Kristen E. Adams

Comparison of visible and near-infrared wavelength-excitable fluorescent dyes for molecular imaging of cancer

Targeted fluorescent molecular imaging probes may provide an optimal means of detecting disease. Stable, organic fluorophores can be repeatedly excited in vivo by propagated light and consequentially can provide large signal-to-noise ratios (SNRs) for image detection of target tissues. In the literature, many small animal imaging studies are performed with a red excitable dye, Cy5.5, conjugated to the targeting component. We report the comparison of the in vivo fluorescent imaging performance of a near-IR (NIR) and a red-excitable dye. Epidermal growth factor (EGF) was conjugated with Cy5.5 [excitation/emission (ex/em), 660710 nm] or IRDye 800CW (ex/em: 785830 nm) for imaging EGF receptor (EGFr) positive (MDA-MB-468) and/or negative (MDA-MB-435) human breast cancer cell lines in subcutaneous xenograft models. The conjugates were injected intravenously at 1-nmol-dye equivalent with and without anti-EGFr monoclonal antibody C225, preadministered 24 h prior as a competitive ligand to EGFr. Our images show that while both agents target EGFr, the EGF-IRDye 800CW evidenced a significantly reduced background and enhanced the tumor-to-background ratio (TBR) compared to the EGF-Cy5.5. Immunohistochemistry shows that EGF causes activation of the EGFr signaling pathway, suggesting that prior to use as a targeting, diagnostic agent, potential deleterious effects should be considered.

Near-Infrared Fluorescence Imaging in Humans with Indocyanine Green: A Review and Update.

Near-infrared (NIR) fluorescence imaging clinical studies have been reported in the literature with six different devices that employ various doses of indocyanine green (ICG) as a non-specific contrast agent. To date, clinical applications range from (i) angiography, intraoperative assessment of vessel patency, and tumor/metastasis delineation following intravenous administration of ICG, and (ii) imaging lymphatic architecture and function following subcutaneous and intradermal ICG administration. In the latter case, NIR fluorescence imaging may enable new discoveries associated with lymphatic function due to (i) a unique niche that is not met by any other conventional imaging technology and (ii) its exquisite sensitivity enabling high spatial and temporal resolution. Herein, we (i) review the basics of clinical NIR fluorescence imaging, (ii) survey the literature on clinical application of investigational devices using ICG fluorescent contrast, (iii) provide an update of non-invasive dynamic lymphatic imaging conducted with our FDPM device, and finally, (iv) comment on the future NIR fluorescence imaging for non-invasive and intraoperative use given recent demonstrations showing capabilities for imaging following microdose administration of contrast agent.

New Horizons for Imaging Lymphatic Function

In this review, we provide a comprehensive summary of noninvasive imaging modalities used clinically for the diagnosis of lymphatic diseases, new imaging agents for assessing lymphatic architecture and cancer status of lymph nodes, and emerging near‐infrared (NIR) fluorescent optical imaging technologies and agents for functional lymphatic imaging. Given the promise of NIR optical imaging, we provide example results of functional lymphatic imaging in mice, swine, and humans, showing the ability of this technology to quantify lymph velocity and frequencies of propulsion resulting from the contractility of lymphatic structures.

Metabolically biotinylated adenovirus for cell targeting, ligand screening, and vector purification

Development of cell-targeting vectors is an important focus for gene therapy. While some ligands can be genetically inserted into virus capsid proteins for cell targeting, for many ligands, this approach can disrupt either ligand function or vector function. To address this problem for adenovirus type 5 vectors, the fiber capsid protein was genetically fused to a biotin acceptor peptide (BAP). Adenovirus particles bearing this BAP were metabolically biotinylated during vector production by the endogenous biotin ligase in 293 cells to produce covalently biotinylated virions. The resulting biotinylated vector could be retargeted to new receptors by conjugation to biotinylated antibodies using tetrameric avidin (K(d) = 10(-15) M). The biotinylated vector could also be purified by biotin-reversible binding on monomeric avidin (K(d) = 10(-7) M). Finally, this vector was used as a ligand screening platform for dendritic cells in which a variety of structurally diverse protein, carbohydrate, and nucleic acid ligands were easily added to the vector using the biotin-avidin interaction. This work demonstrates the utility of metabolically biotinylated viruses for ligand screening, vector targeting, and virus purification applications.

Assessment of Lymphatic Contractile Function After Manual Lymphatic Drainage Using Near-Infrared Fluorescence Imaging

OBJECTIVE To investigate the feasibility of assessing the efficacy of manual lymphatic drainage (MLD), a method for lymphedema (LE) management, by using near-infrared (NIR) fluorescence imaging. DESIGN Exploratory pilot study. SETTING Primary care unit. PARTICIPANTS Subjects (N=10; age, 18-68y) with a diagnosis of grade I or II LE and 12 healthy control subjects (age, 22-59y). INTERVENTION Indocyanine green (25 μg in 0.1 mL each) was injected intradermally in bilateral arms or legs of subjects. Diffused excitation light illuminated the limbs, and NIR fluorescence images were collected by using custom-built imaging systems. Subjects received MLD therapy, and imaging was performed pre- and posttherapy. MAIN OUTCOME MEASURES Apparent lymph velocities and periods between lymphatic propulsion events were computed from fluorescence images. Data collected pre- and post-MLD were compared and evaluated for differences. RESULTS By comparing pre-MLD lymphatic contractile function against post-MLD lymphatic function, results showed that average apparent lymph velocity increased in both the symptomatic (+23%) and asymptomatic (+25%) limbs of subjects with LE and control limbs (+28%) of healthy subjects. The average lymphatic propulsion period decreased in symptomatic (-9%) and asymptomatic (-20%) limbs of subjects with LE, as well as in control limbs (-23%). CONCLUSIONS We showed that NIR fluorescence imaging could be used to quantify immediate improvement of lymphatic contractile function after MLD.

Direct evidence of lymphatic function improvement after advanced pneumatic compression device treatment of lymphedema

Lymphedema affects up to 50% of all breast cancer survivors. Management with pneumatic compression devices (PCDs) is controversial, owing to the lack of methods to directly assess benefit. This pilot study employed an investigational, near-infrared (NIR) fluorescence imaging technique to evaluate lymphatic response to PCD therapy in normal control and breast cancer-related lymphedema (BCRL) subjects. Lymphatic propulsion rate, apparent lymph velocity, and lymphatic vessel recruitment were measured before, during, and after advanced PCD therapy. Lymphatic function improved in all control subjects and all asymptomatic arms of BCRL subjects. Lymphatic function improved in 4 of 6 BCRL affected arms, improvement defined as proximal movement of dye after therapy. NIR fluorescence lymphatic imaging may be useful to directly evaluate lymphatic response to therapy. These results suggest that PCDs can stimulate lymphatic function and may be an effective method to manage BCRL, warranting future clinical trials.

Biotinylated gene therapy vectors

The avidin–biotin system is a fundamental technology in biomedicine for immunolocalisation, imaging, nucleic acid blotting and protein labelling. This technology has recently been adapted for use in gene therapy vector applications to add proteins or cell-targeting ligands to non-viral and viral vectors. Two biotinylation technologies are being used in these applications: chemical biotinylation and metabolic biotinylation. In chemical biotinylation, reactive alkylating agents couple biotin to proteins by random covalent attachment to amino acid side chains. In metabolic biotinylation, proteins are genetically engineered with a biotin acceptor peptide (BAP), such that they are covalently biotinylated by cellular biotin ligases during viral vector production. Both technologies show promise for cell-targeting in vitro and in vivo, and for ligand screening applications. Metabolic biotinylation has the added feature of allowing viruses, vectors and vaccines to be produced from cells already biotinylated, thereby allowing them to purified by affinity chromatography on monomeric avidin columns.

Real-time dynamic imaging of virus distribution in vivo.

The distribution of viruses and gene therapy vectors is difficult to assess in a living organism. For instance, trafficking in murine models can usually only be assessed after sacrificing the animal for tissue sectioning or extraction. These assays are laborious requiring whole animal sectioning to ascertain tissue localization. They also obviate the ability to perform longitudinal or kinetic studies in one animal. To track viruses after systemic infection, we have labeled adenoviruses with a near-infrared (NIR) fluorophore and imaged these after intravenous injection in mice. Imaging was able to track and quantitate virus particles entering the jugular vein simultaneous with injection, appearing in the heart within 500 milliseconds, distributing in the bloodstream and throughout the animal within 7 seconds, and that the bulk of virus distribution was essentially complete within 3 minutes. These data provide the first in vivo real-time tracking of the rapid initial events of systemic virus infection.

Validation of ALFIA: A platform for quantifying near-infrared fluorescent images of lymphatic propulsion in humans

Recently, we demonstrated near-infrared (NIR) fluorescence imaging for quantifying real-time lymphatic propulsion in humans following intradermal injections of microdose amounts of indocyanine green. However computational methods for image analysis are underdeveloped, hindering the translation and clinical adaptation of NIR fluorescent lymphatic imaging. In our initial work we used ImageJ and custom MatLab programs to manually identify lymphatic vessels and individual propulsion events using the temporal transit of the fluorescent dye. In addition, we extracted the apparent velocities of contractile propagation and time periods between propulsion events. Extensive time and effort were required to analyze the 6-8 gigabytes of NIR fluorescent images obtained for each subject. To alleviate this bottleneck, we commenced development of ALFIA, an integrated software platform which will permit automated, near real-time analysis of lymphatic function using NIR fluorescent imaging. However, prior to automation, the base algorithms calculating the apparent velocity and period must be validated to verify that they produce results consistent with the proof-of-concept programs. To do this, both methods were used to analyze NIR fluorescent images of two subjects and the number of propulsive events identified, the average apparent velocities, and the average periods for each subject were compared. Paired Students t-tests indicate that the differences between their average results are not significant. With the base algorithms validated, further development and automation of ALFIA can be realized, significantly reducing the amount of user interaction required, and potentially enabling the near real-time, clinical evaluation of NIR fluorescent lymphatic imaging.

Abstract 5238: Near-infrared fluorescence imaging of human lymphatics in healthy subjects and in cancer survivors

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Near-infrared (NIR) fluorescence imaging was used to non-invasively image the (dys)function of the lymphatics in healthy control subjects and in subjects clinically diagnosed with unilateral lymphedema, a little understood disease characterized by chronic swelling and commonly observed in cancer survivors following therapeutic, complete nodal dissection. As part of an FDA approved feasibility trial and after informed consent, 24 control and 20 lymphedema (both hereditary and acquired) subjects received multiple intradermal injections of 25 µg of indocyanine green (total dose ≤ 400 µg) in bilateral arms or legs. The limbs were illuminated with NIR excitation light and the resulting fluorescent signal was imaged using custom, intensified CCD cameras. Lymphatic architecture and propulsive lymphatic flow from the injection sites to the nodal basin was observed. The apparent lymph velocities and propulsion periods were determined for the control subjects arms and legs as well as for the asymptomatic and symptomatic limbs of the lymphedema subjects. While the lymphatics of the control subjects consisted of well-defined vessels which propelled fluid from the injection sites to the regional nodal basin, diseased lymphatics exhibited distinct architectural malformations, such as (i) extravascular fluorescence in which the ICG diffused away from the injection site or leaked out of the lymphatic vessels, (ii) dense networks of lymphatic capillaries, (iii) tortuous lymphatic vessels, and (iv) lymphatic backflow and/or reflux. Statistical analyses of control subjects found subtle but significant differences in the left and right velocities in both arms (0.84 cm/s and 0.76 cm/s, p=8.1e-5) and legs (0.99 cm/s and 0.87 cm/s, p=0.032) but not in the periods of contralateral limbs. The difference in velocity in control arms (0.79 cm/s) and legs (0.94 cm/s) is small but significant (p=1.22e-7) while the difference in period is not. Significant differences in period exist between control (arms: 48.2 s; legs: 52.2 s) and both asymptomatic (arms: 39.6 s, p=2.68e-6; legs: 65.3 s, p=0.0022) and symptomatic (arms: 33.2 s, p=4.14e-5; legs: 72.1 s, p=0.00014) limbs. Significant differences were also found between the velocities in the control (0.94 cm/s) and both asymptomatic (0.83 cm/s, p=0.0084) and symptomatic (0.78 cm/s, p=0.0036) legs but not in the arms of control and lymphedema subjects. Results demonstrate that non-invasive NIR fluorescence imaging of microgram amounts of fluorophore can detect architectural and functional lymphatic abnormalities. The lack of significant differences between the velocities and periods of the asymptomatic and symptomatic limbs suggests that lymphedema may be a systemic disease regardless of onset etiology. Supported in parts by the American Cancer Society and the Longaberger Foundation (RSG-06-213-01-LR) and the National Institutes of Health (R01 [HL092923][1] and U54 [CA136404][2]). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5238. [1]: /lookup/external-ref?link_type=GEN&access_num=HL092923&atom=%2Fcanres%2F70%2F8_Supplement%2F5238.atom [2]: /lookup/external-ref?link_type=GEN&access_num=CA136404&atom=%2Fcanres%2F70%2F8_Supplement%2F5238.atom