A rationally designed nuclei-targeting FAPI 04-based molecular probe with enhanced tumor uptake for PET/CT and fluorescence imaging.

PURPOSE: Fibroblast activation protein inhibitor (FAPI) -based probes have been widely studied in the diagnosis of various malignant tumors with positron emission tomography/computed tomography (PET/CT). However, current imaging studies of FAPI-based probes face challenges in rapid clearance rate and potential false-negative results. Furthermore, FAPI has been rarely explored in optical imaging. Considering this, further modifications are imperative to improve the properties of FAPI-based probes to address existing limitations and broaden their application scenarios. In this study, we rationally introduced methylene blue (MB) to FAPIs, thereby imparting nuclei-targeting and fluorescence imaging capabilities to the probes. Furthermore, we evaluated the added value of FAPI-based fluorescence imaging to traditional PET/CT, exploring the potential application of FAPI-based probes in intraoperative fluorescence imaging. METHODS: A new FAPI-based probe, namely NOTA-FAPI-MB, was designed for both PET/CT and fluorescence imaging by conjugation of MB. The targeting efficacy of the probe was evaluated on fibroblast activation protein (FAP)-transfected cell line and human primary cancer-associated fibroblasts (CAFs). Subsequently, PET/CT and fluorescence imaging were conducted on tumor-bearing mice. The tumor detection and boundary delineation were assessed by fluorescence imaging of tissues from hepatocellular carcinoma (HCC) patients. RESULTS: NOTA-FAPI-MB demonstrated exceptional targeting ability towards FAP-transfected cells and CAFs in comparison to NOTA-FAPI. This benefit arises from the cationic methylene blue (MB) affinity for anionic nucleic acids. PET/CT imaging of tumor-bearing mice revealed significantly higher tumor uptake of [18F]F-NOTA-FAPI-MB (standard uptake value of 2.20 ± 0.31) compared to [18F]F-FDG (standard uptake value of 1.66 ± 0.14). In vivo fluorescence imaging indicated prolonged retention at the tumor site, with retention lasting up to 24 h. In addition, the fluorescent probes enabled more precise lesion detection and tumor margin delineation than clinically used indocyanine green (ICG), achieving a 100.0% (6/6) tumor-positive rate for NOTA-FAPI-MB while 33.3% (2/6) for ICG. These findings highlighted the potential of NOTA-FAPI-MB in guiding intraoperative surgical procedures. CONCLUSIONS: The NOTA-FAPI-MB was successfully synthesized, in which FAPI and MB simultaneously contributed to the targeting effect. Notably, the nuclear delivery mechanism of the probes improved intracellular retention time and targeting efficacy, broadening the imaging time window for fluorescence imaging. In vivo PET/CT demonstrated favorable performance of NOTA-FAPI-MB compared to [18F]F-FDG. This study highlights the significance of fluorescence imaging as an adjunct technique to PET/CT. Furthermore, the encouraging results obtained from the imaging of human HCC tissues hold promise for the potential application of NOTA-FAPI-MB in intraoperative fluorescent surgery guidance within clinical settings.

Holographically Activatable Nanoprobe via Glutathione/Albumin-Mediated Exponential Signal Amplification for High-Contrast Tumor Imaging.

Glutathione (GSH)-activatable probes hold great promise for in vivo cancer imaging, but are restricted by their dependence on non-selective intracellular GSH enrichment and uncontrollable background noise. Here, a holographically activatable nanoprobe caging manganese tetraoxide is shown for tumor-selective contrast enhancement in magnetic resonance imaging (MRI) through cooperative GSH/albumin-mediated cascade signal amplification in tumors and rapid elimination in normal tissues. Once targeting tumors, the endocytosed nanoprobe effectively senses the lysosomal microenvironment to undergo instantaneous decomposition into Mn2+ with threshold GSH concentration of ≈ 0.12 mm for brightening MRI signals, thus achieving high contrast tumor imaging and flexible monitoring of GSH-relevant cisplatin resistance during chemotherapy. Upon efficient up-regulation of extracellular GSH in tumor via exogenous injection, the relaxivity-silent interstitial nanoprobe remarkably evolves into Mn2+ that are further captured/retained and re-activated into ultrahigh-relaxivity-capable complex by stromal albumin in the tumor, and simultaneously allows the renal clearance of off-targeted nanoprobe in the form of Mn2+ via lymphatic vessels for suppressing background noise to distinguish tiny liver metastasis. These findings demonstrate the concept of holographic tumor activation via both tumor GSH/albumin-mediated cascade signal amplification and simultaneous background suppression for precise tumor malignancy detection, surveillance, and surgical guidance.

First-in-human assessment of the novel LAT1 targeting PET probe 18F-FIMP.

In the first-in-human PET study, we evaluated the biodistribution and tumor accumulation of the novel PET probe, (S)-2-amino-3-[3-(2-18F-fluoroethoxy)-4-iodophenyl]-2-methylpropanoic acid (18F-FIMP), which targets the tumor-related L-type amino acid transporter 1 (LAT1), and compared it with L-[methyl-11C]methionine (11C-MET) and 2-Deoxy-2-18F-fluoro-D-glucose (18F-FDG). 18F-FIMP biodistribution was revealed by whole-body and brain scans in 13 healthy controls. Tumor accumulation of 18F-FIMP was evaluated in 7 patients with a brain tumor, and compared with those of 11C-MET and 18F-FDG. None of the subjects had significant problems due to probe administration, such as adverse effects or abnormal vital signs. 18F-FIMP was rapidly excreted from the kidneys to the urinary bladder. There was no characteristic physiological accumulation in healthy controls. 18F-FIMP PET resulted in extremely clear images in patients with suspected glioblastoma compared with 11C-MET and 18F-FDG. 18F-FIMP could be a useful novel PET probe for LAT1-positive tumor imaging including glioblastoma.

A magnetic resonance nanoprobe with STING activation character collaborates with platinum-based drug for enhanced tumor immunochemotherapy.

BACKGROUND: Immunochemotherapy is a potent anti-tumor strategy, however, how to select therapeutic drugs to enhance the combined therapeutic effect still needs to be explored. METHODS AND RESULTS: Herein, a magnetic resonance nanoprobe (MnP@Lip) with STING (Stimulator of INterferon Genes) activation character was synthesized and co-administered with platinum-based chemotherapeutics for enhanced immunochemotherapy. MnP@Lip nanoparticles was prepared by simple fabrication process with good reproducibility, pH-sensitive drug release behavior and biocompatibility. In vitro experiments elucidated that Mn2+ can promote the polarization of M0 and/or M2 macrophages to M1 phenotype, and promote the maturation of BMDC cells. Upon Mn2+ treatment, the STING pathway was activated in tumor cells, mouse lung epithelial cells, and immune cells. More importantly, anti-tumor experiments in vivo proved that MnP@Lip combined with platinum-based chemotherapeutics increased T cells infiltration in the tumor microenvironment, and inhibited tumor growth in the orthotopic therapeutic and postoperative tumor models. CONCLUSIONS: This kind of therapeutic strategy that combined MnP@Lip nanoparticles with platinum-based chemotherapeutics may provide a novel insight for immunochemotherapy.

Design, Synthesis, and Evaluation of Thienodiazepine Derivatives as Positron Emission Tomography Imaging Probes for Bromodomain and Extra-Terminal Domain Family Proteins.

To better understand the role of bromodomain and extra-terminal domain (BET) proteins in epigenetic mechanisms, we developed a series of thienodiazepine-based derivatives and identified two compounds, 3a and 6a, as potent BET inhibitors. Further in vivo pharmacokinetic studies and analysis of in vitro metabolic stability of 6a revealed excellent brain penetration and reasonable metabolic stability. Compounds 3a and 6a were radiolabeled with fluorine-18 in two steps and utilized in positron emission tomography (PET) imaging studies in mice. Preliminary PET imaging results demonstrated that [18F]3a and [18F]6a have good brain uptake (with maximum SUV = 1.7 and 2, respectively) and binding specificity in mice brains. These results show that [18F]6a is a potential PET radiotracer that could be applied to imaging BET proteins in the brain. Further optimization and improvement of the metabolic stability of [18F]6a are still needed in order to create optimal PET imaging probes of BET family members.

Chemical tools for epichaperome-mediated interactome dysfunctions of the central nervous system.

Diseases are a manifestation of how thousands of proteins interact. In several diseases, such as cancer and Alzheimer's disease, proteome-wide disturbances in protein-protein interactions are caused by alterations to chaperome scaffolds termed epichaperomes. Epichaperome-directed chemical probes may be useful for detecting and reversing defective chaperomes. Here we provide structural, biochemical, and functional insights into the discovery of epichaperome probes, with a focus on their use in central nervous system diseases. We demonstrate on-target activity and kinetic selectivity of a radiolabeled epichaperome probe in both cells and mice, together with a proof-of-principle in human patients in an exploratory single group assignment diagnostic study (ClinicalTrials.gov Identifier: NCT03371420). The clinical study is designed to determine the pharmacokinetic parameters and the incidence of adverse events in patients receiving a single microdose of the radiolabeled probe administered by intravenous injection. In sum, we introduce a discovery platform for brain-directed chemical probes that specifically modulate epichaperomes and provide proof-of-principle applications in their use in the detection, quantification, and modulation of the target in complex biological systems.

SPECT Imaging of Hepatocellular Carcinoma Detection by the GPC3 Receptor.

The glypican-3 (GPC3) receptor is a membrane protein that is highly expressed in tumor tissues but rarely expressed in the normal liver and can be used as a target for early diagnosis of hepatocellular carcinoma (HCC). Herein, we developed a GPC3-targeted 99mTc-labeled probe for SPECT imaging in HCC. 99mTc-HPG was rapidly radiosynthesized within 20 min with an excellent radiochemical purity (>98%), possessing good stability. Results from in vitro cell binding assays indicated that the binding specificity of 99mTc-HPG to GPC3-positive HepG2 cells was acceptable. For SPECT/CT imaging, the HepG2 tumors were clearly visualized with the highest tumor/muscle ratio (11.55 ± 0.54) at 1 h post-injection, and the tumor uptake of 99mTc-HPG reduced from 2.99 ± 0.15 to 1.17 ± 0.09% ID/g in the blocking study. Convenient preparation, excellent GPC3 specificity in HCC, rapid clearance from normal organs, and good biosafety profiles of 99mTc-HPG warrant further investigations for clinical translation.

A Pretargeted Imaging Strategy for EGFR-Positive Colorectal Carcinoma via Modulation of Tz-Radioligand Pharmacokinetics.

PURPOSE: Previously, we successfully developed a pretargeted imaging strategy (atezolizumab-TCO/[99mTc]HYNIC-PEG11-Tz) for evaluating programmed cell death ligand-1 (PD-L1) expression in xenograft mice. However, the surplus unclicked [99mTc]HYNIC-PEG11-Tz is cleared somewhat sluggishly through the intestines, which is not ideal for colorectal cancer (CRC) imaging. To shift the excretion of the Tz-radioligand to the renal system, we developed a novel Tz-radioligand by adding a polypeptide linker between HYNIC and PEG11. PROCEDURES: Pretargeted molecular probes [99mTc]HYNIC-polypeptide-PEG11-Tz and cetuximab-TCO were synthesized. [99mTc]HYNIC-polypeptide-PEG11-Tz was evaluated for in vitro stability and in vivo blood pharmacokinetics. In vitro ligation reactivity of [99mTc]HYNIC-polypeptide-PEG11-Tz towards cetuximab-TCO was also tested. Biodistribution assay and imaging of [99mTc]HYNIC-polypeptide-PEG11-Tz were performed to observe its excretion pathway. Pretargeted biodistribution was measured at three different accumulation intervals to determine the optimal pretargeted interval time. Pretargeted (cetuximab-TCO 48 h/[99mTc]HYNIC-PEG11-Tz 6 h) and (cetuximab-TCO 48 h/[99mTc]HYNIC-Polypeptide-PEG11-Tz 6 h) imagings were compared to examine the effect of the excretion pathway on tumor imaging. RESULTS: [99mTc]HYNIC-polypeptide-PEG11-Tz showed favorable in vitro stability and rapid blood clearance in mice. SEC-HPLC revealed almost complete reaction between cetuximab-TCO and [99mTc]HYNIC-polypeptide-PEG11-Tz in vitro, with the 8:1 Tz-to-mAb reaction providing a conversion yield of 87.83 ± 3.27 %. Biodistribution and imaging analyses showed that the Tz-radioligand was cleared through the kidneys. After 24, 48, and 72 h of accumulation in HCT116 tumor, the tumor-to-blood ratio of cetuximab-TCO was 0.83 ± 0.13, 1.40 ± 0.31, and 1.15 ± 0.21, respectively. Both pretargeted (cetuximab-TCO 48 h/[99mTc]HYNIC-PEG11-Tz 6 h) and (cetuximab-TCO 48 h/[99mTc]HYNIC-polypeptide-PEG11-Tz 6 h) clearly delineated HCT116 tumor. Pretargeted imaging strategy using cetuximab-TCO/[99mTc]HYNIC-polypeptide-PEG11-Tz could be used for diagnosing CRC, as the surplus unclicked [99mTc]HYNIC-polypeptide-PEG11-Tz was cleared through the urinary system, leading to low abdominal uptake background. CONCLUSION: Our novel pretargeted imaging strategy (cetuximab-TCO/[99mTc]HYNIC-polypeptide-PEG11-Tz) was useful for imaging CRC, broadening the application scope of pretargeted imaging strategy. The pretargeted imaging strategy clearly delineated HCT116 tumor, showing that its use could be extended to selection of internalizing antibodies.

Synthesis of [18F]F-γ-T-3, a Redox-Silent γ-Tocotrienol (γ-T-3) Vitamin E Analogue for Image-Based In Vivo Studies of Vitamin E Biodistribution and Dynamics.

Vitamin E, a natural antioxidant, is of interest to scientists, health care pundits and faddists; its nutritional and biomedical attributes may be validated, anecdotal or fantasy. Vitamin E is a mixture of tocopherols (TPs) and tocotrienols (T-3s), each class having four substitutional isomers (α-, ß-, γ-, δ-). Vitamin E analogues attain only low concentrations in most tissues, necessitating exacting invasive techniques for analytical research. Quantitative positron emission tomography (PET) with an F-18-labeled molecular probe would expedite access to Vitamin E's biodistributions and pharmacokinetics via non-invasive temporal imaging. (R)-6-(3-[18F]Fluoropropoxy)-2,7,8-trimethyl-2-(4,8,12-trimethyltrideca-3,7,11-trien-1-yl)-chromane ([18F]F-γ-T-3) was prepared for this purpose. [18F]F-γ-T-3 was synthesized from γ-T-3 in two steps: (i) 1,3-di-O-tosylpropane was introduced at C6-O to form TsO-γ-T-3, and (ii) reaction of this tosylate with [18F]fluoride in DMF/K222. Non-radioactive F-γ-T-3 was synthesized by reaction of γ-T-3 with 3-fluoropropyl methanesulfonate. [18F]F-γ-T-3 biodistribution in a murine tumor model was imaged using a small-animal PET scanner. F-γ-T-3 was prepared in 61% chemical yield. [18F]F-γ-T-3 was synthesized in acceptable radiochemical yield (RCY 12%) with high radiochemical purity (>99% RCP) in 45 min. Preliminary F-18 PET images in mice showed upper abdominal accumulation with evidence of renal clearance, only low concentrations in the thorax (lung/heart) and head, and rapid clearance from blood. [18F]F-γ-T-3 shows promise as an F-18 PET tracer for detailed in vivo studies of Vitamin E. The labeling procedure provides acceptable RCY, high RCP and pertinence to all eight Vitamin E analogues.

The Labeling, Visualization, and Quantification of Hyaluronan Distribution in Tumor-Bearing Mouse Using PET and MR Imaging.

PURPOSE: Hyaluronan (HA) based biomaterials are widely used as tissue scaffolds, drug formulations, as well as targeting ligands and imaging probes for diagnosis and drug delivery. However, because of the presence of abundant endogenous HA presented in various tissues in vivo, the pharmacokinetic behavior and biodistribution patterns of exogenously administered HAs have not been well characterized. METHODS: The HA backbone was modified with Diethylenetriamine (DTPA) to enable the chelation of gadolinium (Gd) and aluminum (Al) ions. Series of PET and MR imaging were taken after the injection of HA-DTPA-Gd and HA-DTPA-Al18F while using18F-FDG and Magnevist(DTPA-Gd) as controls. The Tomographic images were analyzed and quantified to reveal the distribution and locations of HA in tumor-bearing mice. RESULTS: The labeled HAs had good stability in plasma. They retained binding affinity towards CD44s on tumor cell surface. The injected HAs distributed widely in various organs, but were found to be cleared quickly except inside tumor tissues where the signals were higher and persisted longer. CONCLUSION: Medical imaging tools, including MR and PET, can be highly valuable for examining biomaterial distribution non-invasively. The HA tumor accumulation properties may be explored for the development of active targeting drug carriers and molecular probes.

Evaluation of an antibody-PNA conjugate as a clearing agent for antibody-based PNA-mediated radionuclide pretargeting.

Radionuclide molecular imaging of cancer-specific targets is a promising method to identify patients for targeted antibody therapy. Radiolabeled full-length antibodies however suffer from slow clearance, resulting in high background radiation. To overcome this problem, a pretargeting system based on complementary peptide nucleic acid (PNA) probes has been investigated. The pretargeting relies on sequential injections of primary, PNA-tagged antibody and secondary, radiolabeled PNA probe, which are separated in time, to allow for clearance of non-bound primary agent. We now suggest to include a clearing agent (CA), designed for removal of primary tumor-targeting agent from the blood. The CA is based on the antibody cetuximab, which was conjugated to PNA and lactosaminated by reductive amination to improve hepatic clearance. The CA was evaluated in combination with PNA-labelled trastuzumab, T-ZHP1, for radionuclide HER2 pretargeting. Biodistribution studies in normal mice demonstrated that the CA cleared ca. 7 times more rapidly from blood than unmodified cetuximab. Injection of the CA 6 h post injection of the radiolabeled primary agent [131I]I-T-ZHP1 gave a moderate reduction of the radioactivity concentration in the blood after 1 h from 8.5 ± 1.8 to 6.0 ± 0.4%ID/g. These proof-of-principle results could guide future development of a more efficient CA.

Hemoglobin-mediated biomimetic synthesis of paramagnetic O2-evolving theranostic nanoprobes for MR imaging-guided enhanced photodynamic therapy of tumor.

The hypoxic microenvironment in solid tumors severely limits the efficacy of photodynamic therapy (PDT). Therefore, the development of nanocarriers co-loaded with photosensitizers and oxygen, together with imaging guidance ability, is of great significance in cancer therapy. However, previously reported synthetic methods for these multi-functional probes are complicated, and the raw materials used are toxic. Methods: Herein, the human endogenous protein, hemoglobin (Hb), was used for the simultaneous biomimetic synthesis of Gd-based nanostructures and co-loading of Chlorine e6 (Ce6) and oxygen for alleviating the hypoxic environment of tumors and accomplishing magnetic resonance imaging (MRI)-guided enhanced PDT. The Gd@HbCe6-PEG nanoprobes were synthesized via a green and protein biomimetic approach. The physicochemical properties, including relaxivity, oxygen-carrying/release capability, and PDT efficacy of Gd@HbCe6-PEG, were measured in vitro and in vivo on tumor-bearing mice after intravenous injection. Morphologic and functional MRI were carried out to evaluate the efficacy of PDT. Results: The results demonstrated the successful synthesis of compact Gd@HbCe6-PEG nanostructures with desired multi-functionalities. Following treatment with the nanoparticles, the embedded MR moiety was effective in lighting tumor lesions and guiding therapy. The oxygen-carrying capability of Hb after biomimetic synthesis was confirmed by spectroscopic analysis and oxygen detector in vitro. Further, tumor oxygenation for alleviating tumor hypoxia in vivo after intravenous injection of Gd@HbCe6-PEG was verified by photoacoustic imaging and immunofluorescence staining. The potent treatment efficacy of PDT on early-stage was observed by the morphologic and functional MR imaging. Importantly, rapid renal clearance of the particles was observed after treatment. Conclusion: In this study, by using a human endogenous protein, we demonstrated the biomimetic synthesis of multi-functional nanoprobes for simultaneous tumor oxygenation and imaging-guided enhanced PDT. The therapeutic efficacy could be quantitatively confirmed at 6 h post PDT with diffusion-weighted imaging (DWI).

Radionuclide Molecular Imaging of EpCAM Expression in Triple-Negative Breast Cancer Using the Scaffold Protein DARPin Ec1.

Efficient treatment of disseminated triple-negative breast cancer (TNBC) remains an unmet clinical need. The epithelial cell adhesion molecule (EpCAM) is often overexpressed on the surface of TNBC cells, which makes EpCAM a potential therapeutic target. Radionuclide molecular imaging of EpCAM expression might permit selection of patients for EpCAM-targeting therapies. In this study, we evaluated a scaffold protein, designed ankyrin repeat protein (DARPin) Ec1, for imaging of EpCAM in TNBC. DARPin Ec1 was labeled with a non-residualizing [125I]I-para-iodobenzoate (PIB) label and a residualizing [99mTc]Tc(CO)3 label. Both imaging probes retained high binding specificity and affinity to EpCAM-expressing MDA-MB-468 TNBC cells after labeling. Internalization studies showed that Ec1 was retained on the surface of MDA-MB-468 cells to a high degree up to 24 h. Biodistribution in Balb/c nu/nu mice bearing MDA-MB-468 xenografts demonstrated specific uptake of both [125I]I-PIB-Ec1 and [99mTc]Tc(CO)3-Ec1 in TNBC tumors. [125I]I-PIB-Ec1 had appreciably lower uptake in normal organs compared with [99mTc]Tc(CO)3-Ec1, which resulted in significantly (p < 0.05) higher tumor-to-organ ratios. The biodistribution data were confirmed by micro-Single-Photon Emission Computed Tomography/Computed Tomography (microSPECT/CT) imaging. In conclusion, an indirectly radioiodinated Ec1 is the preferable probe for imaging of EpCAM in TNBC.

Synthesis and Evaluation of 68Ga- and 177Lu-Labeled (R)- vs (S)-DOTAGA Prostate-Specific Membrane Antigen-Targeting Derivatives.

Prostate-specific membrane antigen (PSMA) is overexpressed in prostate cancer cells and therefore is an attractive target for prostate cancer diagnosis and radionuclide therapy. Recently, published results from clinical studies using a new PSMA-targeting PET imaging agent, [68Ga]Ga-PSMA-093 ([68Ga]Ga-HBED-CC-O-carboxymethyl-Tyr-CO-NH-Glu), support the development of this agent for the diagnosis of prostate cancer. In this study, the HBED-CC chelating group in PSMA-093 was replaced by stereoselective (R)- or (S)-DOTAGA. This chelating group serves not only for chelating 68Ga but is also amendable for complexing other radioactive metals for radionuclide therapy. The corresponding optically pure (R)- and (S)-[68Ga/177Lu]-DOTAGA derivatives, (R)-[68Ga/177Lu]-13 and (S)-[68Ga/177Lu]-13, were successfully prepared. Comparison of radiolabeling, binding affinity, cell uptake, and biodistribution between the two isomers was performed. Radiolabeling of (R)-[177Lu]Lu-13 and (S)-[177Lu]Lu-13 at 50 °C suggested that rates of complex formation were time-dependent and the formation of (S)-[177Lu]Lu-13 was distinctly faster. The rates of complex formation for the corresponding 68Ga agents were comparable between structural isomers. The natGa and natLu equivalents showed high binding PSMA affinity (IC50 = 24-111 nM), comparable to that of the parent agent, [natGa]Ga-PSMA-093 (IC50 = 34.0 nM). Results of cell uptake and biodistribution studies in PSMA-expressing PC3-PIP tumor-bearing mice appeared to show no difference between the labeled (R)- and (S)-isomers. This is the first time that a pair of [68Ga/177Lu]-(R)- and (S)-DOTAGA isomers of PSMA agents were evaluated. Results of biological studies between the isomers showed no noticeable difference; however, the distinctions on the rate of Lu complex formation should be considered in the development of new 177Lu-DOTAGA-based radionuclide therapy agents in the future.

Molecular Imaging of Inflammation in a Mouse Model of Atherosclerosis Using a Zirconium-89-Labeled Probe.

BACKGROUND: Beyond clinical atherosclerosis imaging of vessel stenosis and plaque morphology, early detection of inflamed atherosclerotic lesions by molecular imaging could improve risk assessment and clinical management in high-risk patients. To identify inflamed atherosclerotic lesions by molecular imaging in vivo, we studied the specificity of our radiotracer based on maleylated (Mal) human serum albumin (HSA), which targets key features of unstable atherosclerotic lesions. MATERIALS AND METHODS: Mal-HSA was radiolabeled with a positron-emitting metal ion, zirconium-89 (89Zr4+). The targeting potential of this probe was compared with unspecific 89Zr-HSA and 18F-FDG in an experimental model of atherosclerosis (Apoe-/- mice, n=22), and compared with wild-type (WT) mice (C57BL/6J, n=21) as controls. RESULTS: PET/MRI, gamma counter measurements, and autoradiography showed the accumulation of 89Zr-Mal-HSA in the atherosclerotic lesions of Apoe-/- mice. The maximum standardized uptake values (SUVmax) for 89Zr-Mal-HSA at 16 and 20 weeks were 26% and 20% higher (P<0.05) in Apoe-/- mice than in control WT mice, whereas no difference in SUVmax was observed for 18F-FDG in the same animals. 89Zr-Mal-HSA uptake in the aorta, as evaluated by a gamma counter 48 h postinjection, was 32% higher (P<0.01) for Apoe-/- mice than in WT mice, and the aorta-to-blood ratio was 8-fold higher (P<0.001) for 89Zr-Mal-HSA compared with unspecific 89Zr-HSA. HSA-based probes were mainly distributed to the liver, spleen, kidneys, bone, and lymph nodes. The phosphor imaging autoradiography (PI-ARG) results corroborated the PET and gamma counter measurements, showing higher accumulation of 89Zr-Mal-HSA in the aortas of Apoe-/- mice than in WT mice (9.4±1.4 vs 0.8±0.3%; P<0.001). CONCLUSION: 89Zr radiolabeling of Mal-HSA probes resulted in detectable activity in atherosclerotic lesions in aortas of Apoe-/- mice, as demonstrated by quantitative in vivo PET/MRI. 89Zr-Mal-HSA appears to be a promising diagnostic tool for the early identification of macrophage-rich areas of inflammation in atherosclerosis.

Functional Genetic Screening Enables Theranostic Molecular Imaging in Cancer.

PURPOSE: Targeted therapies for cancer have accelerated the need for functional imaging strategies that inform therapeutic efficacy. This study assesses the potential of functional genetic screening to integrate therapeutic target identification with imaging probe selection through a proof-of-principle characterization of a therapy-probe pair using dynamic nuclear polarization (DNP)-enhanced magnetic resonance spectroscopic imaging (MRSI). EXPERIMENTAL DESIGN: CRISPR-negative selection screens from a public dataset were used to identify the relative dependence of 625 cancer cell lines on 18,333 genes. Follow-up screening was performed in hepatocellular carcinoma with a focused CRISPR library targeting imaging-related genes. Hyperpolarized [1-13C]-pyruvate was injected before and after lactate dehydrogenase inhibitor (LDHi) administration in male Wistar rats with autochthonous hepatocellular carcinoma. MRSI evaluated intratumoral pyruvate metabolism, while T2-weighted segmentations quantified tumor growth. RESULTS: Genetic screening data identified differential metabolic vulnerabilities in 17 unique cancer types that could be imaged with existing probes. Among these, hepatocellular carcinoma required lactate dehydrogenase (LDH) for growth more than the 29 other cancer types in this database. LDH inhibition led to a decrease in lactate generation (P < 0.001) and precipitated dose-dependent growth inhibition (P < 0.01 overall, P < 0.05 for dose dependence). Intratumoral alanine production after inhibition predicted the degree of growth reduction (P < 0.001). CONCLUSIONS: These findings demonstrate that DNP-MRSI of LDH activity using hyperpolarized [1-13C]-pyruvate is a theranostic strategy for hepatocellular carcinoma, enabling quantification of intratumoral LDHi pharmacodynamics and therapeutic efficacy prediction. This work lays the foundation for a novel theranostic platform wherein functional genetic screening informs imaging probe selection to quantify therapeutic efficacy on a cancer-by-cancer basis.

Syntheses and Preliminary Evaluation of Dual Target PET Probe [18F]-NOTA-Gly3- E (2PEG4-RGD-WH701) for PET Imaging of Breast Cancer.

PURPOSE: Tumor Necrosis Factor Receptor 1 (TNFR1) and integrin αvß3 receptor are overexpressed in breast cancer. We hypothesized that a peptide ligand recognizing both receptors in a single receptor-binding probe would be advantageous. Here, we developed a novel 18F-labeled fusion peptide probe [18F]-NOTA-Gly3- E(2PEG4-RGD-WH701) targeting dual receptors (TNFR1 and αvß3) and evaluated the diagnostic efficacy of this radioactive probe in both MDA-MB-231 and MCF-7 xenograft models in mice. METHODS: The NOTA-conjugated RGD-WH701 analog was radiolabeled with 18F using NOTA-AlF chelation method. We used two PEG4 molecules and Glutamic acid (Glu) to covalently link c(RGDyK) with WH701. Gly3 was also added to further improve the water solubility and pharmacokinetic properties of the probe. The expression of TNFR1 and Integrin αvß3 in MCF-7 and MDA-MB-231 cells was detected by western blot analysis and immunofluorescence staining. The tumor-targeting characteristics of [18F]-NOTA-Gly3-E(2PEG4-RGDWH701) were assessed in nude mice bearing MDA-MB-231 and MCF-7 xenografts. RESULTS: HPLC analysis of the product NOTA-G3-E (2P4-RGD-WH701) revealed a purity >95%. The yield after attenuation correction was approximately 33.5%±2.8% (n=5), and the radiochemical purity was above 95%. The MDA-MB-231 tumor uptake of [18F]-NOTA-Gly3-E(2PEG4-RGD-WH701) was 1.14±0.14%ID/g, as measured by PET at 40min postinjection (p.i.). In comparison, the tumor uptake of [18F]-NOTA-RGD and [18F]- NOTA-WH701 in MDA-MB-231 xenografts was 0.96±0.13%ID/g and 0.93±0.28%ID/g, respectively. The MCF-7 tumor uptake of [18F]-NOTA-Gly3-E(2PEG4-RGD-WH701) was 1.22±0.11%ID/g, as measured by PET at 40min postinjection (p.i.). In comparison, the tumor uptake of [18F]-NOTA-RGD and [18F]-NOTA-WH701 in MCF-7 xenografts was 0.99±0.18%ID/g and 0.57±0.08%ID/g, respectively. CONCLUSION: [18F]AlF-NOTA-Gly3-E(2PEG4-RGD-WH701) was successfully synthesized and labeled with 18F. The results from the microPET/CT and biodistribution studies of [18F]AlF-NOTA-Gly3-E(2PEG4-RGDWH701) showed that the tracer could specifically target TNFR1 and integrin αvß3 receptors.

Noninvasive Classification of Human Triple Negative Breast Cancer by PET Imaging with GRP78-Targeted Molecular Probe [68Ga]DOTA-VAP.

PURPOSE: There is currently no effective noninvasive method for accurate molecular typing of triple negative breast cancer (TNBC) except needle biopsy. Glucoregulated Protein 78 (GRP78) is overexpressed in TNBC cells and tumors which closely related to the invasion, metastasis, and drug resistance of cancer. Meanwhile, it has been verified that VAP peptide bind specifically to GRP78 in vitro and in vivo. In this study, we constructed a GRP78-targeted molecular probe Ga-68-radiolabeled DOTA-VAP conjugate ([68Ga]DOTA-VAP) based on VAP peptide, and evaluated its potential to distinguish TNBC from non-TNBC tumors. PROCEDURES: DOTA-VAP was synthesized and then radiolabeled with Ga-68 to obtain [68Ga]DOTA-VAP. The expression of GRP78 in TNBC MDA-MB-231 and non-TNBC MCF-7 cells was validated by Western Blot, and cell binding or uptake experiments with both [68Ga]DOTA-VAP and 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) were also performed. Biodistribution analysis and positron emission tomography (PET) imaging of [68Ga]DOTA-VAP were carried out in subcutaneous MDA-MB-231 and MCF-7 human breast cancer tumor models with [18F]FDG PET imaging as comparison. RESULTS: [68Ga]DOTA-VAP was prepared with high radiochemical purity which showed excellent stability in vitro. The MDA-MB-231 tumors were clearly visualized by [68Ga]DOTA-VAP PET imaging with a low background, except for the relatively high liver uptake. Cells and tumors of MDA-MB-231 could be distinguished from MCF-7 by [68Ga]DOTA-VAP instead of [18F]FDG. Biodistribution results were consistent with the imaging results. The blocking study with excess cold peptide showed significantly reduced tumor uptake, which indicated the specificity of [68Ga]DOTA-VAP targeting MDA-MB-231 tumors in vivo. CONCLUSIONS: GRP78-targeted PET imaging with [68Ga]DOTA-VAP provided an effective approach for the noninvasive accurate classification of TNBC from other breast cancer subtypes comparing with [18F]FDG. GRP78 may be a potential target for the diagnosis and treatment of TNBC. For clinical transformation, efforts should be made to overcome deficiencies of [68Ga]DOTA-VAP such as relative high uptake in normal tissues.

Monitoring the Kinetics of the Cellular Uptake of a Metal Carbonyl Conjugated with a Lipidic Moiety in Living Cells Using Synchrotron Infrared Spectromicroscopy.

Presented here is the exploitation of synchrotron infrared spectromicroscopy to evaluate the feasibility of monitoring the cellular uptake of rhenium-tris-carbonyl-tagged (Re(CO)3) lipophilic chains in living cells. To this aim, an in-house thermostated microfluidic device was used to limit water absorption while keeping cells alive. Indeed, cells showed a high survival rate in the microfluidic device over the course of the experiment, proving the short-term biocompatibility of the device. We recorded spectra of single, living, fully hydrated breast cancer MDA-MB231 cells and could follow the penetration of the rhenium complexes for up to 2 h. Despite the strong variations observed in the uptake kinetics between individual cells, the Re(CO)3 complex was traced inside the cells at low concentration and shown to enter them on the hour time scale by active transport.

Differentiation and targeting of HT 29 cancer cells based on folate bioreceptor using cysteamine functionalized gold nano-leaf.

Cancer is one of the main causes of death worldwide. To decrease the mortality of cancer, early stage detection of cancer is of great importance. An innovative platform was developed for differentiation and detection of HT 29 cancer cells based on interactions between folate (FA) and folate receptors (FRs) of the membrane of cancer cells. In summary, FA and cysteamine (CA)-functionalized gold nanoparticles (AuNPs) were synthesized and characterized by field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), dynamic light scattering (DLS) and Fourier-transform infrared (IR) spectroscopy. Also, the surface charge was determined by measuring of the zeta potential. Fluorescence imaging and flow cytometry analyses were used to approve the selective uptake of the synthesized probe to the cancer cells. HEK 293 FR-negative cells were applied to assess the selectivity of AuNPs/CA/FA towards FR-negative cells. The differential pulse voltammetry (DPV) technique was used to determine the HT 29 cells from 250 to 5000 cells/mL with a lower limit of quantification (LLOQ) of 250 cells/mL. The produced AuNPs/CA/FA based nanoprobe could not only detect the signaling of HT 29 cells but also improve the specificity of cytosensor towards FR-positive cancer cells. According to the obtained results, the newly developed nano-probe could be used as a portable biomedical device for cancer diagnosis.