Diagnosis of Prostate Cancer with a Neurotensin–Bombesin Radioligand Combination—First Preclinical Results

Prostate cancer cells often express multiple peptide receptors, including the gastrin-releasing peptide receptor (GRPR) and neurotensin subtype 1 receptor (NTS1R). While single-targeted tracers can visualize lesions, diagnostic sensitivity may be limited if receptor expression varies across different disease stages. A dual-targeting approach could overcome this limitation and improve tumor diagnostic sensitivity by targeting receptors with increased tumor expression during different disease stages.

This study used SPECT to investigate whether combining NTS1R- and GRPR-specific radioligands would enhance uptake in prostate cancer xenografts, and whether treatment with the neprilysin inhibitor sacubitrilat (via Entresto®) could further improve tumor targeting.

Two Tc-99m-labeled peptide radioligands were developed and tested in vivo: [99mTc]Tc-DT11, which specifically targets the neurotensin subtype 1 receptor (NTS1R), and [99mTc]Tc-DB7, which specifically targets the gastrin-releasing peptide receptor (GRPR).

To study in vivo performance, biodistribution experiments were performed on SCID mice bearing PC-3 tumor xenografts. The animals were divided into different groups: untreated controls, mice pre-treated with Entresto® to inhibit neprilysin and stabilize the tracers, and mice that received one of two selective receptor-blocking agents or their combination, blocking NTS1R or GRPR.

SPECT/CT imaging was carried out in tumor-bearing mice to visualize and quantify radioligand uptake in tumors and background tissues. For this, tumor-bearing mice first received Entresto® orally, followed 30 minutes later by intravenous injection of an equimolar mixture of [99mTc]Tc-DT11 and [99mTc]Tc-DB7 (containing 185 kBq and 5 pmol of each analog).

SPECT scans were carried out on the γ-CUBE system using a 40-minute acquisition protocol, while CT scans were performed on the x-CUBE system with a high-resolution 50 kVp protocol. The SPECT images were reconstructed with the MLEM method (250 μm voxel size, 100 iterations), and CT images were reconstructed using the ISRA method (100 μm voxel size). This combined protocol enabled both high-contrast functional imaging of tracer distribution and precise anatomical localisation of tumors.

SPECT/CT imaging showed intense and well-defined uptake of the [99mTc]Tc-DT11 + [99mTc]Tc-DB7 mixture in PC-3 tumors, with low background activity in normal tissues. The dual-targeting approach provided clear tumor-to-background contrast and precise anatomical localization. In mice that received receptor-blocking agents, tumor signals were strongly reduced or absent, confirming that uptake was specifically mediated by NTS1R and GRPR. The use of Entresto® further enhanced tumor uptake without increasing off-target activity, supporting its role in stabilizing the tracers for improved imaging.

Adapted from Bibika et al.

Static whole body SPECT/CT images 4 h pi of an equimolar [99mTc]Tc-DT11+ [99mTc]Tc-DB7 mixture alone (a,b) or together with 100 µg NT and 50 µg [Tyr4]BBN for twin NTS1R and GRPR-blockade (c). All animals had orally received Entresto® 30 min in advance. Yellow arrows indicate the position of PC-3 xenografts; images correspond to maximum intensity projections, and intensity of uptake is represented by the color scale on the right of each image. Blue corresponds to the lowest and yellow to the highest value.