Fibroblast activation protein-targeted radiopharmaceuticals: Recent trends in cancer diagnosis

FAP-specific PET radiotracers

¹⁸F-FDG remains the most widely used PET imaging tracer in clinical oncology, providing insights into tumor metabolism as a glucose analog. However, its diagnostic accuracy can be limited due to high physiological uptake in normal tissues, such as the brain, and in sites of inflammation, which may lead to false-positive findings^[4,5] [Table 1]. To overcome these limitations, FAP-based radiotracers [Figure 1] have been developed, offering several advantages, including independence from blood glucose levels, superior tumor-to-background ratios (TBRs) [Figure 2], rapid renal clearance, and favorable pharmacokinetics^[6] [Table 2].

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Figure 1:

This figure illustrates the relationship of cancer-associated fibroblasts to the tumor microenvironment and outlines strategies for the development of FAPI-based radiotracers. (FAPI: Fibroblast activated protein inhibitor)

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Figure 2:

Representative comparison of eight patients with different tumor entities undergoing both [18F]fluoro-D-glucose - (¹⁸F-FDG-PET) and ¹⁸F-FAPI-42 positron emission tomography imaging within <1 week. As shown by the arrows. (FAPI: Fibroblast activated protein inhibitor)

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Fibroblast activation protein inhibitor (FAPI)-02 and FAPI-04 are small organic molecules derived from (4-quinolinyl) glycyl-2-cyanopyrrolidine. FAPI-04 was created by adding fluorine atoms to the proline moiety in FAPI-02, resulting in a higher affinity for FAP. In addition, the inclusion of the 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA) chelator allows FAPI-04 to be labeled with various radionuclides, enabling high-quality imaging across different tumor types. Although both FAPI-02 and FAPI-04 demonstrate similar maximum standardized uptake value (SUV_max) in tumor tissues and only minor differences in normal tissue uptake, FAPI-04 has the advantage of longer tumor retention.^[7]

Most literature on FAPI-04 involves labeling with ⁶⁸Ga, obtained from a ⁶⁸Ge/⁶⁸Ga generator. Using the DOTA chelator, 68Ga is conjugated to FAPI-04 to produce ⁶⁸Ga-FAPI-04, a radiotracer with high specificity and strong affinity for FAP. It exhibits excellent pharmacokinetics and has shown utility in tumor detection, staging, restaging, and treatment response assessment^[8-10]. It produces radionuclide, offering lower positron energy than ¹⁸F-FDG, resulting in higher image resolution and better contrast.

¹⁸F-FAPI-04 PET/computed tomography (CT) has demonstrated superior diagnostic performance over FDG PET/CT in detecting primary tumors, lymph node involvement, and distant metastases across various cancers, including gastric,^[11-13] pancreatic,^[14,15] intrahepatic cholangiocarcinoma,^[16] and lung adenocarcinoma.^[17,18]

To further improve imaging performance, researchers have modified FAPI-04’s molecular structure. One such variant, ¹⁸F-FAPI-42 (¹⁸F-1, 4, 7-triazacyclononane-1, 4, 7-triacetic acid [NOTA]-FAPI-04), substitutes DOTA with NOTA, enabling labeling with ¹⁸F. This tracer shows higher TBR in peritoneal, lymphatic, hepatic, osseous, and pleural lesions compared to ⁶⁸Ga-FAPI-04, while maintaining similar detection rates.^[19]

Another derivative, ¹⁸F-FGlc-FAPI-04, is a glycosylated version of FAPI-04 labeled with ¹⁸F. In preclinical models (HT-1080h FAP and U87MG), it demonstrated high tumor uptake and prolonged circulation. However, its hepatobiliary clearance leads to significant physiological uptake in the liver and intestines, potentially complicating abdominal lesion assessment. It also shows uptake in specific bones and joints, posing additional challenges for clinical use.^[20]

FAPI-46, another FAPI-04 analog, replaces the benzene ether bond with nitrogen, improving pharmacokinetics and reducing radiation exposure. Biodistribution studies have shown that ⁶⁸Ga-FAPI-46 achieves higher and increasing TBR over time compared to ⁶⁸Ga-FAPI-04, suggesting superior diagnostic utility. Its longer tumor retention and higher uptake also make it a potential candidate for therapeutic applications.^[21]

Clinical investigations by Dadgar et al.^[22] confirmed that ⁶⁸Ga-FAPI-46 PET/CT offers greater sensitivity and specificity in detecting metastases than ¹⁸F-FDG, particularly in breast, gastrointestinal, cervical, and thyroid cancers. Based on FAPI-46, several enhanced probes have been developed, including dimeric FAPI-46 (FAPI-46-F1D), tetrameric FAPI-46, and FAPI-46-evans blue (EB), which is albumin-bound through Evans Blue, all demonstrating prolonged tumor retention.^[23,24] In 2022, Zhao et al. synthesized the probe ⁶⁸Ga-DOTA-2P(FAPI)₂ by attaching two identical FAPI-46 targeting motifs to the same molecule through the strategy of constructing dimer derivatives.^[25] In biodistribution experiments in mice with hepatocellular carcinoma patient-derived xenografts (HCC-PDX) tumors, a human HCC xenograft model, 68Ga-DOTA-2P(FAPI)₂ showed higher tumor uptake and tumor-to-kidney ratios, with significant advantages over ⁶⁸Ga-FAPI-46. PET/CT imaging data of patients similarly showed that 68Ga-DOTA-2P(FAPI)2 showed significantly higher uptake at the tumor site than ⁶⁸Ga-FAPI-46. Dual-target probes such as FAPI-RGD and FAPI-LM3, also based on FAPI-46, have further improved tumor uptake and retention compared to their monomeric counterparts.^[26-29]

FAPI-74, which incorporates the NOTA chelator, can be labeled with ¹⁸F through the Al¹⁸F method. Among ¹⁸F-labeled FAPI tracers, it shows the highest tumor accumulation and the lowest radiation dose. In PET/CT imaging of non-small cell lung cancer and widespread liver and bone metastases, it clearly delineates both primary and metastatic lesions due to its rapid blood clearance and low background activity in normal tissues.^[30,31]

A clinical study by Watabe et al.^[32] involving 31 patients demonstrated that ¹⁸F-FAPI-74 achieves higher SUVmax in primary malignant lesions than in benign ones. It also outperforms 18F-FDG in detecting both primary and metastatic tumors, especially in cases where FDG uptake is minimal or absent.^[32] These findings underscore ¹⁸F-FAPI-74 as a highly promising molecular probe for cancer imaging.

In 2024, Liu et al.^[33] constructed a novel cyclic peptide with an N-oxalyl-modified tail coupled to NOTA and ⁶⁴Cu labeled (⁶⁴Cu-FAP-N-oxalyl modified cyclic peptide [NOX]). Compared with ⁶⁴Cu-FAPI-04, ⁶⁴Cu-FAP-NOX exhibited faster and higher rates of cellular uptake and internalization but lower rates of cellular efflux in A549.hFAP cells. In vivo experiments showed more intense tumor accumulation of ⁶⁴Cu-FAP-NOX and slower clearance from the target. ⁶⁴CuFAP-NOX is a promising FAP-targeted tracer and may offer advantages in terms of image contrast, imaging time window, and low uptake in normal tissue in anticipation of large cohort studies.^[33]

In summary, these FAPI-based PET tracers target CAFs, exhibit glucose-independent uptake, high TBR, and favorable pharmacokinetic properties. FAPI-04, particularly its ¹⁸F-and ⁶⁸Ga-labeled forms, outperforms FDG in detecting primary and metastatic lesions in various cancers. Structural derivatives such as ¹⁸F-FAPI-42, FAPI-46 (and its multimers or dual-targeted forms), FAPI-74, and 64Cu-FAP-NOX further enhance tumor retention, image contrast, and detection sensitivity. These advances make FAPI tracers promising alternatives or supplements to first appeared in the introduction section (FDG) in tumor PET imaging.

FAP-specific SPECT radiotracers

Single-photon radionuclides, such as technetium-99m (99mTc) and indium-111 (¹¹¹In), are the most commonly used in clinical practice due to their affordability and widespread availability. In recent years, researchers have successfully achieved ^99mTc labeling of FAPI molecules through various strategies. Experimental data demonstrate that this ^99mTc-labeled FAPI tracer exhibits high selectivity for FAP-expressing cells and tumor xenografts in both in vitro experiments and tumor-bearing mouse models, demonstrating its promising clinical application^[34] [Table 3].