracer that allows noninvasive evaluation of the efficacy of antiangiogenesis therapy, but it should be noted that some angiogenesis inhibitors may directly influence integrin expression on tumor cells, which would make data analysis complicated.

One major disadvantage of [18F]FPPRGD2 and other 18F-labeled RGD peptides is the multistep, time-consuming, and low-yield synthetic procedures, hindering their widespread use as routine tracers in the clinic. A recent report65suggested that [18F]AlF-NOTA-PRGD2 is a promising alternative to [18F]FPPRGD2, with the former radiolabeling process via a recently introduced kit formulation method using an 18F-fluoride–aluminum complex, simplifying the 18F-labeling procedure to facilitate clinical translation. When studied in mice bearing U87MG tumors, [18F]AlF-NOTA-PRGD2 had pharmacokinetics and quantitative parameters for αvβ3 integrin-targeting efficiency comparable to those of [18F]FPPRGD2.


Gallium-68 (68Ga, generator-produced positron emitter, half-life: 68 minutes)-labeled tracer, 68Ga-1,4,7-Triazacyclononane-1,4-7-triaceticacid-isothiocyanatobenzyl-c(Arg-Gly-Asp-D-Tyr-Lys) (68Ga-NOTA-RGD), was introduced by Jeong et al.66 using 2-(p-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4-7-triacetic acid (SCN-Bz-NOTA) as a bifunctional chelator for labeling the c(RGDyK) monomer with 68Ga. 68Ga offers an advantage for clinical translation over other cyclotron-produced positron emitters including the most frequently used 18F because it can be obtained from commercially available 68Ge/68Ga generator systems. The production of 68Ga is also cost effective because the parent nuclide 68Ge has a long half-life of 271 days, allowing its use as a generator for more than 1 year. The short half-life of 68Ga (68 minutes) and its hydrophilic nature makes it suitable for labeling of small peptides like the c(RGDyK) monomer, with fast pharmacokinetics in vivo. Moreover, labeling of NOTA-RGD with 68Ga is simple and straightforward, and may be achieved by incubating the reaction mixture at room temperature for only 10 minutes.

A biodistribution study of 68Ga-NOTA-RGD in human colon cancer SNU-C4 xenograft-bearing mice showed high tumor tracer uptake (5.1 ± 1%ID/g) and high T/B and T/M ratios of 10.3 ± 4.8 and 9.3 ± 3.9 at 1 hour p.i., with predominant renal excretion.66 The highest radioactivity accumulation was in the kidneys (5.3 ± 1.3%ID/g at 1 hour p.i.) and relatively lower levels were observed in the liver, intestines, lung, spleen, and heart (<2.5 %ID/g at 1 hour p.i.). PET imaging clearly visualized tumors at 1 and 2 hour p.i.

Whole-body distribution of 68Ga-NOTA-RGD (172.4 ± 20.5 MBq) in humans and mice was similar, and radiation dosimetry studies indicate the acceptable effective radiation dose of 68Ga-NOTA-RGD in humans.67 The first clinical trial of 68Ga-NOTA-RGD PET was performed in six patients with liver metastasis from colorectal cancer.38 18F-FDG and 68Ga-NOTA-RGD PET/CT were performed before combination therapy with the chemotherapeutic drug FOLFOX and the antiangiogenesis drug bevacizumab. All six patients showed hypermetabolic lesions on 18F-FDG PET/CT. For 68Ga-NOTA-RGD PET, mildly elevated tracer uptake in liver metastases was seen in three of six patients; the others were negative. Finally, only patients with elevated 68Ga-NOTA-RGD uptake showed at least a partial response to therapy indicating the potential of 68Ga-NOTA-RGD PET to predict response to chemotherapy combined with an antiangiogenesis drug. Collectively, these reports suggest that 68Ga-NOTA-RGD should be evaluated further as a promising radiopharmaceutical for tumor angiogenesis imaging.


Technetium-99m (99mTc, generator-produced, widely used γ-ray emitter, half-life: 6 hours)-labeled NC100692, 99mTc-Diamine dioxime-Lys-Cys-Arg-Gly-Asp-Cyc-Phe-Cys-polyethylene glycol has been developed and is in active clinical trials for breast cancer detection. NC100692 is a cyclic RGD-containing peptide with a PEG chain linked to the C-terminal amino acid and a 99mTc-binding chelator linked to the N-terminal amino acid.68 A phase I study of 31 healthy volunteers demonstrated the acceptable pharmacokinetics of 99mTc-NC100692 with rapid blood clearance, prominent renal excretion, and relatively high background activity in the liver and intestines.68 Scintigraphy with 99mTc-NC100692 identified 19 of 22 primary lesions (86%) (561 to 747 MBq, imaged 40 to 150 minutes p.i.)69 and was feasible for detection of lung and brain metastases from breast and lung cancer in 25 patients (800 to 1,100 MBq, imaged 45 to 75 minutes p.i.).70 Although the utility of 99mTc-NC100692 for detection of angiogenesis was shown in murine models of hind leg ischemia,71 its value as a marker of angiogenesis in breast cancer remains to be determined in patients.


99mTc-3PRGD2 is a dimeric cyclic RGD peptide (3PRGD2 = PEG4-E[PEG4-c(RGDfK)]2; PEG4 = 15-amino-4,7,10,13-tetraoxapentadecanoic acid) labeled with 99mTc using HYNIC (6-(2-(2-sulfonatobenzaldehyde)hydrazono)) as a bifunctional coupling agent and tricine and TPPTS (trisodium triphenylphosphine-3,3′,3′′-trisulfonate) as coligands to prepare 99mTc complexes. Optimization of dimeric cyclic RGD peptides has been explored by inserting various pharmacokinetic modifiers (PKMs) between the RGD motifs; Shi et al.72 developed 99mTc-3PRGD2 using two PEG4 linkers as the PKM and with high αvβ3 integrin–binding affinity and high tumor contrast resolution in a nude mouse model. This SPECT tracer was developed in kit form by the Medical Isotopes Research Center of Peking University.73 The first clinical trial of scintigraphic imaging using 99mTc-3PRGD2 was performed in 21 patients with 21 solitary pulmonary nodules (SPNs) (lesion size ranging from 1 to 3 cm) based on CT scans, the current standard imaging procedure for SPN evaluation.74 Thoracic planar and SPECT sequential acquisitions were obtained at 45 and 75 minutes p.i. of 99mTc-3PRGD2 (939 ± 118 MBq). All 15 malignant tumors including adenocarcinoma, squamous cell carcinoma, and small cell carcinoma were detected, and all of them were confirmed to show positive expression of αvβ3 integrin. In one example of lung adenocarcinoma with high tracer uptake, immunohistochemistry demonstrated intense αvβ3 staining in the tumor vessels (Fig. 37.4). Two of the six benign lesions, which were highly suspicious of malignancy on CT scan and showed relatively high tracer uptake, were histopathologically diagnosed as tuberculoma and inflammatory pseudotumor, with αvβ3 integrin expression identified predominantly on the endothelium of newly formed blood vessels. A multicenter study of 70 patients further demonstrated the suitability of this tracer for the detection of lung cancer at 1 hour p.i.75 In addition, a very recently published clinical study showed the potential of 99mTc-3PRGD2 SPECT for detection of radioactive iodine-refractory (RAIR) differentiated thyroid cancer (DTC), and found a correlation between the mean T/B ratios and mean lesion growth rates, which may indicate active angiogenesis and malignant phenotypes of RAIR DTC.76 However, the efficacy of 99mTc-3PRGD2 SPECT for angiogenesis imaging must be validated by exploring the correlations between tumor tracer uptake and αvβ3 expression, and between tracer uptake and MVD.

FIGURE 37.4. Example of a true-positive 99mTc-3P4-RGD2 scintigraphy based on an optimal cutoff value of two by visual scores. A: A solitary pulmonary nodule (SPN; indicated by arrow) was identified on CT scan. B, C: High uptake of the radiotracer was observed on the SPN. The T/N ratio is 2.30. D: Histopathology staining indicated that the SPN is an adenocarcinoma. E: Immunohistochemistry demonstrates intense ανβ3 expression in tumor vessels. (With kind permission from Springer Science and Business Media: Ma Q, Ji B, Jia B, et al. Differential diagnosis of solitary pulmonary nodules using 99mTc-3P4-RGD2 scintigraphy. Eur J Nucl Med Mol Imaging. 2011;38: 2145–2152.)

Promising RGD-Based Tracers Under Preclinical Development


64Cu-cyclam-RAFT-c(-RGDfK-)4 (molecular weight ∼ 5 kDa) is a copper-64 (64Cu, a promising positron emitter, half-life: 12.7 hours)-labeled tetrameric cyclic RGD peptide with four cyclo(-RGDfK-)monomers grafted onto the upper face of a cyclic decapeptide scaffold regioselectively addressable functionalized template (RAFT) and radiolabeling with 64Cu via the bifunctional chelator, cyclam (1,4,8,11-tetraazacyclotetradecane) conjugated onto the opposite face of the RAFT.77,78 This PET probe was developed by a collaboration of the Molecular Imaging Center of the National Institute of Radiological Sciences in Japan (NIRS) and the Département de Chimie Moléculaire at the University Joseph Fourier in France (UJF). RAFT-c(-RGDfK-)4 was designed and developed by Dumy et al.79 at UJF based on the concept that multiple presentation of ligands in a single construct may significantly improve targeting. The advantages of RAFT-c(-RGDfK-)4 over other multimeric cRGD polymers are that the required chemistry is regio- and chemoselective, and the synthesis and purification of the final product are perfectly controlled at gram scale. In addition, the RAFT architecture allows spatial separation between the two functional groups, RGD ligands, and the radiotracer 64Cu. Moreover, it is interesting because four cRGD motifs are simply and separately grafted onto the RAFT without using space linkers, allowing presentation of four RGD motifs in an area of ±0.7 nm2, a high density of ligands on such a small surface.80 The mechanistic study of RAFT-c(-RGDfK-)4 interaction with αvβ3 integrin clearly revealed the relationships between multimeric presentation, increased affinity, and subsequent integrin-mediated and clathrin-dependent co-internalization.81 In addition to the benefits of RAFT-c(-RGDfK-)4, radiolabeling with 64Cu is also an advantage, because 64Cu is a promising biomedical radioisotope. The positron energy spectrum of 64Cu is comparable to that of 18F, allowing high spatial resolution in PET imaging; its relatively longer half-life permits PET evaluation of slow biokinetics of multimeric ligands, and moreover, 64Cu can be produced on a small biomedical cyclotron. In comparison with other 18F-labeled RGD peptides including galacto-RGD, fluciclatide, and FPPRGD2, the radiolabeling procedure for 64Cu-cyclam-RAFT-c(-RGDfK-)4 is easy, mild, and straightforward: The mixture of peptide and 64Cu only needs to be incubated at 37°C for less than 60 minutes. Specific radioactivity as high as ∼37 MBq/nmol with a labeling efficiency >99% can be achieved in the production of 64Cu-cyclam-RAFT-c(-RGDfK-)4, making purification unnecessary and reducing radioactive exposure.82 Although cyclam is not a new chelating agent, it is well suited for labeling with 64Cu in the case of RAFT-c(-RGDfK-)4.

64Cu-cyclam-RAFT-c(-RGDfK-)4 has high affinity and selectivity for αvβ3 in vitro and αvβ3-specific tumor-targeting efficiency with good PET imaging quality.78 A preclinical study in murine tumor xenografts demonstrated a strong and positive linear correlation between tumor uptake of 64Cu-cyclam-RAFT-c(-RGDfK-)4 and corresponding αvβ3 integrin expression levels quantified by SDS-PAGE/autoradiography.78 A subsequent study in mice bearing human hepatocellular carcinoma HuH-7 xenografts, which expressed αvβ3 at negligible levels on the tumor cells but were αvβ3-positive on the endothelial cells of murine origin, demonstrated that 64Cu-cyclam-RAFT-c(-RGDfK-)4PET enables clear visualization of tumor angiogenesis by targeting the αvβ3 expressed on the vasculature and helps monitor the antiangiogenic effect of a novel multitargeted tyrosine kinase inhibitor (TKI), TSU-68.82 TSU-68 significantly slowed tumor growth and reduced MVD; these findings were consistent with a significant reduction in the tumor 64Cu-cyclam-RAFT-c(-RGDfK-)4 uptake (Fig. 37.5A, B, G, H ). Moreover, a linear correlation was observed between tumor MVD and the corresponding SUV (r = 0.829, p= 0.011 for SUVmean; r = 0.776, p = 0.024 for SUVmax) determined by quantitative PET. Autoradiography and immunostaining showed that the distribution of intratumoral radioactivity and tumor vasculature corresponded (see Fig. 37.5C–F). This proof-of-concept study clearly shows that 64Cu-cyclam-RAFT-c(-RGDfK-)4 is a promising candidate for PET imaging of tumor angiogenesis.

RAFT-c(-RGDfK-)4 is also flexible and can be conjugated with a variety of substances such as fluorescent dye for optical imaging,83 other radioisotopes in addition to 64Cu for nuclear medicine imaging,84,85 or oligonucleotides or peptides for drug delivery.86 Indium-111 (111In, γ-ray emitter, half-life: 2.8 days) or 99mTc-labeled RAFT-c(-RGDfK-)4 have been reported for the purpose of SPECT imaging of angiogenesis.84,87

FIGURE 37.5. Transverse and coronal PET images of s.c. HuH-7 tumor-bearing mice at 3 hours after i.v. injection of 64Cu-cyclam-RAFT-c(-RGDfK-)4 (11.1 MBq) on the day after daily intraperitoneal injections of (A) vehicle alone (50 μL of DMSO) or (B) TSU-68 (75 mg/kg/day in 50 μL of DMSO) for 14 days (n = four mice for each group). The arrows indicate the tumor location. Representative autoradiographic examination (C, E) and CD31 immunofluorescence staining (D, F) with the same whole-tumor sections from (C, D) vehicle-treated and (E, F) TSU-68–treated tumors excised after PET imaging. G: Microvessel density (MVD), HA′ SUVmean, and HB′SUVmax were compared in TSU-68–treated and vehicle-treated tumors. All data presented in (AH) are from the same set of experimental groups. SUV, standardized uptake value. (With kind permission from Springer Science and Business Media: Jin ZH, Furukawa T, Claron M, et al. Positron emission tomography imaging of tumor angiogenesis and monitoring of antiangiogenic efficacy using the novel tetrameric peptide probe 64Cu-cyclam-RAFT-c(-RGDfK-)4Angiogenesis. 2012;15(4):569–580.)

Finally, because 64Cu-cyclam-RAFT-c(-RGDfK-)4 is predominantly eliminated through the kidneys, common for radiolabeled peptides or antibodies, relatively high renal radioactivity accumulation because of proximal tubular cell reabsorption of the tracer and retention of radiolabeled metabolite, is a limitation for 64Cu-cyclam-RAFT-c(-RGDfK-)4 when imaging lesions located in the abdominal area. However, this problem can be solved by reducing renal uptake of RAFT-c(-RGDfK-)4 labeled with fluorescent dye or 111In by co- or preinjection of Gelofusine, the succinylated gelatin plasma expander.88

αvβ3 Integrin–Targeted Radiolabeled Nanoparticles

αvβ3 Integrin is highly expressed on activated endothelial cells during angiogenesis, but also on some types of tumor cells. Therefore, αvβ3 integrin expression levels in a tumor may predominantly be associated with its vasculature or with the mixture of vasculature and αvβ3-positive tumor cells. For the latter case, molecular imaging of tumor angiogenesis via detection of αvβ3 expression does not reflect the extent of angiogenesis. αvβ3 Integrin–targeted radiolabeled RGD-conjugated nanoparticles (100 to 250 nm in diameter), which can be confined to the circulatory system when vascular integrity is not severely disrupted, may allow visualization and quantitation of tumor angiogenesis by targeting only the vascular αvβ3 integrin.89,90 αvβ3-Targeted 111In perfluorocarbon nanoparticle,89 64Cu-labeled DOTA-QD (quantum dot)-RGD,91 125I-labeled cyclic RGD-PEGylated gold nanoparticle,92 and 64Cu-labeled RGD-PEGylated single-walled carbon nanotubes (SWNTs)93 have been introduced by different research groups, with the first two agents mainly localized in the tumor vasculature. However, nonspecific tumor uptake because of the enhanced permeability and retention (EPR) effect should also be considered.94 Other issues such as chemical structure definition, quality control, toxicity, stability, pharmacokinetics, and biodistribution have not yet been characterized.


The VEGF family includes VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, snake venom VEGF, and placental growth factor, all of which share a common VEGF homology and are encoded by individual genes.95 These members bind differentially to three subtypes of transmembrane tyrosine kinase VEGF signal-transducing receptors (VEGFRs): VEGFR-1 (also known as Flt-1 [Fms-like tyrosine kinase-1]), VEGFR-2 (also known as Flk-1 [fetal liver kinase-1] and, in humans, kinase insert domain-containing receptor [KDR]), and VEGFR-3.95 VEGF-A (VEGF) is a homodimeric, disulfide-bound glycoprotein that exists in at least four main isoforms of 121, 165, 189, and 206 amino acids generated by alternative mRNA splicing.9597 VEGF121 is acidic, nonheparin and nonheparan sulfate proteoglycan (HSPG) binding, and is freely diffusible. VEGF189 and VEGF206 are highly basic and bind to HSPGs with high affinity, and both are almost completely sequestered in the ECM. VEGF165 is the predominant isoform in vivo. It is also secreted and diffusible, but carries a heparin-binding domain that can result in cell association, leading to a significant portion remaining bound to the cell surface and ECM.

VEGF, synthesized and secreted by many cell types in several embryonic and adult tissues (e.g., the lung, kidney, heart, brain, and spleen) and by a variety of tumor cells, plays key roles in the regulation of developmental vasculogenesis, angiogenesis, and differentiation of progenitor endothelial cells, functioning mainly by interacting with VEGFR-1 and VEGFR-2.97,98 VEGFR-2 is expressed in vascular and lymphatic endothelial cells but several types of nonendothelial cells such as hematopoietic stem cells, megakaryocytes, and some tumor cells also express VEGFR-2.95,98,99 Binding of VEGF to VEGFR-2 on endothelial cells starts a tyrosine kinase signaling cascade that stimulates production of factors that differentially stimulate vascular permeability and activate or regulate endothelial cell proliferation, survival, migration, and differentiation during angiogenesis. Upregulated expression of VEGF/VEGFR-2 by tumor cells/endothelial cells frequently occurs in many human solid tumor types, and is associated with tumor progression, metastasis, and angiogenesis, and is implicated in poor prognosis, thus making them rational targets for cancer detection and therapy.

Radionuclide Tracers for Angiogenesis Imaging Using Radiolabeled Anti-VEGF Antibodies

Radiolabeled VG76e

VG76e is an IgG1-type mouse monoclonal antibody that recognizes the human VEGF121, VEGF165, and VEGF189 isoforms.100 Labeling of VG76e with iodine-124 (124I, positron emitter, half-life: 4.2 days), 125I, samarium-153 (153Sm), or 99mTc was reported for detection of VEGF expression in human tumor xenograft models.100,101 VG76e-based tracers specifically bound VEGF, with maximum tumor uptake at 24 and 48 hours after injection.100 However, high levels of radioactivity accumulated in other organs, especially the blood and plasma. 124I-VG76e PET imaging in HT1080-26.6 human fibrosarcoma-bearing mice clearly showed localization to the tumor 24 hours after tracer injection.100 Additional areas of high radioactivity were visible in the thoracic cavity, abdominal cavity, and bladder.


HuMV833 is a humanized IgG4κ-type mouse monoclonal antibody that recognizes human VEGF121 and VEGF165.102 In a phase I clinical trial, PET imaging with 124I-labeled HuMV833 was performed in 20 patients with progressive solid tumors that were not amenable to standard therapy.103 The data did not clearly indicate whether HuMV833 showed specific targeting of the tumor tissue. Instead, the authors observed a strikingly wide variation of 124I-HuMV833 uptake and/or clearance in tumor tissues between and within patients and individual tumors whereas most normal tissues cleared the antibody at approximately equal rates.

Radiolabeled Bevacizumab and the Fab Fragment, Ranibizumab

Subsequent studies of VEGF imaging have focused on the use of bevacizumab, a humanized IgG1-type variant of anti–VEGF-A monoclonal antibody A.4.6.1 that binds all VEGF isoforms.104 Bevacizumab was approved for first-line treatment of metastatic colorectal cancer in combination with 5-fluorouracil–based chemotherapy, and for initial systemic treatment of non–small-cell lung cancer in combination with carboplatin and paclitaxel.104,105Bevacizumab was labeled with 125I, 111In, or zirconium-89 (89Zr, positron emitter, half-life: 3.27 days) and demonstrated VEGF-specific tumor uptake in human tumor xenograft models.105,106 Scintigraphic imaging using 111In-bevacizumab and PET imaging using 89Zr-bevacizumab provided clear tumor visualization beginning on day 3 after tracer injection, and image quality improved by day 7. 111In-Bevacizumab and 89Zr-bevacizumab showed similar biodistribution in major normal tissues, with high radioactivity accumulation in the blood, lung, and heart at 24 hours p.i. that decreased over time, and high radioactivity accumulation in the spleen and liver at later time points. In addition, radioactivity accumulation in the bone was much higher for 89Zr-bevacizumab than 111In-Bevacizumab at day 7 p.i. In comparison with 89Zr-labeled human IgG and antihuman IgG staining in a tumor slice from mice receiving 89Zr-bevacizumab, the study found that radiolabeled bevacizumab binds primarily to VEGF in tumor blood vessels, but binding to VEGF in the ECM around tumor cells could not be demonstrated.106 Another study examined uptake of 111In-bevacizumab in mouse tumors of human melanoma Mel57 (originally VEGF-negative) transfected with various VEGF isoforms (VEGF121, VEGF165, and VEGF189).107 Tumors expressing VEGF121 did not show specific uptake, whereas tumors expressing VEGF165 and VEGF189 were clearly visualized by γ-camera imaging. This suggests that the accumulation of radiolabeled bevacizumab in the tumor is because of the interaction with VEGF-A isoforms VEGF165 and VEGF189 associated with the tumor cell surface and/or ECM.

One clinical study investigated the correlation between tumor accumulation of 111In-bevacizumab and VEGF expression in 12 patients with colorectal cancer liver metastases.108 Enhanced tracer uptake in the liver metastases was observed in nine of the patients. Antibody accumulation in these lesions varied considerably, and no clear-cut correlation was achieved between the level of tracer accumulation and the level of VEGF expression in the tissue as determined by in situ hybridization and ELISA. But in another clinical setting, 111In-bevacizumab SPECT was performed in nine patients with stage III/IV melanoma, and tracer uptake was compared to VEGF levels in resected melanoma lesions.109 Twelve nodal lesions were detected by both FDG-PET and CT, all of which could be visualized by 111In-bevacizumab SPECT; day 4 after tracer injection was determined to be the optimal timing for visualization and quantification of 111In-bevacizumab uptake in the tumor (Fig. 37.6). At baseline, 111In-bevacizumab tumor uptake varied three-fold and 1.6 ± 0.1-fold between and within patients, respectively. After a therapeutic dose of bevacizumab, there was a 21% ± 4% reduction in tracer uptake. 111In-bevacizumab tumor uptake measured after treatment correlated with VEGF expression in the resected tumor lesions.

FIGURE 37.6. A: Transverse and coronal VEGF-SPECT images at days 0, 2, 4, and 7 p.i. of the tracer. Over time, 111In-bevacizumab accumulates in the tumor with optimal tumor-to-background ratio 4 days p.i. B: Transverse and coronal CT images and VEGF-SPECT/CT fusion 4 days p.i. (Reprinted from Nagengast WB, Hooge MN, van Straten EM, et al. VEGF-SPECT with 111In-bevacizumab in stage III/IV melanoma patients. Eur J Cancer.2011;47:1595–1602, with permission from Elsevier.)

89Zr-bevacizumab PET was useful for monitoring tumor response to VEGF-dependent antiangiogenic treatment with HSP90 inhibitor NVP-AUY922 in mice bearing A2780 human ovarian cancer xenografts.110 Compared to pretreatment values, 2 weeks of NVP-AUY922 treatment decreased tumor uptake of 89Zr-bevacizumab by 44.4% (p = 0.0003), coinciding with a significant reduction in tumor VEGF levels and MVD. The long circulating serum half-life (21 days) of bevacizumab is a limitation for dynamic imaging studies because maximum signal is observed as late as 4 to 7 days p.i. Ranibizumab (molecular weight: 48 kDa), an mAb fragment (Fab) derivative of bevacizumab (molecular weight: 148 kDa) was developed111 and labeled with 89Zr (89Zr-ranibizumab) to improve the pharmacokinetics of 89Zr-bevacizumab.112 PET of mice bearing SKOV-3 (a human adenocarcinoma cell line) tumors performed at 1, 3, 6, and 24 hours p.i. of 89Zr-ranibizumab showed clear tumor visualization within 3 hours; the highest tumor tracer uptake and greatest tumor contrast were observed at 24 hours. A biodistribution study showed rapid blood clearance of 89Zr-ranibizumab from 8.44 ± 2.19%ID/g at 1 hour p.i. to 0.38 ± 0.38%ID/g at 24 hours p.i., at which time the blood accumulation of 89Zr-bevacizumab remained high at 9.54 ± 5.12%ID/g, demonstrating the improved pharmacokinetics of the Fab derivative of bevacizumab.112 The VEGF-binding specificity of 89Zr-ranibizumab was supported in comparison with the low tumor uptake of 89Zr-Fab-IgG control and by a competitive blocking assay using increasing doses of unlabeled ranibizumab. The potential of 89Zr-ranibizumab as a tumor-imaging agent to determine the efficacy of sunitinib was also shown in mice bearing SKOV-3, A2780, or Colo205 (a human colorectal adenocarcinoma cell line) tumors.

Radionuclide Tracers for Angiogenesis Imaging by Targeting VEGFR-2

VEGF-Based Tracers

VEGF165 and VEGF121 are secreted by different cell types and represent the predominant isoforms of VEGF, whereas the other isoforms are primarily expressed in membrane-bound form.113 Compared to VEGF121, VEGF165 is less soluble and contains an extra domain for heparin binding, resulting in increased nonspecific binding and low tumor-to-background ratio. VEGFR-2 is overexpressed in vascular endothelial cells of various human tumors and tumor cells.99 Inhibition of VEGFR-2 function inhibits tumor growth and metastasis. Therefore, determination of VEGFR activity is of central interest for research in the field of tumor angiogenesis and for diagnosis and treatment of tumors. VEGF acts directly on endothelial cells by binding to VEGFR-1 and VEGFR-2. Because VEGF is the natural ligand for VEGFR-2, VEGF or derivative-based tracers may have high receptor-binding affinity and be less immunogenic than exogenous molecules. However, because it is also the ligand for VEGFR-1, which is highly expressed in the kidneys, nonspecific accumulation is a major concern.114

Radioiodinated VEGF121 and VEGF165

Human recombinant VEGF121 and VEGF165 were labeled with 123I by electrophilic radioiodination using the chloramine T method, and the in vitro binding properties were analyzed using human umbilical vein endothelial cells, several human tumor cell lines, a variety of primary human tumors, and adjacent nonneoplastic tissues, as well as normal human peripheral blood cells.115 Significantly greater specific binding was observed for 123I-VEGF165 and 123I-VEGF121 in a variety of human tumor cells/tissues compared with corresponding normal tissues or peripheral blood cells. Compared to 123I-VEGF121123I-VEGF165 bound to a higher number of different tumor cell types with higher capacity. 123I-VEGF165 scintigraphy and SPECT were evaluated in 18 patients with gastrointestinal tumors.116 Intravenous injection of 123I-VEGF165 (184 ± 18 MBq) did not cause side effects. Among the 40 lesions detected by CT/MRI, 23 (58%) could be visualized 0.5 to 3 hours after tracer injection. In patients with pancreatic adenocarcinomas, primary tumors were visualized in seven of nine. Malignant liver lesions can be visualized by 123I-VEGF165SPECT; however, benign liver hyperplasia appeared as a cold spot. In a follow-up study, nine patients with biopsy-proven pancreatic carcinomas received 123I-VEGF165 scintigraphy; seven of nine primary lesions could be clearly visualized within 30 minutes after injection and remained detectable at 3 hours p.i.117 No substantial uptake by normal gastrointestinal tissue was noted. 123I-VEGF165 showed rapid clearance from the circulation, as radioactivity in the blood rapidly decreased to less than 4% ± 2% of the injected activity within 30 minutes, and approximately 86% ± 6% of the injected activity was recovered in the urine by 24 hours p.i. Main 123I-VEGF165-retaining tissues in humans include the thyroid, spleen, lungs, liver, and kidneys. A very recent clinical report suggested 123I-VEGF165 receptor scintigraphy may be useful for visualization of highly malignant osteosarcoma and/or metastasis and the angiogenic activity of the tumor.118 Although promising results have been obtained for 123I-VEGF165 in the clinical setting, another study comparing the biodistribution of 125I-VEGF121 and 123I-VEGF165 in an LS180 human colon cancer xenograft model in mice suggested that VEGF121 would be the more appropriate targeting molecule of VEGFRs in terms of low background activity.119 However, prolonged elevated radioactivity in the circulation is a major limitation of 125I-VEGF121, whereas prominent 125I-VEGF165 radioactivity accumulation was observed in the stomach because of deiodination. The similarities and differences between VEGF121 and VEGF165 in terms of their in vivo behavior may be attributable to their distinct binding affinity to different VEGFRs with different tissue distributions.


Human recombinant VEGF121 was randomly conjugated with DOTA (4.3 ± 0.2 DOTA molecules per VEGF121) and labeled with 64Cu.114 DOTA-VEGF121 has VEGFR-2-binding affinity comparable to that of VEGF12164Cu-labeled DOTA-VEGF121 was stable in mouse serum after 24-hours incubation at 37°C. PET of mice bearing U87MG tumors of different sizes revealed rapid (within 2 hours), specific, and prominent uptake of 64Cu-DOTA-VEGF121(∼15 %ID/g) in small U87MG tumors (tumor volume: 64.9 ± 24.6 mm3; high VEGFR-2 expression and high MVD) but significantly lower and sporadic uptake (∼3 %ID/g) in large U87MG tumors (tumor volume: 1,164.3 ± 179.6 mm3; low VEGFR-2 expression and low MVD). This study demonstrates that even in the same tumor model, VEGFR-2 expression differs dramatically between stages of tumor growth. In another study of similar design, 64Cu-DOTA-VEGF121 PET imaging was performed in mice bearing a relatively larger number (n = 15) of various-sized U87MG tumors, followed by western blotting and immunofluorescence staining of tumor tissues.120 Tumor uptake of 64Cu-DOTA-VEGF121 peaked when the tumor size was about 100 to 250 mm3. Both smaller and larger tumors had lower tracer uptake, indicating a narrow range of tumor size with high VEGFR-2 expression. All tumors shared low VEGFR-1 expression. Most importantly, the tumor uptake value obtained from PET imaging at 4 hours p.i. showed a good linear correlation with the relative tumor tissue VEGFR-2 expression as measured by western blotting. Histology of the frozen tumor tissue verified the imaging results.

The highest accumulation of radioactivity from 64Cu-DOTA-VEGF121 was observed in the kidneys, mainly because of VEGFR-1 binding, as this receptor is highly expressed in the kidneys.64Cu-DOTA-VEGFDEE was developed as a solution for this problem.121 Alanine-scanning mutagenesis revealed that Arg(82), Lys(84), and His(86) are critical for binding of VEGF to VEGFR-2, whereas Asp(63), Glu(64), and Glu(67) are required for binding to VEGFR-1.122 Based on this finding, the D63AE64AE67A mutant of VEGF121 (VEGFDEE), in which Asp(63), Glu(64), and Glu(67) of VEGF121 were mutated to Ala, conjugated with DOTA, and labeled with 64Cu (64Cu-DOTA-VEGFDEE) for PET imaging.121 In comparison with 64Cu-DOTA-VEGF121, 64Cu-DOTA-VEGFDEE had comparable tumor-targeting efficacy but reduced renal accumulation because of its 20-fold lower affinity for VEGFR-1.


99mTc-HYNIC-VEGF was developed based on the construction and expression of VEGF121 fused with a cysteine-containing peptide tag (C-tag).123 C-tagged-VEGF allowed site-specific conjugation of the biofunctional chelator hydrazine nicotinamide (HYNIC)-maleimide for 99mTc labeling. The site-specific strategy may overcome the problem of random labeling usually associated with reduced receptor-binding activity, high liver uptake, or complicated probe design. 99mTc-HYNIC-VEGF SPECT imaging of 4T1 murine mammary carcinoma-bearing mice showed that the tumor can easily be visualized 1 hour after tracer injection, and that 99mTc-HYNIC-VEGF preferentially accumulates at the tumor rim, where the most extensive angiogenesis takes place.123 SPECT imaging with 99mTc-HYNIC-VEGF can also readily detect the effects of cyclophosphamide treatment on 4T1 tumors. One week of cyclophosphamide therapy resulted in reduced tracer uptake, corresponding with reduced VEGFR-2 expression as determined by immunohistochemistry.

scVEGF-Based Radiotracers

scVEGF is an engineered 28-kDa single-chain VEGF composed of two repeated 3- to 112-amino acid fragments of VEGF121 with a 15-amino acid N-terminal Cys tag containing a unique cysteine residue for site-specific attachment of a variety of agents such as 64Cu,124 68Ga,125 18F,126 etc. for nuclear imaging. In addition, incorporation of PEG as a linker between the protein and chelator prolonged blood clearance, leading to higher tumor accumulation and reduced renal uptake. The advantages of radiolabeled scVEGF-based tracers to image tumor VEGFR-2 expression in clinical settings should be determined with respect to in vivo stability, targeting efficiency and specificity, pharmacokinetics, the correlation between tracer uptake and receptor expression levels, and the ease of radiolabeling and cost.

VEGFR-2-Targeting Radiotracers

VEGFR-2 is a transmembrane tyrosine kinase VEGF signal-transducing receptor. Many small molecules have been developed to block the intracellular tyrosine kinase at the adenosine triphosphate–binding site99; some of which, such as sorafenib and sunitinib, have been approved by the FDA as antiangiogenesis drugs for cancer therapy. Preparation and evaluation of radiolabeled TKIs or analogs such as 11C-PAQ,127 11C-labeled Vandetanib or chloro-Vandetanib,128 5-125I-iodo-sunitinib,129 and 18F-SKI-249380130 have been reported for VEGFR-2 imaging. In mice bearing B16F10 melanoma xenografts, enhanced radioactivity accumulation of 11C-PAQ was observed in the VEGFR-2–positive area of the tumor; however, strong accumulation was also found in the lungs, kidneys, and liver.127 Although radiolabeling of TKI is emerging as a new strategy for VEGFR-2 imaging, much more evidence is necessary to confirm the in vivo targeting efficiency and specificity for tumor VEGFR-2 expression.

Radiolabeling of anti–VEGFR-2 antibody is also an approach for VEGFR-2 imaging. DC101, a rat antimouse VEGFR-2 monoclonal antibody, inhibits angiogenesis and suppresses tumor growth and metastasis.131133 DC101 was conjugated with chitosan, a linear polysaccharide composed of D-glucosamine and N-acetylglucosamine subunits with numerous D-glucosamine groups for ligand conjugation, and labeled with 99mTc.134 γ-Camera imaging of mice bearing B16F10 and HeLa tumor models demonstrates the potential of 99mTc-chitosan-DC101 for VEGFR-2 imaging, with T/M ratios >3 a couple of hours after tracer injection135 Human/mouse cross-reactive anti–VEGFR-2 antibodies136 available for radiolabeling are awaiting further evaluation with VEGFR-2 imaging in clinical settings.


Fibronectin is a large glycoprotein widely distributed in the ECM, and exists in several isoforms (e.g., III CS, ED-A, ED-B).137,138 The ED-B (extra domain B), a sequence of 91 amino acids identical in mice, rats, and humans, is a marker of angiogenesis and tissue remodeling because it plays an important role in endothelial cell proliferation and vascular morphogenesis, and is abundantly expressed around the vasculature of tumors in the exponential growth phase, but not in the slow-growth phase or other tissues undergoing angiogenesis, but is undetectable in mature normal tissues.139142 High levels of ED-B expression have been detected in a variety of human cancers and metastases such as breast and lung cancers, high-grade astrocytoma (but not low-grade), glioblastoma, and liver metastases.140,141,143

First identified by Pini et al.,144 the human recombinant single-chain antibody fragment L19, scFv(L19), selectively binds ED-B with high affinity. Microautoradiography of an F9 tumor (a fast-growing murine teratocarcinoma expressing high levels of ED-B) dissected from a mouse after injection with 125I-labeled scFv(L19) clearly showed radioactivity accumulation in the tumor blood vessels but not in the vessels of the normal surrounding tissue.145 No radioactivity accumulation was observed in the vessels of other organs (liver, lung, muscle, spleen, kidney, brain, and skin). Afterward, several other scFv(L19) derivatives were constructed such as a dimeric scFv [(scFv)2], a human bivalent “small immunoprotein” (SIP, approximately 80 kDa), and a complete human IgG1,146 based on which a variety of radiotracers have been designed, synthesized, and characterized for their tumor-targeting efficiency and pharmacokinetics.

An initial clinical study published in 2003 reported scintigraphic imaging of ED-B expression using iodine-123 (123I, γ-ray emitter, half-life: 13.2 hours)-labeled L19(scFv)2 (185 to 518 MBq) in 20 patients with brain, lung, and colorectal cancer.143 No side effects were observed. Sixteen of the patients showed different levels of tracer accumulation in the primary tumor or metastases 6 hours after tracer injection. For example, a strong and selective radioactivity accumulation was observed in the liver lesions of a patient with liver metastases of colorectal cancer, and in the tumor mass of a patient with recurrent glioblastoma (Fig. 37.7A, B). In a small-cell lung carcinoma patient with miliary lesions in both lungs, diffuse activity accumulation was revealed in both lungs. No selective accumulation was observed in the brain of a patient with a benign brain tumor (pilocytic astrocytoma) (see Fig. 37.7C, D). The differences in tracer uptake correlated with the different levels of ED-B expression demonstrated by immunohistochemical staining. This clinical work shows the ability of 123I-labeled L19(scFv)2 to noninvasively detect aggressive primary tumors and metastases in patients by targeting ED-B expression around the tumor vasculature.

Radiotracers for ED-B Imaging in Preclinical Studies


Because of the thiophilic nature of Tc(V), a free sulfhydryl group must be introduced into the protein sequence for stable radiometal binding. In Berndorff et al.’s147 work, the amino acid sequence (Gly)3-Cys-Ala was genetically inserted at the C-terminus of scFv(L19), resulting in a recombinant protein named AP39, which is suitable for direct labeling with 99mTc. In F9 tumor-bearing mice, 99mTc-L19-AP39 showed high tumor uptake with a maximum of 8.7%ID/g after 5 hours, decreasing to 2.8%ID/g 24 hours after injection, and fast blood clearance, leading to a T/B ratio of 6.4, 3 hours after injection, increasing to 17.2 after 24 hours, with very low radioactivity accumulation in nontarget organs with the exception of the kidneys because of the major renal elimination pathway of this radiotracer. Scintigraphic images also showed that the tumor could be clearly visualized at 3, 5, and 24 hours after tracer injection. Their work shows the feasibility of SPECT imaging of tumor angiogenesis with 99mTc-L19-AP39 radiotracer.


L19-SIP was labeled with bromine-76 (76Br, positron emitter, half-life: 16.2 hours) via enzymatic radiobromination using bromoperoxidase/H2O2.148 The labeling yield was >55% under mild reaction conditions (0°C for 80 minutes). Biodistribution and small-animal imaging studies (PET and CT) in mice with F9 tumors showed high and persistent tumor uptake of 18.1 ± 7.6, 9.3 ± 3.5, and 14.3 ± 1.6 %ID/g at 5, 24, and 48 hours p.i., respectively, and clear tumor visualization. Other ED-B–expressing organs, especially the uterus, also showed high tracer uptake of 13.5 ± 6.3, 9.3 ± 3.5, and 6 ± 1 %ID/g at 5, 24, and 48 hours p.i. However, slow renal clearance and persistent radioactivity predominately in the blood and stomach was also observed, partially because of 76Br-L19-SIP debromination in vivo. 76Br-L19-SIP PET is a valuable tool for tumor angiogenesis imaging by targeting the angiogenesis-associated expression of ED-B fibronectin. Further efforts to reduce debromination of 76Br-L19-SIP are necessary.


L19-SIP was labeled with 124I produced in a GMP compliant facility or with commercially available 131I by using a modified version of the IODO-GEN method, and overall labeling efficiency, radiochemical purity, and the immunoreactive fraction were ∼80%, 99.9%, and >90% for both 124I-L19-SIP and 131I-L19-SIP.149 In human SCCHN FaDu xenograft-bearing mice, biodistribution data showed that the tumor uptake of 124I-L19-SIP was 7.3 ± 2.1, 10.8 ± 1.5, 7.8 ± 1.4, 5.3 ± 0.6, and 3.1 ± 0.4%ID/g at 3, 6, 24, 48, and 72 hours p.i., respectively, resulting in increased T/B ratios ranging from 6 at 24 hours to 45.9 at 72 hours p.i., and increased T/M ratios from 22.6 at 24 hours to 119 at 72 hours p.i. At 24 hours p.i., tumor-to-major normal organ ratios were generally >4 except for the bladder; importantly, the tumor-to-liver and tumor-to-kidney ratios were 18.5 and 6.2, revealing the highly sensitive and selective receptor-targeting efficiency and optimum pharmacokinetics of 124I-L19-SIP. Fully concordant labeling and biodistribution results were obtained with 124I- and 131I-L19-SIP. PET imaging with 124I-L19-SIP (3.7 MBq) revealed clear delineation of tumors, even those ∼50 mm3 and no adverse uptake in other organs (Fig. 37.8). This study revealed the promise of 124I-L19-SIP PET for tumor angiogenesis imaging and for predicting 131I-L19-SIP biodistribution as a guide to 131I-L19-SIP radioimmunotherapy. Further studies should be conducted to characterize the correlation between 124I-L19-SIP uptake and ED-B expression levels in tumors.

FIGURE 37.7. Localization of 123I-L19(scFv)2 in brain tumors. SPECT γ-camera transaxial section (A) and MRI (B), from a patient with a recurrent glioblastoma lesion growing around the postoperatory cavity. SPECT transaxial scans (C) and CT (D) from a patient with a low-grade pilocytic astrocytoma, which could be removed only subtotally by surgery. Residual tumor tissue adjacent to the brainstem is indicated by the arrow (D). CT had to be performed for this patient, because a metallic implant prevented MRI. (Reprinted from Santimaria M, Moscatelli G, Viale GL, et al. Immunoscintigraphic detection of the ED-B domain of fibronectin, a marker of angiogenesis, in patients with cancer, with permission from AACR. Clin Cancer Res. 2003;9:571–579.)


CD105 (also known as endoglin) is a homodimeric transmembrane glycoprotein expressed on activated vascular endothelial cells of newly formed blood vessels.150,151 It is an accessory protein of the transforming growth factor-β receptor system. The patterns of CD105 and CD31 expression were evaluated and compared in primary colon adenocarcinomas and normal colonic mucosa.150 Whereas anti-CD31 antibodies equally stained blood vessels in normal and malignant colon, CD105 expression was observed primarily in malignant lesions, with little to no expression in the vessels of nonmalignant mucosa. Several research groups have reported that, compared to some traditional vascular markers such as CD31 and von Willebrand factor, CD105 expression serves as a better prognostic marker of patient outcomes.150 CD105 expression determined by immunohistochemical staining in gastrointestinal, breast, lung, brain, ovarian, endometrial, prostate, and head and neck malignancies was consistently associated with lower patient survival rates, and in gastrointestinal, breast, prostate, and head and neck malignancies, CD105 expression was associated with the presence of distant metastases.150 Overexpression of CD105 was also reported to be a useful predictor of recurrence of resected gastric cancer and may be specifically associated with the development of locoregional recurrence and hematogenous metastasis.152 CD105 expression thus seems to have prognostic value in a variety of solid cancers, and has become a powerful therapeutic target of tumor angiogenesis. Moreover, CD105 expression is mainly restricted to vascular endothelial cells; in solid malignancies, CD105 is almost exclusively expressed on endothelial cells of both peri- and intratumoral blood vessels and on tumor stromal components.153 Hence, CD105 can be considered one of the most suitable markers for evaluating tumor angiogenesis.

Radiotracers for Targeting CD105

Monoclonal anti-CD105 antibodies MAEND3, E9, and MJ7/18 were initially radiolabeled with 125I,154 99mTc,155 and 111In,156 respectively, and tested for tumor angiogenesis imaging in experimental tumor models with encouraging results. TRC105 (also known as c-SN6j) is a human/murine chimeric IgG1 monoclonal antibody that binds human and murine CD105 (with lower affinity for the latter) and inhibits angiogenesis and tumor growth.157,158 Compared to other anti-CD105 antibodies, TRC105 has very high affinity to human CD105. Very recently, a phase I first in human study in 50 patients with advanced refractory solid tumors was reported, and subsequent phase II clinical studies are ongoing to evaluate TRC105 alone and in combination with other agents in a wide variety of cancer types.159 Developing and clinical translation of radiolabeled TRC105-based tracers should be helpful for patient stratification, monitoring treatment response, and drug evaluation for dose and dosing regimen in TRC105-based clinical trials. Meanwhile, efforts to determine the value of CD105-targeted tumor angiogenesis imaging are also necessary. Currently, CD105-targeted radiotracers are mostly focused on the use of TRC105 which is under development by Weibao Cai and colleagues at the University of Wisconsin-Madison. At this time, there have not been clinical trials for CD105 SPECT or PET imaging.

FIGURE 37.8. Serial PET images of FaDu xenograft-bearing nude mouse injected with 124I-L19-SIP (3.7 MBq, 25 μg). At 1 (A, D), 2 (B, E) and 3 (C, F) weeks after tumor implantation 124I-L19-SIP was administered, and 48 hours later coronal images were acquired. Image planes have been chosen where the left tumor (upper, AC) or right tumor (lower, DF) is optimally visible. The thyroid (arrow) is visible because this organ was not blocked by potassium iodide in this experiment. (With kind permission from Springer Science and Business Media: Tijink BM, Perk LR, Budde M, et al. 124I-L19-SIP for immuno-PET imaging of tumour vasculature and guidance of 131I-L19-SIP radioimmunotherapy. Eur J Nucl Med Mol Imaging. 2009;36:1235–1244.)

TRC105-Based Radiotracers in Preclinical Studies

Hong et al.160 reported the first successful PET imaging study of CD105 expression using TRC105 conjugated to the biofunctional chelator DOTA and labeled with 64Cu. 64Cu labeling (reaction temperature: 40°C; reaction time: 30 minutes) was achieved with high yield and specific activity. PET imaging clearly visualized CD105-positive 4T1 tumors at 24 and 48 hours after 64Cu-DOTA-TRC105 injection, and tumor activity uptake was 8 ± 0.5, 10.4 ± 2.8, and 9.7 ± 1.8%ID/g at 4, 24, and 48 hours p.i., higher than most organs at later time points, which provided good tumor contrast. Tumor tracer uptake was CD105-specific, which was validated by blocking experiments, control studies with an isotype-matched 64Cu-DOTA-cetuximab that binds to human epidermal growth factor receptor, and in vitro/ex vivo immunostaining studies. Predominant radioactivity accumulation in normal organs was observed at 24 hours and in the liver and spleen, both of which were CD105-negative, suggesting that tracer uptake in the liver and spleen was largely unrelated to CD105 binding and more likely related to nonspecific capture by the reticuloendothelial system, hepatic clearance, and possible transchelation of 64Cu. Zhang et al.161 from the same group reported the development of 64Cu-labeled TRC105 using a different chelator NOTA which was performed and evaluated in the same experimental settings including the labeling conditions used for 64Cu-DOTA-TRC105. Tumor-targeting efficacy did not significantly differ between the two bifunctional chelators. The major differences between 64Cu-DOTA-TRC105 and 64Cu-NOTA-TRC105 were observed in their accumulation levels in some normal organs, with the latter showing particularly higher activity in the blood but lower activity in the liver and spleen. Detailed comparison of the agents suggests 64Cu-NOTA-TRC105 may have higher stability, longer circulation half-life, and better tumor contrast at 24 or 48 hours. However, the authors also stated that DOTA is a universal chelator which can complex with a wide variety of imaging and therapeutic radioisotopes; the same DOTA-TRC105 conjugate can therefore be employed for imaging and therapeutic applications, with the use of appropriate isotopes, without altering its pharmacokinetics and tumor-targeting efficacy. The choice of 64Cu-DOTA-TRC105 or 64Cu-NOTA-TRC105 may depend on the application, imaging alone or imaging plus radioimmunotherapy (e.g., with yttrium-90 (90Y) and/or lutetium-177 [177Lu]). For imaging alone, the localization of lesions is also a factor in choosing a suitable radiotracer.

FIGURE 37.9. Small-animal PET imaging of 4T1 tumor-bearing mice. A: Serial coronal PET images at 5, 24, 48, 72, and 96 hours after injection of 89Zr-Df-TRC105, 2 mg of TRC105 before 89Zr-Df-TRC105 (i.e., blocking), or 89Zr-Df-cetuximab. Tumors are indicated by arrowheads. B: Representative PET/CT images of 89Zr-Df-TRC105 in 4T1 tumor-bearing mice at 48 hours p.i. (With kind permission from Springer Science and Business Media: Hong H, Severin GW, Yang Y, et al. Positron emission tomography imaging of CD105 expression with 89Zr-Df-TRC105. Eur J Nucl Med Mol Imaging.2012;39:138–148.)

Cai’s group developed another TRC105-based PET probe by conjugating the biofunctional chelator p-isothiocyanatobenzyl-desferrioxamine (Df-Bz-NCS) to TRC105 for 89Zr labeling.162 The longer decay half-life of 89Zr (3.3 days) makes long-term tracing of TRC105 possible. Also evaluated in the same experimental settings as those used for 64Cu-DOTA-TRC105 and 64Cu-NOTA-TRC105,89Zr-Df-TRC105 showed superb stability, excellent tumor contrast at 24 hours and later time points until 96 hours p.i., and much higher tracer uptake in the tumor than in all major organs including the liver and kidney (Fig. 37.9). The CD105 specificity of 89Zr-Df-TRC105 was confirmed by competitive blocking with TRC105 2 hours before the tracer injection, which significantly reduced tracer uptake in the tumor at all time points examined, and further supported in comparison with the isotype-matched control, 89Zr-Df-cetuximab (see Fig. 37.9). Forward quantitative correlation and colocalization study of tracer uptake and CD105 expression (levels and pattern) in tumors is anticipated to explore the clinical potential of CD105-targeted angiogenesis imaging by TRC105-based radiotracers.


Endostatin was discovered in 1997 by O’Reilly et al.163 in Judah Folkman’s laboratory as an endogenous inhibitor of tumor angiogenesis and growth. It was isolated from a murine hemangioendothelioma and identified as a naturally occurring 20-kDa C-terminal proteolytic fragment of type XVIII collagen, an important proteoglycan in epithelial and endothelial basement membranes. Endostatin may bind and interact with several membrane proteins including α5β1, αvβ3, and αvβ5 integrins,5,163,164 heparin/heparan sulfate,164,165 and VEGFR-2,166 which are possible endostatin receptors. The mechanisms of endostatin in tumor angiogenesis are complicated and not fully understood167,168but appear to mediate many aspects of the VEGF/VEGFR-2 pathway,166 cell cycle pathway,169 and proapoptotic pathway.170 Endostatin may significantly affect approximately 12% of genes used by human endothelial cells.171Recombinant human endostatin has been evaluated in clinical trials as a broad-spectrum angiogenesis inhibitor for several types of cancer including pancreatic endocrine tumors and carcinoid tumors,172 as well as stage IIIB–IV non–small-cell lung cancers (NSCLCs)173 without causing severe adverse effects or drug resistance. Addition of endostatin to the standard chemotherapeutic regimen in advanced NSCLC patients resulted in significant and clinically meaningful improvement in response rate, median time to progression, and clinical benefit compared with the chemotherapeutic regimen alone.

As early as in 2002, it was proposed that endostatin imaging may facilitate understanding of antiangiogenic drugs.174 Currently, there are limited reports on endostatin-based molecular imaging in tumor angiogenesis. Yang et al.175reported the 99mTc-labeled endostatin γ-camera imaging of rat with RBA CRL-1747 breast cancer tumors and assessment of the antiangiogenic effect of endostatin or the chemotherapeutic drug paclitaxel. Endostatin was conjugated with L, L-ethylenedicysteine (EC) and labeled with 99mTc to form the endostatin-based SPECT probe. Scintigraphic imaging showed that the tumor could be visualized 0.5 to 4 hours after 99mTc-EC-endostatin injection (3.7 MBq), compared with 99mTc-labeled EC chelator alone (3.7 MBq) (Fig. 37.10). Tumor uptake of 99mTc-EC-endostatin could be efficiently blocked with preinjection of excess unlabeled endostatin, revealing the receptor-binding specificity, which was supported in comparison with a nonspecific peptide tracer,99mTc-EC-K1XaK2tPA. One week of endostatin or paclitaxel treatment resulted in decreased tumor uptake of 99mTc-EC-endostatin and reduced tumor volume. There was a positive correlation between tumor uptake of 99mTc-EC-endostatin and expression of angiogenic factors including VEGF, basic fibroblast growth factor, and interleukin-8. An optical imaging study in mice with Lewis lung carcinoma xenografts demonstrated the efficiency of near-infrared Cy5.5-labeled endostatin for tumor detection.176 The intratumoral binding site for endostatin was located in the tumor vasculature and colocalized with CD31-positive endothelial cells. These studies show the promise of endostatin-based imaging as a surrogate approach for tumor angiogenesis imaging, which would be helpful for unraveling the mysteries of endostatin as reviewed by Fu et al.177 and may be useful for selection of patients who will benefit from endostatin, and for monitoring the efficacy of antiangiogenic treatment. Further development of endostatin-based radiopharmaceuticals are needed.

FIGURE 37.10. Scintigraphic images of mammary-tumor–bearing rats following administration of 99mTc-EC-endostatin and 99mTc-EC (100 μCi/10 μg/rat, i.v.) at 0.5 to 4 hours on day 14 after inoculation of tumor cells. Tumor, located in right hind leg, was well visualized with 99mTc-EC-endostatin. (From Yang DJ, Kim KD, Schechter NR, et al. Assessment of antiangiogenic effect using 99mTc-EC-endostatin. Cancer Biother Radiopharm. 2002;17:233–245. The publisher for this copyrighted material is Mary Ann Liebert, Inc. publishers.)


As angiogenesis in solid tumors plays a critical role in cancer progression, invasion, and metastasis, it presents a primary target for cancer diagnosis and treatment. Now several antiangiogenic drugs are currently available for clinical use. The need for reliable methods to predict treatment outcome and/or evaluate treatment effectiveness at an early stage is ever increasing. Tumor characterization by in vivo imaging is physically easy on patients and nuclear medicine imaging has the potential to provide direct information on the presence and change of neovasculature in tumors that would be valuable for treatment planning. Many radiolabeled tracers for angiogenesis imaging have been developed that target molecules associated with angiogenesis, such as αvβ3 integrin, VEGF, and so on. Some that were developed earlier are already in clinical trials. More refined designs that have been developed recently are in preclinical evaluations. It should be noted, however, that the target molecule is not entirely specific for angiogenesis in tumors, that is, it is expressed physiologically in other tissues. Each labeled tracer undergoes metabolism in vivo and may nonspecifically accumulate in nontarget tissue. Therefore, caution is necessary when interpreting imaging results. Through additional clinical and preclinical studies, further information will be obtained and the most appropriate tracers selected. Angiogenesis imaging, with further development is likely to play an important role in clinical molecular imaging of tumors.


Many radiolabeled tracers have been developed for various targets related to tumor angiogenesis, and some are already in clinical settings. Clinical translation of newly developed probes for angiogenesis imaging needs to be accelerated to obtain proof-of-concept in various clinical scenarios, such as quantification of angiogenesis activity, selection of candidate patients for antiangiogenesis treatment, early evaluation and prediction of treatment outcome, and to select optimal imaging methods for each situation.

Angiogenesis is a microscopic event occurring in a limited portion of the tumor tissue; the total target in the area of angiogenesis is much smaller than the number of total tumor cells. In this regard, further technical improvement is necessary to include signal amplification by clever design of the targeting probes, and improved spatial resolution, while preserving or increasing the sensitivity of the imaging modality.

Application of angiogenesis imaging probes for internal radiation therapy by replacing radionuclides from γ-emitters/positron emitters to β-emitters/α-emitters (e.g., 131I, 90Y, 177Lu, 64Cu/Astatine-211) is also important, as is research on the reduction of normal tissue uptake of the probes to increase therapeutic gain.

The process of angiogenesis plays an important role in tumor progression and many other pathologic states such as atherosclerosis, arthritis, and chronic inflammation. Application of angiogenesis imaging to these pathologies will also be beneficial.


We thank Professor Yasuhisa Fujibayashi and the other members of the Molecular Imaging Center, NIRS, for kindly supporting this work.


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