17-DMAG, a heat shock protein 90 (Hsp90) inhibitor, has been intensively investigated for cancer therapy and is undergoing clinical trials. Human epidermal growth factor receptor 2 (HER-2) is one of the client proteins of Hsp90 and its expression is decreased upon 17-DMAG treatment. In this study, we aimed to non-invasively monitor the HER-2 response to 17-DMAG treatment in xenografted mice.
The sensitivity of human ovarian cancer SKOV-3 cells to 17-DMAG in vitro was measured by MTT assay. HER-2 expression of SKOV-3 cells was determined by flow cytometry. Nude mice bearing SKOV-3 tumors were treated with 17-DMAG and the therapeutic efficacy was evaluated by tumor size measurement. Both treated and control mice were imaged with microPET using 64Cu-DOTA-trastuzumab and 18F-FDG. Biodistribution studies, immunofluorescence staining were performed to validate the microPET results.
SKOV-3 cells are sensitive to 17-DMAG treatment, in a dose dependent manner, with an IC50 value of 68.7 nM after 72 h incubation. The tumor growth curve supported the inhibition effect of 17-DMAG on SKOV-3 tumors. Quantitative microPET imaging showed that 64Cu-DOTA-trastuzumab had prominent tumor activity accumulation in untreated SKOV-3 tumors, which was significantly reduced in 17-DMAG treated tumors. There was no uptake difference detected by FDG PET. Immunofluorescence staining confirmed the significant reduction in tumor HER-2 level upon 17-DMAG treatment.
The early response to anti-Hsp90 therapy was successfully monitored by quantitative PET using 64Cu-DOTA-trastuzumab. This approach may be valuable in monitoring the therapeutic response in HER-2-positive cancer patients under 17-DMAG treatment.
Human epidermal growth factor receptor (HER-2); Positron emission tomography (PET); Heat shock protein 90 (Hsp90); 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG); Trastuzumab
A cell permeable cyclometalated iridium(III) complex has been developed as a phosphorescent probe for cell imaging. The iridium(III) solvato complex [Ir(phq)2(H2O]2)] preferentially stains the cytoplasm of both live and dead cells with a bright luminescence.
Biomedical imaging techniques such as skeletal survey and 18F-fluorodeoxyglucose (FDG)/Positron Emission Tomography (PET) are frequently used to diagnose and stage multiple myeloma (MM) patients. However, skeletal survey has limited sensitivity as it can detect osteolytic lesions only after 30–50% cortical bone destruction, and FDG is a marker of cell metabolism that has limited sensitivity for intramedullary lesions in MM. Targeted, and non-invasive novel probes are needed to sensitively and selectively image the unique molecular signatures and cellular processes associated with MM. Very late antigen-4 (VLA-4; also called α4β1 integrin) is over-expressed on MM cells, and is one of the key mediators of myeloma cell adhesion to the bone marrow (BM) that promotes MM cell trafficking and drug resistance. Here we describe a proof-of-principle, novel molecular imaging strategy for MM tumors using a VLA-4 targeted PET radiopharmaceutical, 64Cu-CB-TE1A1P-LLP2A. Cell uptake studies in a VLA-4-positive murine MM cell line, 5TGM1, demonstrated receptor specific uptake (P<0.0001, block vs. non-block). Tissue biodistribution at 2 h of 64Cu-CB-TE1A1P-LLP2A in 5TGM1 tumor bearing syngeneic KaLwRij mice demonstrated high radiotracer uptake in the tumor (12±4.5%ID/g), and in the VLA-4 rich organs, spleen (8.8±1.0%ID/g) and marrow (11.6±2.0%ID/g). Small animal PET/CT imaging with 64Cu-CB-TE1A1P-LLP2A demonstrated high uptake in the 5TGM1 tumors (SUV 6.6±1.1). There was a 3-fold reduction in the in vivo tumor uptake in the presence of blocking agent (2.3±0.4). Additionally, 64Cu-CB-TE1A1P-LLP2A demonstrated high binding to the human MM cell line RPMI-8226 that was significantly reduced in the presence of the cold targeting agent. These results provide pre-clinical evidence that VLA-4-targeted imaging using 64Cu-CB-TE1A1P-LLP2A is a novel approach to imaging MM tumors.
To prepare and evaluate a new radiotracer for molecular imaging of cell surface receptors for epidermal growth factor (EGF).
Cys tagged EGF (cEGF) was labeled with 18F by coupling the free thiol group of the Cys tag with N-[2-(4-[18F]fluorobenzamido)ethyl]maleimide ([18F]FBEM) to form [18F]FBEM-cEGF. Cell uptake, internalization and efflux of [18F]FBEM-cEGF were tested in human head and neck squamous carcinoma UM-SCC1 cells. In vivo tumor targeting and pharmacokinetics of the radiotracers were evaluated in UM-SCC1 tumor-bearing athymic nude mice by static and dynamic microPET imaging. Ex vivo biodistribution assays were performed to confirm the noninvasive imaging results.
The radiolabeling yield for [18F]FBEM-cEGF was over 60%, based on starting [18F]FBEM. [18F]FBEM-cEGF exhibited rapid blood clearance through both hepatobiliary and renal excretion. UM-SCC1 tumors were clearly visualized and showed modest tracer uptake of 2.60 ± 0.59 %ID/g at 30 min post injection. Significantly higher tumor uptake of [18F]FBEM-cEGF (5.99 ± 1.61 %ID/g at 30 min p.i., p < 0.01) and tumor/non-tumor ratio were achieved by co-injection of 50 μg of unlabeled EGF. Decreased liver uptake of [18F]FBEM-cEGF was observed when unlabeled EGF was co-administered.
With optimized liver blocking, [18F]FBEM-cEGF has the potential to be used in a non-invasive and quantitative manner for detection of malignant lesions and evaluation of EGFR activity.
PET; [18F]FBEM-cEGF; HNSCC; EGFR
Tumors expressing the chemokine receptor CXCR4 have been reported to be more aggressive and to produce more metastatic seeding in specific organs, such as the bone marrow. However, evaluation of tumors for CXCR4 expression requires testing of ex vivo biopsy samples, and is not routinely done in cancer management. In prior work to address this issue, we and others have developed tracers for positron emission tomography (PET) that targeted CXCR4, but in addition to binding to CXCR4 these tracers also bound to red blood cells (and to other unrelated targets) in vivo. Here we report two new tracers based on the CXCR4 peptide antagonist 4F-benzoyl-TN14003 (T140) that bind to CXCR4, but not to undesired targets. These tracers, NOTA-NFB and DOTA-NFB, show slight reductions in both 1) binding affinities for CXCR4 and 2) inhibition of CXCL12 induced migration, compared to T140, in vitro. Both NOTA-NFB and DOTA-NFB specifically accumulate in CXCR4-positive, but not CXCR4-negative, tumor xenografts in mice and allow clear visualization of CXCR4 expression by PET. Evaluation of NOTA-NFB and DOTA-NFB for their potential to mobilize immune cells and progenitor cells from the bone marrow to the peripheral blood revealed slightly reduced, but still comparable, results to the parent molecule T140. The tracers reported here may allow the evaluation of CXCR4 expression in primary tumors and metastatic nodules, and enable better informed, more personalized treatment for patients with cancer.
T140; CXCR4 imaging; PET; copper-64
Arylboronates capture aqueous 18F-fluoride in one step to afford a highly polar 18F-labeled aryltrifluoroborate anion (18F-ArBF3
-) that clears rapidly in vivo. To date however, there is little data to show that a ligand labeled with a prosthetic 18F-ArBF3
- will provide functional images. RGD, a high-affinity ligand for integrins that are present on the cell surface of numerous tumors, has been labeled in many formats with many different radionuclides, and as such represents a well-established ligand that can be used to evaluate new labeling methods. Herein we have labeled RGD with a prosthetic 18F-ArBF3
- via two approaches for the first time: 1) a RGD-boronate bioconjugate is directly labeled in one step and 2) an alkyne-modified arylborimidine is first converted to the corresponding 18F-ArBF3
- which is then conjugated to an RGD-azide via Cu+-mediated [2+3] dipolar cycloaddition in one pot over two steps. RGD-18F-ArBF3
- bionconjugates were produced in reasonable radiochemical yields using low amounts of 18F-fluoride anion (10-50 mCi). Despite relatively low specific activities, good tumor images are revealed in each case.
One-step 18F-labeling; click labeling; RGD; PET imaging
Objectives: Most chemotherapy agents cause tumor cell death primarily by the induction of apoptosis. The ability to noninvasively image apoptosis in vivo could dramatically benefit pre-clinical and clinical evaluation of chemotherapeutics targeting the apoptotic pathway. This study aims to visualize the dynamics of apoptotic process with temporal bioluminescence imaging (BLI) using an apoptosis specific bioluminescence reporter gene. Methods: Both UM-SCC-22B human head and neck squamous carcinoma cells and 4T1 murine breast cancer cells were genetically modified with a caspase-3 specific cyclic firefly luciferase reporter gene (pcFluc-DEVD). Apoptosis induced by different concentrations of doxorubicin in the transfected cells was evaluated by both annexin V staining and BLI. Longitudinal BLI was performed in xenografted tumor models at different time points after doxorubicin or Doxil treatment, to evaluate apoptosis. After imaging, DNA fragmentation in apoptotic cells was assessed in frozen tumor sections using TUNEL staining. Results: Dose- and time-dependent apoptosis induced by doxorubicin in pcFluc-DEVD transfected UM-SCC-22B and 4T1 cells was visualized and quantified by BLI. Caspase-3 activation was confirmed by both caspase activity assay and GloTM luciferase assay. One dose of doxorubicin treatment induced a dramatic increase in BLI intensity as early as 24 h after treatment in 22B-pcFluc-DEVD xenografted tumors. Sustained signal increase was observed for the first 3 days and the fluorescent signal from ex vivo TUNEL staining was consistent with BLI imaging results. Long-term imaging revealed that BLI signal consistently increased and reached a maximum at around day 12 after the treatment with one dose of Doxil. Conclusions: BLI of apoptosis with pcFluc-DEVD as a reporter gene facilitates the determination of kinetics of the apoptotic process in a real-time manner, which provides a unique tool for drug development and therapy response monitoring.
apoptosis; cyclic firefly luciferase; bioluminescence imaging; doxorubicin; caspase-3.
[18F]FPPRGD2, an F-18 labeled dimeric cyclic RGDyK peptide, has favorable properties for PET imaging of angiogenesis by targeting the αvβ3 integrin receptor. This radiotracer has been approved by the FDA for use in clinical trials. However, the time-consuming multiple-step synthetic procedure required for its preparation may hinder the widespread usage of this tracer. The recent development of a method using an F-18 fluoride-aluminum complex to radiolabel peptides provides a strategy for simplifying the labeling procedure. On the other hand, the easy-to-prepare [68Ga]-labeled NOTA-RGD derivatives have also been reported to have promising properties for imaging αvβ3 integrin receptors. The purpose of this study was to prepare [18F]FPPRDG2, [18F]FAl-NOTA-PRGD2, and [68Ga]Ga-NOTA-PRGD2 and to compare their pharmacokinetics and tumor imaging properties using small animal PET. All three compounds showed rapid and high tracer uptake in U87MG tumors with high target-to-background ratios. The uptake in the liver, kidneys and muscle were similar for all three tracers and they all showed predominant renal clearance. In conclusion, [18F]FAl-NOTA-PRGD2 and [68Ga]Ga-NOTA-PRGD2 have imaging properties and pharmacokinetics comparable to those of [18F]FPPRGD2. Considering their ease of preparation and good imaging qualities, [18F]FAl-NOTA-PRGD2 and [68Ga]NOTA-PRGD2 are promising alternatives to [18F]FPPRGD2 for PET imaging of tumor αvβ3 integrin expression.
positron emission tomography (PET); integrin αvβ3; Arg-Gly-Asp (RGD) peptide; fluorine-aluminum complex; Ga-68; F-18
Herein we demonstrate for the first time that a fluorogenic probe can be used as an in vivo imaging agent for visualizing activities of membrane-tethered, membrane-type matrix metalloproteinases (MT-MMPs). An MT-MMP fluorogenic probe that consisted of an MT1-MMP (MMP-14) substrate and near-infrared (NIR) dye-quencher pair exhibited rapid, efficient boosts in fluorescence upon cleavage by MT1-MMP in tumor-bearing mice. In particular, unlike similar fluorogenic probes designed to target extracellular, soluble-type MMPs (EC-MMPs)--which can be cleared from the blood stream after activation--the fluorescence signals activated by MT1-MMP enable clear visualization of MT1-MMP-positive tumors in animal models for up to 24 hours. The results indicate that a simple form of a fluorogenic probe that is less effective in EC-MMP imaging is an effective probe for imaging MT-MMP activities in vivo. These findings can be widely applied to designing probes and to applications targeting various membrane-anchored proteases in vivo.
activatable probe; fluorogenic probe; membrane-type matrix metalloproteinase; optical imaging; protease
Existing two-phase solvent systems for high-speed countercurrent chromatography cover the separation of hydrophobic to moderately polar compounds, but often fail to provide suitable partition coefficient values for highly polar compounds such as sulfonic acids, catecholamines and zwitter ions. The present paper introduces a new solvent series which can be applied for the separation of these polar compounds. It is composed of 1-butanol, ethanol, saturated ammonium sulfate and water at various volume ratios and consists of a series of 10 steps which are arranged according to the polarity of the solvent system so that the two-phase solvent system with suitable K values for the target compound(s) can be found in a few steps. Each solvent system gives proper volume ratio and high density difference between the two phases to provide a satisfactory level of retention of the stationary phase in the spiral column assembly. The method is validated by partition coefficient measurement of four typical polar compounds including methyl green (basic dye), tartrazine (sulfonic acid), tyrosine (zwitter ion) and epinephrine (a catecholamine), all of which show low partition coefficient values in the polar 1-butanol-water system. The capability of the method is demonstrated by separation of three catecholamines.
Organic-high ionic aqueous two-phase solvent systems; high-speed counter-current chromatography (HSCCC); flat-twisted spiral column; sulfonic acids; zwitter ions; catecholamines
The dimeric transmembrane integrin, αvβ3, is a well-investigated target by different imaging modalities through suitably labeled arginine–glycine–aspartic acid (RGD) containing peptides. In this study, we labeled four cyclic RGD peptides with or without PEG functional groups: c(RGDfK) (denoted as FK), PEG3-c(RGDfK) (denoted as FK-PEG3), E[c(RGDfK)]2 (denoted as [FK]2), and PEG4-E[PEG4-c(RGDfK)]2 (denoted as [FK]2-3PEG4), with 89Zr (t1/2=78.4 h), using the chelator desferrioxamine-p-SCN (Df) for imaging tumor integrin αvβ3.
The Df conjugated RGD peptides were subjected to integrin αvβ3 binding assay in vitro using MDA-MB-435 breast cancer cells. The 89Zr-labeled RGD peptides were then subjected to small animal positron emission tomography (PET) and direct tissue sampling biodistribution studies in an orthotopic MDA-MB-435 breast cancer xenograft model.
All four tracers, 89Zr-Df-FK, 89Zr-Df-FK-PEG3, 89Zr-Df-[FK]2, and 89Zr-Df-[FK]2-3PEG4, were labeled in high radiochemical yield (89±4%) and high specific activity (4.07–6 MBq/µg). Competitive binding assay with 125I-echistatin showed that conjugation of the RGD peptides to the Df chelator did not have significant impact on their integrin αvβ3 binding affinity and the dimeric peptides were shown to be more potent than the monomers. In agreement with binding results, tumor uptake of 89Zr-Df-[FK]2 and 89Zr-Df-[FK]2-3PEG4 was significantly higher (4.32±1.73%ID/g and 4.72±0.66%ID/g, respectively, at 2 h post-injection) than the monomers 89Zr-Df-FK and 89Zr-Df-FK-PEG3 (1.97±0.38%ID/g and 1.57±0.49%ID/g, respectively, at 2 h post-injection). Out of the four labeled peptides, 89Zr-Df-[FK]2-3PEG4 gave the highest tumor-to-background ratio (18.21±2.52 at 2 h post-injection and 19.69±3.99 at 4 h post-injection), with the lowest uptake in metabolic organs. Analysis of late time points biodistribution data revealed that the uptake in the tumor was decreased, along with increase in the bone, which implies decomplexation of 89Zr-Df.
Efficient radiolabeling of peptides with an appropriate chelator such as Df-RGD with 89Zr was observed. The 89Zr radiolabeled peptides provided high-quality and high-resolution microPET images in xenograft models. 89Zr-Df-[FK]2-3PEG4 demonstrated the highest tumor-to-background ratio of the compounds tested. Preparation of 89Zr peptides to take advantage of the longer half-life is unwarranted due to the relatively rapid clearance from the tumor region of peptide tracers prepared for this study and the increased uptake in the bone of transchelated 89Zr with time (2.0±0.36%ID/g, 24 h post-injection).
89Zr—zirconium; RGD peptides; Integrin αvβ3; PET
To characterize lymphatic vessel morphology in lower extremity lymphedema using MR lymphography at 3T.
Forty females with lower extremity lymphedema secondary to gynecologic carcinoma treatment underwent MR lymphography (MRL) at 3T. Lymphatic vessel morphology in normal and affected limbs was compared.
The median diameter of the lymphatic vessels in swollen calf and thigh were significantly larger than that in the contralateral calf and thigh, respectively (p<0.05). The median number of lymphatic vessels visualized in normal calf was less than that in the lymphedematous calf (p<0.01), while no significant difference was found between the normal thigh and swollen thigh. Lymphatic vessel number in the affected calf was significantly greater than that in affected thigh and the mean diameter of affected calf was also significantly wider than that of affected thigh (p<0.01). Mean diameter of lymphatic vessels in the affected calf was significantly different between stage I and stage III (p<0.05), but not significantly different between stages I and II, and between stages II and III (p>0.05). The median number of lymphatic vessels for affected calf showed significant difference between stage I and stage III, and between stage II and stage III (p<0.05), but no significant difference between stage I and stage II (p>0.05). There was no significant difference in mean diameter or median number of lymphatic vessels in the affected thigh found between different stages (p>0.05).
There are significant differences in the number or diameter of lymphatic vessels between normal and affected limbs and there are significant differences for affected calf between early and late stages of lymphedema; therefore, MR lymphography can be helpful in diagnosis or clinical staging for lower extremity with gynecologic oncology-related lymphedema.
The risk of endobronchial ultrasound-guided transbronchial biopsy-related pneumothorax is a major concern and warrants further studies. The aim of our study was to estimate the risk of pneumothorax after this procedure and identify its risk factors.
From 2007 to 2011, 399 patients who underwent endobronchial ultrasound-guided transbronchial biopsy for peripheral lung lesions were included in this study. The variables analyzed included patient factors, lesion factors and procedure factors. Multivariate logistic regression analysis was used to identify independent risk factors for pneumothorax.
The incidence of pneumothorax was 3.3% (13/399). Chest tube placement was required for 31% (4/13) of pneumothoraces. Independent risk factors for pneumothorax included pulmonary emphysema (OR, 55.09; 95% CI, 9.37–324.03; p<0.001) and probe position adjacent to the lesion (OR, 17.01; 95% CI, 2.85–101.64; p = 0.002). The number of biopsy specimens, age, sex, history of prior lung surgery and lesion size, location and character did not influence the risk of pneumothorax in our analyses.
The risk of pneumothorax after endobronchial ultrasound-guided transbronchial biopsy is low. To further reduce the risk of pneumothorax, every effort should be made to advance the endobronchial ultrasound probe into the bronchus where it is imaged within the target lesion before embarking on transbronchial biopsy.
To determine the accuracy of MR imaging with Gd-EOB-DTPA for the detection of liver metastases.
Materials and Methods
PUBMED, EMBASE, the Web of Science, and the Cochrane Library were searched for original articles published prior to February 2012. The criteria for the inclusion of articles were as follows: reported in the English language; MR imaging with Gd-EOB-DTPA was performed to detect liver metastases; histopathologic analysis (surgery, biopsy), intraoperative observation (manual palpatation, intraoperative ultrasonography), and/or follow-up US was the reference standard; and data were sufficient for the calculation of true-positive or false-negative values. The methodological quality was assessed by using the quality assessment of diagnostic studies instrument. The data were extracted to calculate sensitivity, specificity, predictive value, diagnostic odds ratio, and areas under hierarchical summary receiver operating characteristic (HSROC) curve to perform heterogeneity test and threshold effect test, as well as publication bias analysis and subgroup analyses.
From 229 citations, 13 were included in the meta-analysis with a total of 1900 lesions. We detected heterogeneity between studies and evidence of publication bias. The methodological quality was moderate. The pooled weighted sensitivity with a corresponding 95% confidence interval (CI) was 0.93 (95% CI: 0.90, 0. 95), the specificity was 0.95 (95% CI: 0.91, 0.97), the positive likelihood ratio was 18.07 (95% CI: 10.52, 31.04), the negative likelihood ratio was 0.07 (95% CI: 0.05, 0.10), and the diagnostic odds ratio was 249.81 (95% CI: 125.12, 498.74). The area under the receiver operator characteristic curve was 0.98 (95% CI: 0.96, 0.99).
MR imaging with Gd-EOB-DTPA is a reliable, non-invasive, and no-radiation-exposure imaging modality with a high sensitivity and specificity for detection of liver metastases. Nonetheless, it should be applied cautiously, and large scale, well-designed trials are necessary to assess its clinical value.
Early evaluation of cancer response to a therapeutic regimen can help increase the effectiveness of treatment schemes and, by enabling early termination of ineffective treatments, minimize toxicity, and reduce expenses. Biomarkers that provide early indication of tumor therapy response are urgently needed. Solid tumors require blood vessels for growth, and new anti-angiogenic agents can act by preventing the development of a suitable blood supply to sustain tumor growth. The purpose of this study is to develop a class of novel molecular imaging probes that will predict tumor early response to an anti-angiogenic regimen with the humanized VEGF antibody bevacizumab.
Using a bevacizumab sensitive LS174T colorectal cancer model and a 12-mer bacteriophage (phage)-display peptide library, a bevacizumab responsive peptide (BRP) was identified after six rounds of biopanning and tested in vitro and in vivo.
This 12-mer peptide was metabolically stable and had low toxicity to both endothelial cells and tumor cells. Near-infrared dye IRDye800-labeled BRP phage showed strong binding to bevacizumab treated tumors, but not to untreated control LS174T tumors. In addition, both IRDye800 and 18F-labeled BRP peptide had significantly higher uptake in tumors treated with bevacizumab than in controls treated with phosphate buffered saline (PBS). Ex vivo histopathology confirmed the specificity of the BRP peptide to bevacizumab-treated tumor vasculature.
In summary, a novel 12-mer peptide BRP selected usmg phage display techniques allowed noninvasive visualization of early responses to anti-angiogenic treatment. Suitably labeled BRP peptide may be potentially useful pre-clinically and clinically for monitoring treatment response.
Phage display; Angiogenesis; Therapy response; Bevacizumab; Molecular imaging
Enormous efforts have been made toward translating nanotechnology into medical practice, including cancer management. The approaches have generally been classifiable into two categories--those for diagnosis and those for therapy. The targets for diagnostic probes and therapy are often the same, however, and separate approaches to develop diagnostic and therapeutic agents can miss opportunities to improve the efficiency and effectiveness of both. A close and continuous linkage between therapy and diagnosis is also important, because a patient’s diagnosis/prognosis will evolve during treatment.
The unique physical properties of nanomaterials enable them to serve as 1) bases for superior imaging probes to locate and report cancerous lesions, and 2) vehicles to deliver therapeutics preferentially to those lesions. These technologies for probes and vehicles have converged in the current efforts to develop nano-theranostics—that is, nanoplatforms with both imaging and therapeutic functionalities. These latest multimodal platforms are highly versatile and valuable components of the emerging beneficial trend toward personalized medicine, which emphasizes tailoring practices to individual needs so as to optimize outcomes. Unlike conventional methods, imaging and therapeutic functions are seamlessly unified in nano-theranostics, thereby permitting updates to diagnosis/prognosis along with treatment, and enabling opportunities to switch to alternative, possibly more suitable, regimens.
Magnetic nanoparticles, especially superparamagnetic iron oxide nanoparticles (hereafter referred to as IONPs), have long been studied as contrast agents for magnetic resonance imaging (MRI). Owing to recent progress in synthesis and surface modification, many new avenues have opened, though, for this class of biomaterials. The idea is to conceptualize the nanoparticles not as merely tiny magnetic crystals, but rather as platforms with large surface-to-volume ratios. By taking advantage of the well developed surface chemistry of these materials, one can load a wide range of functionalities, such as targeting, imaging and therapeutic features, onto their surfaces. This makes magnetic nanoparticles excellent scaffolds to construct theranostic agents and has attracted many efforts toward this goal.
In this account we will summarize the progress made in our recent studies. We will introduce the surface engineering techniques that we and others have developed, with an emphasis on how the techniques affect the role of nanoparticles as imaging or therapeutic agents.
Derived from endocrine pancreatic beta cells, insulinomas express glucagon-like peptide-1 (GLP-1) receptor with high density and incidence. In this study, we labeled a novel GLP-1 analog, EM3106B, with 18F and performed PET imaging to visualize insulinoma tumors in an animal model. A GLP-1 analog that contains multiple lactam bridges, EM3106B, was labeled with 18F through a maleimide-based prosthetic group, N-2-(4-18F-fluorobenzamido) ethylmaleimide (18F-FBEM). The newly developed radiotracer was characterized by cell based receptor-binding assay, cell uptake and efflux assay. The stability in serum was evaluated by radio-HPLC analysis. In vivo PET imaging was performed in nude mice bearing subcutaneous INS-1 insulinoma tumors and MDA-MB-435 tumors of melanoma origin. Ex vivo biodistribution study was performed to confirm the PET imaging data. EM3106B showed high binding affinity (IC50 = 1.38 nM) and high cell uptake (5.25 ± 0.61% after 120 min incubation). 18F-FBEM conjugation of EM3106B resulted in high labeling yield (24.9 ± 2.4%) and high specific activity (>75 GBq/ μmol at the end of bombardment). EM3106B specifically bound and was internalized by GLP-1R positive INS-1 cells. After intravenous injection of 3.7 MBq (100 μCi) of 18F-FBEM-EM3106B, the INS-1 tumors were clearly visible with high contrast in relation to the contralateral background on PET images, and tumor uptake of 18F-FBEM-EM3106B was determined to be 28.5 ± 4.7 and 25.4 ± 4.1 %ID/g at 60 and 120 min, respectively. 18F-FBEM-EM3106B showed low uptake in MB-MDA-435 tumors with low level of GLP-1R expression. Direct tissue sampling biodistribution experiment confirmed high tracer uptake in INS-1 tumors and receptor specificity in both INS-1 tumor and pancreas. In conclusion, 18F-FBEM-EM3106B exhibited GLP-1R-receptor-specific targeting properties in insulinomas. The favorable characteristics of 18F-FBEM-EM3106B, such as high specific activity and high tumor uptake, and high tumor to non-target uptake, demonstrate that it is a promising tracer for clinical insulinoma imaging.
Insulinoma; glucagon-like peptide-1 receptor; bicyclic GLP-1 analog; 18F-FBEM; PET
An ongoing effort in the field of nanomedicine is to develop nanoplatforms with both imaging and therapeutic functions, the “nano-theranostics”. We have previously developed a human serum albumin (HSA) coated iron oxide nanoparticle (HINP) formula and used multiple imaging modalities to validate its tumor targeting attributes. In the current study, we sought to impart doxorubicin (Dox) onto the HINPs and to assess the potential of the conjugates as theranostic agents. In a typical preparation, we found that about 0.5 mg of Dox and 1 mg of iron oxide nanoparticles (IONPs, Fe content) could be loaded into 10 mg of HSA matrices. The resulting D-HINPs (Dox loaded HINPs) have a hydrodynamic size of 50 nm and are able to release Dox in a sustained fashion. More impressively, the HINPs can assist the translocation of Dox across cell membrane and even its accumulation in the nucleus. In vivo, D-HINPs retained a tumor targeting capability of HINPs, as manifested by both in vivo MRI and ex vivo immunostaining results. In a follow-up therapeutic study on a 4T1 murine breast cancer xenograft model, D-HINPs showed a striking tumor suppression effect that was comparable to Doxil and greatly outperformed free Dox. Such a strategy can be readily extended to load other types of small molecules, making HINP a promising theranostic nanoplatform.
Iron oxide nanoparticle; theranostic nanomedicine; magnetic resonance imaging; doxorubicin; drug delivery; breast cancer
Radiotracers play an important role in interrogating molecular processes both in vitro and in vivo. However, current methods are limited to measuring average radiotracer uptake in large cell populations and, as a result, lack the ability to quantify cell-to-cell variations. Here we apply a new technique, termed radioluminescence microscopy, to visualize radiotracer uptake in single living cells, in a standard fluorescence microscopy environment. In this technique, live cells are cultured sparsely on a thin scintillator plate and incubated with a radiotracer. Light produced following beta decay is measured using a highly sensitive microscope. Radioluminescence microscopy revealed strong heterogeneity in the uptake of [18F]fluoro-deoxyglucose (FDG) in single cells, which was found consistent with fluorescence imaging of a glucose analog. We also verified that dynamic uptake of FDG in single cells followed the standard two-tissue compartmental model. Last, we transfected cells with a fusion PET/fluorescence reporter gene and found that uptake of FHBG (a PET radiotracer for transgene expression) coincided with expression of the fluorescent protein. Together, these results indicate that radioluminescence microscopy can visualize radiotracer uptake with single-cell resolution, which may find a use in the precise characterization of radiotracers.
Tumor endothelial marker 8 (TEM8) has been reported to be upregulated in both tumor cells and tumor-associated endothelial cells in several cancer types. TEM8 antagonists and TEM8-targeted delivery of toxins have been developed as effective cancer therapeutics. The ability to image TEM8 expression would be of use in evaluating TEM8-targeted cancer therapy.
A 13-meric peptide, KYNDRLPLYISNP (QQM), identified from the small loop in domain IV of protective antigen of anthrax toxin was evaluated for TEM8 binding and labeled with 18F for small-animal PET imaging in both UM-SCC1 head-and-neck cancer and MDA-MB-435 melanoma models.
A modified ELISA showed that QQM peptide bound specifically to the extracellular vWA domain of TEM8 with an IC50 value of 304 nM. Coupling 4-nitrophenyl 2-18F-fluoropropionate with QQM gave almost quantitative yield and a high specific activity (79.2±7.4 TBq/mmol, n=5) of 18F-FP-QQM at the end of synthesis. 18F-FP-QQM showed predominantly renal clearance and had significantly higher accumulation in TEM8 high-expressing UM-SCC1 tumors (2.96±0.84 %ID/g at 1 h after injection) than TEM8 low-expressing MDA-MB-435 tumors (1.38±0.56 %ID/g at 1 h after injection).
QQM peptide bound specifically to the extracellular domain of TEM8. 18F-FP-QQM peptide tracer would be a promising lead compound for measuring TEM8 expression. Further efforts to improve the affinity and specificity of the tracer and to increase its metabolic stability are warranted.
Tumor endothelial marker 8 (TEM8); Small-animal PET; 18F; Peptide
To investigate radiation-induced carotid and cerebral vascular injury and its relationship with radiation-induced temporal lobe necrosis in nasopharyngeal carcinoma (NPC) patients.
Methods and Materials
Fifty eight NPC patients with radiation-induced temporal lobe necrosis (TLN) were recruited in the study. Duplex ultrasonography was used to scan bilateral carotid arterials to evaluate the intima-media thickness (IMT) and occurrence of plaque formation. Flow velocities of bilateral middle cerebral arteries (MCAs), internal carotid arteries (ICAs) and basal artery (BA) were estimated through Transcranial Color Doppler (TCD). The results were compared with data from 33 patients who were free from radiation-induced temporal lobe necrosis after radiotherapy and 29 healthy individuals.
Significant differences in IMT, occurrence of plaques of ICAs and flow velocities of both MCAs and ICAs were found between patients after radiotherapy and healthy individuals (p<0.05). IMT had positive correlation with post radiation interval (p = 0.049). Compared with results from patients without radiation-induced TLN, the mean IMT was significantly thicker in patients with TLN (p<0.001). Plaques were more common in patients with TLN than patients without TLN (p = 0.038). In addition, flow velocities of MCAs and ICAs in patients with TLN were much faster (p<0.001, p<0.001). Among patients with unilateral TLN, flow velocity of MCAs was significantly different between ipsilateral and contralateral sides to the lesion (p = 0.001).
Thickening of IMT, occurrence of plaque formation and hemodynamic abnormality are more common in patients after radiotherapy, especially in those with TLN, compared with healthy individuals.
Malignant brain tumors are characterized by extensive infiltration into the normal brain tissue. Tumor migration is a complicated process which results from the interplay of a number of mechanisms, and the extent to which anatomic structure determined the migration pattern has not been extensively addressed. In the present study, we labeled C6 glioma cells with iron oxide nanoparticles and monitored the fate of the cells in vivo with magnetic resonance imaging (MRI).
C6 glioma cells were labeled with ferumoxide–poly-L-lysine complexes and their migration in the brains of rats tracked by T2-weighted MRI. The same amount of iron-laden cells were implanted into the caudate nucleus (CN) and at the vicinity of anterior commissure (AC), respectively, and MRI was performed during the course of 20-day monitoring period to track tumor growth and migration.
A clear tendency of tumor migration along the white matter fiber tracts was observed in the AC group, which is consistent with the previous reports; by contrast, tumor expanded to but remained confined within the boundary of right hemisphere in the CN group.
We successfully demonstrated the ability of MRI to investigate the impact of anatomical structure on the glioma migration pathway in vivo.
Superparamagnetic iron oxide (SPIO) nanoparticles; Cell trafficking; Glioma; Magnetic resonance imaging (MRI); Tumor invasion and migration
MicroRNAs (miRNAs) are a novel class of small noncoding RNAs that regulate gene expression by targeting mRNAs for either cleavage or translational repression. They have been shown to play important roles in a broad range of biological processes including development, cellular differentiation, proliferation and apoptosis. Conventional detection methods, such as northern blot, real-time PCR or microarray, have been used to assess miRNA expression. However, these techniques require the fixation or lysis of cells, and thus cannot be used to study the dynamic function of miRNAs in living cells. Recent remarkable advances in molecular imaging techniques have provided the capability of noninvasive repeated quantitative imaging of tumour or stem cells in living animals. The current brief discussion focuses on the reporter and fluorescent beacon imaging approaches to visualize miRNA expression in living subjects.
MicroRNA; Molecular imaging; Reporter gene