In vivo magnetomotive optical molecular imaging using targeted magnetic nanoprobes

Dynamic magnetomotion of magnetic nanoparticles (MNPs) detected with magnetomotive optical coherence tomography (MM-OCT) represents a new methodology for contrast enhancement and therapeutic interventions in molecular imaging. In this study, we demonstrate in vivo imaging of dynamic functionalized iron oxide MNPs using MM-OCT in a preclinical mammary tumor model. Using targeted MNPs, in vivo MM-OCT images exhibit strong magnetomotive signals in mammary tumor, and no significant signals were measured from tumors of rats injected with nontargeted MNPs or saline. The results of in vivo MM-OCT are validated by MRI, ex vivo MM-OCT, Prussian blue staining of histological sections, and immunohistochemical analysis of excised tumors and internal organs. The MNPs are antibody functionalized to target the human epidermal growth factor receptor 2 (HER2 neu) protein. Fc-directed conjugation of the antibody to the MNPs aids in reducing uptake by macrophages in the reticulo-endothelial system, thereby increasing the circulation time in the blood. These engineered magnetic nanoprobes have multifunctional capabilities enabling them to be used as dynamic contrast agents in MM-OCT and MRI.     

In vivo imaging of molecular targets and their function in endocrinology

Imaging is one of the fastest growing fields of study. New technologies and multimodal approaches are increasing the application of imaging to determine molecular targets and functional processes in vivo. The identification of a specific target, transporter, or biological process using imaging has introduced major breakthroughs to the field of endocrinology primarily utilizing computed tomography, magnetic resonance imaging, ultrasonography, positron emission tomography, single-photon emission computed tomography, and optical imaging. This review provides a general background to the specific developments in imaging that pertains to in vivo function and target identification in endocrine-based diseases.     

In vivo phosphorescence imaging of pO2 using planar oxygen sensors

OBJECTIVE: Oxygen-dependent quenching of luminescence of metal porphyrin complexes has been used to image the pO(2) distribution over tumor and normal tissue. METHODS: An experimental setup is described using a platinum(II)-octaethyl-porphyrin immobilized in a polystyrene matrix as transparent planar sensor. RESULTS: Sensitivity over a broad range is high at low pO(2) values (+/- 0.2 mm Hg at 0 mm Hg; +/- 1.5 mm Hg at 160 mm Hg pO(2)). Due to intrinsically referencing via lifetime encoding there was no modification of the sensor response in vivo in the dorsal skinfold chamber model with amelanotic melanoma (A-MEL-3) in awake hamsters when compared to the in vitro calibration. pO(2) measurements over normal tissue (25.8 +/- 5.1 mm Hg) and tumor tissue (9.2 +/- 5.1 mm Hg) were in excellent agreement with previous results obtained in this model using a surface multiwire electrode. CONCLUSIONS: Using the presented method the surface pO(2) distribution can be mapped with a high temporal resolution of approximately 100 ms and a spatial resolution of at least 25 mu m. Moreover, the transparent sensor allows the simultaneous visualization of the underlying microvasculature.     

In vivo measurement of myocardial mass using nuclear magnetic resonance imaging

To examine the accuracy of nuclear magnetic resonance imaging in measuring left ventricular mass, measurements of left ventricular mass made using this technique were compared with left ventricular weight in 10 mongrel dogs. Left ventricular myocardial volume was measured from five short-axis end-diastolic images that spanned the left ventricle. Left ventricular mass was calculated from left ventricular myocardial volume and compared with the left ventricular weight determined after formalin immersion-fixation. Linear regression analysis yielded the following relation in grams: left ventricular mass determined using nuclear magnetic resonance imaging = (0.94) (left ventricular weight) + 9.1 (r = 0.98, SEE = 6.1 g). The small overestimation of left ventricular weight by nuclear magnetic resonance imaging was judged to be secondary to both difficulty with proper border definition and partial volume effects. Hence, this imaging technique can be used to obtain accurate measurements of left ventricular mass in dogs in vivo.     

In vivo magnetic resonance vascular imaging using laser-polarized 3He microbubbles

Laser-polarized gases (³He and ¹²⁹Xe) are currently being used in magnetic resonance imaging as strong signal sources that can be safely introduced into the lung. Recently, researchers have been investigating other tissues using ¹²⁹Xe. These studies use xenon dissolved in a carrier such as lipid vesicles or blood. Since helium is much less soluble than xenon in these materials, ³He has been used exclusively for imaging air spaces. However, considering that the signal of ³He is more than 10 times greater than that of ¹²⁹Xe for presently attainable polarization levels, this work has focused on generating a method to introduce ³He into the vascular system. We addressed the low solubility issue by producing suspensions of ³He microbubbles. Here, we provide the first vascular images obtained with laser-polarized ³He. The potential increase in signal and absence of background should allow this technique to produce high-resolution angiographic images. In addition, quantitative measurements of blood flow velocity and tissue perfusion will be feasible.     

In vivo measurement of atrial natriuretic peptide receptors using nuclear imaging.

We have successfully visualized atrial natriuretic peptide (ANP) receptors in vivo using nuclear imaging. 123I-Labelled ANP, injected in green vervet monkeys, was rapidly bound to ANP receptors in the kidneys and lungs. That the observed uptake was receptor mediated was demonstrated with competition studies using simultaneous injection of unlabelled ANP 99-126. It was possible to distinguish between the ANP receptor subtypes by the use of selective antagonists. Thus coinjection of ANP 102-121-des[Gln, Ser, Gly, Leu, Gly] (C-ANP), an ANP analog that selectively binds to the ANP C-receptor, decreased uptake in the kidneys by 50% but increased relative uptake in the lungs and soft tissues. This method permits for the first time, the dynamic in vivo analysis of ANP receptors and their interaction with endogenous ligand. Differences and changes in local ANP receptor concentrations and occupancy could be detected. Since ANP receptor density and affinity are influenced by various physiological and pathological conditions, clinical and diagnostic applications seem possible.     

In vivo diagnosis of murine pancreatic intraepithelial neoplasia and early-stage pancreatic cancer by molecular imaging

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with poor patient outcome often resulting from late diagnosis in advanced stages. To date methods to diagnose early-stage PDAC are limited and in vivo detection of pancreatic intraepithelial neoplasia (PanIN), a preinvasive precursor of PDAC, is impossible. Using a cathepsin-activatable near-infrared probe in combination with flexible confocal fluorescence lasermicroscopy (CFL) in a genetically defined mouse model of PDAC we were able to detect and grade murine PanIN lesions in real time in vivo. Our diagnostic approach is highly sensitive and specific and proved superior to clinically established fluorescein-enhanced imaging. Translation of this endoscopic technique into the clinic should tremendously improve detection of pancreatic neoplasia, thus reforming management of patients at risk for PDAC.     

In vivo imaging of cellular proliferation in colorectal cancer using positron emission tomography

BACKGROUND: and aims: Positron emission tomography (PET) using (18)F labelled 2-fluoro-2-deoxy-D-glucose ((18)FDG) is an established imaging tool, although the recent development of a biologically stable thymidine analogue [18F] 3'-deoxy-3-fluorothymidine ((18)FLT) has allowed PET to image cellular proliferation by utilising the salvage pathway of DNA synthesis. In this study, we have compared uptake of (18)FLT and (18)FDG with MIB-1 immunohistochemistry to evaluate the role of PET in quantifying in vivo cellular proliferation in colorectal cancer (CRC). PATIENTS AND METHODS: Patients with resectable, primary, or recurrent CRC were prospectively studied. Thirteen lesions from 10 patients (five males, five females), median age 68 years (range 54-87), were evaluated. Patients underwent (18)FDG and (18)FLT PET scanning. Tracer uptake within lesions was quantified using standardised uptake values (SUVs). Histopathological examination and MIB-1 immunohistochemistry were performed on all lesions, and proliferation quantified by calculating a labelling index (% of MIB-1 positively stained nuclei within 1500 tumour cells). RESULTS: Histology confirmed adenocarcinoma in 12 of 13 lesions; the remaining lesion was reactive. All eight extrahepatic lesions were visualised using both (18)FLT and (18)FDG. Three of the five resected liver metastases were also avid for (18)FLT and showed high proliferation, while the remaining two lesions which demonstrated no uptake of (18)FLT had correspondingly very low proliferation. There was a statistically significant positive correlation (r =0.8, p<0.01) between SUVs of the tumours visualised with (18)FLT and the corresponding MIB-1 labelling indices. No such correlation was demonstrated with (18)FDG avid lesions (r =0.4). CONCLUSIONS: (18)FLT PET correlates with cellular proliferation markers in both primary and metastatic CRC. This technique could provide a mechanism for in vivo grading of malignancy and early prediction of response to adjuvant chemotherapy.     

In vivo imaging of the bronchial wall microstructure using fibered confocal fluorescence microscopy

RATIONALE: Fibered confocal fluorescence microscopy (FCFM) is a new technique that produces microscopic imaging of a living tissue through a 1-mm fiberoptic probe that can be introduced into the working channel of the bronchoscope. OBJECTIVES: To analyze the microscopic autofluorescence structure of normal and pathologic bronchial mucosae using FCFM during bronchoscopy. METHODS: Bronchial FCFM and spectral analyses were performed at 488-nm excitation wavelength on two bronchial specimens ex vivo and in 29 individuals at high risk for lung cancer in vivo. Biopsies of in vivo FCFM-imaged areas were performed using autofluorescence bronchoscopy. RESULTS: Ex vivo and in vivo microscopic and spectral analyses showed that the FCFM signal mainly originates from the elastin component of the basement membrane zone. Five distinct reproducible microscopic patterns were recognized in the normal areas from the trachea down to the more distal respiratory bronchi. In areas of the proximal airways not previously biopsied, one of these patterns was found in 30 of 30 normal epithelia, whereas alterations of the autofluorescence microstructure were observed in 19 of 22 metaplastic or dysplastic samples, five of five carcinomas in situ, and two of two invasive lesions. Disorganization of the fibered network could be found on 9 of 27 preinvasive lesions, compatible with early disruptions of the basement membrane zone. FCFM alterations were also observed in a tracheobronchomegaly syndrome and in a sarcoidosis case. CONCLUSIONS: Endoscopic FCFM represents a minimally invasive method to study specific basement membrane alterations associated with premalignant bronchial lesions in vivo. The technique may also be useful to study the bronchial wall remodeling in nonmalignant chronic bronchial diseases.     

In vivo imaging of cerebral "peripheral benzodiazepine binding sites" in patients with hepatic encephalopathy

BACKGROUND AND AIMS: One proposed mechanism whereby hepatic encephalopathy (HE) leads to loss of brain function is dysregulated synthesis of neurosteroids. Mitochondrial synthesis of neurosteroids is regulated by "peripheral benzodiazepine binding sites" (PBBS). Expressed in the brain by activated glial cells, PBBS can be measured in vivo by the specific ligand [11C](R)-PK11195 and positron emission tomography (PET). Recently, it has been suggested that PBBS expressing glial cells may play a role in the general inflammatory responses seen in HE. Therefore, we measured PBBS in vivo in the brains of patients with minimal HE using [11C](R)-PK11195 PET. METHODS: Five patients with minimal HE and biopsy proven cirrhosis of differing aetiology were assessed with a neuropsychometric battery. Regional expression of PBBS in the brain was detected by [11C](R)-PK11195 PET. RESULTS: All patients showed brain regions with increased [11C](R)-PK11195 binding. Significant increases in glial [11C](R)-PK11195 binding were found bilaterally in the pallidum, right putamen, and right dorsolateral prefrontal region. The patient with the most severe cognitive impairment had the highest increases in regional [11C](R)-PK11195 binding. CONCLUSION: HE is associated with increased cerebral binding of [11C](R)-PK11195 in vivo, reflecting increased expression of PBBS by glial cells. This supports earlier experimental evidence in rodent models of liver failure, suggesting that an altered glial cell state, as evidenced by the increase in cerebral PBBS, might be causally related to impaired brain functioning in HE.     

In vivo imaging of graft-versus-host-disease in mice

We have developed a mouse system by which to track the migration and homing of cells in a setting of bone marrow transplantation (BMT)-induced graft-versus-host disease (GVHD) after systemic infusion using enhanced green fluorescence protein (eGFP) transgenic (Tg) cells and a simple application of a fluorescence stereomicroscope outfitted with a color charge-coupled device (CCD) camera. Whole body images of anesthetized mice taken at various time points after cell infusion revealed the early migration of allogeneic cells to peripheral lymphoid organs, with later infiltration of GVHD target organs. Localization of eGFP Tg cells could be seen through the skin of shaved mice, and internal organs were easily discernible. After allogeneic or syngeneic eGFP Tg cell infusion, representative mice were dissected to better visualize deeper internal organs and tissues. Infusion of different cell populations revealed distinct homing patterns, and this method also provided a simple way to identify the critical time points for expansion of the transplanted cells in various organs. This simple application of the fluorescence stereomicroscope will be valuable for GVHD and graft-versus-tumor studies in which visualization of cellular migration, expansion, and cell-cell interactions will be more informative when analyzed by such an intravital method.     

Surgery with molecular fluorescence imaging using activatable cell-penetrating peptides decreases residual cancer and improves survival

The completeness of tumor removal during surgery is dependent on the surgeon’s ability to differentiate tumor from normal tissue using subjective criteria that are not easily quantifiable. A way to objectively assess tumor margins during surgery in patients would be of great value. We have developed a method to visualize tumors during surgery using activatable cell-penetrating peptides (ACPPs), in which the fluorescently labeled, polycationic cell-penetrating peptide (CPP) is coupled via a cleavable linker to a neutralizing peptide. Upon exposure to proteases characteristic of tumor tissue, the linker is cleaved, dissociating the inhibitory peptide and allowing the CPP to bind to and enter tumor cells. In mice, xenografts stably transfected with green fluorescent protein show colocalization with the Cy5-labeled ACPPs. In the same mouse models, Cy5-labeled free ACPPs and ACPPs conjugated to dendrimers (ACPPDs) delineate the margin between tumor and adjacent tissue, resulting in improved precision of tumor resection. Surgery guided by ACPPD resulted in fewer residual cancer cells left in the animal after surgery as measured by Alu PCR. A single injection of ACPPD dually labeled with Cy5 and gadolinium chelates enabled preoperative whole-body tumor detection by MRI, intraoperative guidance by real-time fluorescence, intraoperative histological analysis of margin status by fluorescence, and postoperative MRI tumor quantification. Animals whose tumors were resected with ACPPD guidance had better long-term tumor-free survival and overall survival than animals whose tumors were resected with traditional bright-field illumination only.     

In vivo molecular imaging of HER2 expression in a rat model of Barrett's esophagus adenocarcinoma

Human epidermal growth factor receptor 2 (HER2) is involved in the malignant progression of several human cancers, including esophageal adenocarcinoma (EAC). The purpose of this study was to evaluate HER2 overexpression and to explore the feasibility of confocal laser endomicroscopy for in vivo molecular imaging of HER2 status in an animal model of Barrett's‐related EAC. Rats underwent esophagojejunostomy with gastric preservation. At 30 weeks post‐surgery, the esophagus of 46 rats was studied; endoscopic and histological findings were correlated with HER2 immunofluorescence on excised biopsies and gross specimens. At this age, 23/46 rats developed Barrett's esophagus (BE), and 6/46 had cancer (four EAC and two squamous cell carcinomas). A significant overexpression of HER2 was observed in esophageal adenocarcinoma compared with normal squamous esophagus (9.4‐fold) and BE (6.0‐fold). AKT and its phosphorylated form were also overexpressed in cancer areas. Molecular imaging was performed at 80 weeks post‐surgery in four rats after tail injection of fluorescent‐labeled anti‐HER2 antibody. At this age, 3/4 rats developed advance adenocarcinoma and showed in vivo overexpression of HER2 by molecular confocal laser endomicroscopy with heterogeneous distribution within cancer; no HER2 signal was observed in normal or Barrett's tissues. Therefore, HER2 overexpression is a typical feature of the surgical induced model of EAC that can be easily quantified in vivo using an innovative mini‐invasive approach including confocal endomicroscopy; this approach may avoid limits of histological evaluation of HER2 status on ‘blinded’ biopsies.     

In vivo imaging of pituitary tumours using a radiolabelled dopamine D2 receptor radioligand

OBJECTIVE Knowledge of the dopamine D2 receptor status of pituitary tumours may play a predictive role in differential diagnosis and therapeutic decisions. This study was performed to evaluate the value of pituitary dopamine D2 receptor scintigraphy with (S)-2-hydroxy-3-¹²³I-iodo-6-methoxy-N-[(1-ethyl-2-pyrrolidinyl)methyl]benzamide (¹²³I-IBZM) in the diagnostic evaluation of patients with pituitary tumours. DESIGN AND PATIENTS Scintigraphy using ¹²³I-IBZM was performed in 5 patients with PRL-secreting macroadenomas, 2 patients with PRL-secreting microadenomas, 17 patients with clinically non- functioning pituitary adenomas (NFPAs), 12 patients with GH-secreting adenomas and 1 patient with a TSH-secreting macroadenoma. RESULTS Single-photon emission tomography (SPECT) showed significant uptake of ¹²³I-IBZM in the pituitary region in 3/5 macroprolactinoma patients. These results closely correlated with the response of plasma PRL levels to the dopamine D2 receptor agonist quinagolide. In two scan-positive prolactinoma patients, repeated SPECTs during therapy with quinagolide showed a reduction in the pituitary uptake of ¹²³I-IBZM. Pituitary SPECT was negative in the 2 microprolactinoma patients, who responded to quinagolide administration. In 4/17 patients with NFPA, significant uptake of the radioligand in the pituitary region was observed. In 2/3 scan-positive NFPA patients, who were treated with quinagolide, shrinkage of the pituitary tumours was observed. Treatment with quinagolide resulted in stabilization of tumour growth in the other scan- positive patients. Four out of 17 patients with NFPA and a negative SPECT were treated with quinagolide. Tumour growth was observed in 1 patient, and tumour size did not change in the other 3 patients. The pituitary region of none of the 12 acromegaly patients showed significant uptake of ¹²³I-IBZM. Sensitivity of the GH-secreting adenomas to quinagolide was demonstrated in 8/12 patients in vivo by an acute test, and in 6/9 of the tumours in vitro. Pituitary SPECT was negative in the patient with the TSH-secreting macroadenoma and this tumour also showed no sensitivity to quinagolide in vivo or in vitro. CONCLUSIONS We conclude that ¹²³I-IBZM is a ligand for in vivo imaging of dopamine agonist- sensitive macroprolactinomas, but not for microprolactinomas or GH-secreting adenomas. The technique potentially provides a means of predicting the dopamine agonist-responses of non-functioning pituitary adenomas in vivo.     

In vivo albumin labeling and lymphatic imaging

The ability to accurately and easily locate sentinel lymph nodes (LNs) with noninvasive imaging methods would assist in tumor staging and patient management. For this purpose, we developed a lymphatic imaging agent by mixing fluorine-18 aluminum fluoride-labeled NOTA (1,4,7-triazacyclononane-N,N'',N''''-triacetic acid)-conjugated truncated Evans blue (¹⁸F-AlF-NEB) and Evans blue (EB) dye. After local injection, both ¹⁸F-AlF-NEB and EB form complexes with endogenous albumin in the interstitial fluid and allow for visualizing the lymphatic system. Positron emission tomography (PET) and/or optical imaging of LNs was performed in three different animal models including a hind limb inflammation model, an orthotropic breast cancer model, and a metastatic breast cancer model. In all three models, the LNs can be distinguished clearly by the apparent blue color and strong fluorescence signal from EB as well as a high-intensity PET signal from ¹⁸F-AlF-NEB. The lymphatic vessels between the LNs can also be optically visualized. The easy preparation, excellent PET and optical imaging quality, and biosafety suggest that this combination of ¹⁸F-AlF-NEB and EB has great potential for clinical application to map sentinel LNs and provide intraoperative guidance.The lymphatic system plays a key role in maintaining tissue interstitial pressure by collecting protein-rich fluid that is extracted from capillaries (1). The lymphatic system is also a critical component of the immune system. Many types of malignant tumors such as breast cancer, melanoma, and prostate cancer are prone to metastasize in regional lymph nodes (LNs), possibly through tumor-associated lymphatic channels (2, 3). The status of these sentinel LNs (SLNs) not only provides a marker for tumor staging but also serves as an indicator of prognosis (4). Consequently, detection and mapping of SLNs is a key step in therapeutic decision-making (5).One common method used in the clinic is a two-step procedure that consists of local administration of radionuclide-labeled colloids, mostly with technetium-99m, several hours before the injection of a vital dye such as Patent blue (isosulfan blue). SLNs can be visualized either by gamma scintigraphy or single photon emission computed tomography. The SLNs during surgery can be located with a hand-held gamma ray counter and visual contrast of the blue dye (6–8). However, this method requires separate administration of two agents because of different rates of local migration of the colloidal particles and blue dye molecules (9). Due to the relatively low sensitivity and poor spatial resolution of scintigraphy and single photon emission computed tomography, it is highly desirable to develop new imaging probes for other imaging modalities. The objective is to improve the detection of SLNs for either noninvasive visualization or intrasurgical guidance (10–14).Recently, imaging-guided surgery, especially with fluorescent probes, has been intensively studied due to its low cost, simplicity, and adaptability (15). The limited tissue penetration of light is less critical because of the open field of view during surgery (16). For example, NIR fluorescence dyes, such as indocyanine green, have been investigated for sentinel node navigation during surgery either alone or in combination with nanoformulations (13, 14). Owing to the nanometer-scale size, stability, and strong fluorescence, various nanoparticles and nanoformulations have been applied for SLN imaging and showed promising results in preclinical models (10–12). However, most of these probes are composed of heavy metals, making their clinical translation difficult due to the acute and chronic toxicity (17). In addition, scattering and tissue attenuation cause poor results for presurgical evaluation of SLNs using optical imaging.Evans blue (EB) is an azo dye, which can bind quantitatively to serum albumin, and has been used for nearly a century to determine blood plasma volume and extravascular protein leakage in patients (18). Indeed, EB also showed promise in LN mapping in both clinical practice and preclinical studies (19, 20). In this respect, ^99mTc–EB appears to be better than ^99mTc–antimony trisulfide colloid/Patent Blue V dual injection in discriminating the SLN (21, 22). Recently, we synthesized a NOTA (1,4,7-triazacyclononane-N,N′,N″-triacetic acid)-conjugated truncated EB (NEB). ¹⁸F-labeling was achieved through the formation of the ¹⁸F-aluminum fluoride (¹⁸F-AlF) complex. The whole labeling process takes only around 20–30 min without the need of HPLC purification (23). After i.v. injection, ¹⁸F-AlF-NEB complexed with serum albumin very quickly, and thus, most of the radioactivity was restrained to the blood circulation. ¹⁸F-AlF-NEB has been successfully applied to evaluate cardiac function in a myocardial infarction model and vascular permeability in inflammatory and tumor models (23).Although primarily an intravascular protein, albumin cyclically leaves the circulation through the endothelial barrier at the level of the capillaries. The albumin concentration in the interstitial fluid is around 20–30% of that in the plasma, and back transport of proteins from the interstitial fluid to the plasma is accomplished mainly by the lymphatic system (24). After local administration, NEB forms a complex with albumin in the interstitial fluid and travels through the lymphatic system. In this study, we mixed ¹⁸F-AlF-NEB with EB (¹⁸F-AlF-NEB/EB) to perform multimodal imaging of LNs in different animal models via a single administration. Although PET provides a presurgical evaluation of SLNs, fluorescence signal and visible blue color afford guidance for surgery.