Impact of FAPI Molar Dose on Targeted PET Imaging and Therapy in Mouse Syngeneic Tumor Models

Oncology Medical Imaging Medicine
Molar dose Targeted imaging Mouse models Radiotherapy FAPI Cancer-associated fibroblasts Preclinical research

Background

In recent years, significant progress has been made in the field of cancer diagnosis and treatment, particularly in technologies based on molecular imaging and targeted radiotherapy. Among these, Fibroblast Activation Protein (FAP), a biomarker highly expressed in the tumor microenvironment, has garnered widespread attention. FAP is primarily expressed in cancer-associated fibroblasts (CAFs), which play a critical role in tumor growth, invasion, and metastasis. Consequently, FAP has become an important target for tumor diagnosis and therapy.

However, despite the tremendous potential shown by FAP-targeted radiolabeled ligands (FAPIs) in preclinical and clinical studies, the relatively low level of FAP expression in tumor models during preclinical research poses challenges. This results in poor imaging quality and susceptibility to saturation effects when using radiolabeled FAPIs with low molar activity. Furthermore, the effect of FAPI molar doses on targeted PET imaging and radiotherapy remains unclear. To address this, the study aims to investigate how FAPI molar doses influence FAP-targeted PET imaging and radiotherapy to enhance the reliability and reproducibility of preclinical research.

Paper Source

The paper was jointly authored by Luoxia Liu, Yifan Shi, Shujie He, and others, from the Department of Nuclear Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology and the National Center for Major Public Health Events. It was published in 2025 in the journal European Journal of Nuclear Medicine and Molecular Imaging.

Study Workflow and Findings

1. Preparation and Quality Control of Radiolabeled FAPI

The study began with radiolabeling of FAPI-04, utilizing different radionuclides (e.g., 68Ga and 177Lu) and chelators (e.g., DOTAGA). By optimizing the labeling process, the research team employed an anion exchange cartridge (QMA) adsorption strategy to remove unbound cold FAPI precursors, thereby improving the apparent molar activity (AM) of radiolabeled FAPI. Results showed that after QMA adsorption, the ultraviolet absorption peak of cold FAPI precursors significantly decreased, while the radioactive peak of radiolabeled FAPI showed only a slight decline, indicating the effectiveness of the QMA adsorption strategy in enhancing apparent molar activity.

2. Cell Binding Assays

To verify the specific binding of FAPI-04 to FAP, the research team conducted cell binding assays. These used 4T1 and U87MG cell lines, and results revealed that 4T1 cells do not express FAP, while U87MG cells displayed significant FAP expression. The cell binding assays confirmed the specificity of [68Ga]Ga-FAPI-04 binding to FAP and further validated that 4T1 cell-derived tumor models primarily express murine FAP (muFAP) during tumorigenesis, with muFAP mainly localized in CAFs.

3. PET/CT Imaging and Biodistribution Study

The research investigated [68Ga]Ga-FAPI-04 PET/CT imaging in 4T1 tumor-bearing mice and analyzed how varying molar doses of FAPI-04 affected tumor and organ uptake. Results showed that as the molar dose of FAPI-04 increased, uptake in the tumor, joints, and vertebrae gradually declined, showing a gradient blocking effect. Using sigmoidal curve fitting, the team identified a significant correlation between the uptake in tumors, joints, and vertebrae and the molar dose of FAPI-04. Additionally, biodistribution studies revealed that low molar doses of FAPI-04 displayed higher uptake in tumors and the osteoarticular system, while higher molar doses significantly decreased tumor uptake.

4. Targeted Radiotherapy Experiments

The team further evaluated therapeutic outcomes using [177Lu]Lu-DOTAGA.(SA.FAPI)2 at varying molar doses. Results indicated that although both high and low molar doses of [177Lu]Lu-DOTAGA.(SA.FAPI)2 inhibited 4T1 tumor growth, the therapeutic effects were more pronounced at lower molar doses. Biodistribution and autoradiography findings confirmed that lower molar doses of [177Lu]Lu-DOTAGA.(SA.FAPI)2 accumulated more effectively in tumors, enhancing radiotherapy outcomes.

5. PET Imaging in Different Tumor Models

The team also performed [68Ga]Ga-FAPI-04 PET imaging in various tumor models, including Huh7, Hepa1-6, PC3, and MC38. Results demonstrated significant tumor and osteoarticular uptake in all models, further validating the repeatability and reliability of FAPI PET imaging across different tumor types.

Conclusions and Implications

The study showed that the molar dose of FAPI has a significant impact on FAP-targeted PET imaging and radiotherapy. Lower molar doses of FAPI improved the specificity of tumor uptake, thereby enhancing imaging and radiotherapy effectiveness. The research also emphasized the importance of precisely controlling the molar dose of radiolabeled FAPI to increase reliability and reproducibility in preclinical studies. Additionally, the QMA adsorption strategy developed by the research team provides a novel method for the preparation of radiolabeled FAPI, which holds promise for broader application in future preclinical and clinical research.

Research Highlights

  1. First Systematic Study on FAPI Molar Dose Effects: This study is the first to systematically explore the impact of FAPI molar doses on PET imaging and radiotherapy in preclinical models, addressing a significant research gap in this field.
  2. QMA Adsorption Strategy Enhances Apparent Molar Activity: The QMA adsorption strategy developed by the team effectively improved the apparent molar activity of radiolabeled FAPI, providing new insights into radiopharmaceutical preparation.
  3. Validation in Multiple Tumor Models: The reliability and repeatability of FAPI PET imaging were validated across multiple tumor models, strongly supporting FAPI’s application in tumor diagnosis and therapy.

Additional Insight

The study also found high physiological expression of FAP in the osteoarticular system of mice, which may explain the high osteoarticular uptake observed in FAPI PET imaging. This discovery suggests that the physiological differences in FAP expression between mice and humans should be considered during preclinical-to-clinical translation. Moreover, single-cell RNA sequencing databases confirmed significant FAP expression in bone marrow mesenchymal cells, further revealing its physiological role in the osteoarticular system.

This study provides crucial theoretical and practical guidance for preclinical research on FAP-targeted PET imaging and radiotherapy, offering substantial scientific and translational value.