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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Cancer Res. Author manuscript; available in PMC 2011 November 15.
Published in final edited form as:
PMCID: PMC3057960

PET Imaging of Tumor Neovascularization in a Transgenic Mouse Model Using a Novel 64Cu-DOTA-Knottin Peptide


Due to the high mortality of lung cancer, there is a critical need to develop diagnostic procedures enabling early detection of the disease while at a curable stage. Targeted molecular imaging builds on the positive attributes of PET/CT to allow for a non-invasive detection and characterization of smaller lung nodules thus increasing the chances of positive treatment outcome.

In this study, we investigate the ability to characterize lung tumors that spontaneously arise in a transgenic mouse model. The tumors are first identified with small animal CT followed by characterization using small animal PET with a novel 64Cu-DOTA-knottin peptide that targets integrins up-regulated during angiogenesis on the tumor associated neo-vasculature. The imaging results obtained with the knottin peptide are compared to standard 18F-FDG PET small animal imaging.

Lung nodules as small as 3 mm in diameter were successfully identified in the transgenic mice by small animal CT, and both 64Cu-DOTA-knottin 2.5F and FDG were able to differentiate lung nodules from the surrounding tissues. Uptake and retention of the 64Cu-DOTA-knottin 2.5F tracer in the lung tumors combined with a low background in the thorax resulted in a statistically higher tumor to background (normal lung) ratio compared to FDG (6.01±0.61 versus 4.36±0.68, p<0.05). Ex vivo biodistribution showed 64Cu-DOTA-knottin 2.5F to have a fast renal clearance combined with low non-specific accumulation in the thorax.

Collectively, these results demonstrate 64Cu-DOTA-knottin 2.5F to be a promising candidate for clinical translation for earlier detection and improved characterization of lung cancer.

Keywords: molecular imaging, angiogenesis, lung cancer, integrins, transgenics


Despite advances in cancer management and treatment, the mortality due to lung cancer is still strikingly high with an average 5-year survival of only 15% (1). The major contributor to the high mortality is diagnosis at a stage when the likelihood of effective treatment is low (2-4). Therefore, there is a need to develop technologies that will aid in the earlier detection of lung nodules, e.g. molecular imaging probes that are able to detect small tumors early in disease.

Low dose Computed Tomography (CT) is increasingly utilized for screening patients with a high risk of developing lung cancer. CT screening has a high sensitivity (median 96%, range 81-100% (5)) and can detect lung lesions in the sub-centimeter range but is limited by a relatively low specificity (median 82%, range 50-95% (5)) resulting in frequent false-positive screening results (5, 6). Combined positron emission tomography and CT (PET/CT) with 18Fluorodeoxyglucose (FDG) can differentiate benign from malignant lesions larger than 1 cm in diameter with high sensitivity and reasonable good specificity (6-8). However, non-invasive characterization of smaller lesions with 18FDG-PET/CT remains challenging due to the low volume of tumor, the partial volume effect (PVE), and inherent background from metabolic active tissues in the region of interest. Invasive procedures, such as thin-needle biopsy, are difficult to perform because of the small lesion size, number of potential lesions to biopsy, and challenging due to the associated high risk of missed sampling (7, 9-12). As clinical PET-CT scanners continue to improve in spatial resolution and sensitivity there is an opportunity to improve tumor detection/management with better tracers. A targeted imaging modality offers the opportunity to expand on the positive attributes of PET/CT and allows for a non-invasive detection and characterization of smaller lung nodules thus increasing the chances of positive treatment outcome.

Once a tumor grows beyond 1-2 mm in diameter it becomes dependent on angiogenesis to support its growth (13, 14). Imaging probes that target cell surface markers related to angiogenesis, such as integrins, show potential for early cancer detection (15, 16). Integrins are a family of transmembrane bound receptors consisting of α and β subunits non-covalently linked as heterodimers. Integrins are involved in cell-to-cell and cell-to-extra cellular matrix adhesion and play a major role in cell migration processes such as angiogenesis and metastasis. Expression of integrins, particularly, αvβ3, αvβ5, and α5β1, are significantly up-regulated on tumor neovasculature and many tumor cells compared to quiescent endothelium and normal tissue (14-17) making them a promising target for differentiating tumor from normal vasculature.

In this study, we investigated the ability to image and characterize lung tumors that spontaneously arise in a transgenic mouse model. The tumors were first identified with small animal CT followed by characterization using small animal PET with our novel 64Cu-DOTA-knottin peptide engineered to bind integrins receptors with high affinity. Knottins, also known as cystine-knots, are 20-60 amino acid peptides that consist of at least three interwoven disulfide bonds, imparting high thermal and proteolytic stability (18). Previously, we used the Ecballium elaterium trypsin inhibitor II (EETI-II), a knottin from the squash family of protease inhibitors, as a molecular scaffold to engineer peptides that bind with high affinity to tumor-related integrin receptors (17). We showed that the EETI-II based knottin peptide 2.5F, which contains a Arg-Gly-Asp (RGD) motif, binds a range of integrins subtypes (αvβ3, αvβ5, and α5β1) with affinities in the low nM range, and developed it as a promising new probe for molecular imaging applications in living subjects (19).

The conditional transgenic mouse model used in this study spontaneously develops lung tumors upon activation of the K-Ras and MYC oncogenes upon the administration of doxycycline (20). Lung tumor status was serially monitored by small animal CT screening and mice with CT positive lung lesions were further examined using small animal PET. Comparative PET imaging was performed using 64Cu-DOTA-knottin 2.5F and 18F-FDG (Figure 1). Both probes were able to identify lung tumors seen on CT. Uptake and retention of the 64Cu-DOTA-knottin 2.5F tracer in the lung tumor combined with a low background in the thorax resulted in a statistically higher tumor to background (normal lung) ratio compared to FDG, which demonstrate the potential of 64Cu-DOTA-knottin 2.5F to be a promising candidate for clinical translation for earlier detection and improved characterization of lung cancer.

Figure 1
Schematic representation of experimental design


Transgenic mouse model

A detailed description of the generation of the transgenic mouse model and genotyping by PCR has been previously described (20, 21). Briefly, the transgenic mouse model used in the study was established by crossing transgenic lines containing the Clara cell secretory protein (CCSP) promoter driving the reverse tetracycline transactivating protein (rtTA), a MYC line under the control of the tetracycline-responsive operon (TetO) and a mutant KrasG12D line under control of TetO. The desired result was the bi-conditional model CCSP-rtTa / TetO- MYC / TetO-KrasG12D

Expression of the MYC and KrasG12D oncogenes was activated by weekly administration of doxycycline 100 mg/ml (Sigma) to the drinking water. Activation was initiated after weaning, typically 4-6 weeks of age, and tumors developed with an average latency of 36 weeks. All animal procedures were carried out according to a protocol approved by Stanford University Administrative Panels on Laboratory Animal Care.

Small animal computed tomography

Small animal CT scans were performed using a custom GE Medical Systems eXplore RS MicroCT System cone-beam scanner (GE Medical Systems). Seven transgenic mice with activated MYC and KrasG12D underwent serial small animal CT-screening once a week for 3 weeks. Mice were anesthetized with 2% isofluorane in 2 L/min oxygen and positioned prone. Images were acquired at 97μm resolution with a 70kV (40 μA) beam, 12 ms expose, and 400 radial views over a 360 degrees rotation. The respiration was monitored during the scan with BioVet monitoring system (m2m Imaging Corp.) and the acquisitions triggered to the end of the inspiratory phase.


Synthesis and radiolabeling of the knottin 2.5F probe was performed as previously described by us (19). 18F-FDG was supplied by the cyclotron and radiochemistry facility at Stanford University with high specific activity.

Stability of 64Cu-DOTA-knottin 2.5F

64Cu-DOTA-knottin 2.5F in PBS was incubated in an equal volume of mouse serum at 37°C and analyzed at 1 and 4 hours after incubation. At the indicated time points, one volume of solvent A (99.9% H2O and 0.1 % TFA) and one volume of DMF (Sigma) was added and the resulting solution was centrifuged and filtered with a 0.2 micron spin filter (Corning). This mixture was analyzed by radio HPLC. Urine samples collected from mice 1 hour post-injection of 64Cu-DOTA-knottin 2.5F was diluted to 1 mL with solvent A and analyzed by radio HPLC.

Ex vivo biodistribution

Healthy mice (N=5) were anesthetized and injected with 66.3-70.6 μCi (2.45-2.61 MBq) of 64Cu-DOTA-knottin 2.5F in 150 μl PBS via the tail vein and were euthanized 1 hour post-injection. The urine from 3 mice was collected for metabolite analysis by radio HPLC. Blood, heart, liver, lungs, muscle kidneys, spleen, brain, intestine, stomach, pancreas, and bone were removed and weighed. The radioactivity of each organ was measured by gamma counting and the activity level expressed as %ID/g or %ID/cm3 for soft tissue and the lungs. Soft tissue was assumed to have a density of 1 g/cm3. Prior to injection of the tracer, the mice (N=5) underwent small animal CT. The densities of the lungs were calculated by dividing the weight of the lungs with the volumes estimated by CT.

Small animal PET/CT

A R4 microPET (Siemens Medical Solutions USA, Inc., Malvern, PA) was used for small animal PET imaging. Mice with CT-positive nodules underwent PET imaging with 18F-FDG on day 0 (relatve to CT scan) and with 64Cu-DOTA-knottin 2.5F on day 1. Mice were anesthetized with 2% isofluorane in 2 l/min oxygen and injected with 93-115 μCi (2.44-4.26 MBq) of either 18F-FDG or 64Cu-DOTA-knottin 2.5F in 150 μl PBS via the tail vein. Mice were placed in a holder with four fiducial markers each containing ~0.5 μCi of either 18F-FDG or 64Cu-DOTA-knottin 2.5F in 10 μl iodine solution (30 mg/ml). Prior to the PET scan mice underwent small animal CT scanning as described above followed by a 15 minutes prone PET acquisition approximately 60 min post-injection of tracer. For 18F-FDG-PET, mice were maintained under anesthesia in between injection and imaging. During anesthesia mice were kept warm on a heating pad. For knottin 2.5F, mice were allowed to recover from anesthesia in between injection and scanning. PET images were reconstructed using the 2D ordered-subsets expectation maximization (2D-OSEM) algorithm with a spatial resolution of 1.66 to 1.85 mm (22). No attenuation correction or partial volume corrections were applied.

Data Analysis

All imaging data were analyzed offline using software developed in our laboratory (AMIDE version 0.8.22) (23) and 3D volume viewer and analysis software (MicroView). Small animal PET/CT images were co-registered by manually identifying the center of the fiducial markers in the CT and PET images and applying a built in function for co-registration in the software. ROIs were subsequently drawn over the nodules and healthy lung tissue based on the CT images. Voxel values were converted to μCi/cm3 by multiplying with a cylindrical factor obtained by scanning a cylindrical phantom with a known concentration of activity. For each ROI, the mean uptake as percent-injected dose per gram tissue (%ID/g) was calculated assuming a tissue density of 1g/cm3. For each nodule the tumor to background ratio was calculated. The volumes of the lungs were estimated by drawing a rough ROI over the thorax of the mouse followed by simple binning to include only voxels with CT values corresponding to lung tissue.


A subset of the mice (N=4) were sacrificed immediately after 64Cu-DOTA-knottin 2.5F PET imaging. The lungs were removed and dissected into pieces containing tumor-tissue and lung tissue without visible tumor mass. The tissue pieces were embedded in optical cutting temperature (OCT) compound, and flash frozen on dry ice. Frozen blocks were sectioned at 10 microns and mounted on charged glass slides for immunofluorescence. A double-staining procedure was applied to visualize endothelial cells (CD31) and integrins (αv-subunit). The following were used to stain for CD31: A rat anti-mouse CD31 primary antibody diluted 1:100 (BD Biosciences); a goat anti-rat biotinylated secondary antibody diluted 1:500 (Jackson ImmunoResearch Laboratories); and streptavidin conjugated AlexaFluor 488 diluted 1:200 (Invitrogen). The following were used to stain for αv: A rat anti-mouse αv biotinylated primary antibody diluted 1:200 (Jackson ImmunoResearch Laboratories); and streptavidin conjugated AlexaFluor 594 diluted 1:200 (Invitrogen). Fluorescent images were acquired with a microscope (Axiophot; Carl Zeiss, Thornwood, NY).

Statistical Analysis

Data is given as mean ± standard error mean unless stated otherwise. The difference between the knottin 2.5F and FDG tumor to background ratios was assessed using the two sided Mann-Whitney test. A p-value <0.05 was considered statistically significant.


Small animal CT screening

All seven transgenic that underwent serial small animal CT-screening for 3 weeks were identified to have positive CT lung lesions. Next we investigated the stability and biodistribution of the 64Cu-DOTA-knottin 2.5F tracer before using it in a comparative study to characterized the lung lesions using FDG and 64Cu-DOTA-knottin 2.5F PET.

Serum and in vivo stability of knottin 2.5F

64Cu-DOTA-knottin 2.5F incubated in mouse serum and urine samples from mice injected with 64Cu-DOTA-knottin 2.5F were analyzed by radio HPLC to test the stability of the probe under physiologically relevant environments. All samples show a major elution peak at 14 min corresponding to the intact probe. The probe is mainly excreted intact in urine, and it is highly stable after 4 hours of serum incubation (Supplementary Figure 1). Overall, the radio-HPLC analysis showed that the probe is stable at the conditions tested and is suitable as an in vivo imaging probe.

Ex vivo biodistribution

The biodistribution of 64Cu-DOTA-knottin 2.5F was investigated in healthy mice 1-hour post injection (Figure 2). The tracer cleared rapidly through the kidneys (11.43 ±1.45 %ID/cm3). Modest levels of background activity were observed in the stomach, intestine, spleen, and liver (1.28-2.53 %ID/cm3) in agreement with previous reports (19). Lower background signal was seen in the brain, blood, muscle, pancreas, lungs, and heart (0.04-0.50 %ID/cm3).

Figure 2
Ex vivo biodistribution of 64Cu-DOTA-knottin 2.5F

Small animal PET/CT

Mice with CT-positive tumors underwent PET imaging with 18F-FDG and with 64Cu-DOTA-knottin 2.5F. For both tracers, differentiation of lung nodules from surrounding lung tissue was possible based on PET images obtained 1 hour post-injection. However, in a few cases (N=3), where the nodules were in close proximity to the heart, delineation of nodule boundaries was very difficult with FDG-PET. This was due to relatively high 18F-FDG uptake in the heart (26.92 %ID/g ±2.74) leading to a spillover effect into the surrounding tissue. Importantly however, nodules that were obscured by high 18F-FDG heart spillover were clearly visible on the 64Cu-DOTA-knottin 2.5F images (Figure 3). These images were made possible because of the low thoracic background signal that is characteristic of knottin peptides [18].

Figure 3
Small animal PET/CT

Overall, the radioactivity uptake in the nodules, measured as %ID/g by ROI analysis, was higher for 18F-FDG compared to 64Cu-DOTA-knottin 2.5F, Supplementary Figure 2. However, ROI analysis of 10 distinguishable tumors showed a higher tumor to background ratio for the 64Cu-DOTA-knottin 2.5F tracer compared to FDG (Table 1). The tumor to background ratio was 6.01±0.61 (range 3.51-9.05) for 64Cu-DOTA-knottin 2.5F, which was significantly higher than the ratio obtained with FDG, 4.36 ±0.68 (range 1.70-8.30, p<0.05 by two sided Mann-Whitney test, Figure 4). Higher tumor to background ratios of 64Cu-DOTA-knottin 2.5F result from low background in the thorax, which facilitates nodule identification in regions made inaccessible by higher background 18F-FDG uptake (0.30 %ID/g ±0.03 for 64Cu-DOTA-knottin 2.5F versus 1.35 %ID/g ±0.13 for FDG), Figure 5 and Supplementary Figure 2.

Figure 4
Tumor to background ratio of knottin 2.5F and FDG
Figure 5
(A) Volume renderings of the thorax of a mouse imaged with FDG (left) and knottin 2.5F (right). A nodule is present immediately adjacent to the heart and is clearly visible when knottin 2.5F is used. The intense FDG uptake in the heart makes it impossible ...
Table 1
Results of quantitative ROI analysis


Mouse lung with nodules identified using 64Cu-DOTA-knottin 2.5F PET, were double stained for mouse CD31, a marker expressed on endothelial cells, and αv-integrin subunit to confirm the presence of the imaging target. Immunofluorescence showed co-localization of CD31 and αv, (Figure 5) which confirms the presence of the imaging target on the tumor vasculature.


Despite advances in diagnostic clinical imaging, there is a critical need for new imaging agents and new methods that allow non-invasive characterization of lung lesions in the sub-centimeter range. Engineered knottin peptides have recently been validated by our group to specifically image integrin-expressing tumors in xenograft mouse models (17, 19, 24-27). Here we expand on our previous work and show that we can characterize spontaneous lung tumors developing in a conditional transgenic mouse model using 64Cu-DOTA-knottin 2.5F and combined small animal PET/CT. These transgenic mouse models provide a unique opportunity to study tumorigenesis in a lung cancer model that more closely resembles the “natural” development and progression of human cancer. Since there is always an underlying concern using artificial xenografts models (28, 29), these studies provide additional information which will aid in clinical translation of newly developed molecular imaging probes.

The performance of knottin 2.5F, a peptide engineered to target integrin receptors, was compared to that of 18F-FDG. We were able to obtain a higher tumor to background ratio with 64Cu-DOTA-knottin 2.5F compared to 18F-FDG (6.01±0.61 versus 4.36 ±0.68, p<0.05) due to a low background signal in the area of interest. Is has previously been reported that anesthesia does not influence the tumor uptake of FDG (30). It is unlikely that the difference in tumor to background ratio can be addressed to the difference in anesthetic regiment between the two probes during PET imaging. Lung lesions imaged with 64Cu-DOTA-knottin 2.5F are clearly visible allowing for easier image interpretation easier compared to 18F-FDG due to large spillover effect from uptake in the heart. However, it is important to note that in a few cases the quantification of 18F-FDG uptake is biased towards a higher tumor uptake because of this spillover from the heart to the adjacent lung tumors. The presence of the αv integrin imaging target on the tumor neovasculature was confirmed by ex vivo fluorescence microscopy. Furthermore, in a previous study we determined the specificity of integrin-binding knottin probes by competition with an excess of unlabeled peptide (19). Collectively, our results indicate that engineered knottin peptides are able to provide detailed molecular information about receptors expressed on vascular endothelium and the surface of tumors. Studies using dynamic PET imaging and compartment modeling are in progress to investigate the fraction of the signal originating from knottins bound to integrins on the endothelium compared to the surface of the tumor.

Clinical studies with radiolabeled RGD peptides (18F-galacto-RGD and 18F-AH111585) for PET imaging have demonstrated the feasibility of imaging integrin receptors expressed on tumor cells and tumor neovasculature in cancer patients (17, 31-35). In addition, recent studies show that the pharmacokinetics of integrin targeting peptides may easily be fine-tuned (19, 36, 37). For example, in an orthotopic lung cancer mouse model, PET imaging with 64Cu-DOTA labeled PEGylated dimeric cyclic RGD peptide showed better tumor delineation compared to imaging with 18F-FDG. These studies demonstrate the potential of integrin imaging for early diagnosis and primary staging of lung cancer (15).

An important characteristic of any imaging probe is its uptake efficiency at the tumor site and its lack of accumulation at non-tumor sites. The optimal probe will have high affinity for the target of interest leading to high uptake and at the same time show minimal background accumulation (38). Significantly work has been put into engineering multimeric RDG peptide probes with increased affinity towards integrins. Multimeric RGD peptide probes show higher tumor accumulation and retention in vivo, but are also accompanied by and increase uptake in non-tumor tissues (36, 39, 40). In contrast, engineering of the knottin scaffold with the monomeric RGD binding motif shows comparable affinity towards integrins as the tetrameric RGD peptide 64Cu-DOTA-E{E[c(RGDfK)]2}2, while maintaining a low accumulation in normal tissues leading to a higher tumor to non-tumor tissues ratio (Supplementary Table 1).

A common criterion for diagnosing malignancy with FDG-PET clinically is the standardized uptake value (SUV), which is a measure of the absolute uptake in a given region normalized for injected dose and body mass. However, it has been reported that using the tumor to background ratio improves the sensitivity of FDG-PET for diagnosis small pulmonary nodules (7). In the current study, absolute radiotracer uptake in the lung tumors was higher for FDG than for knottin 2.5F. This example illustrates that the comparatively better contrast demonstrated by knottin 2.5F was due to lower background signal in the thorax for 64Cu-DOTA-knottin 2.5F compared to 18F-FDG. Together, the favorable biodistribution and tumor accumulation merits further investigation to see if these findings translate to the clinical setting.

Knottins have previously been shown to be non-immunogenic and synthetic version of the knottin MVIIA (Ziconotide) is approved for treatment of chronic pain (19, 41). We have successfully performed up to 6 imaging sessions over 8 months with 64Cu-DOTA-knottin 2.5F in immunocompetent mice without the observation of an acute immune response (data not shown). This demonstrates that repeated imaging with 64Cu-DOTA-knottin 2.5F in mice is possible. However, rigorous toxicity studies will be required before clinical translation to minimize the possibility of an unexpected immunologic reaction against 64Cu-DOTA-knottin 2.5F.

The transgenic mouse model allows us to investigate if imaging of integrin expression with 64Cu-DOTA-knottin 2.5F can be used to characterize the lesions identified on CT as malignant. However, the difficulties of generating adequate numbers of transgenic mice with the right genotype and the inability to control the exact latency of tumor development after administration of doxycycline made a rigorous investigation the tumor development at the earliest time impossible. Therefore, we were not able to investigate in detail the minimal detectable size of lung nodules that can be characterized as malignant by integrin imaging, but based on our data we were able to characterize nodules as small as 3 mm in diameter as malignant (Figure 3). Some of the mice (N=2) had lesions visible on CT smaller than 3 mm in diameter (N=5). 64Cu-DOTA-knottin 2.5F were able to delineate one lesion with a diameter of 2.5 mm but missed the other small lesions, whereas FDG missed all of the lesions smaller than 3 mm in diameter. Assuming that all lesions detected by CT are true positive findings, the sensitivity of 64Cu-DOTA-knottin 2.5F and FDG for all lesions is 73.3% and 66.7% respectively, and 100% for both probes for lesions larger than 3 mm in diameter. Although we do not have histology to confirm the malignancy of the smaller lesions we know from previous work that lung lesions detected by CT correspond with lung cancer (20). A possible explanation for failed detection of those small lesions by PET could be loss of contrast in the PET images due to blurring originating from respiratory motion during the PET acquisitions and PVE. Unlike CT, no respiratory gating was applied during the PET acquisitions.

Because of the relative poor resolution of clinical PET scanners the PVE becomes substantial for tumors in the sub-centimeter range. For small tumors, PVE will lead to a under estimation of the true uptake and decreased sensitivity; however, using mathematical models, it is possible to correct for PVE. Using a combination of morphological information obtained from CT images, correction for respiratory motion, and low background signal from engineered peptides such 64Cu-DOTA-knottin 2.5F, PVE-corrective algorithms will likely lead to better estimations of true uptake in lung nodules (42). Furthermore, clinical scanners with improved spatial resolution continue to be developed, which if married to the right tracers has significant potential for improved cancer diagnostics/management.

The clinical utility of 64Cu-DOTA-knottin 2.5F imaging will have to be carefully studied in patients with different types of lung cancer. If knottin 2.5F imaging is able to outperform FDG it could replace FDG-PET. Alternatively, if the knottin 2.5F imaging agent is not able to perform as well as FDG, then it could be used in cases where FDG uptake is borderline as indicated by an intermediate SUV. In these cases, FDG is unable to distinguish inflammation vs. tumor and the knottin 2.5F may help to do so.

In summary our results demonstrate that engineered peptides, such as 64Cu-DOTA-knottin 2.5F, have the potential to be used in early detection of lung cancer. However, further studies are needed in order to address the smallest detectable tumor size by 64Cu-DOTA-knottin 2.5F. Further studies will also be needed to understand 64Cu-DOTA-knottin 2.5F uptake in sites of pulmonary infection and inflammation, which could lead to up-regulation of integrins during the inflammatory process. Together with improvements in scanner spatial resolution/sensitivity, PVE correction, and further engineering of knottins to improve the tracer performance, PET imaging with engineered knottins might prove to be more sensitive than FDG-PET for primary diagnosis of lung lesions.

Supplementary Material


We thank Pauline Chu for her kind help with tissue staining, the Stanford University Cyclytron and Radiochemistry Facility for synthesis of 18F-FDG, and Dr. Zhe Liu for his help with 64Cu radiolabeling of the knottin peptide. This work was supported in part by National Institutes of Health Grants NCI ICMIC P50 CA114747 (SSG), CCNE U54 CA119367 (SSG), and the Canary Foundation (SSG). Carsten Haagen Nielsen acknowledges the support received from the Lundbeck Foundation, the Denmark American Foundation, BioCampus University of Copenhagen, Department of Clinical Physiology, Nuclear Medicine and PET, Rigshospitalet, and the Danish National Advanced Technology Foundation.


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