Purpose: This study aims to develop and evaluate a novel glypican-3 (GPC3)–targeted, single-chain variable fragment–based PET radiotracer for noninvasive assessment of GPC3 expression and precise diagnosis of hepatocellular carcinoma (HCC) in both preclinical models and a first-in-human clinical study. Experimental Design: The novel anti-GPC3 single-chain variable fragment, namely, XH-06, was labeled with gallium-68 to obtain [68Ga]Ga-XH-06. Cell uptake assays, small-animal PET imaging, and biodistribution studies were performed to evaluate its targeting ability. In the first-in-human study, eight patients with suspected HCC underwent [68Ga]Ga-XH-06 PET/MRI. Radiotracer uptake in tumors and normal tissues was quantified, and tumor-to-blood ratios and tumor-to-liver ratios were calculated. Results: [68Ga]Ga-XH-06 was synthesized with high radiochemical purity and exhibited specific uptake and efficient internalization in GPC3-positive cells. In subcutaneous and orthotopic animal models, [68Ga]Ga-XH-06 effectively visualized HCC tumors. Eight patients underwent [68Ga]Ga-XH-06 PET/MRI scans, and no adverse events were observed. The radiotracer successfully detected HCC lesions, including subcentimeter tumors, with high imaging contrast. Among seven patients with HCC, the median maximum standardized uptake value of the lesions was 16.9 (range, 8.2–51.8), the median tumor-to-liver ratio was 6.2 (range, 2.8–24.7), and the median tumor-to-blood ratio was 16.6 (range, 3.0–57.6) at 2.5 hours after injection. Conclusions: [68Ga]Ga-XH-06 is clinically promising for detecting GPC3-positive HCC with a favorable safety profile. Further investigation is warranted to validate the clinical value of GPC3-targeted PET imaging in HCC management. See related commentary by Seo and Flavell, p. 445

Fibroblast activation protein (FAP) is a type II transmembrane serine protease that is highly expressed in cancer-associated fibroblasts (CAFs) but absent in quiescent fibroblasts. Its overexpression is associated with poor prognosis in various cancers and contributes to treatment resistance. In recent years, radiolabeled FAP inhibitors (FAPI) for PET imaging have shown promising clinical value across a range of cancers. Gastrointestinal (GI) malignancies, which often exhibit a desmoplastic reaction with a high density of FAP-expressing CAFs, are particularly well-suited for FAPI PET. Given the limitations of [¹⁸F]FDG PET in GI cancers, such as low sensitivity in certain histological subtypes and high physiological background uptake, FAPI PET is expected to serve as a complementary method, potentially enhancing both diagnostic accuracy and treatment guidance. This review provides a comprehensive comparison of the clinical applications of FAPI PET and [¹⁸F]FDG PET in various GI cancers, including their value in diagnosis, staging, and treatment guidance. Additionally, this review summarizes studies on the expanding role of FAPI PET, including its use in assessing treatment response and predicting prognosis, aiming to provide insights into its potential contribution to the improved management of GI malignancies.

Despite significant progress in targeted cancer therapies and conventional imaging methodologies, the effective detection and treatment of solid tumours remain a major clinical challenge. This is thought to be caused by the complexity and heterogeneity found in the tumour microenvironment (TME), which significantly effects drug delivery and therapeutic response. Different levels of fibrosis, varying immune-cell infiltration, and disorganized vasculature form barriers for therapeutic approaches. However, in the next decade, radiotheranostics, defined here as the combined use of matched diagnostic and therapeutic radiopharmaceuticals, could present a targeted and flexible strategy for addressing some of the challenges caused by the TME. By combining molecular imaging with therapeutic delivery, it enables the in vivo visualization of TME features and the selective treatment of tumour and stromal compartments. This provides the unique opportunity to target tumour regions resistant to conventional therapies, including those shaped by (extracellular matrix) ECM stiffness, immune infiltration, or hypoxia. However, new strategies are needed to identify targets and evaluate their efficacy for more precise therapies. In this review, we will discuss why radiotheranostics is an ideal field for advancing the therapeutic approaches to solid tumours by incorporating the growing understanding of the TME. We will discuss how key microenvironmental features affect radiotracer distribution and treatment outcomes. We will highlight emerging tools including ECM- and immune-targeted imaging, patient-derived organoids, and organ-on-chip models which will be instrumental in developing physiologically relevant radiopharmaceutical therapies. Finally, we will discuss how spatial/single-cell transcriptomic approaches can support target discovery and allow for patient outcome assessment, with the aim of integrating microenvironment-aware insights into the development of novel radiotheranostic agents.

Supplementary Figure S3. Quadrupole time-of-flight spectra of anti-GPC3 scFv (XH-06). The mass spectrum showed that the molecular weight of anti-GPC3 scFv was 26741 Da.

Supplementary Figure S6. Quantitative ROI data in [68Ga]Ga-XH-06 immunoPET imaging of Hep3B orthotopic xenograft models.

Supplementary Figure S5. Hematoxylin and eosin (H&E) staining and GPC3 immunohistochemistry (IHC) of the resected subcutaneous xenografts.