PET imaging of VPAC1 expression in experimental and spontaneous prostate cancer

Abstract

Among U.S. men, prostate cancer (PC) accounts for 29% of all newly diagnosed cancers. A reliable scintigraphic agent to image PC and its metastatic or recurrent lesions and to determine the effectiveness of its treatment will contribute to the management of this disease. All PC overexpresses VPAC1 receptors. This investigation evaluated a probe specific for a (64)Cu-labeled receptor for PET imaging of experimental human PC in athymic nude mice and spontaneously grown PC in transgenic mice.

Methods: The probe, TP3939, was synthesized, purified, and labeled with (64)Cu and (99m)Tc. Using a muscle relaxivity assay, biologic activity was assessed and inhibitory concentrations of 50% calculated. Receptor affinity (Kd) for human PC3 cells was determined using (99m)Tc-TP3939 and (64)CuCl(2.) Blood clearance and in vivo stability were studied. After intravenous administration of either (64)Cu-TP3939 or (64)CuCl(2) in PC3 xenografts and in transgenic mice, PET/CT images were acquired. Prostate histology served as the gold standard. Organ distribution studies (percentage injected dose per gram [%ID/g]) in normal prostate were performed. The ratios of tumor to muscle, tumor to blood, normal prostate to muscle, and tumor to normal prostate were determined.

Results: Chemical and radiochemical purities of TP3939 were 96.8% and 98% +/- 2%, respectively. Inhibitory concentrations of 50% and affinity constants were 4.4 x 10(-8) M and 0.77 x 10(-9) M, respectively, for TP3939 and 9.1 x 10(-8) M and 15 x 10(-9) M, respectively, for vasoactive intestinal peptide 28. Binding of (64)CuCl(2) to PC3 was nonspecific. Blood clearance was rapid. In vivo transchelation of (64)Cu-TP3939 to plasma proteins was less than 15%. (64)Cu-TP3939 uptake in PC was 7.48 +/- 3.63 %ID/g at 4 h and 5.78 +/- 0.66 %ID/g at 24 h after injection and was significantly (P < 0.05) greater than with (64)CuCl(2) (4.79 +/- 0.34 %ID/g and 4.03 +/- 0.83 %ID/g at 4 and 24 h, respectively). The ratios of PC to normal prostate at 4 and 24 h were 4 and 2.7, respectively. (64)Cu-TP3939 distinctly imaged histologic grade IV prostate intraepithelial neoplasia in transgenic mice, but (18)F-FDG and CT did not.

Conclusion: Data indicate that TP3939, with its uncompromised biologic activity, delineated xenografts and cases of occult PC that were not detectable with (18)F-FDG. (64)Cu-TP3939 is a promising probe for PET imaging of PC. It may also be useful for localizing recurrent lesions and for determining the effectiveness of its treatment.

FIGURE 1

Schematic presentation of Cu-TP3939 (A) and HPLC eluates of ⁶⁴Cu-TP3939 at 6.7 min and free ⁶⁴Cu at 3.9 min (B). Diagonal line represents gradient.

FIGURE 2

Muscle relaxivity assays as function of concentration for VIP₂₈ and its analogue TP3939. IC₅₀ values are calculated as concentration at which 50% relaxivity occurred.

FIGURE 3

Scatchard plots for ^99mTc-TP3939 binding to PC3 cells known to express VPAC1 oncogene receptors. Kd value was 0.77 × 10⁻⁹ M.

FIGURE 4

⁶⁴CuCl₂ binding assays for PC3 cells performed with added copper carrier (B and D) and with no ⁶⁴CuCl₂ carrier added (A and C). In either case, data reveal nonsaturable, nonspecific binding.

FIGURE 5

Blood clearance curve of ⁶⁴Cu-TP3939 in athymic nude mice (n = 3).

FIGURE 6

Autoradiography (A) and gel stained with Coomassie blue (B) after PAGE analysis of serum isolated from mouse after intravenous administration of ⁶⁴Cu-TP3939. Lane 1 (L1) = ⁶⁴Cu-TP3939 in mouse serum; lane 2 (L2) = ⁶⁴Cu-TP3939 with human serum albumin; lane 3 (L3) = ⁶⁴CuCl₂ with human serum albumin; lane 4 (L4) = ⁶⁴CuCl₂; STD = molecular weight standard.

FIGURE 7

(A) Transaxial PET images demonstrate high uptake of ⁶⁴Cu-TP3939 in xenografted PC3 tumor in right flank of athymic nude mouse (dashed arrows). Images were taken 4 and 24 h after injection of ⁶⁴Cu-TP3939. (B) Presented from left to right are composite of ¹⁸F-FDG, CT, and ⁶⁴Cu-TP3939 images obtained from TRAMP I and TRAMP II transgenic mice and histology of their corresponding prostate glands. One-hour ¹⁸F-FDG images show only bladder activity (solid arrow). In 4-h CT images, dashed and solid arrows show prostate and bladder, respectively. Four-hour ⁶⁴Cu-TP3939 transaxial PET images show only colon activity (oval-head arrow) in TRAMP I, whereas in TRAMP II, significant PC uptake (dashed arrow) and negligible colon uptake are seen. Prostate histology (×40) indicates grade II and grade IV prostate intraepithelial neoplasia in TRAMP I and TRAMP II, respectively (black arrows). Tumor with histology grade IV is visible with ⁶⁴Cu-TP3939 but not with ¹⁸F-FDG.