Noninvasive Visualization of the Activated αvβ3 Integrin in Cancer Patients by Positron Emission Tomography and [(18)F]Galacto-RGD

BACKGROUND: The integrin αvβ3 plays an important role in angiogenesis and tumor cell metastasis, and is currently being evaluated as a target for new therapeutic approaches. Several techniques are being studied to enable noninvasive determination of αvβ3 expression. We developed [(18)F]Galacto-RGD, a (18)F-labeled glycosylated αvβ3 antagonist, allowing monitoring of αvβ3 expression with positron emission tomography (PET). METHODS AND FINDINGS: Here we show by quantitative analysis of images resulting from a small-animal PET scanner that uptake of [(18)F]Galacto-RGD in the tumor correlates with αvβ3 expression subsequently determined by Western blot analyses. Moreover, using the A431 human squamous cell carcinoma model we demonstrate that this approach is sensitive enough to visualize αvβ3 expression resulting exclusively from the tumor vasculature. Most important, this study shows, that [(18)F]Galacto-RGD with PET enables noninvasive quantitative assessment of the αvβ3 expression pattern on tumor and endothelial cells in patients with malignant tumors. CONCLUSIONS: Molecular imaging with [(18)F]Galacto-RGD and PET can provide important information for planning and monitoring anti-angiogenic therapies targeting the αvβ3 integrins and can reveal the involvement and role of this integrin in metastatic and angiogenic processes in various diseases

Noninvasive Visualization of the Activated alphavbeta3 Integrin in Cancer Patients by Positron Emission Tomography and [¹⁸F]Galacto-RGD

Background The integrin alphavbeta3 plays an important role in angiogenesis and tumor cell metastasis, and is currently being evaluated as a target for new therapeutic approaches. Several techniques are being studied to enable noninvasive determination of alphavbeta3 expression. We developed [18F]Galacto-RGD, a 18F-labeled glycosylated alphavbeta3 antagonist, allowing monitoring of alphavbeta3 expression with positron emission tomography (PET). Methods and Findings Here we show by quantitative analysis of images resulting from a small-animal PET scanner that uptake of [18F]Galacto-RGD in the tumor correlates with alphavbeta3 expression subsequently determined by Western blot analyses. Moreover, using the A431 human squamous cell carcinoma model we demonstrate that this approach is sensitive enough to visualize alphavbeta3 expression resulting exclusively from the tumor vasculature. Most important, this study shows, that [18F]Galacto-RGD with PET enables noninvasive quantitative assessment of the alphavbeta3 expression pattern on tumor and endothelial cells in patients with malignant tumors. Conclusions Molecular imaging with [18F]Galacto-RGD and PET can provide important information for planning and monitoring anti-angiogenic therapies targeting the alphavbeta3 integrins and can reveal the involvement and role of this integrin in metastatic and angiogenic processes in various diseases.</p

Preclinical Evaluation of [¹⁸F]Galacto-RGD

<div><p>(A) Transaxial images of nude mice bearing tumors with increasing amounts of αvβ3-positive M21 cells (0% [M21-L], 25%, 75%, and 100% [M21]) 90 min p. i. provided by a prototype small-animal PET scanner show an increasing tracer uptake in the tumor and low background activity.</p> <p>(B) Immunohistochemical staining of tumor tissue sections prepared after PET imaging with an anti-human αvβ3 monoclonal antibody (LM 609) indicate that there is a correlation between tracer uptake and αvβ3 expression.</p> <p>(C) Western blots of the dissected tumors show a band at 25 kDa that corresponds with the mass of the αv subunit under reductive conditions, and indicate the increasing αvβ3 density in the murine tumor model used.</p> <p>(D) The correlation between receptor expression and [¹⁸F]Galacto-RGD accumulation is confirmed by quantitative analysis based on the tumor/background ratios and tumor/muscle ratios calculated from the PET images and from the biodistribution studies, respectively, and by the relative αv expression in Western blot analyses.</p></div

Correlation of Tracer Accumulation and αvβ3 Expression

<div><p>(A–C) patient with a soft tissue sarcoma dorsal of the right knee joint. (A) The sagittal section of a [¹⁸F]Galacto-RGD PET acquired 170 min p. i. shows circular peripheral tracer uptake in the tumor with variable intensity and a maximum SUV of 10.0 at the apical-dorsal aspect of the tumor (arrow). (B) The image fusion of the [¹⁸F]Galacto-RGD PET and the corresponding computed tomography scan after intravenous injection of contrast medium shows that the regions of intense tracer uptake correspond with the enhancing tumor wall, whereas the non-enhancing hypodense center of the tumor shows no tracer uptake. (C) Immunohistochemistry of a peripheral tumor section using the anti-αvβ3 monoclonal antibody LM609 demonstrates intense staining predominantly of tumor vasculature.</p> <p>(D–F) patient with malignant melanoma and a lymph node metastasis in the right axilla. (D) The axial section of a [¹⁸F]Galacto-RGD PET acquired 140 min p. i. shows intense focal uptake in the lymph node (arrow). (E) Image fusion of the [¹⁸F]Galacto-RGD PET and the corresponding computed tomography scan after intravenous injection of contrast medium. (F) Immunohistochemistry of the lymph node using the anti-αvβ3 monoclonal antibody LM609 demonstrates intense staining predominantly of tumor cells and also blood vessels.</p></div

Noninvasive Monitoring of αvβ3 Expression on the Tumor Vasculature

<div><p>(A) Immunohistochemical staining of tumor section using the anti-αvβ3 monoclonal antibody LM609 demonstrates that squamous cell carcinoma cells of human origin do not express the αvβ3 integrin. In contrast, staining of section with an antibody against the murine β3 subunit indicates that the tumor vasculature is αvβ3-positive.</p> <p>(B) Transaxial images of a nude mouse bearing a human squamous cell carcinoma at the right shoulder (left) acquired at the small-animal PET 90 min after tracer injection show a clearly contrasting tumor. Tracer accumulation in the tumor (right, top image) can be blocked by injecting 18 mg of cyclo(-Arg-Gly-Asp-DPhe-Val-) per kilogram of mouse 10 min prior to tracer injection (right, bottom image), indicating receptor-specific accumulation.</p></div

Comparison of [¹⁸F]FDG and [¹⁸F]Galacto-RGD Scans

<div><p>Coronal image sections, acquired 60 min p. i.</p> <p>(A) Patient with malignant melanoma stage IV and multiple metastases in liver, skin, and lower abdomen (arrows): marked uptake of [¹⁸F]FDG in the lesions (left), but no uptake of [¹⁸F]Galacto-RGD (right).</p> <p>(B) Patient with malignant melanoma stage IIIb and a solitary lymph node metastasis in the right axilla (arrow): intense uptake of both [¹⁸F]FDG (left) and [¹⁸F]Galacto-RGD (right).</p></div