Non-invasive visualization of liver fibrosis with [68Ga]Ga-DOTA-FAPI-04 PET from preclinical insights to clinical translation.

PURPOSE: The reversibility of early liver fibrosis highlights the need for improved early detection and monitoring techniques. Fibroblast activation protein (FAP) is a promising theranostics target significantly upregulated during fibrosis. This preclinical and preliminary clinical study investigated a FAP-targeted probe, gallium-68-labeled FAP inhibitor 04 ([68Ga]Ga-DOTA-FAPI-04), for its capability to visualize liver fibrosis. METHODS: The preclinical study employed [68Ga]Ga-DOTA-FAPI-04 micro-positron emission tomography (PET)/computed tomography (CT) on carbon tetrachloride-induced mice model (n = 34) and olive oil-treated control group (n = 26), followed by validation of the probe's biodistribution. Hepatic uptake was correlated with fibrosis and inflammation levels, quantified through histology and serum assays. FAP and α-smooth muscle actin expression were determined by immunohistochemistry, as well as immunofluorescence. The subsequent clinical trial enrolled 26 patients with suspected or confirmed liver fibrosis to undergo [68Ga]Ga-DOTA-FAPI-04 PET/magnetic resonance imaging or PET/CT. Key endpoints included correlating [68Ga]Ga-DOTA-FAPI-04 uptake with histological inflammation grades and fibrosis stages, and evaluating its diagnostic and differential efficacy compared to established serum markers and liver stiffness measurement (LSM). RESULTS: [68Ga]Ga-DOTA-FAPI-04 mean uptake in mice livers was notably higher than in control mice, increasing from week 6 [0.70 ± 0.11 percentage injected dose per cubic centimeter (%ID/cc)], peaking at week 10 (0.97 ± 0.15%ID/cc) and slightly reducing at week 12 (0.89 ± 0.28%ID/cc). The hepatic biodistribution and FAP expression showed a consistent trend. In the patient cohort, hepatic [68Ga]Ga-DOTA-FAPI-04 uptake presented moderate correlations with inflammation grades (r = 0.517 to 0.584, all P < 0.05) and fibrosis stages (r = 0.653 to 0.698, all P < 0.01). The average SUVmax to background ratio in the liver showed superior discriminative ability, especially between stage 0 and stage 1, outperforming LSM (area under curve 0.984 vs. 0.865). CONCLUSION: [68Ga]Ga-DOTA-FAPI-04 PET shows significant potential for non-invasive visualization and dynamic monitoring of liver fibrosis in both preclinical experiment and preliminary clinical trial, especially outperforming other common clinical indicators in the early stage. TRIAL REGISTRATION: NCT04605939. Registered October 25, 2020, https://clinicaltrials.gov/study/NCT04605939.

[68Ga]FAPI PET for Imaging and Treatment Monitoring in a Preclinical Model of Pulmonary Fibrosis: Comparison to [18F]FDG PET and CT.

PURPOSE: This study aimed to evaluate the feasibility of using [68Ga]-fibroblast-activating protein inhibitor (FAPI) positron emission tomography (PET) imaging for diagnosing pulmonary fibrosis in a mouse model. We also examined its value in monitoring treatment response and compared it with traditional [18F]-fluorodeoxyglucose (FDG) PET and computed tomography (CT) imaging. METHODS: A model of idiopathic pulmonary fibrosis was established using intratracheal injection of bleomycin (BLM, 2 mg/kg) into C57BL/6 male mice. For the treatment of IPF, a daily oral dose of 400 mg/kg/day of pirfenidone was administered from 9 to 28 days after the establishment of the model. Disease progression and treatment efficacy were assessed at different stages of the disease every week for four weeks using CT, [18F]FDG PET, and [68Ga]FAPI PET (baseline imaging performed at week 0). Mice were sacrificed and lung tissues were harvested for hematoxylin-eosin staining, picrosirius red staining, and immunohistochemical staining for glucose transporter 1 (GLUT1) and FAP. Expression levels of GLUT1 and FAP in pathological sections were quantified. Correlations between imaging parameters and pathological quantitative values were analyzed. RESULTS: CT, [18F]FDG PET and [68Ga]FAPI PET revealed anatomical and functional changes in the lung that reflected progression of pulmonary fibrosis. In untreated mice with pulmonary fibrosis, lung uptake of [18F]FDG peaked on day 14, while [68Ga]FAPI uptake and mean lung density peaked on day 21. In mice treated with pirfenidone, mean lung density and lung uptake of both PET tracers decreased. Mean lung density, [18F]FDG uptake, and [68Ga]FAPI uptake correlated well with quantitative values of picrosirius red staining, GLUT1 expression, and FAP expression, respectively. Conclusions: Although traditional CT and [18F]FDG PET reflect anatomical and metabolic status in fibrotic lung, [68Ga]FAPI PET provides a means of evaluating fibrosis progression and monitoring treatment response.

Development and Evaluation of a Peptide Heterodimeric Tracer Targeting CXCR4 and Integrin αvß3 for Pancreatic Cancer Imaging.

Nowadays, pancreatic cancer is still a formidable disease to diagnose. The CXC chemokine receptor 4 (CXCR4) and integrin αvß3 play important roles in tumor development, progression, invasion, and metastasis, which are overexpressed in many types of human cancers. In this study, we developed a heterodimeric tracer 68Ga-yG5-RGD targeting both CXCR4 and integrin αvß3, and evaluated its feasibility and utility in PET imaging of pancreatic cancer. The 68Ga-yG5-RGD could accumulate in CXCR4/integrin αvß3 positive BxPC3 tumors in a high concentration and was much higher than that of 68Ga-yG5 (p < 0.001) and 68Ga-RGD (p < 0.001). No increased uptake of 68Ga-yG5-RGD was found in MX-1 tumors (CXCR4/integrin αvß3, negative). In addition, the uptake of 68Ga-yG5-RGD in BxPC3 was significantly blocked by excess amounts of AMD3100 (an FDA-approved CXCR4 antagonist) and/or unlabeled RGD (p < 0.001), confirming its dual-receptor targeting properties. The ex vivo biodistribution and immunohistochemical results were consistent with the in vivo imaging results. The dual-receptor targeting strategy achieved improved tumor-targeting efficiency and prolonged tumor retention in BxPC3 tumors, suggesting 68Ga-yG5-RGD is a promising tracer for the noninvasive detection of tumors that express either CXCR4 or integrin αvß3 or both, and therefore may have good prospects for clinical translation.

Noninvasive Monitoring of Reparative Fibrosis after Myocardial Infarction in Rats Using 68Ga-FAPI-04 PET/CT.

Noninvasively monitoring activated fibroblasts is of great value for understanding the dynamic process of myocardial fibrosis after myocardial infarction (MI). This study aimed to evaluate the feasibility of 68Ga-labeled fibroblast activation protein inhibitor 04 (68Ga-FAPI-04) for monitoring reparative fibrosis and reactive fibrosis after MI. MI models were prepared by ligation of the left anterior descending (LAD) coronary artery and validated by electrocardiogram and 18F-FDG PET/CT 1 day after MI and hematoxylin and eosin (HE) staining. 68Ga-FAPI-04 PET/CT scans (1, 3, 6, 9, 12, 15, 18, 21, 28, and 35 days after MI) were carried out in MI rats and sham-operated rats without ligation of LAD. Blocking experiments were carried out on MI rats on day 7 after MI with 68Ga-FAPI-04 and excessive FAPI-04. Autoradiography, HE staining, Masson's trichrome staining, and immunofluorescence staining were carried out for ex vivo validation. The infarcted area with decreased or defective myocardial metabolic activity in 18F-FDG PET/CT correspondingly showed high 68Ga-FAPI-04 uptake in the MI rats. The myocardial tracer uptake was significantly different between MI and sham-operated rats from day 1 to 28 after MI and reached peak value 6 days after MI (0.806 ± 0.257%ID/cc vs 0.199 ± 0.012%ID/cc, P < 0.05). Tracer uptake at the infarcted myocardium and normal tissues in MI rats decreased significantly after blocking. Obvious tracer uptake was confirmed by autoradiography, and immunofluorescence staining showed FAP+ cells in the infarcted myocardium and border zone. Masson's trichrome staining of the heart sections of MI rats at different times suggested the presence of myocardial fibrosis. 68Ga-FAPI-04 uptake was not observed in the distal uninjured myocardium throughout the observation period. In conclusion, 68Ga-FAPI-04 PET could noninvasively monitor the activated fibroblasts in the early stage post acute MI and may be helpful for evaluating the degree of reparative fibrosis, while reactive fibrosis monitoring still needs further study.

Visualizing γδ T cells by very late antigen-4-targeted positron emission tomography.

PURPOSE: γδ T cell-based immunotherapy has been rolled out as a promising treatment strategy for malignant tumors due to their potent anti-tumor cytotoxicity, ease of expansion, and unrestricted MHC feature. However, the dynamics and outcomes of γδ T cells in tumor sites are poorly understood. Reported strategies rely on ex vivo biolabeling, significantly limiting the application of γδ T cell molecular imaging. Herein, we investigated whether VLA-4 (very late antigen-4), a crucial component in the effective trafficking of lymphocytes, could serve as a biomarker to non-invasively visualize γδ T cells. METHODS: VLA-4-targeted tracer, 68 Ga-LLP2A, was evaluated in MDA-MB-231- and A549-bearing mice with adoptive transfer of γδ T cells by longitudinal PET/CT imaging. Imaging data were verified by ex vivo biodistribution studies, and the co-localization of CD3 and VLA-4 was validated by immunohistochemistry studies. RESULTS: 68 Ga-LLP2A showed high specificity to VLA-4-expressing γδ T cells in both in vitro and tumor-bearing mice with adoptive transfer of γδ T cells. Longitudinal PET imaging of 68 Ga-LLP2A in tumor-bearing mice with adoptive transfer of γδ T cells showed an increasing tumor tracer uptake, revealing the tumor-specific homing of γδ T cells. The presence of VLA-4-expressing γδ T cells in tumors was confirmed via histological analysis. CONCLUSION: To the best of our knowledge, we reported the first molecular probe, 68 Ga-LLP2A, for in vivo imaging of γδ T cells in live tumors, which advances PET imaging of γδ T cells and supports the translation of imaging agents for immunotherapeutic monitoring.

A Novel Radioimmune 99mTc-Labeled Tracer for Imaging Sphingosine 1-Phosphate Receptor 1 in Tumor Xenografts: An In Vitro and In Vivo Study.

Sphingosine-1-phosphate (S1P) is a phospholipid that regulates pleiotropic biological activities and exerts extracellular functions by binding to five specific G-protein-coupled receptors, S1P receptors (S1PR) 1-5. When activated by S1P, S1PR promote the proliferation and invasion of tumor cells by inducing the formation of new blood vessels. We developed and assessed a new monoclonal antibody imaging probe 99mTc-HYNIC-S1PR1mAb, to explore the feasibility of targeting the S1PR1 in vitro and in vivo. S1PR1mAb was prepared and followed by technetium-99m labeling with succinimidyl 6-hydraziniumnicotinate hydrochloride. Cell uptake and blocking studies were performed to investigate the binding specificity of 99mTc-HYNIC-S1PR1mAb in vitro. 99mTc-HYNIC-S1P1mAb was also tested in vivo in mice xenografted with SK-HEP-1 (high-expression of S1PR1) and MCF-7 (low-expression of S1PR1) using single-photon emission-computed tomography (SPECT). Ex vivo gamma counting of tissues from tumor-bearing mice was used to evaluate 99mTc-HYNIC-S1PR1mAb biodistribution. The biodistribution study results showed significantly higher uptake in SK-HEP-1 tumors than in MCF-7 tumors (P < 0.001). Reduced uptake of 99mTc-HYNIC-S1PR1mAb in SK-HEP-1 was observed in tumor-bearing nude mice pretreated with fingolimod, which binds competitively to the receptors, especially S1PR1. 99mTc-HYNIC-S1PR1mAb can be synthesized and specifically targeted to S1PR1 in vitro and in vivo, allowing S1PR1 expression assessment with SPECT imaging.