Identification of a Novel Vascular Endothelial Growth Factor Receptor-3-Targeting Peptide for Molecular Imaging of Metastatic Lymph Nodes.

Because of the insidious nature of lymphatic metastatic cancer, accurate imaging tracing is very difficult to achieve in the clinic. Previous studies have developed the LARGR peptide (named TMVP1) as a radiotracer for vascular endothelial growth factor receptor-3 (VEGFR-3) imaging in cancer. However, its affinity for the target remains insufficient, resulting in low imaging sensitivity. In this study, we identified a high-affinity VEGFR-3 targeting peptide, named TMVP1446, using a multiplex screening platform. TMVP1446 demonstrated a dissociation constant of 8.97 × 10-8 M. Both in vitro and in vivo assays confirmed that fluorescently labeled TMVP1446 specifically bound to VEGFR-3. In a 4T1-luciferase tumor mouse model, cyanine 7-labeled TMVP1446 effectively discriminated between contralateral normal lymph nodes (c-LN) and cancer-metastatic sentinel lymph nodes (m-SLN). To evaluate the potential of TMVP1446, we developed a novel VEGFR-3 positron emission tomography radiotracer ([68Ga]Ga-DOTA-TMVP1446) for cancer-m-SLN imaging. [68Ga]Ga-DOTA-TMVP1446 accurately detected and assessed the status of lymph node metastasis, even in micrometastatic tumors, in the B16-F10 mouse tumor model. These findings suggest that TMVP1446 has great potential for advancing VEGFR-3 molecular imaging and metastatic sentinel lymph node imaging.

Peptide-based PET tracer targeting LAG-3 for evaluating the efficacy of immunotherapy in melanoma.

BACKGROUND: Lymphocyte activation gene 3 (LAG-3) is expressed on activated immune cells and has emerged as a promising target for immune checkpoints blockade. However, conflicting findings have been reported regarding the association between LAG-3 expression in tumors and patient prognosis, indicating the need for further investigation into the significance of LAG-3 expression levels in tumor therapies. In this study, 68Ga-NOTA-XH05, a novel peptide-based positron emission tomography (PET) tracer targeting LAG-3, was constructed to non-invasively detect LAG-3 expression in melanoma after CpG oligonucleotide (CpG) treatment and explore the relationship between LAG-3 expression and therapeutic effect. METHODS: The tracer 68Ga-NOTA-XH05 was identified by high-performance liquid chromatography after being prepared and purified. Cell uptake and blocking essays were performed to verify the specificity of the tracer in vitro. The expression of LAG-3 in B16-F10 subcutaneous tumors was monitored by flow cytometry, and its correlation with the tracer uptake was analyzed to evaluate the tracer specificity. PET imaging and biodistribution studies were conducted after CpG treatment of unilateral or bilateral B16-F10 subcutaneous tumor models to assess the ability of 68Ga-NOTA-XH05 in monitoring immunotherapy efficacy and the abscopal effect of CpG. RESULTS: Following purification, 68Ga-NOTA-XH05 exhibited high radiochemical purity and specificity. Flow cytometry analysis revealed a positive correlation between LAG-3 expression in tumors and the uptake of 68Ga-NOTA-XH05. In B16-F10 bearing mice treated with CpG, PET imaging using 68Ga-NOTA-XH05 demonstrated a higher tumor to blood ratio (TBR) compared with the control group. Furthermore, TBR values obtained from CpG-treated mice allowed for differentiation between responders and non-responders. In a bilateral subcutaneous tumor model where only right-sided tumors were treated with intratumoral injection of CpG, TBR values of left-sided tumors were significantly higher than those in the control group, indicating that 68Ga-NOTA-XH05 could effectively monitor the systemic effect of local CpG injection. CONCLUSION: Our findings highlight the detection capability of 68Ga-NOTA-XH05 in assessing LAG-3 expression levels within tumors and evaluating response to immunotherapy, thereby suggesting promising clinical translational prospects.

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 albumin binder-conjugated heterodimeric radiopharmaceuticals targeting integrin αvß3 and CD13 for cancer therapy.

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 αvß3, 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 αvß3 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.

Tau induces inflammasome activation and microgliosis through acetylating NLRP3.

BACKGROUND: Alzheimer's disease (AD) and related Tauopathies are characterised by the pathologically hyperphosphorylated and aggregated microtubule-associated protein Tau, which is accompanied by neuroinflammation mediated by activated microglia. However, the role of Tau pathology in microglia activation or their causal relationship remains largely elusive. METHODS: The levels of nucleotide-binding oligomerisation domain (NOD)-like receptor pyrin domain containing 3 (NLRP3) acetylation and inflammasome activation in multiple cell models with Tau proteins treatment, transgenic mice with Tauopathy, and AD patients were measured by Western blotting and enzyme-linked immunosorbent assay. In addition, the acetyltransferase activity of Tau and NLRP3 acetylation sites were confirmed using the test-tube acetylation assay, co-immunoprecipitation, immunofluorescence (IF) staining, mass spectrometry and molecular docking. The Tau-overexpressing mouse model was established by overexpression of human Tau proteins in mouse hippocampal CA1 neurons through the adeno-associated virus injection. The cognitive functions of Tau-overexpressing mice were assessed in various behavioural tests, and microglia activation was analysed by Iba-1 IF staining and [18F]-DPA-714 positron emission tomography/computed tomography imaging. A peptide that blocks the interaction between Tau and NLRP3 was synthesised to determine the in vitro and in vivo effects of Tau-NLRP3 interaction blockade on NLRP3 acetylation, inflammasome activation, microglia activation and cognitive function. RESULTS: Excessively elevated NLRP3 acetylation and inflammasome activation were observed in 3xTg-AD mice, microtubule-associated protein Tau P301S (PS19) mice and AD patients. It was further confirmed that mimics of 'early' phosphorylated-Tau proteins which increase at the initial stage of diseases with Tauopathy, including TauT181E, TauS199E, TauT217E and TauS262E, significantly promoted Tau-K18 domain acetyltransferase activity-dependent NLRP3 acetylation and inflammasome activation in HEK293T and BV-2 microglial cells. In addition, Tau protein could directly acetylate NLRP3 at the K21, K22 and K24 sites at its PYD domain and thereby induce inflammasome activation in vitro. Overexpression of human Tau proteins in mouse hippocampal CA1 neurons resulted in impaired cognitive function, Tau transmission to microglia and microgliosis with NLRP3 acetylation and inflammasome activation. As a targeted intervention, competitive binding of a designed Tau-NLRP3-binding blocking (TNB) peptide to block the interaction of Tau protein with NLRP3 inhibited the NLRP3 acetylation and downstream inflammasome activation in microglia, thereby alleviating microglia activation and cognitive impairment in mice. CONCLUSIONS: In conclusion, our findings provide evidence for a novel role of Tau in the regulation of microglia activation through acetylating NLRP3, which has potential implications for early intervention and personalised treatment of AD and related Tauopathies.

Noninvasive longitudinal PET/CT imaging of CAR T cells using PSMA reporter gene.

PURPOSE: Chimeric antigen receptor (CAR) T cell therapy has achieved great success in treating hematologic malignancies. However, it is yet to prove effective in the treatment of solid tumors. Thus, it is necessary to develop appropriate methodology for the long-term, accurate, and quantitative evaluation of the distribution and activities of CAR T cells in solid tumors. In the present study, we engineered TfR ΔPSMA CAR (CAR-ΔPSMA) T cells, which targeted the transferrin receptor (TfR) expressed by tumor cells and could be tracked in vivo via a reporter gene encoding the truncated prostate specific membrane antigen (ΔPSMA). We then quantitatively monitored these CAR T cells in vitro and in vivo using [68Ga]Ga-PSMA-617 positron emission tomography (PET)/computed tomography (CT). METHODS: The CAR-ΔPSMA T cells were genetically engineered by transducing T cells with a lentiviral vector encoding TfR41BBζ-T2A-ΔPSMA. Firstly, the target expression, activation, and cytotoxicity of CAR-ΔPSMA T cells were validated in vitro. Secondly, the minimum thresholds of CAR-ΔPSMA T cells detection for [68Ga]Ga-PSMA-617 PET/CT were also determined in vitro and in vivo respectively. Lastly, the feasibility of monitoring the biodistribution and infiltration of CAR-ΔPSMA T cells after systematic administration was evaluated in the breast cancer subcutaneous xenograft model. RESULTS: The CAR-ΔPSMA T cells retained activation and tumor killing capacity after transduction of the ΔPSMA-encoding reporter gene. Next, the CAR-ΔPSMA T cells could be reliably tracked by [68Ga]Ga-PSMA-617 PET/CT, the detection sensitivity of which was 250 cells/mm3 in vitro and 100 cells/mm3 in vivo. Next, the sequential imaging assays revealed that [68Ga]Ga-PSMA-617 PET/CT could be used to specifically visualize ΔPSMA+ CAR T cells at the tumor site. The increase in the [68Ga]Ga-PSMA-617 signal intensity over time allowed us to effectively detect CAR T cells in vivo. CONCLUSION: Our findings preliminarily confirmed that [68Ga]Ga-PSMA-617 PET/CT could reliably detect CAR-ΔPSMA T cells in vitro and in vivo in solid tumors, laying the foundation for the monitoring CAR T cell therapy in the future.

Melanin-targeted [18F]-PFPN PET imaging may shed light for clear cell sarcoma.

PURPOSE: Intracytoplasmic melanin pigment is a characteristic of clear cell sarcoma (CCS), which is a particularly deadly type of soft-tissue sarcoma. [18F]-N-(2-(diethylamino)ethyl)-5-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)picolinamide ([18F]-PFPN) is a positron emission tomography (PET) probe characterized by high melanin affinity. Therefore, this study aimed to investigate the feasibility of melanin-targeted [18F]-PFPN PET in patients with CCS. METHODS: This prospective single-centre study recruited patients with pathologically confirmed CCS. [18F]-FDG PET/computed tomography and [18F]-PFPN PET/magnetic resonance imaging scans were performed within 1 week of each other. The lesion numbers and [18F]-FDG and [18F]-PFPN PET parameters (maximum standardized uptake value [SUVmax], mean standardized uptake value [SUVmean], metabolic/melanotic tumour volume [MTV/MLTV], and total lesion glycolysis/melanin [TLG/TLM]) were collected. RESULTS: Three patients with CCS were recruited and received PET imaging. A total of 56 lesions were detected on [18F]-PFPN PET, including primary tumour and distant metastases. Identical lesions were not detected on [18F]-PFPN and [18F]-FDG PET. Twelve lesions (12/39, 30.77%) on [18F]-FDG imaging were missed on [18F]-PFPN, and 20 lesions (20/47, 42.55%) on [18F]-PFPN imaging were missed on [18F]-FDG. In quantitative analysis, the [18F]-FDG SUVmean (4.60 ± 3.24) was higher than the [18F]-PFPN SUVmean (3.0 ± 2.63) in all lesions (P = 0.01). No significant correlations were found between the SUVmax, SUVmean, MLTV/MTV, and TLM/TLG values of [18F]-PFPN and [18F]-FDG (P > 0.05). CONCLUSION: Melanin-targeted [18F]-PFPN PET imaging is feasible for the diagnosis of CCS. Different imaging features were displayed on [18F]-PFPN and [18F]-FDG PET imaging, demonstrating the complementary role of the tracers. Combined use of the two imaging modalities would be preferred in patients with CCS. CLINICAL TRIAL REGISTRATION: NCT05963035.

Preclinical evaluation of a dual-receptor targeted tracer [68Ga]Ga-HX01 in 10 different subcutaneous and orthotopic tumor models.

PURPOSE: The integrin αvß3 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 αvß3 and CD13, respectively. In this study, we optimized our previously developed probe and preclinically evaluated the new integrin αvß3 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.

Melanin-targeted [18F]-PFPN PET imaging for prognosticating patients with melanoma.

PURPOSE: Positron emission tomography (PET) using [18F]-PFPN, a melanin-targeted imaging tracer, has excellent diagnostic performance in patients with melanoma. This study aimed to investigate its value in prognostication and determine predictors of progression-free survival (PFS) and overall survival (OS). METHODS: We reviewed melanoma patients who underwent [18F]-PFPN and [18F]-FDG PET from February 2021 to July 2022. Clinical characteristics, follow-up data, and the following [18F]-PFPN PET parameters were recorded: maximum standardized uptake value (SUVmax), whole-body melanotic tumoral volume (WBMTV), and whole-body total lesion melanin (WBTLM). Receiver operating characteristic (ROC), Kaplan-Meier and Cox regression analyses were performed. RESULTS: Seventy-six patients (47 men and 29 women; mean age, 57.99 ± 10.72 years) were included for analysis. Median follow-up was 12.0 months (range: 1-22 months). Eighteen patients died and 38 experienced progression. Median OS was 17.60 months (95% confidence interval, 15.89-19.31). In the ROC analysis, [18F]-PFPN PET parameters were superior to those of [18F]-FDG PET in prognosticating death and disease progression. PFS and OS were significantly better in patients with lower SUVmax, WBMTV, and WBTLM on [18F]-PFPN PET (log-rank, P < 0.05). In the univariate analyses, distant metastasis, SUVmax, WBMTV, and WBTLM were significantly associated with cumulative incidence of PFS and OS (P < 0.05). In the multivariate analysis, SUVmax was an independent predictor of PFS and OS. CONCLUSIONS: [18F]-PFPN PET has a role in prognostication of melanoma patients. Patients with higher [18F]-PFPN SUVmax have worse prognosis. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, NCT05645484. Registered 9 December, 2022, https://clinicaltrials.gov/ct2/show/NCT05645484?cond=The+Prognostic+Value+of+18F-PFPN+PET+Imaging+in+Patients+With+Malignant+Melanoma&draw=2&rank=1.

Characterizing Early-Onset Alzheimer Disease Using Multiprobe PET/MRI: An AT(N) Framework-Based Study.

PURPOSE: Early-onset Alzheimer disease (EOAD) is rare, highly heterogeneous, and associated with poor prognosis. This AT(N) Framework-based study aimed to compare multiprobe PET/MRI findings between EOAD and late-onset Alzheimer disease (LOAD) patients and explore potential imaging biomarkers for characterizing EOAD. METHODS: Patients with AD who underwent PET/MRI in our PET center were retrospectively reviewed and grouped according to the age at disease onset: EOAD, younger than 60 years; and LOAD, 60 years or older. Clinical characteristics were recorded. All study patients had positive ß-amyloid PET imaging; some patients also underwent 18 F-FDG and 18 F-florzolotau PET. Imaging of the EOAD and LOAD groups was compared using region-of-interest and voxel-based analysis. Correlation of onset age and regional SUV ratios were also evaluated. RESULTS: One hundred thirty-three patients were analyzed (75 EOAD and 58 LOAD patients). Sex ( P = 0.515) and education ( P = 0.412) did not significantly differ between groups. Mini-Mental State Examination score was significantly lower in the EOAD group (14.32 ± 6.74 vs 18.67 ± 7.20, P = 0.004). ß-Amyloid deposition did not significantly differ between groups. Glucose metabolism in the frontal, parietal, precuneus, temporal, occipital lobe, and supramarginal and angular gyri was significantly lower in the EOAD group (n = 49) than in the LOAD group (n = 44). In voxel-based morphometry analysis, right posterior cingulate/precuneus atrophy was more obvious in the EOAD ( P < 0.001), although no voxel survived family-wise error correction. Tau deposition in the precuneus, parietal lobe, and angular, supramarginal, and right middle frontal gyri was significantly higher in the EOAD group (n = 18) than in the LOAD group (n = 13). CONCLUSIONS: Multiprobe PET/MRI showed that tau burden and neuronal damage are more severe in EOAD than in LOAD. Multiprobe PET/MRI may be useful to assess the pathologic characteristics of EOAD.

[177Lu]Lu-PSMA-617 theranostic probe for hepatocellular carcinoma imaging and therapy.

PURPOSE: This study aimed to explore the feasibility of using [177Lu]Lu-prostate-specific membrane antigen (PSMA)-617 and [177Lu]Lu-Evans blue (EB)-PSMA-617 for in vivo radioligand therapy by single-dose administration in a PSMA-positive hepatocellular carcinoma (HCC) xenograft mouse model. METHODS: [177Lu]Lu-PSMA-617 and [177Lu]Lu-EB-PSMA-617 were prepared, and labelling efficiency and radiochemical purity were determined. A HepG2 human HCC subcutaneous xenograft mouse model was established. After intravenous injection of [177Lu]Lu-PSMA-617 or [177Lu]Lu-EB-PSMA-617 (37 MBq) into the mouse model, single-photon emission computed tomography/computed tomography (SPECT/CT) was performed. Biodistribution studies were conducted to verify targeting specificity and pharmacokinetics. In the radioligand therapy study, mice were randomized into 4 groups: 37 MBq [177Lu]Lu-PSMA-617, 18.5 MBq [177Lu]Lu-PSMA-617, 7.4 MBq [177Lu]Lu-EB-PSMA-617, and saline (control). A single-dose administration was applied at the beginning of therapy studies. Tumor volume, body weight, and survival were monitored every 2 days. After the end of therapy, mice were euthanized. Tumors were then weighed, and systemic toxicity was evaluated via blood testing and histological examination of healthy organs. RESULTS: [177Lu]Lu-PSMA-617 and [177Lu]Lu-EB-PSMA-617 were successfully prepared with high purity and stability. SPECT/CT and biodistribution showed that tumor uptake was higher and persisted longer for [177Lu]Lu-EB-PSMA-617 compared with [177Lu]Lu-PSMA-617. [177Lu]Lu-PSMA-617 was rapidly cleared from the blood, while [177Lu]Lu-EB-PSMA-617 persisted for significantly longer. In radioligand therapy studies, tumor growth was significantly suppressed in the 37 MBq [177Lu]Lu-PSMA-617, 18.5 MBq [177Lu]Lu-PSMA-617, and 7.4 MBq [177Lu]Lu-EB-PSMA-617 groups compared to the saline group. Median survival was 40, 44, 43, and 30 days, respectively. No healthy organ toxicity was observed in safety and tolerability evaluation. CONCLUSIONS: Radioligand therapy using [177Lu]Lu-PSMA-617 and [177Lu]Lu-EB-PSMA-617 significantly suppressed tumor growth and prolonged survival time in PSMA-positive HCC xenograft mice without obvious toxicity. These radioligands appear promising for clinical use in humans, and future studies are warranted.

Q.Clear reconstruction for reducing the scanning time for 68 Ga-DOTA-FAPI-04 PET/MR imaging.

PURPOSE: This study aims to determine whether Q.Clear positron emission tomography (PET) reconstruction may reduce tracer injection dose or shorten scanning time in 68Gallium-labelled fibroblast activation protein inhibitor (68 Ga-FAPI) PET/magnetic resonance (MR) imaging. METHODS: We retrospectively collected cases of 68 Ga-FAPI whole-body imaging performed on integrated PET/MR. PET images were reconstructed using three different methods: ordered subset expectation maximization (OSEM) reconstruction with full scanning time, OSEM reconstruction with half scanning time, and Q.Clear reconstruction with half scanning time. We then measured standardized uptake values (SUVs) within and around lesions, alongside their volumes. We also evaluated image quality using lesion-to-background (L/B) ratio and signal-to-noise ratio (SNR). We then compared these metrics across the three reconstruction techniques using statistical methods. RESULTS: Q.Clear reconstruction significantly increased SUVmax and SUVmean within lesions (more than 30%) and reduced their volumes in comparison with OSEM reconstruction. Background SUVmax also increased significantly, while background SUVmean showed no difference. Average L/B values for Q.Clear reconstruction were only marginally higher than those from OSME reconstruction with half-time. SNR decreased significantly in Q.Clear reconstruction compared with OSEM reconstruction with full time (but not half time). Differences between Q.Clear and OSEM reconstructions in SUVmax and SUVmean values within lesions were significantly correlated with SUVs within lesions. CONCLUSIONS: Q.Clear reconstruction was useful for reducing PET injection dose or scanning time while maintaining the image quality. Q.Clear may affect PET quantification, and it is necessary to establish diagnostic recommendations based on Q.Clear results for Q.Clear application.

Preparation and In Vitro Evaluation of a Gadolinium-Containing Vitamin E TPGS Micelle as a Potential Contrast Agent for MR Imaging.

The application of many currently evaluated macromolecular contrast agents for magnetic resonance imaging (MRI) has been limited because of their bio-incompatibility and toxicity. The aim of this study is to synthesize and characterize a new micelle-based TPGS gadolinium chelate as a biocompatible MRI contrast agent for prolonged blood circulation time and good tumor imaging contrast. The TPGS-gadolinium conjugate was prepared through the conjugation between TPGS-SA and bifunctional L-NETA-Gd chelate. The conjugate was characterized with regard to molecular weight, critical micellar concentration and particle sizes, cellular uptake, and in vitro cell MRI. Distributions of the MRI contrast agent in various organs were determined via intravenous injection of the agent into mice bearing xenograft tumors. The successfully prepared TPGS-L-NETA-Gd micelle exhibited improved cellular uptake in HepG2 cells and xenografts and high in vivo safety. Distributions of TPGS-L-NETA-Gd in mice showed enhanced cellular uptake up to 2 h after the contrast agent injection. Its in vitro and in vivo properties make it a favorable macromolecular MRI contrast agent for future in vivo imaging.

Nanomedicine for renal cell carcinoma: imaging, treatment and beyond.

The kidney is a vital organ responsible for maintaining homeostasis in the human body. However, renal cell carcinoma (RCC) is a common malignancy of the urinary system and represents a serious threat to human health. Although the overall survival of RCC has improved substantially with the development of cancer diagnosis and management, there are various reasons for treatment failure. Firstly, without any readily available biomarkers, timely diagnosis has been greatly hampered. Secondly, the imaging appearance also varies greatly, and its early detection often remains difficult. Thirdly, chemotherapy has been validated as unavailable for treating renal cancer in the clinic due to its intrinsic drug resistance. Concomitant with the progress of nanotechnological methods in pharmaceuticals, the management of kidney cancer has undergone a transformation in the recent decade. Nanotechnology has shown many advantages over widely used traditional methods, leading to broad biomedical applications ranging from drug delivery, prevention, diagnosis to treatment. This review focuses on nanotechnologies in RCC management and further discusses their biomedical translation with the aim of identifying the most promising nanomedicines for clinical needs. As our understanding of nanotechnologies continues to grow, more opportunities to improve the management of renal cancer are expected to emerge.

A prognostication system based on clinical parameters and [18F]-FDG PET/CT in patients with newly diagnosed multiple myeloma.

PURPOSE: This study aimed to assess prognosis of patients with newly diagnosed multiple myeloma (NDMM) by combining [18F]-FDG positron emission tomography (PET)/CT parameters and clinical indices. METHODS: Clinical data and PET/CT parameters of 133 NDMM patients were retrospectively analyzed for associations between clinical indices and PET/CT parameters. Independent predictors of progression-free survival (PFS) and overall survival (OS) were determined. A new prognostic prediction system (NPPS) was constructed based on our findings. Prediction effectiveness was compared among the NPPS, International Staging System (ISS), Revised ISS (R-ISS), and R2-ISS. RESULTS: Prevalence of elevated ß2-microglobulin, serum creatinine (sCr), serum calcium (sCa), and C-reactive protein concentrations was higher in patients with higher SUVmax (≥ 5.3). Prevalence of elevated sCa, sCr, and extramedullary disease (EMD) was higher in patients with a higher number of focal lesions (≥ 10). SUVmax, serum free-light chain (sFLC) ratio, and EMD were independent predictors of PFS and OS. The NPPS used SUVmax, sFLC ratio, and EMD could effectively predict OS and was more effective at prognostication than the ISS, R-ISS, and R2-ISS. CONCLUSIONS: [18F]-FDG PET/CT parameters play a significant role in predicting prognosis in NDMM patients. The NPPS based on SUVmax, sFLC ratio, and EMD outperformed the ISS, R-ISS, and R2-ISS in prognostication.

Chlorin e6-loaded goat milk-derived extracellular vesicles for Cerenkov luminescence-induced photodynamic therapy.

PURPOSE: Photodynamic therapy (PDT) is a promising cancer treatment strategy with rapid progress in preclinical and clinical settings. However, the limitations in penetration of external light and precise delivery of photosensitizers hamper its clinical translation. As such, the internal light source such as Cerenkov luminescence (CL) from decaying radioisotopes offers new opportunities. Herein, we show that goat milk-derived extracellular vesicles (GEV) can act as a carrier to deliver photosensitizer Chlorin e6 (Ce6) and tumor-avid 18F-FDG can activate CL-induced PDT for precision cancer theranostics. METHODS: GEV was isolated via differential ultracentrifugation of commercial goat milk and photosensitizer Ce6 was loaded by co-incubation to obtain Ce6@GEV. Tumor uptake of Ce6@GEV was examined using confocal microscopy and flow cytometry. To demonstrate the ability of 18F-FDG to activate photodynamic effects against cancer cells, apoptosis rates were measured using flow cytometry, and the production of 1O2 was measured by reactive oxygen species (ROS) monitoring kit. Moreover, we used the IVIS device to detect Cherenkov radiation and Cerenkov radiation energy transfer (CRET). For animal experiments, a small-animal IVIS imaging system was used to visualize the accumulation of the GEV drug delivery system in tumors. PET/CT and CL images of the tumor site were performed at 0.5, 1, and 2 h. For in vivo antitumor therapy, changes of tumor volume, survival time, and body weight in six groups of tumor-bearing mice were monitored. Furthermore, the blood sample and organs of interest (heart, liver, spleen, lungs, kidneys, and tumor) were collected for hematological analysis, immunohistochemistry, and H&E staining. RESULTS: Confocal microscopy of 4T1 cells incubated with Ce6@GEV for 4 h revealed strong red fluorescence signals in the cytoplasm, which demonstrated that Ce6 loaded in GEV could be efficiently delivered into tumor cells. When Ce6@GEV and 18F-FDG co-existed incubated with 4T1 cells, the cell viability plummeted from more than 88.02 ± 1.30% to 23.79 ± 1.59%, indicating excellent CL-induced PDT effects. In vivo fluorescence images showed a peak tumor/liver ratio of 1.36 ± 0.09 at 24 h after Ce6@GEV injection. For in vivo antitumor therapy, Ce6@GEV + 18F-FDG group had the best tumor inhibition rate (58.02%) compared with the other groups, with the longest survival rate (35 days, 40%). During the whole treatment process, neither blood biochemical analysis nor histological observation revealed vital organ damage, suggesting the biosafety of this treatment strategy. CONCLUSIONS: The simultaneous accumulation of 18F-FDG and Ce6 in tumor tissues is expected to overcome the deficiency of traditional PDT. This strategy has the potential to extend PDT to a variety of tumors, including metastases, using targeted radiotracers to provide internal excitation of light-responsive therapeutics. We expect that our method will play a critical role in precision treatment of deep solid tumors.

68Ga-FAPI PET visualize heart failure: from mechanism to clinic.

PURPOSE: Heart failure (HF) is a chronic progressive clinical syndrome associated with structural and/or functional heart abnormalities. Active fibroblasts and ventricular remodelling play an essential role in HF progression. 68Ga-labelled fibroblast activation protein (FAP) inhibitor (68Ga-FAPI) binds to FAP. This study aimed to examine the feasibility of using 68Ga-FAPI positron emission tomography (PET)/computed tomography (CT) to visualize changes in cardiac fibrosis and function over time in the HF setting. METHODS: After establishing an isoproterenol (ISO)-induced HF rat model (14 consecutive days of intraperitoneal ISO injections), echocardiography and 68Ga-FAPI PET/CT were performed weekly in experimental and control groups. Rat hearts were examined weekly for biodistribution analysis; autoradiography; and haematoxylin and eosin, FAP immunofluorescence and Masson's trichrome staining analysis. Rat blood was sampled weekly for enzyme-linked immunosorbent assay analysis of various plasma indicators. A preliminary clinical study was also performed in seven HF patients who underwent both 13N-amino (NH3) perfusion and 68Ga-FAPI cardiac PET imaging. RESULTS: In the animal experiments, myocardial 68Ga-FAPI uptake, expression of FAP and myocardial contractility peaked on day 7 after the initial ISO injection. Only slight fibrotic changes were observed on histopathological examination. 68Ga-FAPI uptake and ventricular wall motion decreased over time as cardiac fibrosis and degree of myocardial injury gradually increased. In the human HF patient study, 68Ga-FAPI PET imaging identified varying degrees of 68Ga-FAPI uptake in the myocardium that did not precisely match with 13N-NH3 myocardial perfusion. CONCLUSION: As HF progresses, 68Ga-FAPI uptake is high in the early stages and then gradually decreases. Although preliminary, our findings suggest that 68Ga-FAPI PET can be used to demonstrate active myocardial fibrosis. Active myocardial FAP expression is followed by myocardial remodelling and fibrosis. Detection of early active FAP expression may assist treatment decision making in HF patients. CLINICAL TRIAL REGISTRATION: NCT04982458.

An infection-microenvironment-targeted and responsive peptide-drug nanosystem for sepsis emergency by suppressing infection and inflammation.

Sepsis is a life-threatening emergency that causes millions of deaths every year due to severe infection and inflammation. Nevertheless, current therapeutic regimens are inadequate to promptly address the vast diversity of potential pathogens. Omiganan, an antimicrobial peptide, has shown promise for neutralizing endotoxins and eliminating diverse pathogens. However, its clinical application is hindered by safety and stability concerns. Herein, we present a nanoscale drug delivery system (Omi-hyd-Dex@HA NPs) that selectively targets infectious microenvironments (IMEs) and responds to specific stimuli for efficient intervention in sepsis. The system consists of omiganan-dexamethasone conjugates linked by hydrazone bonds which self-assemble into nanoparticles coated with a hyaluronic acid (HA). The HA coating not only facilitates IMEs-targeting through interaction with intercellular-adhesion-molecule-1 on inflamed endotheliocytes, but also improves the biosafety of the nanosystem and enhances drug accumulation in primary infection sites triggered by hyaluronidase. The nanoparticles release dual drugs in IMEs through pH-sensitive cleavage of hydrazone bonds to eradicate pathogens and suppress inflammation. In multiple tissue infection and sepsis animal models, Omi-hyd-Dex@HA NPs exhibited rapid source control and comprehensive inflammation reduction, thereby preventing subsequent fatal complications and significantly improving survival outcomes. The bio-responsive and self-delivering nanosystem offers a promising strategy for systemic sepsis treatment in emergencies.