A heterodimeric radioligand labeled with gallium-68 targeting fibroblast activation protein

Abstract

Background

Fibroblast activation protein (FAP) targeting radiotracers have emerged as promising agents for cancer imaging and therapy. Recent advancements have focused on optimizing these agents for better tumor targeting and enhanced theranostic efficacy. In this study, we introduced a novel heterodimeric radioligand labeled with gallium-68, which targets FAP. We aimed to evaluate its in vitro and in vivo performance, comparing its efficacy with monomeric FAPI derivatives.

Results

The heterodimeric ligand BiFAPI was synthesized by conjugating a cyclic peptide with a quinoline-based motif via a DOTA chelator. [⁶⁸ Ga]Ga-BiFAPI demonstrated high radiochemical purity (> 95%) and exceptional stability in physiological conditions, as well as in both PBS and serum. In vitro studies revealed that the binding affinity of BiFAPI was comparable to that of FAP2286 and FAPI-04. Notably, [⁶⁸ Ga]Ga-BiFAPI exhibited superior cellular uptake, with rapid internalization and slower efflux rates. Micro-PET/CT imaging in tumor-bearing mice demonstrated significantly higher tumor uptake than [⁶⁸ Ga]Ga-FAP2286 and [⁶⁸ Ga]Ga-FAPI-04. Co-injection with a FAP inhibitor reduced tumor uptake, confirming the tracer’s FAP specificity. In vitro autoradiography, immunohistochemistry, and Western blotting confirmed the correlation between radioactive tracer accumulation and FAP-positive regions. Biodistribution studies revealed high tumor-to-blood ratios and rapid clearance from non-target tissues, further supporting the tracer’s favorable pharmacokinetics.

Conclusion

[⁶⁸ Ga]Ga-BiFAPI demonstrated superior tumor-targeting properties, higher tumor uptake, and favorable pharmacokinetics compared to [⁶⁸ Ga]Ga-FAP2286 and [⁶⁸ Ga]Ga-FAPI-04. Its promising performance in preclinical models positioned it as a potentially valuable agent for FAP-targeted PET imaging and cancer theranostics.

Introduction

Cancer-associated fibroblasts (CAFs) are pivotal components of the tumor microenvironment, often constituting a significant portion of the tumor mass across various malignancies [1, 2]. CAFs play crucial roles in fostering tumor progression, immune evasion, and the invasive capabilities of cancer cells; as a result, they have emerged as prominent targets for therapeutic interventions and diagnostic approaches in oncology [3]. A key molecular marker that distinguishes CAFs is fibroblast activation protein (FAP), a type II transmembrane serine protease from the prolyl oligopeptidase family [4]. FAP is frequently overexpressed in CAFs within over 90% of epithelial cancers, whereas its expression in normal tissues is minimal or absent [5]. This tumor-specific distribution and its functional roles have positioned FAP as an attractive target for broad diagnostic and therapeutic applications. Additionally, FAP-positive CAFs are associated with poor prognosis, as they contribute to tumor growth and an immunosuppressive microenvironment [6]. These factors underscore the potential of FAP as a critical target for the development of innovative cancer therapies and imaging strategies.

A promising approach to targeting FAP involves the use of theranostic radioligands labeled with radioisotopes [7]. The introduction of the first FAP-specific inhibitor-based radioligand, FAPI-04 [8], in 2018 initiated the development of a series of quinoline derivatives, such as FAPI-02 [7], FAPI-21 [9], and FAPI-46 [10]. These tracers, labeled with gallium-68, have demonstrated superior diagnostic performance compared to traditional [¹⁸F]FDG in certain tumor types. However, radionuclide therapies using ¹⁷⁷Lu-labeled FAPI derivatives, such as [¹⁷⁷Lu]Lu-FAPI-04 and [¹⁷⁷Lu]Lu-FAPI-46, face challenges due to rapid clearance and limited tumor retention [11, 12]. Many of these FAPI tracers are based on the potent FAP inhibitor UAMC1110, a quinoline derivative. Furthermore, FAP-2286, a novel class of radiopharmaceuticals utilizing cyclic peptides for binding, has emerged as a promising candidate for FAP-targeted imaging and therapy [13]. Despite these advancements, further clinical validation is necessary to assess the efficacy of these next-generation tracers in FAP-positive tumors.

To address the limitations of current FAPI-based radioligands, researchers have focused on optimizing pharmacokinetics through structure–activity relationship studies [14,15,16,17]. These efforts have led to the development of modified tracers conjugated with fatty acid/albumin binders and bivalent FAPI ligands, which exhibit improved tumor accumulation and extended circulation times. Preclinical studies in tumor-bearing mice have demonstrated promising anti-tumor effects, suggesting the therapeutic potential of these optimized ligands. The concept of multivalency has gained significant attraction in the design of molecular imaging probes, as it enhances detection sensitivity and improves binding specificity and affinity [18]. Multivalent imaging agents, typically developed by conjugating targeting moieties to bifunctional chelators such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), exhibit notably higher tumor uptake and prolonged retention times compared to their monovalent counterparts. This enhanced retention within tumors not only leads to improved imaging contrast but also offers potential therapeutic benefits. Numerous radiopharmaceuticals have been successfully developed through multivalent approaches, with notable examples including those targeting integrin αvβ3 [19,20,21], prostate-specific membrane antigen (PSMA) [22,23,24], and FAP [25,26,27,28,29]. These examples highlight the effectiveness of this strategy in enhancing molecular imaging and therapeutic applications.

In this study, we designed and synthesized a novel heterodimeric FAP-targeting imaging probe, BiFAPI, incorporating a bifunctional DOTA chelator. This agent combines two previously reported substructures: a quinoline-based motif and cyclic peptides, both of which showed high binding affinity towards FAP. Utilizing a heteromultivalent design, BiFAPI was developed to address the limitations of earlier monomeric compounds, such as rapid in vivo metabolism and insufficient tumor retention. Experimental results revealed that [⁶⁸ Ga]Ga-BiFAPI exhibited significantly enhanced cellular uptake, and greater tumor accumulation compared to [⁶⁸ Ga]Ga-FAPI-04 and [⁶⁸ Ga]Ga-FAPI-2286. These findings demonstrated the potential of [⁶⁸ Ga]Ga-BiFAPI as a promising positron emission computed tomography (PET) imaging agent for cancer diagnosis and its prospective role in future radiotherapeutic applications.

Materials and methods

Synthesis of the precursor

All reagents were commercially available and used without further purification unless otherwise indicated. The synthesis route of BiFAPI were described in detail in the Supplementary Information (Supplemental Scheme. S1).

Radiolabeling and quality control

The radiolabeling of [⁶⁸ Ga]Ga-BiFAPI01, [⁶⁸ Ga]Ga-FAPI-04 or [⁶⁸ Ga]Ga-FAP2286 was conducted by adding 0.5 mL of a 0.05 N hydrochloric acid solution containing [⁶⁸ Ga]GaCl₃ (74–148 MBq) to a reaction vial. Sodium acetate (60 μL, 0.5 N) was then added to adjust the pH to 3.5, followed by the addition of either 15 μL of BiFAPI01 precursor solution (1 mg in 1 mL DMSO), or 10 μL of FAP2286/FAPI-04 precursor solution (1 mg in 1 mL DMSO). The mixture was vortexed to ensure homogeneity and subsequently heated at 95 °C for 10 min. The radiochemical purity (RCP) of the radiolabeled products was analyzed using a high-performance liquid chromatography (HPLC) system. The chromatographic analysis was performed on a C18 column (Agilent, ZORBAX SB-C18, 5 μm, 4.6 × 150 mm). The mobile phase consisted of a gradient of acetonitrile (ACN) and water (containing 0.1% trifluoroacetic acid): 0–10 min, 5%–100% ACN and 95%–0% water; 10–15 min, 100% ACN.

In vitro and in vivo stability studies

For the in vivo stability study, normal mice were intravenously injected with 37 MBq of the radiolabeled compound. At 0.5 h post-injection, blood samples were collected by enucleation. Equal volumes of acetonitrile were added to the blood samples, followed by centrifugation to separate the supernatant. The supernatant was filtered and analyzed using the HPLC system.

For in vitro stability studies, 10 μL of the radiolabeled compound was added to 90 μL of PBS or murine serum, and the mixture was incubated at 37 °C. After 2 h, the RCP was determined using HPLC.

Determination of the partition coefficient (Log P)

To determine the partition coefficient (Log P), the radiolabeled compound was mixed with equal volumes of n-octanol and PBS. The mixture was vortexed and centrifuged to separate the organic and aqueous phases. The aqueous phase was re-extracted with fresh n-octanol. Equal volumes of the aqueous and organic phases were collected in triplicate, and their radioactivity was measured using a gamma counter. The average counts were used to calculate the Log P value.

In vitro cellular evaluation

Cell lines and quantification of FAP expression

The FTC-133 thyroid cancer cell line was purchased from the Kunming Cell Bank, Chinese Academy of Sciences. The HT-1080 human fibrosarcoma cell line and its FAP-transfected derivative, HT-1080-hFAP, were provided by Fenghui Biotechnology Co., Ltd. FTC-133 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. HT-1080 and HT-1080-FAP cells were cultured in DMEM medium with 10% FBS and 1% penicillin–streptomycin. All cells were maintained at 37 °C in a humidified incubator with 5% CO₂.

FAP protein expression levels in the three cell lines were quantified by Western blot analysis. Cells were lysed in lysis buffer containing PMSF (1:100 dilution). After centrifugation, the supernatant containing total protein was collected. Protein concentrations were measured using a BCA kit, and samples were diluted to the same concentration. Denatured protein samples were loaded onto SDS-PAGE gels and transferred to PVDF membranes. Membranes were blocked and incubated with primary antibodies against FAP (1:1000, Abcam), GLUT1 (1:1500, Abcam), and GAPDH (1:10,000, Proteintech), followed by secondary antibody incubation. Chemiluminescent detection was performed using an imaging system, and target band densities were quantified using IPWIN60 software.

Cellular studies

HT-1080 and HT-1080-FAP cells were seeded in 24-well plates and incubated at 37°C for 24–48 h. Cells were washed twice with D-PBS containing calcium and magnesium, then incubated with 0.5 mL of DMEM containing 74 kBq of [⁶⁸ Ga]Ga-BiFAPI, [⁶⁸ Ga]Ga-FAPI-04 or [⁶⁸ Ga]Ga-FAP2286, with or without the FAP inhibitor UAMC1110. After incubation for 10, 30, 60, and 120 min, the medium was removed, and cells were washed twice with ice-cold PBS. Surface-bound radioactivity was stripped using glycine–HCl solution (50 mM, pH 2.8), and the remaining cells were lysed with NaOH (1 M). Radioactivity was measured using a gamma counter. Each group included three replicates, and each experiment was repeated three times.

Efflux assays were performed similarly to cellular uptake sudies except cells were incubated with the radiolabeled compounds for 1 h, followed by washing with cold D-PBS and further incubation in DMEM without radiotracers. At various time points, cells were lysed with NaOH, and radioactivity was measured.

Affinity assays were conducted by incubating HT1080-hFAP cells with 74 kBq of [⁶⁸ Ga]Ga-FAP2286 and varying concentrations (10⁻³ to 10⁴ nM) of FAP2286, FAPI-04 or BiFAPI in 0.5 mL of DMEM. After 60 min of incubation, cells were washed, lysed with NaOH, and radioactivity was measured using a gamma counter.

In vivo evaluation in animal models

Tumor-bearing mouse model

Female Balb/c-nu/nu nude mice (4–6 weeks old) were purchased from the Laboratory Animal Center of the First Affiliated Hospital of Sun Yat-sen University. All animal experiments were conducted under approval from the Institutional Animal Care and Use Committee (IACUC). Mice were housed in an IVC system with standard conditions. Approximately 5 × 10⁶ FTC-133 cells were injected subcutaneously into the right flank of each mouse, while 5 × 10⁶ HT-1080 cells were injected into the left flank. Mice were used for subsequent experiments once tumors reached a diameter of 8–10 mm.

Micro-PET/CT imaging

Mice were fasted for 6–8 h prior to imaging. After weighing, mice were anesthetized with chloral hydrate (10%, 0.1 mL/10 g) and injected via the tail vein with 37 MBq of [⁶⁸ Ga]Ga-BiFAPI, [⁶⁸ Ga]Ga-FAP2286 or [⁶⁸ Ga]Ga-FAP-04. The blocking study was carried out by co-injection with 3 μL of FAPI-04 (1 mg/mL). Micro-PET/CT imaging was conducted 1 h post-injection with a scan time of 10 min. Dynamic PET scans were performed immediately after injecting 37 MBq of the radiotracer via the tail vein, with a total scan time of 2 h and frame collection every 5 min.

Biodistribution studies

Mice were injected with 11.1 MBq of [⁶⁸ Ga]Ga-BiFAPI, [⁶⁸ Ga]Ga-FAP2286 or [⁶⁸ Ga]Ga-FAP-04. At 10, 30, 60, and 120 min post-injection, mice were anesthetized and euthanized. Tissues, including heart, spleen, pancreas, brain, liver, blood, kidney, intestine, stomach, muscle, skin, bone, and tumor, were collected, weighed, and measured for radioactivity using a gamma counter. The blocking study was carried out by co-injection with 3 μL of FAPI-04 (1 mg/mL).

Autoradiography

For in vivo autoradiography, mice were injected with 11.1 MBq of [⁶⁸ Ga]Ga-BiFAPI and euthanized 1 h post-injection. Tumors were rapidly frozen in liquid nitrogen, embedded in OCT, and sectioned at 60 μm. Sections were exposed to a phosphor screen for 12 h and scanned. Adjacent sections underwent FAP immunohistochemistry.

Statistical analysis

Quantitative data are expressed as mean ± SD. Statistical analyses were performed using GraphPad Prism 9.3.1 and IBM SPSS 21. A two-tailed t-test was used to assess significance. P-values < 0.05 were considered statistically significant. Significance levels are denoted as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Results

Synthesis and radiolabeling

BiFAPI was successfully synthesized by conjugating the cyclic peptide with quinoline motif via a DOTA chelator, enabling efficient radiolabeling with ⁶⁸ Ga (Fig. 1). The preparation of BiFAPI followed the procedure detailed in the Supplementary Information (Supplementary Scheme S1). HPLC analysis revealed a purity exceeding 97% (Supplementary Fig. S1). High-resolution mass spectrometry (HRMS) analysis (Supplementary Fig. S2) provided the following results: calculated for C₁₀₁H₁₄₂F₂N₂₀O₂₆S₃ (M + 2H)⁺²/2, 1092.46; found 1092.46.

Fig. 1

Chemical structure of BiFAPI

The RCP of [⁶⁸ Ga]Ga-BiFAPI consistently exceeded 95% after incubation in PBS and serum for 2 h, demonstrating its stability under physiological conditions (Supplementary Fig. S3). In vivo stability assessments via radio-HPLC analysis of blood samples from mice indicated that [⁶⁸ Ga]Ga-BiFAPI remained intact for at least 30 min post-injection (Supplementary Fig. S3). The logP value of [⁶⁸ Ga]Ga-BiFAPI was determined to be − 2.70 ± 0.40, confirming its hydrophilic nature.

In vitro evaluation

The binding affinity of BiFAPI (IC50: 11.43 ± 1.66 nmol/L) in HT1080-hFAP cells was comparable to that of FAP2286 (IC50: 6.58 ± 1.60 nmol/L) and FAPI-04 (IC50: 4.04 ± 1.64 nmol/L) (Fig. 2a). In cellular uptake experiments, [⁶⁸ Ga]Ga-BiFAPI demonstrated significantly higher uptake in HT1080-hFAP cells than [⁶⁸ Ga]Ga-FAP2286 and [⁶⁸ Ga]Ga-FAPI-04 (Fig. 2c). Importantly, this uptake was effectively inhibited by the FAP-specific inhibitor UAMC1110, confirming the selective targeting of FAP by [⁶⁸ Ga]Ga-BiFAPI (Fig. 2b). Furthermore, negligible uptake of [⁶⁸ Ga]Ga-BiFAPI was observed in FAP-negative HT1080 cells at all time points, highlighting its specificity (Fig. 2b). As shown in Fig. 2d and Fig. 2e, [⁶⁸ Ga]Ga-BiFAPI exhibited a markedly faster internalization rate and a consistently lower efflux rate at all evaluated time points compared to those of [⁶⁸ Ga]Ga-FAPI-04 and [⁶⁸ Ga]Ga-FAP2286. These findings suggested that [⁶⁸ Ga]Ga-BiFAPI held considerable promise for further exploration in FAP-targeted applications, owing to its superior binding affinity, rapid cellular uptake, and specific FAP-targeting capabilities.

Fig. 2

In vitro characterization of FAPI radioligands. a IC50 of BiFAPI, FAPI-04 and FAPI-2286. b Cell uptake assays of [⁶⁸ Ga]Ga-BiFAPI in HT-1080-FAP (FAP positive) and HT-1080 (FAP negative) cells compared with c [⁶⁸ Ga]Ga-FAPI-04 or [⁶⁸ Ga]Ga-FAPI-2286, respectively, with and without the competitor UAMC1110. d The efflux of [⁶⁸ Ga]Ga-BiFAPI in HT-1080-FAP cells compared with [⁶⁸ Ga]Ga-FAPI-04 or [⁶⁸ Ga]Ga-FAPI-2286, respectively. e The internalization of [⁶⁸ Ga]Ga-BiFAPI in HT-1080-FAP cells compared with [⁶⁸ Ga]Ga-FAPI-04 or [.⁶⁸ Ga]Ga-FAPI-2286, respectively. *P < 0.05. **P < 0.01. ****P < 0.0001

Micro PET/CT imaging

Micro-PET/CT imaging was conducted in mice bearing FTC-133 and HT1080 tumors to evaluate the biodistribution and FAP specificity of [⁶⁸ Ga]Ga-BiFAPI (Fig. 3). Dynamic imaging over 120 min post-injection revealed rapid and sustained uptake of [⁶⁸ Ga]Ga-BiFAPI in FTC133 tumors, with uptake peaking at 3.8%ID/g at 2 h post-injection (Fig. 3a and b). In contrast, minimal uptake was observed in FAP negative HT-1080 tumors, with only 1.0%ID/g at 2 h post-injection. High kidney uptake was noted, indicating primary renal excretion, along with minor liver accumulation, suggesting partial hepatobiliary clearance. Over time, the tumor-to-muscle ratio in FTC133 tumor-bearing mice increased, reaching a peak of 11.42 due to rapid clearance from non-target tissues (Fig. S4).

Fig. 3

a Dynamic PET imaging of [⁶⁸ Ga]Ga-BiFAPI in the FTC-133 (FAP positive, red arrow) and HT-1080 (FAP negative, white arrow) tumor-bearing mouse. b Corresponding time–activity curves for tumors and major organs of [⁶⁸ Ga]Ga-BiFAPI. c [⁶⁸ Ga]Ga-BiFAPI, [⁶⁸ Ga]Ga-FAPI-04 and [⁶⁸ Ga]Ga-FAPI-2286 PET imaging in the FTC-133 (FAP positive, red arrow) and HT-1080 (FAP negative, white arrow) tumor-bearing mouse 1 h post-injection, with and without the competitor FAPI-04. d The corresponding ROI analysis of tumor uptake

Comparative imaging studies demonstrated that FTC-133 tumors exhibited significantly higher uptake of [⁶⁸ Ga]Ga-BiFAPI compared to that of [⁶⁸ Ga]Ga-FAP2286 and [⁶⁸ Ga]Ga-FAPI-04 at 1 h post-injection (Fig. 3c). Specifically, [⁶⁸ Ga]Ga-BiFAPI achieved a tumor uptake of 4.00 ± 0.30%ID/g, which was markedly higher than [⁶⁸ Ga]Ga-FAP2286 (1.53 ± 0.15%ID/g, P < 0.0001) and [⁶⁸ Ga]Ga-FAPI-04 (0.73 ± 0.03%ID/g, P < 0.0001) (Fig. 3d). Co-injection of [⁶⁸ Ga]Ga-BiFAPI with an excess of FAP inhibitor significantly reduced tumor uptake in FTC133-bearing mice to 0.80 ± 0.05%ID/g (P < 0.0001), further confirming FAP specificity of [⁶⁸ Ga]Ga-BiFAPI (Fig. 3d).

Biodistribution

Biodistribution studies were conducted in tumor-bearing mice to evaluate the uptake and clearance kinetics of [⁶⁸ Ga]Ga-BiFAPI (Fig. 4). Tumor uptake of [⁶⁸ Ga]Ga-BiFAPI reached its peak at 15.2 ± 2.03%ID/g at 30 min post-injection and maintained a high level over the observed time period. The tumor-to-blood ratio increased progressively over time, reached a maximum of 4.71 ± 1.37 at 120 min (Fig. S5). Consistent with the imaging results, co-injection of [⁶⁸ Ga]Ga-BiFAPI with an excess of FAP inhibitor reduced FTC133 tumor uptake to 2.49 ± 0.85%ID/g. Comparative biodistribution analysis showed that [⁶⁸ Ga]Ga-BiFAPI achieved significantly higher tumor uptake (12.75 ± 0.78%ID/g) than both [⁶⁸ Ga]Ga-FAP2286 (7.05 ± 0.85%ID/g) and [⁶⁸ Ga]Ga-FAPI-04 (9.00 ± 2.02%ID/g) at 1 h post-injection. However, [⁶⁸ Ga]Ga-BiFAPI exhibited higher uptake in non-target organs, including the liver and kidneys, compared to [⁶⁸ Ga]Ga-FAP2286 and [⁶⁸ Ga]Ga-FAPI-04, and further investigation is required to address this issue.

Fig. 4

Biodistribution of ⁶⁸ Ga-labeled FAPI radioligands in FTC-133 tumor-bearing mice. a Biodistribution of [⁶⁸ Ga]Ga-BiFAPI at different time points. b Biodistribution of [⁶⁸ Ga]Ga-BiFAPI, [⁶⁸ Ga]Ga-FAPI-04 and [⁶⁸ Ga]Ga-FAPI-2286 at 60 min pi. n = 3/group

Quantification of FAP protein expression

In vitro autoradiography, immunohistochemistry, and Western blotting confirmed the correlation between radioactive tracer accumulation and FAP-positive regions. The expression levels of FAP in FTC133 and HT1080FAP cells were observed to be significantly elevated when compared to those in HT1080 cells (Fig. 5b). Moreover, FTC133 tumors exhibited significantly enhanced FAP staining in IHC analysis (Fig. 5a and Fig. S6), as well as elevated radioactivity levels in autoradiography studies, when compared to HT1080 tumors. Notably, the regions exhibiting high FAP expression corresponded with areas of increased radioactivity accumulation. These findings are consistent with the results obtained from micro-PET imaging and biodistribution studies, further validating the FAP-targeting capability of [⁶⁸ Ga]Ga-BiFAPI.

Fig. 5

a In vitro autoradiography and IHC result of FTC-133 and HT1080 tumor slide. b Expression levels of FAP in FTC-133, HT1080-hFAP and HT1080 cell lines

Discussion

In this study, [⁶⁸ Ga]Ga-BiFAPI, a novel FAPI-based radiotracer with heterodimeric structural properties, was successfully synthesized and radiolabeled, showing promising potential for FAP-targeted applications. The high radiochemical purity (> 95%) and stability of [⁶⁸ Ga]Ga-BiFAPI in PBS and serum highlight its robustness under physiological conditions. Its hydrophilic nature (log P: -2.70 ± 0.40) facilitates rapid clearance from non-target tissues, further supporting its favorable pharmacokinetics.

Compared to monomeric FAPI analogs, the heterodimeric structure of BiFAPI offers distinct advantages via the multivalent effect, a phenomenon previously shown to enhance tumor targeting in multimeric peptides, such as RGD dimers. This effect allows one FAPI motif to bind FAP, while simultaneously increasing the local concentration of the second motif near the target, thus enhancing tumor uptake and prolonging retention. Consistent with this mechanism, [⁶⁸ Ga]Ga-BiFAPI exhibited significantly higher tumor uptake in FAP-positive HT1080-hFAP cells and FTC133 xenografts compared to [⁶⁸ Ga]Ga-FAP2286 and [⁶⁸ Ga]Ga-FAPI-04. The increased tumor accumulation was also associated with rapid internalization and slower efflux, a key combination that contributes to improved imaging contrast and may enhance therapeutic efficacy. Considering that although IC50 of BiFAPI was comparable to that of FAP2286 and FAPI-04, the cellular uptake of BiFAPI was increased and its efflux rate was reduced, both of which were superior to those of FAP2286 and FAPI-04, we attributed this advantage to the multivalent effect, which concurrently increased the local concentration of the second motif near the target. Furthermore, the observed uptake was effectively inhibited by co-incubation with a FAP-specific inhibitor, confirming its specificity for FAP targeting. Importantly, negligible uptake was noted in FAP-negative HT1080 cells, suggesting minimal off-target binding of [⁶⁸ Ga]Ga-BiFAPI.

Micro-PET/CT imaging further confirmed the superior performance of [⁶⁸ Ga]Ga-BiFAPI. FTC133 tumors demonstrated a peak uptake of 3.8%ID/g at 2 h post-injection, which was markedly higher than that of [⁶⁸ Ga]Ga-FAP2286 and [⁶⁸ Ga]Ga-FAPI-04. This high tumor uptake resulted in an excellent tumor-to-muscle ratio, further emphasizing its imaging superiority. The specificity of [⁶⁸ Ga]Ga-BiFAPI was validated through blocking experiments with a FAP inhibitor, where significant reductions in tumor uptake were observed, reaffirming its selective targeting. Additionally, the results from IHC analysis and autoradiography experiments on adjacent tumor sections further corroborated the in vivo specificity of [⁶⁸ Ga]Ga-BiFAPI targeting FAP. These findings suggested that [⁶⁸ Ga]Ga-BiFAPI retained the advantageous pharmacokinetics of monomeric tracers while leveraging the enhanced tumor-targeting properties of heterodimeric structures. Although the heterodimeric design indeed enhances tumor targeting, it also leads to an increase in non-specific binding in organs such as the kidneys and liver. Future optimization strategies for the compound may involve modifications to the linkers, such as the incorporation of multiple glutamic acid residues or PEG chains, which are commonly employed chemical structures in the structure–activity relationship studies of radiopharmaceuticals. We observed that the tumor uptake of [⁶⁸ Ga]BiFAPI in PET imaging studies was relatively lower than the uptake in biodistribution experiments. A similar trend was noted for the concurrently evaluated [⁶⁸ Ga]FAPI-04 and [⁶⁸ Ga]FAPI-2286. We hypothesized that this discrepancy might be attributed to two primary factors: Firstly, inherent methodological differences between PET imaging and biodistribution sampling protocols might introduce variability in uptake measurements. Secondly, the mice utilized for PET imaging and biodistribution studies were not from the same batch, potentially resulting in variations in tumor size and physiological state, which could further influence uptake values. Recently, radiopharmaceuticals incorporating bifunctional chelators, such as DOTA—exemplified by compounds like PSMA617 and DOTATOC—have gained significant traction in cancer theranostics. Building on this progress, [⁶⁸ Ga]Ga-BiFAPI, which also features a DOTA moiety, has demonstrated promising preclinical performance in this study. However, its elevated uptake in non-target organs, particularly the liver and kidneys, remains a critical challenge. To address this, future research should focus on structural optimization to minimize off-target accumulation. Subsequently, the therapeutic potential of [⁶⁸ Ga]Ga-BiFAPI can be further explored through radionuclide therapy using therapeutic radionuclides such as ¹⁷⁷Lu, with the aim of evaluating its efficacy and clinical applicability.

In this study, xenograft mouse models derived from cancer cell lines were utilized for the evaluation of [⁶⁸ Ga]Ga-BiFAPI. Previous research has predominantly employed similar xenograft models to assess the pharmacokinetics of FAP-targeting agents. However, patient-derived xenograft (PDX) models, which are established by directly implanting fresh surgical tumor tissue into immunodeficient mice, present a more compelling alternative. PDX models are particularly advantageous as they retain the tumor microenvironment and molecular characteristics of the original patient tumors, offering a more clinically relevant platform for drug evaluation. Consequently, future investigations aimed at clinical translation will prioritize the biological assessment of [⁶⁸ Ga]Ga-BiFAPI in PDX mouse models to ensure a more accurate prediction of therapeutic efficacy and safety.

However, several limitations of this study should be acknowledged. First, the study was conducted in murine models, and further validation in human subjects is necessary to assess the clinical translation of [⁶⁸ Ga]Ga-BiFAPI. Second, while [⁶⁸ Ga]Ga-BiFAPI exhibited favorable tumor uptake and retention, the biodistribution data revealed higher background uptake in certain organs compared to other tracers. Optimization of the molecular structure to reduce nonspecific uptake could further improve its imaging performance.

Conclusion

In summary, the novel radiotracer [⁶⁸ Ga]Ga-BiFAPI exhibited enhanced FAP-targeting capabilities and demonstrated superior tumor uptake and retention compared to [⁶⁸ Ga]Ga-FAP2286 and [⁶⁸ Ga]Ga-FAPI-04. Its heterodimeric design and favorable pharmacokinetic profile positioned it as a promising candidate for FAP-targeted PET imaging. These findings establish a strong foundation for further investigation into the clinical potential of [⁶⁸ Ga]Ga-BiFAPI, particularly in cancer diagnosis and, potentially, in theranostics.