Preclinical Study of a Dual-Target Molecular Probe Labeled with 68Ga Targeting SSTR2 and FAP

Abstract

Objective: Currently, ⁶⁸Ga-labeled somatostatin analogs (SSAs) are the most commonly used imaging agents for patients with neuroendocrine tumors (NETs) in clinical practice, demonstrating good results in tumor diagnosis. For applications in peptide receptor radionuclide therapy (PRRT), targeted drugs should have high tumor uptake and prolonged tumor retention time. To enhance the uptake and retention of tracers in NETs, our goal is to design a ⁶⁸Ga-labeled heterodimer for optimizing pharmacokinetics and assess whether this form is more efficacious than its monomeric equivalents.

Methods: Using the somatostatin analog TATE and quinoline-based compound FAPI-46 as raw materials, we designed and synthesized ⁶⁸Ga-labeled TATE-46. The labeling efficiency and stability were verified by Radio-HPLC. The receptor binding properties and tumor targeting were examined both in vitro and in vivo by using NCI-H727 (SSTR2/FAP, positive) and Mc38 (SSTR2/FAP, negative) cell lines and tumor-bearing mouse models. Preclinical evaluation was performed through cell uptake, pharmacokinetics, Micro PET, and biodistribution studies, and the results were compared with [⁶⁸Ga]Ga-DOTA-TATE and [⁶⁸Ga]Ga -FAPI-46. Immunohistochemistry and HE staining were performed on tumor tissues from tumor-bearing mice for further validation.

Results: [⁶⁸Ga]Ga-TATE-46 showed comparable SSTR2 and FAP targeting ability to monomeric TATE and FAPI-46 in cell uptake and PET imaging studies. [⁶⁸Ga]Ga-TATE-46 exhibited significantly higher uptake in NCI-H727 (SSTR2/FAP, positive) tumors compared to [⁶⁸Ga]Ga-DOTA-TATE (p < 0.001) and [⁶⁸Ga]Ga-FAPI-46 (p < 0.001). No increased uptake of [⁶⁸Ga]Ga-TATE-46 was observed in MC38 tumors (SSTR2/FAP, negative). Additionally, excess DOTA-TATE and/or unlabeled FAPI-46 significantly blocked the uptake of [⁶⁸Ga]Ga-TATE-46 in NCI-H727 tumors (p < 0.001), confirming its dual-receptor targeting characteristics. The ex vivo biodistribution, immunofluorescence and immunohistochemistry results were in line with the in vivo imaging findings.

Conclusion: Compared with ⁶⁸Ga-labeled FAPI-46 and DOTA-TATE mono-specific tracers, the dual-target tracer [⁶⁸Ga]Ga-TATE-46 improves tumor uptake, extends tumor retention, and enhances pharmacokinetics. It is an effective probe for non-invasive detection of tumors expressing FAP and SSTR2, and it is worth further studying its application in the expression of sstr2 and FAP-related tumors.

Keywords: PET; fibroblast activation protein; heterodimer; somatostatin receptor 2.

Figure 1

The chemical structures of [⁶⁸Ga]Ga-TATE-46 (A), [⁶⁸Ga]Ga-DOTA-TATE (C), and [⁶⁸Ga]Ga-FAPI-46 (B).

Figure 2

(A) Radiochemical HPLC analysis of [⁶⁸Ga]Ga-TATE-46. (B) In vitro stability of [⁶⁸Ga]Ga-TATE-46 in physiological saline over 1 and 2 h. (C) In vitro stability of [⁶⁸Ga]Ga-TATE-46 in FBS over 1 and 2 h.

Figure 3

In vitro studies. (A) Uptake of [⁶⁸Ga]Ga-TATE-46 by NCI-H727 and MC38 cells at 30 min, 1 h, 2 h, and 4 h. (B) Uptake of [⁶⁸Ga]Ga-TATE-46, [⁶⁸Ga]Ga-DOTA-TATE, and [⁶⁸Ga]Ga-FAPI-46 by NCI-H727 cells at 30 min, 1 h, 2 h, and 4 h. (C) Blocking of [⁶⁸Ga]Ga-TATE-46 uptake by NCI-H727 cells with unlabeled DOTA-TATE, FAPI-46, and TATE + FAPI-46 (data are expressed as mean ± SD, n = 3; ** p < 0.01, *** p < 0.001).

Figure 4

Time–activity curves for [⁶⁸Ga]Ga-TATE-46 (A), [⁶⁸Ga]Ga-DOTA-TATE (B), and [⁶⁸Ga]Ga-FAPI-46 (C) in BALB/C mice, respectively.

Figure 5

(A) Biodistribution of [⁶⁸Ga]Ga-TATE-46 in NCI-H727 tumor-bearing mice at 0.5, 1, 2, and 4 h post-injection (each mouse received 5.55–7.4 MBq). (B) Biodistribution of [⁶⁸Ga]Ga-TATE-46, [⁶⁸Ga]Ga-DOTA-TATE, and [⁶⁸Ga]Ga-FAPI-46 in NCI-H727 tumor-bearing mice at 1h p.i. (*** p < 0.001).

Figure 6

Micro PET scans of NCI-H727 tumor-bearing mice with [⁶⁸Ga]Ga-TATE-46. (A) [⁶⁸Ga]Ga-TATE-46, (B) [⁶⁸Ga]Ga-FAPI-46, and (C) [⁶⁸Ga]Ga-DOTA-TATE (each injected with 5.55–7.4 MBq) at 30 min, 1 h, 2 h, and 4 h post-injection. (D) Quantitative analysis of tumor uptake from PET images using ROIs, n = 3; ***p < 0.001.

Figure 7

Micro PET imaging and quantitative analysis demonstrate the specificity of [⁶⁸Ga]Ga-TATE-46 for SSTR2 and FAP expression in tumors. (A) PET images of NCI-H727 and MC38 tumor-bearing mice at 1 h post-injection of [⁶⁸Ga]Ga-TATE-46 (5.55–7.4 MBq) with blocking agents DOTA-TATE (10 μg/g), FAPI-46 (10 μg/g), or DOTA-TATE (10 μg/g) + FAPI-46 (10 μg/g). The red arrows are tumors. (B) ROI quantitative analysis of tumor uptake from PET images. (** p < 0.01, *** p < 0.001).

Figure 8

Immunofluorescence (A) and immunohistochemistry/HE staining (B) analyses of SSTR2 and FAP in NCI-H727 and MC38 tumors.