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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 [ABSTRACT FROM AUTHOR]

Purpose: The advancement of heterodimeric tracers, renowned for their high sensitivity, marks a significant trend in the development of radiotracers for cancer diagnosis. Our prior work on [68Ga]Ga-HX01, a heterodimeric tracer targeting CD13 and integrin αvβ3, led to its approval for phase I clinical trials by the China National Medical Production Administration (NMPA). However, its fast clearance and limited tumor retention pose challenges for broader clinical application in cancer treatment. This study aims to develop a new radiopharmaceutical with increased tumor uptake and prolonged retention, rendering it a potential therapeutic candidate. Methods: New albumin binder-conjugated compounds were synthesized based on the structure of HX01. In vitro and in vivo evaluation of these new compounds were performed after labelling with 68Ga. Small-animal PET/CT imaging were conducted at different time points at 0.5–6 h post injection (p.i.) using BxPC-3 xenograft mice models. The one with the best imaging performance was further radiolabeled with 177Lu for small-animal SPECT/CT and ex vivo biodistribution investigation. Results: We have synthesized novel albumin binder-conjugated compounds, building upon the structure of HX01. When radiolabeled with 68Ga, all compounds demonstrated improved pharmacokinetics (PK). Small-animal PET/CT studies revealed that these new albumin binder-conjugated compounds, particularly [68Ga]Ga-L6, exhibited significantly enhanced tumor accumulation and retention compared with [68Ga]Ga-L0 without an albumin binder. [68Ga]Ga-L6 outperformed [68Ga]Ga-L7, a compound developed using a previously reported albumin binder. Furthermore, [177Lu]Lu-L6 demonstrated rapid clearance from normal tissues, high tumor uptake, and prolonged retention in small-animal SPECT/CT and biodistribution studies, positioning it as an ideal candidate for radiotherapeutic applications. Conclusion: A new integrin αvβ3 and CD13 targeting compound was screened out. This compound bears a novel albumin binder and exhibits increased tumor uptake and prolonged tumor retention in BxPC-3 tumors and low background in normal organs, making it a perfect candidate for radiotherapy when radiolabeled with 177Lu. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In solid tumors, there are multiple barriers for a chimeric antigen receptor (CAR) T cell to surmount in order to reach the tumor site. For better understanding whether CAR T cells effectively infiltrate into tumor site, and simultaneously, whether there are off-target effects, real-time monitoring technologies need to be established. Cell-based positron emission tomography reporter genes have been developed to monitor engineered cells in living subjects. In this study, we reported the construction of a novel reporter gene truncated prostate-specific membrane antigen (ΔPSMA) pending for monitoring CAR T cells using 68Ga-PSMA-617 and a method for tracking the distribution of CAR T cells in vivo was developed. Data were provided to demonstrate that ΔPSMA was predominantly localized on the plasma membrane and could take up 68Ga-PSMA-617 in vitro in a time-dependent manner. And the expression of ΔPSMA did not affect CAR expression and cytolytic capacity of CAR T cells. CAR-ΔPSMA T cell xenografts in nude mice were clearly imaged by positron emission tomography 60 min after injection of 68Ga-PSMA-617. PSMA paired with 68Ga-PSMA-617 was capable of identifying approximately 1 × 104 engineered CAR T cells. The ability to image small numbers of CAR T cells in vivo would be helpful to accelerate the translation of cell-based therapies into the clinic, and it may reinforce our understanding of treatment success, failure, and toxicity. [ABSTRACT FROM AUTHOR]

Purpose: The integrin αvβ3 and aminopeptidase N (APN/CD13) play vital roles in the tumor angiogenesis process. They are highly expressed in a variety of tumor cells and proliferating endothelial cells during angiogenesis, which have been considered as highly promising targets for tumor imaging. Arginine-glycine-aspartic (RGD) and asparagine-glycine-arginine (NGR) are two peptides specifically binding to the integrin αvβ3 and CD13, respectively. In this study, we optimized our previously developed probe and preclinically evaluated the new integrin αvβ3 and CD13 dual-targeted probe, NOTA-RGD-NGR (denoted as HX01) radiolabeled with 68Ga, in 10 different subcutaneous and orthotopic tumor models. Methods: The specific activity and radiochemical purity of [68Ga]Ga-HX01 were identified. The dual-receptor targeting ability was confirmed by a series of blocking studies and partly muted tracers using BxPC-3 xenograft model. The dynamic imaging study and dose escalation study were explored to determine the optimal imaging time point and dosage in the BxPC-3 xenograft model. Next, we established a variety of subcutaneous and orthotopic tumor models including pancreas (BxPC-3), breast (MCF-7), gallbladder (NOZ), lung (HCC827), ovary (SK-OV-3), colorectal (HCT-8), liver (HuH-7), stomach (NUGC-4), and glioma (U87) cancers. All models underwent [68Ga]Ga-HX01 PET/CT imaging about 2 weeks post-inoculation, with a subset of them undergoing [18F]FDG PET/CT scan performed concurrently, and their results were compared. In addition, ex vivo biodistribution studies were also performed for verifying the semi-quantitative results of the non-invasive PET images. Results: [68Ga]Ga-HX01 significantly outperformed single target probes in the BxPC-3 xenograft model. All blocking and single target groups exhibited significantly descending tumor uptake. The high tumor uptakes were found in BxPC-3, MCF-7, and NOZ subcutaneous tumors (%ID/g > 1.1), while middle uptakes were observed in HCC827, SK-OV-3, HCT-8, and HuH-7 subcutaneous tumor (%ID/g 0.7–1.0). Due to the low background, the tumor-to-muscle and tumor-to-blood ratios of [68Ga]Ga-HX01 were higher than that of [18F]FDG. Conclusions: [68Ga]Ga-HX01, as a dual target imaging agent, exhibited superior in vivo performance in different subcutaneous and orthotopic mice models of human tumors over [18F]FDG and its respectively mono-receptor targeted agents, which warrants the future clinical translation for tumor imaging. [ABSTRACT FROM AUTHOR]

Ovarian cancer has the highest mortality rate of gynecologic malignancy. 18F-FDG positron emission tomography (PET) adds an important superiority over traditional anatomic imaging modalities in oncological imaging but has drawbacks including false negative results at the early stage of ovarian cancer, and false positives when inflammatory comorbidities are present. Aminopeptidase N (APN, also known as CD13) and integrin αvβ3 are two important targets overexpressed on tumor neo-vessels and frequently on ovarian cancerous cells. In this study, we used subcutaneous and metastatic models of ovarian cancer and muscular inflammation models to identify 68Ga-NGR-RGD, a heterodimeric tracer consisting of NGR and RGD peptides targeting CD13 and integrin αvβ3, respectively, and compared it with 18F-FDG. We found that 68Ga-NGR-RGD showed greater contrast in SKOV3 and ES-2 tumors than 18F-FDG. Low accumulation of 68Ga-NGR-RGD but avid uptake of 18F-FDG were observed in inflammatory muscle. In abdominal metastasis models, PET imaging with 68Ga-NGR-RGD allowed for rapid and clear delineation of both peritoneal and liver metastases (3-6 mm), whereas, 18F-FDG could not distinguish the metastasis lesions due to the relatively low metabolic activity in tumors and the interference of intestinal physiological 18F-FDG uptake. Due to the high tumor-targeting efficacy, low inflammatory uptake, and higher tumor-to-background ratios compared to that of 18F-FDG, 68Ga-NGR-RGD presents a promising imaging agent for diagnosis, staging, and follow-up of ovarian tumors. [ABSTRACT FROM AUTHOR]

Ovarian cancer has the highest mortality rate of gynecologic malignancy. 18 F-FDG positron emission tomography (PET) adds an important superiority over traditional anatomic imaging modalities in oncological imaging but has drawbacks including false negative results at the early stage of ovarian cancer, and false positives when inflammatory comorbidities are present. Aminopeptidase N (APN, also known as CD13) and integrin α v β 3 are two important targets overexpressed on tumor neo-vessels and frequently on ovarian cancerous cells. In this study, we used subcutaneous and metastatic models of ovarian cancer and muscular inflammation models to identify 68 Ga-NGR-RGD, a heterodimeric tracer consisting of NGR and RGD peptides targeting CD13 and integrin α v β 3 , respectively, and compared it with 18 F-FDG. We found that 68 Ga-NGR-RGD showed greater contrast in SKOV3 and ES-2 tumors than 18 F-FDG. Low accumulation of 68 Ga-NGR-RGD but avid uptake of 18 F-FDG were observed in inflammatory muscle. In abdominal metastasis models, PET imaging with 68 Ga-NGR-RGD allowed for rapid and clear delineation of both peritoneal and liver metastases (3-6 mm), whereas, 18 F-FDG could not distinguish the metastasis lesions due to the relatively low metabolic activity in tumors and the interference of intestinal physiological 18 F-FDG uptake. Due to the high tumor-targeting efficacy, low inflammatory uptake, and higher tumor-to-background ratios compared to that of 18 F-FDG, 68 Ga-NGR-RGD presents a promising imaging agent for diagnosis, staging, and follow-up of ovarian tumors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Long, Shao, Ji, Song, Lv, Xia, Liu, Zhang, Zeng, Lan and Gai.)