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American Journal of Nuclear Medicine and Molecular Imaging logoLink to American Journal of Nuclear Medicine and Molecular Imaging
. 2025 Aug 15;15(4):130–139. doi: 10.62347/IRKD4863

Dual-receptor PET imaging of ovarian cancer using a 68Ga-labeled heterodimer targeting folate receptor and HER2

Xiaodan Shi 1,2, Zhangxin Wu 1,2, Yifeng Yuan 1,2, Ying Wang 1,2, Shuo Yang 1,2, Rong Li 1,2
PMCID: PMC12444400  PMID: 40980736

Abstract

Radiolabeled folate derivatives have been extensively investigated for positron emission tomography (PET) imaging of ovarian cancer due to the frequent overexpression of folate receptor α (FRα). However, clinical translation has been hindered, at least in part, by suboptimal tumor uptake of FRα-targeted radiotracers. In this study, we developed and characterized a 68Ga-labeled heterodimeric radiotracer, 68Ga-folate-KR, designed to concurrently target FRα and human epidermal growth factor receptor 2 (HER2), another receptor commonly overexpressed in ovarian cancer. Transcriptomics analysis confirmed the co-upregulation of FOLR1 and HER2 in ovarian cancer tissues relative to normal ovarian tissue, supporting the rationale for dual-receptor targeting. In vitro binding assays demonstrated specific binding of 68Ga-folate-KR to both receptors. PET imaging and biodistribution studies in SKOV3 tumor-bearing mice revealed significantly enhanced tumor uptake and improved tumor-to-nontumor contrast compared to the monomeric radiotracers 68Ga-folate and 68Ga-KR. Competitive blocking experiments further confirmed the in vivo dual-receptor targeting capability of 68Ga-folate-KR. Collectively, our results highlight that 68Ga-folate-KR enables more sensitive PET detection of ovarian cancer xenografts. With further optimization, dual-receptor-targeted radiotracers hold promise for clinical translation in both lesion detection and therapy response monitoring in ovarian cancer.

Keywords: Ovarian cancer, positron emission tomography, radiopharmaceuticals, molecular imaging, dual-targeting

Introduction

Ovarian cancer remains one of the most lethal gynecological malignancies worldwide, largely due to its high mortality rate and frequent diagnosis at late-stages [1,2]. The disease typically presents with vague or nonspecific symptoms, leading to diagnosis at advanced stages in the majority of patients, where the 5-year survival rate falls below 30% [2,3]. Current diagnostic approaches for ovarian cancer, such as pelvic ultrasound, CT, MRI, and serum biomarkers like cancer antigen 125 (CA-125) and human epididymis protein 4 (HE4), offer clinically useful information but suffer from limited sensitivity and specificity, particularly for early-stage disease [4,5]. These limitations underscore the urgent need for more effective imaging strategies that enable early, accurate, and noninvasive detection of ovarian cancer. Timely and precise diagnosis can not only improve clinical outcomes but also guide surgical planning and support fertility-preserving interventions when appropriate.

The emergence of molecular imaging, particularly positron emission tomography (PET), has opened new avenues for noninvasive tumor characterization, early detection, and response monitoring [6,7]. While 18F-fluorodeoxyglucose (18F-FDG) PET is widely used in oncology, including for ovarian cancer, its diagnostic performance is limited by its dependence on elevated glucose metabolism - a hallmark that is not exclusive to malignant cells and may also be observed in benign inflammatory or infectious processes [8]. Moreover, 18F-FDG PET exhibits reduced sensitivity in detecting low-grade or mucinous ovarian cancers and can be confounded by physiological uptake in the intestines and reproductive organs, particularly in premenopausal women [9,10]. These limitations highlight the pressing need for alternative PET radiotracers that selectively target molecular markers overexpressed in ovarian cancer, thereby enhancing diagnostic specificity and clinical accuracy.

Efforts to improve ovarian cancer imaging have focused on radiotracers targeting overexpressed cell-surface receptors. One of the most promising targets is folate receptor α (FRα), which is highly expressed in the majority of epithelial ovarian cancers while showing limited expression in normal tissues. A variety of FRα-targeted radiotracers, including folate derivatives labeled with radionuclides such as 99mTc, 111In, 18F, and 68Ga, have shown encouraging results in both preclinical and early clinical studies [11-14]. Moreover, folate-conjugated fluorescent probes have been used intraoperatively to assist tumor visualization during surgery [15]. However, despite decades of research, FRα-targeted radiotracers have yet to achieve widespread clinical application, at least in part, due to their limited tumor accumulation and the variable expression of FRα across different histological subtypes of ovarian cancer.

Beyond FRα, human epidermal growth factor receptor 2 (HER2) is another key molecular target under investigation for ovarian cancer imaging. HER2 is overexpressed in approximately 20-30% of ovarian cancers, particularly in serous, clear cell, and mucinous subtypes, and is often associated with poor prognosis [16,17]. Various HER2-targeted PET imaging agents, including peptides, affibodies, and monoclonal antibodies, have been developed and evaluated across several tumor types [18-21]. Nevertheless, radiotracers targeting a single molecular marker may be insufficient due to tumor heterogeneity and variability in receptor expression [22]. To address these limitations, dual-targeting strategies that concurrently engage two distinct receptors offer a compelling alternative. Such approaches may enhance tumor detection sensitivity, improve radiotracer retention, and provide better tumor-to-background contrast by ensuring binding even when one of the targets is variably expressed [22,23]. Recent studies have demonstrated that heterodimeric radiotracers can outperform their monovalent counterparts in various cancer models [24-26]. However, to our knowledge, no dual-receptor PET radiotracer targeting both FRα and HER2 has been systematically evaluated for PET imaging of ovarian cancer.

In this study, we developed a 68Ga-labeled heterodimeric PET radiotracer that targets both FRα and HER2. The radiotracer incorporates a folate moiety and a HER2-binding peptide (denoted as the KR peptide; sequence: KLRLEWNR [27]), designed to enable concurrent binding to both receptors. We performed in vitro binding studies to confirm target specificity and conducted in vivo small-animal PET imaging in an ovarian cancer xenograft model to evaluate pharmacokinetics and tumor-targeting performance. Our findings provide preclinical proof-of-concept supporting the feasibility of dual-receptor PET imaging for ovarian cancer, with potential implications for early detection, patient stratification, and image-guided interventions.

Materials and methods

Reagents

The Lys-PEG4-folate (PEG4 = 15-amino-4,7,10,13-tetraoxapentadecanoic acid), PEG4-Lys(Dde)-Leu-Arg-Leu-Glu-Trp-Asn-Arg (PEG4-K(Dde)R), and the heterodimer Lys[PEG4-folate][PEG4-K(Dde)R] (Lys-2PEG4-folate-K(Dde)R) were custom synthesized by WuXi AppTec (Shanghai, China). DOTA-NHS-ester was purchased from Macrocyclics (Dallas, TX). 68Ga was obtained from a 68Ge/68Ga generator (Isotope Technologies Garching GmbH, Germany) eluted with 0.1 N HCl. All water and buffers were passed through a Chelex 100 column prior to use to remove metal ions.

Transcriptomics analyses

The transcriptomics data for FOLR1 (gene coding for FRα) and HER2 (gene coding for HER2) were obtained from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) project. All data were normalized using log2(TPM+1) transformation, and expression levels were analyzed using the Gene Expression Profiling Interactive Analysis (GEPIA) platform [28].

DOTA conjugation

Lys-PEG4-folate, PEG4-K(Dde)R, and Lys-2PEG4-folate-K(Dde)R were conjugated with DOTA-NHS using a previously described method [24]. Briefly, each precursor was reacted with DOTA-NHS ester at a molar ratio of 1:5 in 0.1 M sodium bicarbonate buffer (pH 9.0) and stirred at 4°C for 4 h. The resulting DOTA-conjugated intermediates were purified by semi-preparative high-performance liquid chromatography (HPLC) to remove excess reagents and byproducts, followed by lyophilization. The Dde protecting group on the lysine side chain was then removed using 2% hydrazine in DMF (v/v) for 15 min at room temperature under gentle stirring. The deprotected products were further purified by semi-preparative HPLC, lyophilized, and characterized using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). DOTA-Lys-PEG4-folate was obtained in 62% yield with m/z 1203.5 for [MH]+ (C52H78N14O19, calculated molecular weight 1202.6). DOTA-PEG4-KR was obtained in 56% yield with m/z 1747.9 for [MH]+ (C77H130N22O24, calculated molecular weight 1747.0). DOTA-Lys-2PEG4-folate-KR was obtained in 65% yield with m/z 2546.1 for [MH]+ (C113H180N32O35, calculated molecular weight 2545.3).

68Ga radiolabeling

10 nmol of DOTA-Lys-PEG4-folate, DOTA-PEG4-KR, or DOTA-Lys-2PEG4-folate-KR was dissolved in 350 μL of 0.1 M sodium acetate buffer (pH 4.5) buffer and incubated with 370 MBq of 68GaCl3 at 95°C for 15 min with constant agitation. The resulting compounds, 68Ga-labled DOTA-Lys-PEG4-folate (68Ga-folate), 68Ga-labled DOTA-PEG4-KR (68Ga-KR), and 68Ga-labeled DOTA-Lys-2PEG4-folate-KR (68Ga-folate-KR), were purified using analytical HPLC equipped with a radiation detector. The radioactive peaks containing the desired products were collected and rotary evaporated to remove the solvent. The products were formulated in phosphate-buffered saline (PBS), sterilized by passage through a 0.22-μm Millipore filter, and used for subsequent in vitro and in vivo studies. Radiochemical purity and in vitro stability were assessed in PBS or fetal bovine serum (FBS) at 0, 30, 60, and 120 min post-incubation using radio-HPLC.

Cell culture and animal models

SKOV3 human ovarian cancer and HEK293 human embryonic kidney cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS at 37°C in a humidified atmosphere containing 5% CO2.

All animal procedures were approved by the Institutional Animal Care and Use Committee of Peking University Third Hospital. Female BALB/c nude mice (4-5 weeks old) were obtained from the Department of Experimental Animal, Peking University Health Science Center. The SKOV3 tumor-bearing mouse model was established by subcutaneous injection of 5×106 SKOV3 cells into the right flank. Tumor growth was monitored with caliper measurements, and tumor volume was calculated using the formula: volume = (length × width2)/2.

Western blotting

Cells were lysed, and total protein concentrations were determined using a BCA assay kit (Thermo Fisher Scientific, Waltham, MA). Proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. Membranes were blocked in 5% skimmed milk for 2 h, and then incubated overnight at 4°C with primary antibody against folate receptor 1 (60307-1-Ig; Proteintech, Chicago, IL) or HER2 primary antibody (60311-1-Ig; Proteintech). After washing with Tris-buffered saline containing 0.1% Tween-20, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Abclonal, Wuhan, China) for 1 h. Bands were visualized using a Tanon 5200 Multi imager and quantified using ImageJ (NIH, Bethesda, MD).

Flow cytometry and cell staining

The expression of FRα and HER2 on SKOV3 cells was assessed by flow cytometry and immunofluorescence staining. HEK293 cells were used as controls. For flow cytometry, cells were incubated with 5 μg/mL of anti-folate receptor 1 (60307-1-Ig; Proteintech) or anti-HER2 primary antibody (60311-1-Ig; Proteintech) for 1 h at room temperature. After washing with PBS, cells were incubated with fluorophore-conjugated secondary antibodies (Abclonal). Flow cytometry was performed using a BD cytometer (Becton Dickinson, Germany).

For cell staining, SKOV3 cells grown on 35 mm MatTek glass-bottom culture dishes were stained with 5 μg/mL of anti-folate receptor 1 (Proteintech) or anti-HER2 primary antibody (Proteintech) for 1 h at room temperature, followed by dye-labeled secondary antibodies. Cells were visualized using a confocal microscope (Leica, Wetzlar, Germany).

Cell binding assay

The receptor binding specificity of 68Ga-folate, 68Ga-KR, and 68Ga-folate-KR was determined by in vitro cell binding assay. Briefly, SKOV3 cells were incubated with 74 kBq of each radiotracer with or without an excess dose (5 nmol) of cold competitor (Lys-PEG4-folate, PEG4-KR, Lys-2PEG4-folate-KR, or a combination of Lys-PEG4-folate and PEG4-KR). After incubation at 4°C for 2 h, cells were washed with PBS. Cells were then harvested, and cell-associated radioactivity was measured using a γ-counter (Packard, Meriden, CT). The results were expressed as the percentage of the total added dose per million cells (%AD/106 cells).

Small-animal PET imaging

Small-animal PET/CT imaging and image analysis were performed using a Super Nova small-animal PET/CT scanner (PINGSENG Healthcare, Shanghai, China). BALB/c nude mice bearing SKOV3 tumors (tumor volume: 200-300 mm3) were intravenously injected with 5.55 MBq of either 68Ga-folate, 68Ga-KR, or 68Ga-folate-KR. At 30, 60, and 120 min after injection of each radiotracer, 10-min static PET scanning was performed. For the blocking studies, mice were coinjected with 1 nmol of cold Lys-PEG4-folate, PEG4-KR, or Lys-PEG4-folate plus PEG4-KR along with 5.55 MBq of 68Ga-folate-KR. Ten-min static PET scans were then acquired at 30 min after injection. For each PET scan, region-of-interest (ROIs) were drawn over the tumor and major organs, and the ROI-derived percentage injected dose per gram of tissue (%ID/g) was calculated as previously described [29,30].

Biodistribution

Female BALB/c nude mice bearing subcutaneous SKOV3 tumors were injected with 3.7 MBq of 68Ga-folate, 68Ga-KR, or 68Ga-folate-KR to evaluate the distribution of the radiotracers in the main organs. At 30, 60, and 120 min postinjection, mice were euthanized and blood, tumor, and main organs and tissues were collected, weighed, and measured using a γ-counter (Packard). The uptake of radiotracers were calculated and presented as %ID/g.

In vivo metabolic stability

To assess the in vivo metabolic stability of 68Ga-folate-KR, female BALB/c normal mice were intravenously injected with 37 MBq of 68Ga-folate-KR via the tail vein. At 30 min postinjection, serum and urine samples were collected and centrifuged. The supernatant was diluted with 50% aqueous acetonitrile, filtered through a 0.22-mm Millipore filter (Millipore, Billerica, MA), and analyzed by radio-HPLC.

Immunofluorescence staining

The expression of FRα and HER2 in tumor tissues was analyzed by immunofluorescence staining. Briefly, 5-μmthick frozen tissue sections were fixed with ice-cold acetone and then blocked with 2% bovine serum albumin (in PBS) for 1 h. Tissues were then incubated with 10 μg/mL of anti-folate receptor 1 (Proteintech) or anti-HER2 primary antibody (Proteintech) for 1 h at room temperature, and then visualized with dye-labeled secondary antibodies under a confocal microscope (Leica).

Statistical analysis

Quantitative data are expressed as mean ± SD. Statistical analysis was performed using a one-way ANOVA and an unpaired Student’s t test. P values of < 0.05 were considered statistically significant.

Results

Receptor expression patterns of ovarian cancer

Transcriptomics analysis of FOLR1 and HER2 (encoding FRα and HER2, respectively) revealed that both receptors are significantly upregulated in ovarian cancer tissues compared to normal ovarian tissues (Figure 1A), supporting the rationale for designing dual-targeted radiotracers against FRα and HER2. Based on these findings, we selected a SKOV3 ovarian cancer cell line for further in vitro studies. Dual expression of FRα and HER2 in SKOV3 cells was validated by Western blotting (Figure 1B), flow cytometry (Figure 1C), and immunofluorescence staining (Figure 1D).

Figure 1.

Figure 1

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FRα and HER2 expression patterns of ovarian cancer and SKOV3 cells. A. Box plot comparing FOLR1 and HER2 messenger RNA expression levels in healthy ovarian tissues and ovarian cancer tissues. ****, P < 0.0001. B. Western blot analysis showing the expression of FRα and HER2 in SKOV3 cells. C. Flow cytometry analysis confirms co-expression of FRα and HER2 on SKOV3 cells. D. Immunofluorescence staining of SKOV3 cells demonstrates colocalization of FRα and HER2. Scale bar: 20 μm.

Chemistry and radiochemistry

The DOTA conjugates of Lys-PEG4-folate, PEG4-KR, and Lys-2PEG4-folate-KR (Figure 2A) were confirmed by mass spectroscopy. The radiolabeling of 68Ga was completed within 45 min, yielding a decay-corrected radiochemical yield of 90%-95%. After purification, the radiochemical purity of all three radiotracers exceeded 98%, with a specific activity of 30-40 MBq/nmol. 68Ga-folate, 68Ga-KR, and 68Ga-folate-KR showed favorable in vitro stability, retaining over 80% radiochemical purity after 2 h of incubation in PBS or FBS (Figure 2B-D).

Figure 2.

Figure 2

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In vitro characterizations of 68Ga-folate, 68Ga-KR, and 68Ga-folate-KR. (A) Chemical structures of 68Ga-folate, 68Ga-KR, and 68Ga-folate-KR. (B-D) Radiochemical purity (RCP) values of 68Ga-folate (B), 68Ga-KR (C), and 68Ga-folate-KR (D) after incubating for 0, 30, 60, and 120 min in PBS or FBS. (E) Competitive binding assay of 68Ga-folate to SKOV3 cells in the presence of excess Lys-PEG4-folate. (F) Competitive binding assay of 68Ga-KR to SKOV3 cells in the presence of excess PEG4-KR. (G) Competitive binding assay of 68Ga-folate-KR to SKOV3 cells in the presence of excess PEG4-KR (KR), Lys-PEG4-folate (folate), Lys-PEG4-folate plus PEG4-KR (folate + KR), or Lys-2PEG4-folate-KR (folate-KR). All numerical data are presented as mean ± SD (n = 4). ***, P < 0.001.

Cell binding assay

The binding specificity of 68Ga-folate, 68Ga-KR, and 68Ga-folate-KR to the dual receptor-positive SKOV3 cells was confirmed by cell binding assay. The binding of 68Ga-folate and 68Ga-KR to SKOV3 cells was significantly inhibited by excess doses of Lys-PEG4-folate and PEG4-KR, respectively (Figure 2E, 2F). Co-incubation with an excess of either Lys-PEG4-folate or PEG4-KR alone partially inhibited the binding of 68Ga-folate-KR to SKOV3 cells, whereas co-incubation with both Lys-PEG4-folate and PEG4-KR, or with Lys-2PEG4-folate-KR almost completely abolished its binding (Figure 2G). These results indicate that 68Ga-folate-KR binds specifically to both FRα and HER2 via receptor-mediated mechanisms, while 68Ga-folate and 68Ga-KR exhibit specificity for their respective single targets.

Small-animal PET imaging

Representative PET images at different time points postinjection of 68Ga-folate, 68Ga-KR, and 68Ga-folate-KR in SKOV3 tumor-bearing mice are shown in Figure 3A. The SKOV3 tumors after injection of 68Ga-folate, 68Ga-KR, or 68Ga-folate-KR were visible at 30 min postinjection, followed by signal clearance at 60 and 120 min postinjection. Prominent kidney uptake was observed for all radiotracers, indicating renal-urinary clearance. The ROI-derived tumor uptake of 68Ga-folate-KR was determined to be 1.33 ± 0.08, 0.94 ± 0.13, and 0.55 ± 0.07 %ID/g at 30, 60, and 120 min postinjection, respectively, which are significantly higher than that of the monomeric radiotracers at corresponding time points (P < 0.001; Figure 3B). 68Ga-folate-KR also showed elevated liver uptake at 30 min, which decreased to levels comparable with other radiotracers at later time points (Figure 3C). Renal accumulation was highest for 68Ga-folate-KR across all time points (Figure 3D). Tumor-to-muscle and tumor-to-liver ratios of 68Ga-folate-KR were also significantly higher than that of 68Ga-folate and 68Ga-KR at 30 min postinjection (Figure 3E).

Figure 3.

Figure 3

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PET imaging of 68Ga-folate, 68Ga-KR, and 68Ga-folate-KR. A. Representative PET/CT images of SKOV3 tumor-bearing mice at 30, 60, and 120 min after injection of 68Ga-folate, 68Ga-KR, and 68Ga-folate-KR. Tumors are indicated by white arrows. B. Quantification of tumor uptake (%ID/g) at indicated time points. C. Liver uptake profiles of the three radiotracers. D. Kidney uptake kinetics of the three radiotracers. E. Tumor-to-muscle (T/muscle), tumor-to-liver (T/liver), and tumor-to-kidney (T/kidney) uptake ratios of the radiotracers at 30 min postinjection. All numerical data are presented as mean ± SD (n = 4 per group). ***, P < 0.001; ****, P < 0.0001.

The in vivo FRα and HER2 dual receptor-binding capability of 68Ga-folate-KR was confirmed by several blocking studies (Figure 4A). The tumor uptake of 68Ga-folate-KR was only partially reduced by excess folate (from 1.12 ± 0.15 %ID/g to 0.39 ± 0.08 %ID/g) or KR (from 1.12 ± 0.15 %ID/g to 0.61 ± 0.09 %ID/g) blocking. However, the tumor uptake of 68Ga-folate-KR was nearly abolished when both excess Lys-PEG4-folate and PEG4-KR were co-injected (from 1.12 ± 0.15 %ID/g to 0.24 ± 0.09 %ID/g) (Figure 4B). The tumor-to-muscle, tumor-to liver, and tumor-to-kidney ratios of 68Ga-folate-KR was also significantly blocked by Lys-PEG4-folate plus PEG4-KR (Figure 4C).

Figure 4.

Figure 4

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PET imaging of 68Ga-folate-KR with or without blocking. A. Representative PET/CT images showing receptor blocking effects on 68Ga-folate-KR uptake in SKOV3 tumors following co-injection with excess Lys-PEG4-folate (folate), PEG4-KR (KR), or Lys-PEG4-folate plus PEG4-KR (folate + KR). Tumors are indicated by white arrows. B. Tumor uptake quantification (%ID/g) in each blocking group. C. Quantified tumor-to-muscle (T/muscle), tumor-to-liver (T/liver), and tumor-to-kidney (T/kidney) ratios of 68Ga-folate-KR under blocking conditions. All numerical data are presented as mean ± SD (n = 4 per group). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Biodistribution studies

Biodistribution profiles of the 68Ga-folate, 68Ga-KR, and 68Ga-folate-KR were examined in SKOV3 tumor-bearing nude mice. As shown in Figure 5A-C, predominant kidney uptake was observed for all three radiotracers, which is consistent with the results observed in PET imaging (Figure 3A). The tumor uptake of 68Ga-folate-KR was significantly higher than that of 68Ga-folate and 68Ga-KR at all examined time points (Figure 5D), confirming the improved tumor uptake of the dual receptor targeting radiotracer. A positive correlation was found between tumor uptake values obtained from PET imaging and ex vivo biodistribution (Figure 5E), supporting the quantitative accuracy of noninvasive PET imaging for evaluating in vivo radiotracer behavior. Ex vivo histological staining validated the co-expression of FRα and HER2 in SKOV3 tumor tissues (Figure 5F).

Figure 5.

Figure 5

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Biodistribution of 68Ga-folate, 68Ga-KR, and 68Ga-folate-KR and ex vivo validate of FRα and HER2 expression in tumor tissues. (A-C) Biodistribution of 68Ga-folate (A), 68Ga-KR (B), and 68Ga-folate-KR (C) in SKOV3 tumor-bearing BALB/c nude mice at 30, 60, and 120 min postinjection. (D) Comparison of tumor uptake between dual- and mono-targeted radiotracers at different time points. *, P < 0.05; ***, P < 0.001. All numerical data are presented as mean ± SD (n = 4 per group). (E) Correlation between PET-derived and ex vivo biodistribution-derived tumor uptake values of 68Ga-folate-KR. (F) Immunofluorescence staining of FRα and HER2 for the SKOV-3 tumors harvested from BALB/c nude mice. Scale bar, 20 μm.

Metabolic stability of 68Ga-folate-KR

The metabolic stability of 68Ga-folate-KR in mice serum and urine at 30 min after injection were determined by HPLC. As shown in Figure 6A-C, both the serum and urine detected minimal detectable metabolite formation, indicating that 68Ga-folate-KR possesses excellent metabolic stability in vivo.

Figure 6.

Figure 6

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Metabolic stability of 68Ga-folate-KR analyzed by HPLC. (A) HPLC chromatogram of purified 68Ga-folate-KR. (B, C) Representative HPLC chromatograms of mouse serum (B) and urine (C) collected 30 min after intravenous injection of 68Ga-folate-KR in BALB/c mice. Data are representative of three independent experiments.

Discussion

Tumor receptor-targeted PET imaging has emerged as a powerful tool in precision oncology, enabling noninvasive visualization of tumor-associated biomarkers to facilitate early diagnosis, patient stratification, and real-time monitoring of therapeutic response [31]. Most clinically employed PET radiotracers are designed to target a single receptor, providing valuable information on receptor expression and guiding receptor-targeted therapies. However, the diagnostic performance of single-receptor targeting can be limited by the inherent heterogeneity of tumors [32]. Loss or downregulation of a single receptor, which is common in advanced or treatment-resistant tumors, may lead to false-negative results, thereby reducing diagnostic sensitivity and compromising clinical decision-making. To address these limitations, dual-targeting strategies have gained increasing interest as a means to enhance radiotracer accumulation, increase binding avidity, and expand the spectrum of detectable lesions [33,34]. In this study, we developed and evaluated a heterodimeric PET radiotracer, 68Ga-folate-KR, designed to simultaneously target FRα and HER2, two clinically relevant targets frequently co-expressed in ovarian cancer. Our data demonstrate that 68Ga-folate-KR retains high specificity and affinity for both targets, offering a promising alternative to 18F-FDG by enabling more selective PET imaging of ovarian cancer.

Folate-based PET radiotracers have long been explored for ovarian cancer imaging due to the overexpression of FRα in many epithelial ovarian carcinomas [35,36]. However, the clinical translation of radiolabeled folate analogs has been hampered, at least in part, by their suboptimal tumor uptake and unfavorable pharmacokinetics. Consistent with previous studies [37,38], our PET imaging of the monomeric 68Ga-folate revealed limited tumor accumulation (Figure 3), underscoring the need for enhanced targeting strategies. By incorporating the HER2-targeting KR peptide, we generated a heterodimeric radiotracer with significantly improved tumor uptake and tumor-to-background contrast in the SKOV3 xenograft model, as confirmed by PET imaging and ex vivo biodistribution studies. A series of in vitro and in vivo blocking experiments further validated the specific binding of 68Ga-folate-KR to both FRα and HER2. These findings support the dual-targeting capability of 68Ga-folate-KR and suggest its potential to detect tumors expressing either target alone.

Despite these improvements, the absolute tumor uptake of 68Ga-folate-KR remained moderate, and kidney accumulation was relatively high. These limitations may result from the intrinsic affinities of the individual ligands and rapid renal clearance. Therefore, further molecular optimization is warranted to fully realize the potential of this dual-targeting approach. For instance, replacing folate with 5-methyltetrahydrofolate (MTHF), a derivative with higher specificity and binding affinity for FRα [12], could improve receptor targeting efficiency. Similarly, chemical modifications to the KR peptide, such as cyclization or the incorporation of D-amino acid, may enhance HER2 binding affinity and proteolytic resistance, ultimately improving tumor retention [39,40].

In addition to optimizing binding affinity and enzymatic resistance, pharmacokinetic properties of the radiotracer could be tuned through various structure modification strategies. These include the incorporation of hydrophilic linkers to reduce off-target accumulation, multimerization to increase valency [41,42], conjugation with albumin-binding moieties to prolong circulation half-life [43,44], and enzymolysis-based clearance mechanisms to facilitate rapid renal clearance [45,46]. Notable, despite the moderate affinities of folate and KR as monovalent ligands, their combination in the heterodimeric format synergistically improved in tumor targeting, highlighting the value of dual-receptor engagement in enhancing PET imaging performance.

Beyond FRα and HER2, several alternative molecular targets have shown promise in ovarian cancer imaging, including fibroblast activation protein (FAP) [47] and poly(ADP-ribose) polymerase-1 (PARP1) [48]. The modularity of the heterodimer design allows for flexible replacement or combination of different targeting ligands, enabling the development of customized PET radiotracers tailored to specific tumor phenotypes. Integration of transcriptomic, proteomic, and spatial profiling data from patient-derived tumor samples could further guide the rational selection of dual- or multi-targeting strategies.

Peptide-based radiotracers are generally characterized by low immunogenicity and favorable safety profiles. Several such radiotracers, including radiolabeled heterodimeric peptides, have been evaluated in clinical studies without reports of significant immune responses or adverse events [49,50]. These observations support the translational potential of heterodimeric PET radiotracers and underscore their promise as next-generation agents for precision oncology.

Conclusion

We synthesized and evaluated a new PET radiotracer, 68Ga-folate-KR, capable of concurrently targeting FRα and HER2 in an ovarian cancer mouse model. This dual-targeted radiotracer demonstrated high specificity for both receptors, robust in vivo stability, and markedly enhanced tumor PET imaging compared to its monovalent analogs. Our results highlight the potential of dual-receptor PET imaging to address the challenges posed by receptor heterogeneity in ovarian cancer. Further refinement of 68Ga-folate-KR could pave the way for its clinical application as a precision PET imaging agent in the management of ovarian malignancies.

Acknowledgements

This work was supported by the National Key R&D Program of China (2024YFA1014102) and the Key Clinical Project of Peking University Third Hospital (BYSYZD2024005 and BYSYZD2023016).

Disclosure of conflict of interest

None.

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