Trop2 immuno-PET/CT imaging of prostate cancer: a proof-of-concept translational study

Abstract

Background: Prostate cancer (PCa) is the second leading cause of death in men and is highly prone to metastasis. This study aims to develop the novel immuno-PET/CT tracer targeting trophoblast cell surface antigen 2 (Trop2), a transmembrane protein overexpressed in aggressive prostate malignancies, and to investigate its diagnostic value in preclinical studies as well as its potential utility in detecting metastases in PCa patients.

Methods: Integrated analysis of TCGA, GEO, and HPA datasets revealed Trop2 overexpression in prostate adenocarcinoma. By labeling two Trop2-targeted nanobodies (His-tagged T4 and His-tag-free RT4) with ⁶⁸Ga and ¹⁸F, we synthesized three radiotracers, [⁶⁸Ga]Ga-NOTA-T4, [¹⁸F]AlF-RESCA-T4, and [¹⁸F]AlF-RESCA-RT4. Preclinical validation included cellular assays and xenograft studies for ⁶⁸Ga/¹⁸F-T4 variants, followed by a first-in-human trial evaluating [¹⁸F]AlF-RESCA-RT4 in ten treatment-naïve PCa patients.

Results: Bioinformatics analysis demonstrated Trop2 as a promising target for PCa diagnosis and treatment. In preclinical studies, both [⁶⁸Ga]Ga-NOTA-T4 and [¹⁸F]AlF-RESCA-T4 illustrated high affinity to Trop2, specific tumor uptake (4.33 ± 0.38 %ID/g and 5.50 ± 0.69 %ID/g at 60 min, respectively) in Trop2-positive xenograft mouse models with minimal background distribution. In the translational study, [¹⁸F]AlF-RESCA-RT4 outperformed standard [¹⁸F]-FDG imaging in metastatic prostate cancer patients, detecting 62.4% more lymph node metastases (SUVmax 21.58 ± 13.12 vs. 4.80 ± 1.77) and 69.4% more bone lesions (SUVmax 7.83 ± 4.32 vs. 4.72 ± 1.22). No adverse events were observed.

Conclusions: This work successfully establishes that Trop2 is a new biomarker for PCa, and Trop2 immuno-PET/CT imaging can detect PCa lymph node and osseous metastases. The superior tumor-to-background contrast and clinical safety of [¹⁸F]AlF-RESCA-RT4 support its translational potential to guide Trop2-targeted therapies and enhance personalized treatment strategies.

Trial registration: NCT06851663. Retrospectively registered 02/24/2025.

Keywords: Immuno-PET/CT; Nanobody; Nuclear medicine; Prostate cancer; Trop2.

Fig. 1

Expression pattern of Trop2 in human prostate cancer (PCa). A Trop2 (encoded by the TACSTD2 gene) expression levels in various cancer types were analyzed using the TIMER database, which is based on the TCGA database. B The differential mRNA level of Trop2 was analyzed between 492 PCa tissues (red) and 152 normal prostate tissues (gray) from the TCGA and GTEx databases using the GEPIA database. *P < 0.05. C The difference in Trop2 protein expression at the protein level between PCa tissue and normal prostate tissue was demonstrated by immunohistochemical results obtained from the HPA database (antibody CAB072852). D–F Trop2 expression levels in the single-cell transcriptome. The UMAP dimensional plot showed the cell types (D) and the Trop2 expression level (E) in each cell. F The violin plot exhibited the expression of Trop2 in different cell types

Fig. 2

Synthesis of Trop2-targeted radiotracers and cellular studies. A Radiochemistry purity of [⁶⁸Ga]Ga-NOTA-T4 (left) and B [¹⁸F]AlF-RESCA-T4 (right) after purification. C Trop2 and PSMA expression assay in prostate cancer cells PC3, DU145, and 22RV1 using western blot. Uncropped gel images shown in Additional file 2. D Saturation binding curve illustrating interaction of [⁶⁸Ga]Ga-NOTA-T4 with PC3 tumor cells (n = 4). E Competitive binding curve demonstrating displacement of [⁶⁸Ga]Ga-NOTA-T4 by unlabeled T4 on PC3 tumor cells (n = 4). F Uptake with [⁶⁸Ga]Ga-NOTA-T4 in PC3 cells and 22RV1 cells (n = 3). ****P < 0.0001. G Uptake with [¹⁸F]AlF-RESCA-T4 and blocking experiment with unlabeled T4 in PC3 cells (n = 3). ****P < 0.0001

Fig. 3

[⁶⁸Ga]Ga-NOTA-T4 Immuno-PET/CT Imaging in prostate cancer models. A, D Representative [⁶⁸Ga]Ga-NOTA-T4 immuno-PET/CT images of Trop2-positive PC3 tumor (n = 3) and Trop2-negative 22RV1 tumor (n = 3) bearing mice at 60-min points after injection of the tracer. The tumors were displayed on the images with yellow arrows. B, E The ROI analysis of the Trop2-positive group and the Trop2-negative group. C, F Ex vivo biodistribution data of 2 groups. G, H The Trop2 immunohistochemistry (IHC) staining results of PC3 and 22RV1 tumor tissues. I The PSMA IHC staining results of PC3 tumor tissue

Fig. 4

[¹⁸F]AlF-RESCA-T4 immuno-PET/CT imaging of Trop2-positive PC3 model. A, D At 60-min points, images of the non-blocking (n = 3) and 400 µg-blocking (n = 3) groups after [¹⁸F]AlF-RESCA-T4 injection. B, E ROI quantitative analysis of 2 groups. C, F Ex vivo biodistribution data of 2 groups. Yellow arrows: tumor

Fig. 5

Physiological high uptake of [¹⁸F]AlF-RESCA-RT4 by prostate affects the diagnosis of primary tumor. A 58-year-oldman with prostate cancer. A–C Multi-parametric magnetic resonance imaging suggested a nodule in the left peripheral band of the prostate, with high signal intensity on T2-weighted image and markedly restricted diffusion (red arrows). D [¹⁸F]AlF-RESCA-RT4 showed intense physiological uptake in prostate (red dotted line), which significantly affected identification of primary lesion

Fig. 6

The comparison of [¹⁸F]AlF-RESCA-RT4 and [¹⁸F]-FDG uptake in prostate cancer metastases. A The number and B SUVmax distribution of lymph node and bone metastases of [¹⁸F]AlF-RESCA-RT4 and [¹⁸F]-FDG. C The relationship between [¹⁸F]AlF-RESCA-RT4 SUVmax and [¹⁸F]-FDG SUVmax was analyzed with curve fittings on lesion-based analysis. All metastases were detected from 10 PCa patients. ****P < 0.0001

Fig. 7

Representative PET/CT images in a patient with prostate cancer. A 53-year-old man with prostate cancer underwent PET/CT for initial staging. A [¹⁸F]AlF-RESCA-RT4 showed intense tracer uptake in widespread bone metastases (b and c, red arrowheads) and multiple regional lymph nodes (including left clavicular region, mediastinum, posterior diaphragm, para-abdominal aorta, and bilateral para-iliac vessel lymph nodes; e and g, blue arrowheads). B Corresponding lesions showed low or mild FDG uptake