Recently, we demonstrated that radiolabeled RGD peptides allow receptor-specific monitoring of αvβ3 expression in murine tumor models [24
]. Here we have translated these findings to the clinical setting and for the first time, to our knowledge, demonstrated noninvasive imaging of αvβ3 expression in patients with malignant tumors. Furthermore, we have shown that the activity accumulation in the tumor correlates with the receptor density, determined by immunohistochemistry and Western blotting. This indicates that a noninvasive quantitative determination of αvβ3 expression is feasible. Furthermore, we have demonstrated in a squamous cell carcinoma model that the sensitivity of PET is adequate to image expression of αvβ3 in the tumor vasculature. This indicates that PET with [18
F]Galacto-RGD can be applied to study αvβ3 expression during angiogenesis.
The correlation between [18F]Galacto-RGD uptake in the tumor and αv expression shows considerable scattering. This is probably due to several factors. As for any imaging probe, tumor uptake of [18F]Galacto-RGD is not only influenced by the expression of the αvβ3 integrin, but also by unspecific factors such as perfusion and vascular permeability. Furthermore, heterogeneous tracer uptake within a tumor, e.g., due to the presence of necrotic areas, will influence the correlation between [18F]Galacto-RGD uptake and αv expression, since separate samples were used for measurements of tracer uptake and quantitative assessment of αv expression. Finally, the present study evalutated [18F]Galacto-RGD uptake only at a fixed time, 90 min p. i. Imaging of the dynamics of [18F]Galacto-RGD accumulation in the tumor tissue and tracer kinetic modeling may allow a better quantitative assessment of αvβ3 expression by PET imaging, and this approach should be evaluated in animal models as well as in patients. Nevertheless, the significant correlation between the uptake of [18F]Galacto-RGD at a fixed time after injection and αvβ3 expression is very important for clinical studies, since it suggests that estimates of αvβ3 expression levels may be obtained from simple whole-body PET scans.
It has been shown that the highly bent integrin conformation is physiological and has low affinity for biological ligands, such as fibrinogen and vitronectin. Inside-out and outside-in signaling involve a switchblade-like opening to an extended structure with high affinity for endogenous ligands, as well as integrin antagonists (for overview see [36
]). The inside-out activation is induced by conformational changes in the membrane-proximal regions of the α and β subunit (e.g., by intracellular proteins like talin). Outside-in signaling is triggered by Mn2+
, which defines by quaternary rearrangements a pathway for communication from the ligand-binding site to the cytoplasmatic proximal segments. However, it is also reported that cyclo(-Arg-Gly-Asp-D
Phe-Val-), in addition to binding to the high-affinity conformer, can bind to the low-affinity conformation when used at concentrations far above its dissociation constant, resulting in a similar activation as found for Mn2+
. The nanomolar concentration used in our radiotracer approach is approximately 10,000-fold lower than that reported for the activation of the low-affinity conformation. Thus, PET with [18
F]Galacto-RGD is expected to provide information not only about the expression of αvβ3 but also about the functional status of this integrin.
The glycopeptide [18
F]Galacto-RGD showed high metabolic stability in patients and rapid, predominantly renal elimination, resulting in good tumor/background ratios and, thus, in high-quality images. Moreover, this finding confirms the general advantage of the glycosylation approach [24
] in designing peptide-based tracers with favorable imaging properties for clinical applications. Another approach to optimize pharmacokinetics is based on the conjugation of polyethyleneglycol [40
]. It has been demonstrated that such polyethyleneglycolated peptides also improve pharmacokinetics and tumor retention. However, a direct comparison of tracers resulting from the different strategies has not yet been carried out.
The correlation between regional tracer uptake in the lesion and density of αvβ3-positive vessels confirms that this technique allows not only visualization but also noninvasive quantitative assessment of the integrin expression. Interestingly, our study demonstrated high both inter- and intraindividual variances in tracer accumulation in the different lesions, indicating a great diversity in receptor expression. This finding demonstrates the value of noninvasive techniques for appropriate selection of patients entering clinical trials of αvβ3-targeting therapies. This is further emphasized by the fact that in some cases no [18F]Galacto-RGD uptake was found, despite viable tumor cells being detected via a [18F]FDG scan.
Furthermore, PET imaging with [18
F]Galacto-RGD can be applied to assess successful blocking of αvβ3 by therapeutic agents, thereby providing essential information for the dose and dose scheduling of αvβ3 antagonists. Further studies are needed to demonstrate the impact of this new technique as a novel prognostic indicator in cancer. However, the first evidence of the prognostic value is given by Gasparini et al. [46
], who found αvβ3 expression in tumor vasculature “hot spots” to be a significant prognostic factor predictive of relapse-free survival in both node-negative and node-positive patients.
αvβ3 is also found on endothelial cells during wound healing, in restenosis, in rheumatoid arthritis, and in psoriatic plaques. Thus, radiolabeled RGD peptides may be used to characterize not only malignant tumors but also inflammatory diseases. Most recently, we demonstrated in a murine model for cutaneous delayed-type hypersensitivity reaction that [18
F]Galacto-RGD allows noninvasive assessment of αvβ3 expression in inflammatory processes [47
]. Our preliminary data from a villonodular synovitis show that αvβ3 expression on endothelial cells in this lesion can be monitored in patients. Altogether, these findings indicate that [18
F]Galacto-RGD might also become a new biomarker for disease activity in inflammatory processes.
The primary advantage of PET in imaging molecular processes is its high sensitivity combined with high penetration of the gamma radiation resulting from positron decay. Thus, PET imaging allows quantification of regional radioactivity concentrations in human studies. The optical imaging approach has an even higher sensitivity, but suffers from the low penetration of light in most tissues. This results in a very limited ability to carry out tomographic imaging and to quantify the optical signal. Thus, optical imaging is currently limited to preclinical studies in mice, whereas PET can be performed in preclinical as well as in clinical studies. Magnetic resonance imaging provides high spatial resolution and can combine morphological and functional imaging, but has approximately 1,000-fold lower sensitivity compared with PET. Thus, PET is the most appropriate technique for noninvasive determination of molecular processes in patients at the current time. Obviously, the patient is exposed to high-energy γ-rays during this procedure. However, based on our radiation dose estimates, the effective radiation dose for a [18
F]Galacto-RGD scan is in the same range as for a [18
F]FDG scan, an approved routine examination in the clinic in many countries [48
]. In preclinical studies, different targeted magnetic resonance contrast agents have been evaluated, using either anti-αvβ3 antibody-conjugated polymerized liposomes [49
] or nanoparticles [50
], or nanoparticles linked with an αvβ3 peptidomimetic antagonist [51
]. In those studies, depending on the contrast agent and animal model used, an average magnetic resonance signal intensity enhancement between approximately 20% and 120% was found, a finding which has not yet been confirmed in clinical studies. In our patient study using [18
F]Galacto-RGD and PET, a 9-fold higher activity accumulation, on average, was found in the tumor than in muscle, further indicating the currently superior properties of this radiotracer for molecular imaging. Moreover, recent developments in combining PET with computed tomography or future possibilities to combine PET with magnetic resonance imaging will allow correlation of these processes with the corresponding morphology.
To further improve tumor retention of αvβ3 radioligands, multimeric RGD peptides were recently introduced. Our group developed different series of multimeric structures with up to eight RGD units linked via different spacers [40
]. These multimeric RGD peptides showed increased binding affinities in vitro and improved tumor accumulation and tumor/background ratios in rodents compared with the monomeric compounds. These data and data from other groups [52
] indicate that the multimeric ligand approach may be used for optimization of the performance of peptide-based tracers. However, studies in patients will be necessary to demonstrate the potential of this approach in clinical settings.
In summary, this new class of PET tracer may offer insights into molecular processes during tumor development and dissemination in preclinical as well as clinical settings, and will be a helpful tool in planning and controlling novel αvβ3-directed therapies.