64Cu-Labeled Alpha-Melanocyte-Stimulating Hormone Analog for MicroPET Imaging of Melanocortin 1 Receptor Expression

The alpha-melanocyte-stimulating hormone (α-MSH) receptor (melanocortin type 1 receptor, or MC1R) plays an important role in the development and growth of melanoma cells. It was found that MC1R was overexpressed on most murine and human melanoma, making it a promising molecular target for melanoma imaging and therapy. Radiolabeled α-MSH peptide and its analogs that can specifically bind with MC1R have been extensively explored for developing novel agents for melanoma detection and radionuclide therapy. The goal of this study was to evaluate a (64)Cu-labeled α-MSH analog, Ac-Nle-Asp-His-d-Phe-Arg-Trp-Gly-Lys(DOTA)-NH(2) (DOTA–NAPamide), as a potential molecular probe for microPET imaging of melanoma and MC1R expression in melanoma xenografted mouse models. 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugated NAPamide was synthesized and radiolabeled with (64)Cu (t(1/2)=12 h) in NH(4)OAc (0.1 M; pH 5.5) buffered solution for 60 min at 50 °C. Cell culture studies reveal rapid and high uptake and internalization of (64)Cu–DOTA–NAPamide in B16F10 cells. Over 90% of receptor-bound tracer is internalized at 3 h incubation. A cellular retention study demonstrates that the receptor-bound (64)Cu–DOTA–NAPamide is slowly released from the B16F10 cells into the medium; 66% of the radioactivity is still associated with the cells even after 3 h incubation. The biodistribution of (64)Cu–DOTA–NAPamide was then investigated in C57BL/6 mice bearing subcutaneous murine B16F10 melanoma tumors with high capacity of MC1R and Fox Chase Scid mice bearing human A375M melanoma with a relatively low number of MC1R receptors. Tumor uptake values of (64)Cu–DOTA–NAPamide are found to be 4.63 ± 0.45% and 2.49 ± 0.31% ID/g in B16F10 and A375M xenografted melanoma at 2 h postinjection (pi), respectively. The B16F10 tumor uptake at 2 h pi is further inhibited to 2.29 ± 0.24% ID/g, while A375M tumor uptake at 2 h pi remains 2.20 ± 0.41% ID/g with a coinjection of excess α-MSH peptide. MicroPET imaging of (64)Cu–DOTA–NAPamide in B16F10 tumor mice clearly shows good tumor localization. However, low A375M tumor uptake and poor tumor to normal tissue contrast were observed. This study demonstrates that (64)Cu–DOTA–NAPamide is a promising molecular probe for α-MSH receptor positive melanoma PET imaging as well as MC1R expression imaging in living mice

18F-EF5 PET Is Predictive of Response to Fractionated Radiotherapy in Preclinical Tumor Models.

We evaluated the relationship between pre-treatment positron emission tomography (PET) using the hypoxic tracer 18F-[2-(2-nitro-1-H-imidazol-1-yl)-N-(2,2,3,3,3- pentafluoropropyl) acetamide] (18F-EF5) and the response of preclinical tumor models to a range of fractionated radiotherapies. Subcutaneous HT29, A549 and RKO tumors grown in nude mice were imaged using 18F-EF5 positron emission tomography (PET) in order to characterize the extent and heterogeneity of hypoxia in these systems. Based on these results, 80 A549 tumors were subsequently grown and imaged using 18F-EF5 PET, and then treated with one, two, or four fraction radiation treatments to a total dose of 10-40 Gy. Response was monitored by serial caliper measurements of tumor volume. Longitudinal post-treatment 18F-EF5 PET imaging was performed on a subset of tumors. Terminal histologic analysis was performed to validate 18F-EF5 PET measures of hypoxia. EF5-positive tumors responded more poorly to low dose single fraction irradiation relative to EF5-negative tumors, however both groups responded similarly to larger single fraction doses. Irradiated tumors exhibited reduced 18F-EF5 uptake one month after treatment compared to control tumors. These findings indicate that pre- treatment 18F-EF5 PET can predict the response of tumors to single fraction radiation treatment. However, increasing the number of fractions delivered abrogates the difference in response between tumors with high and low EF5 uptake pre-treatment, in agreement with traditional radiobiology

¹⁸F-EF5 PET Is Predictive of Response to Fractionated Radiotherapy in Preclinical Tumor Models

<div><p>We evaluated the relationship between pre-treatment positron emission tomography (PET) using the hypoxic tracer ¹⁸F-[2-(2-nitro-1-H-imidazol-1-yl)-N-(2,2,3,3,3- pentafluoropropyl) acetamide] (¹⁸F-EF5) and the response of preclinical tumor models to a range of fractionated radiotherapies. Subcutaneous HT29, A549 and RKO tumors grown in nude mice were imaged using ¹⁸F-EF5 positron emission tomography (PET) in order to characterize the extent and heterogeneity of hypoxia in these systems. Based on these results, 80 A549 tumors were subsequently grown and imaged using ¹⁸F-EF5 PET, and then treated with one, two, or four fraction radiation treatments to a total dose of 10–40 Gy. Response was monitored by serial caliper measurements of tumor volume. Longitudinal post-treatment ¹⁸F-EF5 PET imaging was performed on a subset of tumors. Terminal histologic analysis was performed to validate ¹⁸F-EF5 PET measures of hypoxia. EF5-positive tumors responded more poorly to low dose single fraction irradiation relative to EF5-negative tumors, however both groups responded similarly to larger single fraction doses. Irradiated tumors exhibited reduced ¹⁸F-EF5 uptake one month after treatment compared to control tumors. These findings indicate that pre- treatment ¹⁸F-EF5 PET can predict the response of tumors to single fraction radiation treatment. However, increasing the number of fractions delivered abrogates the difference in response between tumors with high and low EF5 uptake pre-treatment, in agreement with traditional radiobiology.</p></div

Plots of response of more and less hypoxic tumors to (A) single fraction and (B) multiple fraction treatments.

<p>Volumes are normalized to pre-treatment volumes.</p

(A) Representative axial slices from microCT (top row), ¹⁸F-EF5 microPET (middle row), and anti-EF5 immunohistochemistry on (bottom row) of HT29, A549 and RKO subcutaneous tumors. CT image intensities represent Hounsfield Units (HU). PET images have been normalized by the mean background muscle %ID/g to yield tumor/muscle ratio (T/M) images. T on microCT and microPET images denotes tumor location. Brown stained areas in IHC images denote regions of bound EF5. (B) Scatterplot of the distribution of ¹⁸F-EF5 uptake in individual HT29, A549 and RKO subcutaneous tumors using three different metrics, T/M measured using CT-derived ROIs (T/MCT), T/M measured using PET-derived ROIs (T/MPET) and %ID/g values measured using CT-derived ROIs (%ID/gCT). (C) Scatterplot of the relationship between T/MCT for HT29, A549 and RKO tumors and their volume.

<p>(A) Representative axial slices from microCT (top row), ¹⁸F-EF5 microPET (middle row), and anti-EF5 immunohistochemistry on (bottom row) of HT29, A549 and RKO subcutaneous tumors. CT image intensities represent Hounsfield Units (HU). PET images have been normalized by the mean background muscle %ID/g to yield tumor/muscle ratio (T/M) images. T on microCT and microPET images denotes tumor location. Brown stained areas in IHC images denote regions of bound EF5. (B) Scatterplot of the distribution of ¹⁸F-EF5 uptake in individual HT29, A549 and RKO subcutaneous tumors using three different metrics, T/M measured using CT-derived ROIs (T/MCT), T/M measured using PET-derived ROIs (T/MPET) and %ID/g values measured using CT-derived ROIs (%ID/gCT). (C) Scatterplot of the relationship between T/MCT for HT29, A549 and RKO tumors and their volume.</p

Effect of radiation on tumor ¹⁸F-EF5 uptake.

<p>(A) Representative axial ¹⁸F-EF5 PET images of two tumors at 1 day pre-treatment and 13 / 32 days post-treatment. Solid arrow denotes unirradiated control tumor, and dashed arrow denotes tumor which was treated with 20 Gy. (B,C) Plots showing changes in ¹⁸F-EF5 uptake and volume, respectively, in control (n = 6) and irradiated (n = 6) tumors during the treatment timecourse.</p