Imaging Of Tumor-bearing Mice Research Articles

Development and Preclinical Evaluation of [68Ga]BMSH as a New Potent Positron Emission Tomography Tracer for Imaging Programmed Death-Ligand 1 Expression.

Immunotherapy targeting the programmed death-ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1) pathway has shown remarkable efficacy against various cancers, but the overall response rate (ORR) is still low. PD-L1 expression in tumors may predict treatment response to immunotherapy. Indeed, ongoing clinical studies utilize a few PD-L1 radiotracers to assess PD-L1 expression as a predictive biomarker for immunotherapy. Here, we present a novel positron emission tomography (PET) radiotracer called [68Ga]BMSH, which is derived from a small molecule inhibitor specifically targeting the binding site of PD-L1. The inhibitor was modified to optimize its in vivo pharmacokinetic properties and enable chelation of 68Ga. In vitro evaluation revealed [68Ga]BMSH possessed a strong binding affinity, high specificity, and rapid internalization in PD-L1 overexpressing cells. Biodistribution studies showed that PD-L1 overexpressing tumors had an uptake of [68Ga]BMSH at 4.22 ± 0.65%ID/g in mice, while the number was 2.23 ± 0.41%ID/g in PD-L1 low-expressing tumors. Micro-PET/CT imaging of tumor-bearing mice further confirmed that, compared to [18F]FDG, [68Ga]BMSH can specifically identify tumors with varying levels of PD-L1 expression. Our findings suggest that the [68Ga]BMSH is a PD-L1 radioligand with ideal imaging properties, and its further application in the clinical screening of PD-L1 overexpressing tumors may improve ORR for immunotherapy.

124I-labeled anti-CD147 antibody for noninvasive detection of CD147-positive pan-cancers: construction and preclinical studies.

Extracellular matrix metalloproteinase inducer CD147 is a glycoprotein on the cell surface. There is minimal expression of CD147 in normal epithelial and fetal tissues, but it is highly expressed in a number of aggressive tumors. CD147 has been implicated in pan-cancer immunity and progression. With the development of CD147-targeting therapeutic strategy, accurate detection of CD147 expression in tumors and its changes during the therapy is necessary. In this study we constructed a novel radiotracer by labeling the anti-CD147 mAb with radionuclide 124/125I (124/125I-anti-CD147) for noninvasive detection of CD147 expression in pan-cancers, and characterized its physicochemical properties, affinity, metabolic characteristics, biodistribution and immunoPET imaging with 124I-IgG and 18F-FDG as controls. By examining the expression of CD147 in cancer cell lines, we found high CD147 expression in colon cancer cells LS174T, FADU human pharyngeal squamous cancer cells and 22RV1 human prostate cancer cells, and low expression of CD147 in human pancreatic cancer cells ASPC1 and human gastric cancer cells BGC823. 124/125I-anti-CD147 was prepared using N-bromine succinimide (NBS) as oxidant and purified by PD-10 column. Its radiochemical purity (RCP) was over 99% and maintained over 85% in saline or 5% human serum albumin (HSA) for more than 7 d; the RCP of 125I-anti-CD147 in blood was over 90% at 3 h post injection (p.i.) in healthy mice. The Kd value of 125I-anti-CD147 to CD147 protein was 6.344 nM, while that of 125I-IgG was over 100 nM. 125I-anti-CD147 showed much greater uptake in CD147 high-expression cancer cells compared to CD147 low-expression cancer cells. After intravenous injection in healthy mice, 125I-anti-CD147 showed high initial uptake in blood pool and liver, the uptake was decreased with time. The biological half-life of distribution and clearance phases in healthy mice were 0.63 h and 19.60 h, respectively. The effective dose of 124I-anti-CD147 was estimated as 0.104 mSv/MBq. We conducted immunoPET imaging in tumor-bearing mice, and demonstrated a significantly higher tumor-to-muscle ratio of 124I-anti-CD147 compared to that of 124I-IgG and 18F-FDG in CD147 (+) tumors. The expression levels of CD147 in cells and tumors were positively correlated with the maximum standardized uptake value (SUVmax) (P < 0.01). In conclusion, 124/125I-anti-CD147 displays high affinity to CD147, and represents potential for the imaging of CD147-positive tumors. The development of 124I-anti-CD147 may provide new insights into the regulation of tumor microenvironment and formulation of precision diagnosis and treatment programs for tumors.

Afterglow/Fluorescence Dual-Emissive Ratiometric Oxygen Probe for Tumor Hypoxia Imaging

Hypoxia is a common feature of many diseases such as solid tumors. The measurement and imaging of oxygen (O2) are extremely important for disease diagnosis and therapy evaluation. In this work, the afterglow/fluorescence dual-emissive ratiometric O2 probe based on a photochemical reaction-based afterglow system is reported. The afterglow is highly sensitive to O2 because the O2 content is directly related to the 1O2 yield and eventually affects the afterglow intensity. The O2-insensitive fluorescence of an emitter can serve as an internal reference. As the O2 concentration changes from 0.08 to 18.5 mg L-1, the ratio value shows a remarkable 53-fold increase. Compared with the intensity of a single peak, the ratiometric signal can eliminate the interference of the probe concentration to achieve higher accuracy. This afterglow/fluorescence dual-emissive ratiometric O2 probe is successfully applied to hypoxia imaging in tumor-bearing mice, which may further promote the development of O2 sensing in the biomedical field.

Bimodal Imaging of Tumors via Genetically Engineered Escherichia coli.

Although there are emerging innovations of molecular imaging probes to detect and image tumors, most of these molecular dyes and nanoparticles have limitations of low targetability in tumors and fast clearance when administered systemically. In contrast, some bacteria, such as Escherichia coli MG1655, can selectively proliferate in a hypoxic environment inside of a tumor for several days, which highlights the potential for the development of a genetically encoded multimodal imaging probe to monitor the progress of the tumor. Here, we developed bimodal imaging tumor-homing bacteria (GVs-miRFP680 MG1655) that allow both optical and acoustic imaging in tumor-bearing mice. An in vivo optical image system and a Vevo 2100 imaging system were applied to detect different imaging properties of the engineered bacteria in vivo. Our results show that the GVs-miRFP680 MG1655 bacteria can effectively integrate the advantages of low tissue absorbance from near-infrared fluorescent proteins and non-invasiveness from gas vesicles. We successfully developed GVs-miRFP680 MG1655 bacteria, which have both acoustic and optical imaging abilities in vitro and in vivo. The acoustic signal can last for up to 25 min, while the near-infrared fluorescence signal can last for up to 96 h. The combination of different imaging modalities in the tumor-homing bacteria may contribute to the non-invasive monitoring of the therapeutic effect of bacterial therapy in the future.

Silver@quercetin Nanoparticles with Aggregation-Induced Emission for Bioimaging In Vitro and In Vivo.

Fluorescent materials based on aggregation-induced emission luminogens (AIEgens) have unique advantages for in situ and real-time monitoring of biomolecules and biological processes because of their high luminescence intensity and resistance to photobleaching. Unfortunately, many AIEgens require time-consuming and expensive syntheses, and the presence of residual toxic reagents reduces their biocompatibility. Herein, silver@quercetin nanoparticles (Ag@QCNPs), which have a clear core–shell structure, were prepared by redox reaction of quercetin (QC), a polyphenolic compound widely obtained from plants, including those used as foods, and silver ions. Ag@QCNPs show both aggregation-induced luminescence and the distinct plasma scattering of silver nanoparticles, as well as good resistance to photobleaching and biocompatibility. The Ag@QCNPs were successfully used for cytoplasmic labeling of living cells and for computerized tomography imaging in tumor-bearing mice, demonstrating their potential for clinical applications.

A tumor-targeting near-infrared fluorescent probe for real-time imaging ATP in cancer cells and mice

Adenosine triphosphate (ATP) is an important biomolecule, which is the primary source of cellular energy. In particular, an abnormal metabolism of ATP level has been took part in many diseases, such as cancer. Thus, developing an effective fluorescent probe for tumor-targeting imaging of ATP is great importance for in-depth understanding the functions of ATP in tumor invasion and matastasis. In this work, we present the design and synthesis of a tumor-targeting near-infrared (NIR) fluorescent probe named Bio-SiR. Bio-SiR is mainly composed of three parts: si-rhodamine-based fluorophore, diethylenetriamine-based recognition group and biotin-based tumor-targetable group. When Bio-SiR reacts with ATP, a turn-on fluorescence at 675 nm (NIR region) is observed clearly, which is suitable for its application in mice. In addition, due to a concurrent effect from dual recognition sites, the probe Bio-SiR displays excellent selectivity for ATP over other potential biological analytes. Under the guidance of biotin group, Bio-SiR can be successfully used for imaging ATP in cancer cells. Furthermore, live-cell imaging allows us to directly real-time monitor the dynamic change of ATP in cancer cells. In particular, this is the first tumor-targeting NIR small-molecule fluorescent probe for endogenous ATP imaging in tumor-bearing mice. These features demonstrate that this probe is a useful imaging tool for expounding the function of ATP in cancer.

Lanthanide europium MOF nanocomposite as the theranostic nanoplatform for microwave thermo-chemotherapy and fluorescence imaging

BackgroundsMicrowave sensitization nanoplatform, integrating multiple functional units for improving tumor selectivity, is of great significance for clinical tumor microwave treatment. Lanthanide europium metal organic framework (EuMOF) is expected to be a theranostic nanoplatform owing to its unique luminescent and microwave sensitization properties. However, it is difficult to be applied to complicated biological systems for EuMOF due to its rapid degradation induced by the solvent molecular and ionic environment. In this work, a luminescent EuMOF nanocomposite (EuMOF@ZIF/AP-PEG, named EZAP) was designed, which brought the multifunctional characteristics of microwave sensitization, fluorescence imaging and drug loading.ResultsLamellar EuMOF was synthesized by a hydrothermal method. Through the charge adsorption mechanism, the zeolite imidazole framework (ZIF) structure was intensively assembled on the surface of EuMOF to realize the protection. Then, through in-situ Apatinib drug loading and PEG modification, EZAP nanocomposite was finally obtained. Apatinib (AP) was a kind of chemotherapy drug approved by Food and Drug Administration for targeted therapy of tumors. PEG modification increased long-term circulation of EZAP nanocomposite. The physical and chemical structure and properties of EuMOF@ZIF (EZ) were systematically represented, indicating the successful synthesis of the nanocomposite. The toxic and side effects were negligible at a safe dose. The growth of human liver cancer cells and murine liver cancer cells in vitro was significantly inhibited, and the combined microwave-thermal therapy and chemotherapy in vivo achieved high anti-cancer efficacy. Moreover, EZAP nanocomposite possessed bright red fluorescence, which can be applied for tumor imaging in tumor-bearing mice in vivo.ConclusionTherefore, EZAP nanocomposite showed high microwave sensitization, excellent fluorescence properties and outstanding drug loading capacity, establishing a promising theranostic nanoplatform for tumor therapy and fluorescence imaging. This work proposes a unique strategy to design for the first time a multifunctional nanoplatform with lanthanide metal organic frameworks for biological applications in tumor therapy and diagnosis.Graphical

A comparison of the monomeric [68Ga]NODAGA-NGR and dimeric [68Ga]NOTA-(NGR)2 as aminopeptidase N ligand for positron emission tomography imaging in tumor-bearing mice

The aminopeptidase N (APN/CD13) is a key protein specifically expressed on activated endothelial cells and by various tumors, representing a promising target for molecular imaging and therapy of malignant diseases. It is known that the tripeptide NGR is a specific ligand for CD13, therefore radiolabeled NGR peptides are auspicious radiotracers for non-invasive imaging of CD13-positive tumors. From previous studies, it is known that the target affinity could be improved by molecules with multiple ligand sequences. Therefore, the aim of this study was to compare two NGR radioligands [68Ga]NODAGA-NGR (NGR monomer) and [68Ga]NOTA-(NGR)2 (NGR dimer), the latter with two NGR ligand motifs, in vitro and in vivo.CD13 expression was determined by FACS in the human tumor cells A549, SKHep-1, and MDA-MB-231, followed by the investigation of the cell uptake of [68Ga]NODAGA-NGR and [68Ga]NOTA-(NGR)2. For in vivo evaluation of [68Ga]NODAGA-NGR and [68Ga]NOTA-(NGR)2, microPET and biodistribution were carried out in A549- and SKHep-1-bearing mice. After the final examination, tumors were cryo-conserved, cut, and stained against CD13 and CD31.A549 and SKHep-1 cells were identified as CD13 positive, whereas no CD13 expression was detected in MDA-MB-231 cells. The cell uptake study showed relatively low accumulation of both the NGR monomer and dimer in all tumor cell lines examined, with consistently higher cell uptake observed for the dimer than for the monomer. In vivo, [68Ga]NODAGA-NGR and [68Ga]NOTA-(NGR)2 accumulated in the tumors, with slightly higher tumor-to-muscle ratio for the NGR dimer in A549 and SKHep-1. The tumor-to-liver ratio of the NGR dimer was diminished in comparison to the NGR monomer. This finding was confirmed by biodistribution, which revealed higher accumulation in liver and spleen for the NGR dimer. Immunohistochemical staining confirmed the CD13 expression in the tumors and tumor-associated vessels.In conclusion, both the [68Ga]NODAGA-NGR and the [68Ga]NOTA-(NGR)2 were found to be suitable for PET imaging of CD13-positive tumors. Despite slight differences in tumor-to-background ratio and organ accumulation, both radiotracers can be considered comparable.

Rational Linker Design to Accelerate Excretion and Reduce Background Uptake of Peptidomimetic PSMA-Targeting Hybrid Molecules.

The evolution of peptidomimetic hybrid molecules for preoperative imaging and guided surgery targeting the prostate-specific membrane antigen (PSMA) significantly progressed over the past few years, and some approaches are currently being evaluated for further clinical translation. However, accumulation in nonmalignant tissue such as kidney, bladder, spleen, or liver might limit tumor-to-background contrast for precise lesion delineation, particularly in a surgical setting. To overcome these limitations, a rational linker design aims at the development of a second generation of PSMA-11-based hybrid molecules with an enhanced pharmacokinetic profile and improved imaging contrast. Methods: A selection of rationally designed linkers was introduced to the PSMA-targeting hybrid molecule Glu-urea-Lys-HBED-CC-IRDye800CW, resulting in a second-generation peptidomimetic hybrid molecule library. The biologic properties were investigated in cell-based assays. In a preclinical proof-of-concept study with the radionuclide 68Ga, the impact of the modifications was evaluated by determination of specific tumor uptake, pharmacokinetics, and fluorescence imaging in tumor-bearing mice. Results: The modified hybrid molecules carrying various selected linkers revealed high PSMA-specific binding affinity and effective internalization. The highest tumor-to-background contrast of all modifications investigated was identified for the introduction of a histidine- (H) and glutamic acid (E)-containing linker ((HE)3-linker) between the PSMA-binding motif and the chelator. In comparison to the parental core structure, uptake in nonmalignant tissue was significantly reduced to a minimum, as exemplified by an 11-fold reduced spleen uptake from 38.12 ± 14.62 percentage injected dose (%ID)/g to 3.47 ± 1.39 %ID/g (1 h after injection). The specific tumor uptake of this compound (7.59 ± 0.95 %ID/g, 1 h after injection) was detected to be significantly higher than that of the parental tracer PSMA-11. These findings confirmed by PET and fluorescence imaging are accompanied by an enhanced pharmacokinetic profile with accelerated background clearance at early time points after injection. Conclusion: The novel generation of PSMA-targeting hybrid molecules reveals fast elimination, reduced background organ enrichment, and high PSMA-specific tumor uptake meeting the key demands for potent tracers in nuclear medicine and fluorescence-guided surgery. The approach's efficacy in improving the pharmacokinetic profile highlights the strengths of rational linker design as a powerful tool in strategic hybrid-molecule development.

Visualization of platelet recruitment to tumor lesions using highly sensitive and stable radioiodine studded gold nanoprobes.

In vivo imaging of platelets will provide a better understanding of their critical roles in arterial cardiovascular disease, hemostasis, inflammation, and cancer. Here, we demonstrate the feasibility of using radioiodine studded gold nanoprobes (RIS-GNPs) as a platelet tracker for nuclear medicine imaging in tumor-bearing mice using positron emission tomography and computed tomography (PET/CT). Platelet labeling with RIS-GNPs did not alter the platelet functions, such as cellular proliferation and aggregation. PET/CT imaging clearly revealed the migration of platelets into tumor sites at 1 to 5 h post-transfer of RIS-GNP-labeled platelets, which was consistent with the biodistribution data. Our findings suggest that the imaging approach using RIS-GNPs makes it feasible to visualize the biological behavior of platelets in living organisms with cancer.

Self-assembled heptamethine cyanine dye dimer as a novel theranostic drug delivery carrier for effective image-guided chemo-photothermal cancer therapy

Near-infrared (NIR)-induced dye-based theranostic drug delivery carriers are used for critical image-guided chemo-photothermal cancer therapy. However, most carriers fail to deliver sufficient heat and fluorescence efficiently due to direct π-π stacking of the aromatic rings of the NIR dye and drug. In the work reported herein, we examined a self-assembled heptamethine cyanine dye dimer (CyD) with improved heat and fluorescence delivery that was developed by manipulating the unique structural and optical properties of the dimer. The H-aggregation of CyD in an aqueous solution generated a great amount of heat by transforming the energy of the excited electrons into non-radiative energy. Moreover, the disulfide bond of CyD assisted nanoparticles with a drug by minimizing the interaction between the NIR dye and drug, and also by releasing the drug in a redox environment. As a result, DOX encapsulated within CyD (CyD/DOX) showed strong heat generation and fluorescence imaging in tumor-bearing mice, allowing detection of the tumor site and inhibition of tumor growth by chemo-photothermal therapy. The multiplicity of features supplied by the newly developed CyD demonstrated the potential of CyD/DOX as an NIR dye-based theranostic drug-delivery carrier for effective chemo-photothermal cancer therapy.

Abstract 3837: Selective ezh2 functional activation alters the metastatic landscape of triple negative breast cancer

Abstract India is the TNBC capital of the world due to its highest (31%) prevalence and globally it has huge unmet medical need for its high mortality rate. Aggressive early metastasis is the key for such poor clinical outcome. So, there is a dire need to tackle this deadly disease worldwide by targeting its metastatic process. Epigenetic modulator Enhancer of Zeste homolog 2 (EZH2) promotes hetero-chromatinization of target genes by tri-methylating lysine at 27 of histone H3 (H3K27me3) and it has been shown to be associated with poor prognosis of TNBC. Besides its canonical function as a transcriptional repressor, EZH2 protein has recently been observed to perform H3K27me3 independent functions. Contribution of EZH2 protein (non-canonical) versus H3K27me3 dependent (canonical) function in TNBC pathogenesis remains poorly understood. Here, we aim to dissect the differential role of EZH2 protein versus H3K27me3 in the context of TNBC growth and metastasis. Utilizing stable EZH2 (protein versus hyperactivated H3K27me3) TNBC cells, we discovered that selective hyperactivation of H3K27me3 function of EZH2 but not its protein overexpression markedly promotes cell migration and invasion though cellular proliferation remains unaltered in both the cases. EZH2 protein overexpression (EPOE) and hyperactivation of H3K27me3 (EFHA) in TNBC cells resulted in similar tumor volume and growth pattern in-vivo in an orthotopic TNBC animal model but mice bearing EFHA tumors died significantly earlier than control and EPOE mice. Sequential Luc-GFP/Td tomato based live animal imaging of tumor bearing mice confirmed that EFHA profoundly augments TNBC metastasis compared to control and interestingly, it changes metastatic landscape of TNBC. Control tumors tend to metastasize to lung whereas; EFHA tumors metastasize to spleen first. Splenic metastasis of TNBC shows selective hyperactivation of H3K27me3 as compared to lung and liver metastasis at basal state. EZH2 functional inhibitor EPZ-6438 (currently in Phase-II clinical trial) though did not alter TNBC primary tumor growth but significantly abrogated TNBC metastasis and increased the life expectancy of tumor bearing mice. The high-throughput Illumina RNA-sequencing data identified unique gene signature of EFHA mediated TNBC metastasis corroborating with patient samples and TCGA database. In summary, our data indicate that splenic metastasis of TNBC may be driven by trimethylation function of EZH2 and targeting the methyltransferase activity of EZH2 could be a wonderful strategy to prevent TNBC metastasis in the clinic. Citation Format: Ayushi Verma, Akhilesh Singh, Anup Kumar Singh, Priyank Chaturvedi, Mushtaq Ahmad Nengroo, Krishan kumar Saini, abhipsa Sinha, Dipak Datta. Selective ezh2 functional activation alters the metastatic landscape of triple negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3837.

Simultaneous realization of persistent luminescence and CT dual-mode imaging by x-ray recharged Bi2Ga4O9:Cr nanoprobes in depth-independent tumors

Synthesis of excellent imaging nanoparticles plays a critical role in disease detection. Here, novel Bi2Ga4O9:Cr (BGOC) nanoparticles were synthesized for persistent luminescence and computed tomography dual-mode imaging. The Cr3+ ions in the BGOC luminescent nanoparticles were coped as the luminescent center, and the afterglow emission was located in the first biological imaging window. Bi3+ ions served as the computed tomography (CT) imaging contrast agent. The NH2 groups were modified on the surface of the nanoparticles to make them highly biocompatible, and folic acid molecules were modified to facilitate the specificity. This modification facilitated simultaneous persistent luminescence and CT imaging in tumor-bearing mice due to the overexpression of the folic acid molecules’ receptors. Furthermore, instead of visible light, x-rays were used as the excitation light source to activate the BGOC-folic acid nanoprobes and realize long time and high sensitivity imaging of tumors in vivo because of their capacity for deep tissue penetration. Therefore, such nanoprobes can provide a promising depth-independent detection mode for high sensitivity diagnosis and long-term monitoring of diseases.

Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody.

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few decades, the field of antibody-drug conjugates, bispecific antibodies, chimeric antigen receptors (CAR) and cancer immunotherapy has emerged as the most promising area of basic and therapeutic investigations. With numerous successful human trials targeting immune checkpoint receptors and CAR-T cells in leukemia and melanoma at a breakthrough pace, it is highly exciting times for oncologic therapeutics derived from variations of antibody engineering. Regrettably, a significantly large numbers of antibody and CAR based therapeutics have also proven disappointing in human trials of solid cancers because of the limited availability of immune effector cells in the tumor bed. Importantly, nonspecific distribution of therapeutic antibodies in tissues other than tumors also contribute to the lack of clinical efficacy, associated toxicity and clinical failure. As faithful translation of preclinical studies into human clinical trails are highly relied on mice tumor xenograft efficacy and safety studies, here we highlight a method to test the tumor and general tissue distribution of therapeutic antibodies. This is achieved by labeling the protein-A purified antibody with near Infrared fluorescent dye followed by live imaging of tumor bearing mice.

In Situ One-Step Fluorescence Labeling Strategy of Exosomes via Bioorthogonal Click Chemistry for Real-Time Exosome Tracking In Vitro and In Vivo.

Exosomes are cellular components with promising uses in cancer diagnostics and therapeutics, and their imaging and tracking are essential to study their biological properties. Herein, we report on an in situ one-step fluorescence labeling strategy for exosomes via bioorthogonal click chemistry. First, exosome donor cancer cells were treated with tetraacetylated N-azidoacetyl-d-mannosamine (Ac4ManNAz) to generate unnatural azide groups (-N3) on their surface via metabolic glycoengineering. Then, the azide groups were labeled with near-infrared fluorescent dye-conjugated dibenzylcyclooctyne (DBCO-Cy5) via bioorthogonal click chemistry. After 2 days of incubation, the DBCO-Cy5-labeled exosomes (Cy5-Exo) were successfully secreted from the donor cancer cells and were isolated via classical ultracentrifugation, providing a high-yield of fluorescent dye-labeled exosomes. This in situ one-step bioorthogonal click chemistry offers improved labeling efficiency, biocompatibility, and imaging sensitivy compared to standard exosomes (ST-Exo), purified with classical ultracentrifugation or carbocyanine lipophilic dye (DiD)-labeled exosomes (DiD-Exo) in vitro. In particular, the Cy5-Exo were successfully taken up by A549 cells in a time-dependent manner, and they could escape from lysosome confinement, showing their possible use as a delivery carrier of therapeutic drugs or imaging agents. Finally, intraveneously injected Cy5-Exo were noninvasively tracked and imaged via near-infrared fluorescence (NIRF) imaging in tumor-bearing mice. This new fluorescence labeling strategy for natural exosomes may be useful to provide better understanding of their theranostic effects in many biomedical applications.

Trans-Cyclooctene-Functionalized PeptoBrushes with Improved Reaction Kinetics of the Tetrazine Ligation for Pretargeted Nuclear Imaging.

Tumor targeting using agents with slow pharmacokinetics represents a major challenge in nuclear imaging and targeted radionuclide therapy as they most often result in low imaging contrast and high radiation dose to healthy tissue. To address this challenge, we developed a polymer-based targeting agent that can be used for pretargeted imaging and thus separates tumor accumulation from the imaging step in time. The developed targeting agent is based on polypeptide-graft-polypeptoid polymers (PeptoBrushes) functionalized with trans-cyclooctene (TCO). The complementary 111In-labeled imaging agent is a 1,2,4,5-tetrazine derivative, which can react with aforementioned TCO-modified PeptoBrushes in a rapid bioorthogonal ligation. A high degree of TCO loading (up to 30%) was achieved, without altering the physicochemical properties of the polymeric nanoparticle. The highest degree of TCO loading resulted in significantly increased reaction rates (77-fold enhancement) compared to those with small molecule TCO moieties when using lipophilic tetrazines. Based on computer simulations, we hypothesize that this increase is a result of hydrophobic effects and significant rearrangements within the polymer framework, in which hydrophobic patches of TCO moieties are formed. These patches attract lipophilic tetrazines, leading to increased reaction rates in the bioorthogonal ligation. The most reactive system was evaluated as a targeting agent for pretargeted imaging in tumor-bearing mice. After the setup was optimized, sufficient tumor-to-background ratios were achieved as early as 2 h after administration of the tetrazine imaging agent, which further improved at 22 h, enabling clear visualization of CT-26 tumors. These findings show the potential of PeptoBrushes to be used as a pretargeting agent when an optimized dose of polymer is used.

Endoscopic molecular imaging of early gastric cancer using fluorescently labeled human H-ferritin nanoparticle

Optical imaging technologies improve clinical diagnostic accuracy of early gastric cancer (EGC). However, there was a lack of imaging agents exhibiting molecular specificity for EGCs. Here, we employed the dye labeled human heavy-chain ferritin (HFn) as imaging nanoprobe, which recognizes tumor biomarker transferrin receptor 1 (TfR1), to enable specific EGC imaging using confocal laser endomicroscopy (CLE). TfR1 expression was initially examined in vitro in gastric tumor cells and in vivo through whole-body fluorescence and CLE imaging in tumor-bearing mice. Subsequently, dye labeled HFn was topically applied to resected human tissues for EGC detection. CLE analysis of TfR1-targeted fluorescence imaging allowed distinction of neoplastic from non-neoplastic tissues (P < 0.0001), and TfR1 expression level was found to correlate with EGC differentiation degrees (P < 0.0001). Notably, the CLE evaluation correlated well with the immunohistochemical findings (κ-coefficient: 0.8023). Our HFn-nanoprobe-based CLE increases the accuracy of EGC detection and enables visualization of tumor margins and endoscopic resection.

In vivo tumor imaging by a γ-glutamyl transpeptidase-activatable near-infrared fluorescent probe.

γ-Glutamyl transpeptidase (GGT), overexpressed in various cancer cells, has been perceived as a latent tumor biomarker. Thus, developing near-infrared (NIR) fluorescent GGT probes is highly desired for in vivo tumor imaging and studies. To our knowledge, however, such a GGT probe is still rare. Herein, we construct a new GGT-activatable NIR fluorescent probe HCAGlu by incorporating γ-glutamyl group as a recognition unit directly into a NIR hemicyanine fluorophore. HCAGlu exhibits a highly sensitive and selective NIR fluorescence off-on response to GGT. The probe has been applied to cell and histological section imaging, which demonstrates the ability of HCAGlu in distinguishing different GGT-expression levels in situ in biological samples. Notably, in vivo fluorescence imaging in tumor-bearing mice has been performed, verifying that probe HCAGlu can rapidly produce a distinct fluorescence signal in the tumor site via both intravenous and intratumoral injections. The simplicity and excellent performance of HCAGlu make it of high potential in studying the physiological function of GGT in vivo. Graphical abstract A near-infrared fluorescent probe for imaging γ-glutamyl transpeptidase in biological samples.

Development of a SPECT Tracer to Image c-Met Expression in a Xenograft Model of Non-Small Cell Lung Cancer.

Elevated expression of the c-Met receptor plays a crucial role in cancers. In non-small cell lung cancer (NSCLC), aberrant activation of the c-Met signaling pathway contributes to tumorigenesis and cancer progression and may mediate acquired resistance to epidermal growth factor receptor-targeted therapy. c-Met is therefore emerging as a promising therapeutic target for NSCLC, and methods for noninvasive in vivo assessment of c-Met expression would improve NSCLC treatment and diagnosis. Methods: We developed a new c-Met-binding peptide (cMBP) radiotracer, 99mTc-hydrazine nicotinamide (HYNIC)-cMBP, for SPECT imaging. Cell uptake assays were performed on 2 NSCLC cell lines with different c-Met expressions: H1993 (high expression) and H1299 (no expression). In vivo tumor specificity was assessed by SPECT imaging in tumor-bearing mice at 0.5, 1, 2, and 4 h after injection of the probe. Blocking assays, biodistribution, and autoradiography were also conducted to determine probe specificity. Results:99mTc-HYNIC-cMBP was prepared with high efficiency and showed higher uptake in H1993 cells than in H1299 cells. Biodistribution and autoradiography also showed significantly higher percentages of the injected dose for 99mTc-HYNIC-cMBP in H1993 tumors than in H1299 tumors at 0.5 h (4.74 ± 1.43%/g and 1.00 ± 0.37%/g, respectively; P < 0.05). H1993 tumors were clearly visualized at 0.5 h in SPECT images, whereas H1299 tumors were not observed at any time. The specificity of 99mTc-HYNIC-cMBP for c-Met was demonstrated by a competitive block with an excess of nonradiolabeled peptide. Conclusion: For c-Met-targeted SPECT imaging of NSCLC, we developed 99mTc-HYNIC-cMBP, a tracer that specifically binds to c-Met with favorable pharmacokinetics in vitro and in vivo.

Immuno-PET in Pontine Glioma: More Than Meets the Eye?

610 Objectives: The success of PET imaging with 68Ga-labeled somatostatin and PSMA ligands has created a markedly increased demand for 68Ga that is challenging to meet with existing 68Ge/68Ga generators. In addition, preparation of patient doses is relatively expensive, because only small batches of the ligands can be prepared that each need to undergo QC procedures. The objective of this study was therefore to investigate the cyclotron production of 68Ga from a solid 68Zn target and the feasibility of using cyclotron produced 68Ga for preparing 68Ga-PSMA-11. We also evaluated how a decrease of specific activity - as would occur during the shipment of larger batches of 68Ga-PSMA-11 - affects tumor and normal organ uptake of [asterisk]Ga-PSMA-11 in mouse models of prostate cancer. Methods: Enriched 68Zn foils were irradiated were irradiated with 11.5 MeV protons for 1-3 h at a beam current of 30-75 µA in a GET PETTRACE cyclotron. The irradiated foils were dissolved in 10 N HCl and the solution passed through a cation exchanging resin to trap 68Ga and 68Zn. 68Zn was then eluted from the column using HBr in 80% acetone. Finally, 68Ga was eluted with 3 N HCl and used to label 68Ga-PSMA-11. In order to study the impact of injected mass on tumor and normal organ of [asterisk]Ga-PSMA-11 we injected mice bearing PSMA expressing PC3-PIP or LNCaP tumors with 3.3 - 10000 pmol of total ligand (67Ga-PSMA-11 and PSMA-11) labeled with 100-200 µCi of (67Ga) and performed a biodistribution study 1 h post injection. We also injected tumor bearing with cyclotron produced and generator produced 68Ga-PSMA-11 and performed PET imaging studies 1h post injection. Results: Up to 4.2 Ci of 68Ga were produced from the 68Zn foil before purification and, 1.8 Ci after purification. The 67Ga content was less than 0.1%. Specific activity of the produced 68Ga-PSMA-11 was 2.2-7.0 mCi/µg and radiochemical purity > 99%. The 68Ga-PSMA-11 met all the usual criteria for PET radiopharmaceuticals (bubble, LAL, and sterility test). There was no significant change in tumor uptake of 67Ga-PSMA-11 up to an injected mass of 1000 pmol for PC3-PIP and LNCaP tumors (333-fold range). PET images of tumor bearing mice injected with 800 pmol of 68Ga-PSMA11 demonstrated identical biodistribution in normal organs and tumors. Conclusions: 68Ga can be produced in Ci amounts using a medical cyclotron. The cyclotron produced 68Ga product appears suitable for labeling PSMA11 for future clinical trials.The injected mass of PSMA-11 (on a per weight basis) had no significant impact on tumor uptake in mice up to a more than 300-fold higher mass than used for human PET/CT studies with 68Ga-PSMA11. This suggests that batches of cyclotron produced 68Ga-PSMA11 may be shipped for several hours after production.