IRDye 800CW Research Articles

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, ((111)In-DTPA)(n)-trastuzumab-(IRDye 800CW)(m), that is dual labeled with (111)In, a gamma-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. 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 ((111)In-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 ((111)In-DTPA)(n)-trastuzumab-(IRDye800)(m). Nonspecific uptake in the SKBr3-luc tumors was analyzed by injecting the mice with IRDye 800CW and ((111)In-DTPA)(p)-IgG-(IRDye800)(q), where "p" and "q" are the stoichiometric ratios of DTPA and IRDye 800CW bound per IgG antibody, respectively. (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 ((111)In-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 ((111)In-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 ((111)In-DTPA)(n)-trastuzumab-(IRDye800)(m) bound only in tumor tissue and not to muscle. Dual-labeled ((111)In-DTPA)(n)-trastuzumab-(IRDye800)(m) may be an effective diagnostic biomarker capable of tracking HER2 overexpression in breast cancer patients.

Conformation State-sensitive Antibodies to G-protein-coupled Receptors

A growing body of evidence indicates that G-protein-coupled receptors undergo complex conformational changes upon agonist activation. It is likely that the extracellular region, including the N terminus, undergoes activation-dependent conformational changes. We examined this by generating antibodies to regions within the N terminus of micro-opioid receptors. We find that antibodies to the midportion of the N-terminal tail exhibit enhanced recognition of activated receptors, whereas those to the distal regions do not. The enhanced recognition is abolished upon treatment with agents that block G-protein coupling or deglycosylate the receptor. This suggests that the N-terminal region of mu receptors undergoes conformational changes following receptor activation that can be selectively detected by these region-specific antibodies. We used these antibodies to characterize micro receptor type-specific ligands and find that the antibodies accurately differentiate ligands with varying efficacies. Next, we examined if these antibodies can be used to investigate the extent and duration of activation of endogenous receptors. We find that peripheral morphine administration leads to a time-dependent increase in antibody binding in the striatum and prefrontal cortex with a peak at about 30 min, indicating that these antibodies can be used to probe the spatio-temporal dynamics of native mu receptors. Finally, we show that this strategy of targeting the N-terminal region to generate receptor conformation-specific antisera can be applied to other G(alpha)(i)-coupled (delta-opioid, CB1 cannabinoid, alpha(2A)-adrenergic) as well as G(alpha)(s)-(beta(2)-adrenergic) and G(alpha)(q)-coupled (AT1 angiotensin) receptors. Taken together, these studies describe antisera as tools that allow, for the first time, studies probing differential conformation states of G-protein-coupled receptors, which could be used to identify molecules of therapeutic interest.

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.

Hyaluronidase Expression Induces Prostate Tumor Metastasis in an Orthotopic Mouse Model

Molecular mechanisms of prostate cancer progression are frequently studied in mice by orthotopic injection of aggressive cell lines, which yield primary tumors that spontaneously metastasize to lymph nodes. In this report, we characterized the human prostate carcinoma cell line 22Rv1 in an orthotopic system and evaluated the functional relevance of the hyaluronidase Hyal1, a correlate of invasive human prostate cancer, to progression in this model. To provide real-time insights into these processes, we first validated use of an epidermal growth factor-conjugated fluorophore to illuminate orthotopic prostate tumors and their metastases in whole animal imaging. Animals receiving intraprostatic injections were tracked throughout a 6-week period. Tumor sizes were correlated 92% with total fluorescence intensities of 22 prostate tumors. In contrast to the highly tumorigenic and metastatic PC3M-LN4 cells, the 22Rv1 line was orthotopically tumorigenic but not metastatic, despite larger tumor sizes. Lymph node metastasis was successfully imaged in animals with PC3M-LN4 tumors on endpoint dissection. Stable transfection of 22Rv1 cells with Hyal1 did not alter growth kinetics of primary orthotopic tumors, but all animals implanted with Hyal1 transfectants exhibited tumor-positive para-aortic lymph nodes. Hyal1 is implicated as an inducer of prostate cancer metastatic progression.