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Reproducibility study of [18F]FPP(RGD)2 uptake in murine models of human tumor xenografts

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European Journal of Nuclear Medicine and Molecular Imaging Aims and scope Submit manuscript

Abstract

Purpose

An 18F-labeled PEGylated arginine-glycine-aspartic acid (RGD) dimer {[18F]FPP(RGD)2} has been used to image tumor αvβ3 integrin levels in preclinical and clinical studies. Serial positron emission tomography (PET) studies may be useful for monitoring antiangiogenic therapy response or for drug screening; however, the reproducibility of serial scans has not been determined for this PET probe. The purpose of this study was to determine the reproducibility of the integrin αvβ3-targeted PET probe, [18F]FPP(RGD)2, using small animal PET.

Methods

Human HCT116 colon cancer xenografts were implanted into nude mice (n = 12) in the breast and scapular region and grown to mean diameters of 5–15 mm for approximately 2.5 weeks. A 3-min acquisition was performed on a small animal PET scanner approximately 1 h after administration of [18F]FPP(RGD)2 (1.9–3.8 MBq, 50–100 μCi) via the tail vein. A second small animal PET scan was performed approximately 6 h later after reinjection of the probe to assess for reproducibility. Images were analyzed by drawing an ellipsoidal region of interest (ROI) around the tumor xenograft activity. Percentage injected dose per gram (%ID/g) values were calculated from the mean or maximum activity in the ROIs. Coefficients of variation and differences in %ID/g values between studies from the same day were calculated to determine the reproducibility.

Results

The coefficient of variation (mean±SD) for %IDmean/g and %IDmax/g values between [18F]FPP(RGD)2 small animal PET scans performed 6 h apart on the same day were 11.1 ± 7.6% and 10.4 ± 9.3%, respectively. The corresponding differences in %IDmean/g and %IDmax/g values between scans were −0.025 ± 0.067 and −0.039 ± 0.426. Immunofluorescence studies revealed a direct relationship between extent of ανβ3 integrin expression in tumors and tumor vasculature with level of tracer uptake. Mouse body weight, injected dose, and fasting state did not contribute to the variability of the scans; however, consistent scanning parameters were necessary to ensure accurate studies, in particular, noting tumor volume, as well as making uniform: the time of imaging after injection and the ROI size. Reanalysis of ROI placement displayed variability for %IDmean/g of 6.6 ± 3.9% and 0.28 ± 0.12% for %IDmax/g.

Conclusion

[18F]FPP(RGD)2 small animal PET mouse tumor xenograft studies are reproducible with relatively low variability.

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Acknowledgments

This work was supported, in part, by National Cancer Institute (NCI) In Vivo Cellular Molecular Imaging Center (ICMIC) grant P50 CA114747 (SSG), NCI 5R01 CA119053 (ZC), and a grant from Bayer HealthCare (Berlin, Germany). The Stanford University Radiochemistry Facility is acknowledged for radionuclide production and radiochemistry support.

Conflicts of interest

None.

Author information

Authors and Affiliations

  1. Molecular Imaging Program at Stanford, Department of Radiology, School of Medicine, Stanford University, 1201 Welch Road, Lucas Center, P020A, Stanford, CA, 94305-5484, USA

    Edwin Chang, Shuangdong Liu, Gayatri Gowrishankar, Shahriar Yaghoubi, Frederick Chin, Sanjiv S. Gambhir & Zhen Cheng

  2. Molecular Imaging Program at Stanford, Department of Bioengineering, School of Medicine, Stanford University, Stanford, CA, 94305, USA

    Gayatri Gowrishankar, Shahriar Yaghoubi, James Patrick Wedgeworth & Sanjiv S. Gambhir

  3. Global Drug Discovery, Bayer Schering Pharma AG, Berlin, Germany

    Dietmar Berndorff & Volker Gekeler

  4. Departments of Radiology and Bioengineering, Molecular Imaging Program at Stanford, Canary Center at Stanford for Cancer Early Detection, Nuclear Medicine, Stanford, CA, 94305, USA

    Sanjiv S. Gambhir

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  1. Edwin Chang
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Corresponding authors

Correspondence to Sanjiv S. Gambhir or Zhen Cheng.

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Supplemental Figure 1

a Representative immunofluorescent pictographs (×100) of 6 μm ectopic tumor (frozen) sections that were stained with antibodies against human ανβ3 integrins (FITC or green signal) and against murine VEGF-R2 (Flt-1: PE or red signal). Cell nuclei were visualized by DAPI (blue signal). Sections were taken from tumors with high [18F]FPP(RGD)2 uptake (top row, 2.6 ± 0.3) and with low probe uptake (bottom row, 1.0 ± 0.3). Each panel represents a unique tumor. b The extent of integrin signaling was computed with the use of Image J Software, and the samples with high (n = 3) vs low (n = 3) probe uptake were compared. c An immunofluorescent pictograph showing patterns of murine CD31 and human integrin expression around a tumor vasculature (×200) magnification. Correlation of a v b 3 integrin expression to [18F]FPP(RGD)2uptake. In order to validate that changes in %ID/g values reflects changes in integrin expression, tumors with both high %IDmax/g (mean = 2.6 ± 0.3, n = 6) and low %IDmax/g (mean = 1.0 ± 0.3, n = 6) were isolated, 6-μm sections were cut, and then monitored via immunofluorescence staining for the extent of ανβ3 expression. Representative examples of these immunofluorescent photomicrographs are shown in a. Although initial qualitative assessment of integrin distribution and expression (green signal) showed considerable heterogeneity, there did seem to be a trend towards more extensive distribution of green signal in tumor sections with high %IDmax/g samples compared to those with low %IDmax/g. The extent of positive antibody staining was determined by assessing the total area of the field of view at ×40. A region of interest (ROI) was then drawn with best judgment around those regions that were positive for integrin fluorescence staining. A ratio of ROI area for integrin over area of total field of view was then compared to [18F]FPP(RGD)2 uptake. ROI measurements of integrin positive regions (b) revealed that there was indeed a significant difference (p < 0.05) between the extent of integrin expression and high vs low [18F]FPP(RGD)2 uptake values (as represented by %IDmax/g). A plot of this ROI ratio vs %IDmax/g yielded a moderately linear function with R2 = 0.45, p < 0.05, and slope = 0.29. Although most of the signal appeared on tumor tissue directly, some of the signal surrounded murine anti-VEGF-R2-PE positive tubules (red signals, arrow, c). This observation suggests that the tumor tissue around murine neovasculature tends to express ανβ3 and this facilitates the interaction of tumor cells to the neovasculature. (PDF 193 kb)

Supplemental Figure 2

a Full image coronal section scan of mouse shown in Fig. 1 for the first scan (a) and the second scan 6 h later (b). a First full-body coronal scans of nude mouse with a scapular and breast xenograft of HCT116 tumors. Injected tracer is [18F]FPP(RGD)2. Scan was performed 1 h after injection. Note small necrotic region in scapular tumor. (PDF 96 kb)

Supplemental Figure 2

b Full image coronal section scan of mouse shown in Fig. 1 for the first scan (a) and the second scan 6 h later (b). b Second full-body coronal scans of nude mouse with a scapular and breast xenograft of HCT116 tumors. Injected tracer is [18F]FPP(RGD)2 and was injected 5 h after first scan. Second scan was performed 1 h after injection. (PDF 92 kb)

Supplemental Figure 3

Chemical structure of [18F]FPP(RGD)2 (Arg-Gly-Asp) radiotracer used in the reproducibility study. Note multiple repeats of the cyclic RGD motif at the active binding site of the tracer as well as the extended PEGylated “tail.” (PDF 37 kb)

Supplemental Table 1

Table showing lack of change in %ID/g uptake as a function of input of high versus low amount of [18F]FPP(RGD)2. (PDF 20 kb)

Supplemental Table 2

Similar rate of change in %IDmean/g and %IDmax/g parameters of the tracer [18F]FPP(RGD)2 for small (126 ± 50 mm3) vs large (739 ± 150 mm3) HCT116 tumor xenografts at 1 to 4 h post-tail vein injection. A total of four mice werecompared with four large tumors and four small ones. (PDF 20 kb)

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Chang, E., Liu, S., Gowrishankar, G. et al. Reproducibility study of [18F]FPP(RGD)2 uptake in murine models of human tumor xenografts. Eur J Nucl Med Mol Imaging 38, 722–730 (2011). https://doi.org/10.1007/s00259-010-1672-1

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