Eva M. Sevick-Muraca

Imaging of Lymph Flow in Breast Cancer Patients after Microdose Administration of a Near-Infrared Fluorophore: Feasibility Study

PURPOSEnTo prospectively demonstrate the feasibility of using indocyanine green, a near-infrared (NIR) fluorophore at the minimum dose needed for noninvasive optical imaging of lymph nodes (LNs) in breast cancer patients undergoing sentinel lymph node mapping (SLNM).nnnMATERIALS AND METHODSnInformed consent was obtained from 24 women (age range, 30-85 years) who received intradermal subcutaneous injections of 0.31-100 microg indocyanine green in the breast in this IRB-approved, HIPAA-compliant, dose escalation study to find the minimum microdose for imaging. The breast, axilla, and sternum were illuminated with NIR light and the fluorescence generated in the tissue was collected with an NIR-sensitive intensified charged-coupled device. Lymphoscintigraphy was also performed. Resected LNs were evaluated for the presence of radioactivity, blue dye accumulation, and fluorescence. The associations between the resected LNs that were fluorescent and (a) the time elapsed between NIR fluorophore administration and resection and (b) the dosage of NIR fluorophores were tested with the Spearman rank and Pearson product moment correlation tests, respectively.nnnRESULTSnLymph imaging consistently failed with indocyanine green microdosages between 0.31 and 0.77 microg. When indocyanine green dosages were 10 microg or higher, lymph drainage pathways from the injection site to LNs were imaged in eight of nine women; lymph propulsion was observed in seven of those eight. When propulsion in the breast and axilla regions was present, the mean apparent velocities ranged from 0.08 to 0.32 cm/sec, the time elapsed between packets of propelled fluid varied from 14 to 92 seconds. In patients who received 10 microg of indocyanine green or more, a weak negative correlation between the fluorescence status of resected LNs and the time between NIR fluorophore administration and LN resection was found. No statistical association was found between the fluorescence status of resected LNs and the dose of NIR fluorophore.nnnCONCLUSIONnNIR fluorescence imaging of lymph function and LNs is feasible in humans at microdoses that would be needed for future molecular imaging of cancer-positive LNs.

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.

Dual-Labeled Trastuzumab-Based Imaging Agent for the Detection of Human Epidermal Growth Factor Receptor 2 Overexpression in Breast Cancer

Overexpression of the human epidermal growth factor receptor (HER) family has been implicated in cancer because of its participation in signaling pathways regulating cellular proliferation, differentiation, motility, and survival. In this work, we exploited the extracellular binding property of trastuzumab, a clinically therapeutic monoclonal antibody to the second member of the HER family (HER2), to design a diagnostic imaging agent, (111In-DTPA)n-trastuzumab-(IRDye 800CW)m, that is dual labeled with 111In, a γ-emitter, and a near-infrared (NIR) fluorescent dye, IRDye 800CW, to detect HER2 overexpression in breast cancer cells. The stoichiometric ratios “n” and “m” refer to the number of diethylenetriaminepentaacetic acid dianhydride (DTPA) and IRDye 800CW molecules bound per trastuzumab molecule, respectively. Methods: Fluorescence microscopy and confocal microscopy were used to determine the molecular specificity of (DTPA)n-trastuzumab-(IRDye800)m in vitro in SKBr3 (HER2-positive) and MDA-MB-231 (HER2-negative) breast cancer cells. SKBr3 cells were incubated with (DTPA)n-trastuzumab-(IRDye800)m or IRDye800CW or pretreated with trastuzumab or human IgG followed by (DTPA)n-trastuzumab-(IRDye800)m and examined under a fluorescence microscope. For in vivo characterization, athymic nude mice bearing HER2-overexpressing SKBr3-luc subcutaneous xenografts were injected intravenously with (111In-DTPA)n-trastuzumab-(IRDye800)m and imaged with SPECT and NIR fluorescence imaging at 48 h. Tumor-bearing mice were also injected intravenously with trastuzumab 24 h before administration of (111In-DTPA)n-trastuzumab-(IRDye800)m. Nonspecific uptake in the SKBr3-luc tumors was analyzed by injecting the mice with IRDye 800CW and (111In-DTPA)p-IgG-(IRDye800)q, where “p” and “q” are the stoichiometric ratios of DTPA and IRDye 800CW bound per IgG antibody, respectively. Results: (DTPA)n-trastuzumab-(IRDye800)m showed significantly greater binding to SKBr3 cells than to MDA-MB-231 cells. Confocal imaging revealed that this binding occurred predominantly around the cell membrane. Competitive binding studies with excess trastuzumab before incubation with (DTPA)n-trastuzumab-(IRDye800)m abolished this binding affinity, but pretreatment with nonspecific IgG did not alter binding. In vivo nuclear and optical imaging of SKBr3-luc xenografts injected with (111In-DTPA)n-trastuzumab-(IRDye800)m revealed significantly more uptake in the tumor region than in the contralateral muscle region. The tumor-to-muscle ratio decreased in mice pretreated with trastuzumab and in mice injected with IRDye 800CW and (111In-DTPA)p-IgG-(IRDye800)q. Ex vivo imaging of dissected organs confirmed these results. Finally, coregistration of histologic hematoxylin–eosin stains with autoradiography signals from tumor and muscle tissue slices indicated that (111In-DTPA)n-trastuzumab-(IRDye800)m bound only in tumor tissue and not to muscle. Conclusion: Dual-labeled (111In-DTPA)n-trastuzumab-(IRDye800)m may be an effective diagnostic biomarker capable of tracking HER2 overexpression in breast cancer patients.

Single-Dose Intravenous Toxicity Study of IRDye 800CW in Sprague-Dawley Rats

ObjectiveFluorophore-labeled contrast imaging agents are moving toward clinical use for a number of applications. The near-infrared dye IRDye 800CW is frequently used in its N-hydroxysuccinamide (NHS) ester form for labeling these agents. Following conjugation or breakdown of a labeled ligand, excess NHS ester is converted to the carboxylate form. To prepare for clinical use as a near-infrared fluorophore, a toxicity study was conducted on IRDye 800CW carboxylate.MethodsMale and female Sprague–Dawley rats were given a single intravenous or intradermal administration of IRDye 800CW carboxylate; Indocyanine Green was used as a comparative control. Animals were injected with varying doses of the test and control articles and observed for up to 14xa0days. Clinical chemistry, hematological, and pharmacokinetic analyses were performed on subgroups of animals. Organs were analyzed for content of the test article. Tissues were analyzed microscopically for pathological changes.ResultsBased on hematologic, clinical chemistry, and histopathologic evaluation, single administration of IRDye 800CW carboxylate intravenously at dose levels of 1, 5, and 20xa0mg/kg or 20xa0mg/kg intradermally produced no pathological evidence of toxicity.ConclusionA dose of 20xa0mg/kg was identified as the no observed adverse effect level following IV or ID routes of administration of IRDye 800CW.

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.

Molecular imaging with optics: primer and case for near-infrared fluorescence techniques in personalized medicine

We compare and contrast the development of optical molecular imaging techniques with nuclear medicine with a didactic emphasis for initiating readers into the field of molecular imaging. The nuclear imaging techniques of gamma scintigraphy, single-photon emission computed tomography, and positron emission tomography are first briefly reviewed. The molecular optical imaging techniques of bioluminescence and fluorescence using gene reporter/probes and gene reporters are described prior to introducing the governing factors of autofluorescence and excitation light leakage. The use of dual-labeled, near-infrared excitable and radio-labeled agents are described with comparative measurements between planar fluorescence and nuclear molecular imaging. The concept of time-independent and -dependent measurements is described with emphasis on integrating time-dependent measurements made in the frequency domain for 3-D tomography. Finally, we comment on the challenges and progress for translating near-infrared (NIR) molecular imaging agents for personalized medicine.

Characterization and performance of a near-infrared 2-deoxyglucose optical imaging agent for mouse cancer models

Malignant neoplasms exhibit an elevated rate of glycolysis over normal cells. This characteristic can be exploited for optical imaging of tumors in mice. A near-infrared fluorophore, IRDye 800CW, emission maximum 794 nm, was conjugated to 2-deoxyglucose (2-DG). An immunofluorescent cell-based assay was used to evaluate specificity and sensitivity of the conjugate in cultured cell monolayers. Dose-dependent uptake was established with increasing concentrations of IRDye 800CW 2-DG for epithelial and prostate carcinomas. IRDye 800CW 2-DG was specifically blocked by an antibody against GLUT1 glucose transporter, and by excess unlabeled 2-DG or d-glucose. Signal was increased by a phorbol ester activator of glucose transport. Fluorescence microscopy data confirmed localization of the conjugate in the cytoplasm. Subsequent in vivo studies optimized dose, clearance, and timing for signal capture in nude mouse xenografts. In all cases, tumors were clearly imaged with good signal-to-noise characteristics. These data indicate that IRDye 800CW 2-DG is a broadly applicable optical imaging agent for in vivo imaging of neoplasms in mice.

Noninvasive quantitative imaging of lymph function in mice

BACKGROUNDnWhereas functional lymph imaging in rodents is imperative for drug discovery of lymph therapeutics, noninvasive imaging of propulsive lymph function in rodents has not been reported previously. Herein, we present a noninvasive and rapid approach to measure lymphatic function in a rodent model using a near-infrared (NIR) dye to minimize background autofluorescence and maximize tissue penetration.nnnMETHODS AND RESULTSnMice were dynamically imaged following intradermal (i.d.) injection of 2 to 10 microL of 1.3 mM of indocyanine green (IC-Green) into the tail and the limb. Our results demonstrate the ability to image the IC-Green trafficking from the lymph plexus, through lymph vessels and lymphangions, to the ischial nodes in the tail, and to the axillary nodes in the limb. Our results show that lymph flow velocity from the propelled IC-Green packet in the lymph vessels in the tail ranged from 1.3 to 3.9 mm/s and the fluorescence intensity peaks repeated on an average of every 51.3 +/- 17.4 seconds in five animals. While pulsatile lymph flow was detected in the deep lymph vessels, lymph propulsion was not visualized in the superficial lymphatic network in the tail. In axillary lymphatic imaging, propulsive lymph flow was also detected. The intensity profile shows that the lymph flow velocity ranged from 0.28 to 1.35 mm/s at a frequency ranging from 0.72 to 11.1 pulses per minute in five animals.nnnCONCLUSIONSnOur study demonstrates the ability to noninvasively and quantitatively image propulsive lymph flow, which could provide a new method to investigate lymph function and its change in response to potential therapeutics.

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

OBJECTIVEnTo 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.nnnDESIGNnExploratory pilot study.nnnSETTINGnPrimary care unit.nnnPARTICIPANTSnSubjects (N=10; age, 18-68y) with a diagnosis of grade I or II LE and 12 healthy control subjects (age, 22-59y).nnnINTERVENTIONnIndocyanine 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.nnnMAIN OUTCOME MEASURESnApparent 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.nnnRESULTSnBy 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%).nnnCONCLUSIONSnWe showed that NIR fluorescence imaging could be used to quantify immediate improvement of lymphatic contractile function after MLD.

Virus-like particle (VLP) lymphatic trafficking and immune response generation after immunization by different routes

Virus-like particles (VLPs) have gained increasing interest for their use as vaccines due to their repetitive antigenic structure that is capable of efficiently activating the immune system. The efficacy of VLP immunization may lie in its ability to traffic into draining lymph nodes while activating antigen-presenting cells to initiate the orchestration of signals required for the development of a robust immune response. Currently, there is no comprehensive study showing the correlation of different VLP vaccination routes to immune outcome. In this study, we took an optical imaging approach to directly visualize the trafficking of simian-human immunodeficiency (SHIV) VLPs after immunization by commonly used routes and analyzed the corresponding humoral and cellular immune responses generated. We found that VLPs can easily enter the subcapsular sinus of draining lymph nodes with quantitative differences in the number of lymph node involvement depending on the immunization route used. Intradermal immunization led to the largest level of lymph node involvement for the longest period of time, which correlated with the strongest humoral and cellular immune responses. Flow cytometry analysis from extracted splenocytes showed that intradermal immunization led to the largest population of germinal center and activated B cells, which translated into higher antibody levels and antigen-specific cytotoxic T lymphocyte responses. Our results indicate that VLPs traffic into lymph nodes upon immunization and can be directly visualized by optical imaging techniques. Intradermal immunization showed improved responses and might be a preferable delivery route to use for viral and cancer immunotherapeutic studies involving VLPs.