Haibiao Gong

β-Galactosidase activity assay using far-red-shifted fluorescent substrate DDAOG

beta-Galactosidase (beta-gal) is commonly used as a reporter gene in biological research, and a wide variety of substrates have been developed to assay its activity. One substrate, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) beta-d-galactopyranoside (DDAOG), can be cleaved by beta-gal to produce 7-hydroxy-9H(I,3-dichloro-9,9-dimethylacridin-2-one) (DDAO). On excitation, DDAO generates a far-red-shifted fluorescent signal. Using this substrate, we developed a beta-gal activity assay method. The DDAO signal was stable for at least 18h. The signal intensity was linearly related to both the enzyme amount and substrate concentration. An optimized buffer for the beta-gal/DDAOG assay was also formulated. When compared with the colorimetric substrate o-nitrophenyl-beta-d-galactopyranoside (ONPG), the signal-to-background ratio of the DDAOG method was approximately 12-fold higher. The beta-gal/DDAOG assay method was also tested in transiently transfected cells employing both pharmacologically and genetically inducible gene expression systems. The ability to detect signal induction is comparable to a similar assay using luciferase as the signal generating moiety. The beta-gal/DDAOG assay method should provide a fluorescent reporter assay system for the wide variety of beta-gal systems currently in use.

Near-Infrared Fluorescence Imaging of Mammalian Cells and Xenograft Tumors with SNAP-Tag

Fluorescence in the near-infrared (NIR) spectral region is suitable for in vivo imaging due to its reduced background and high penetration capability compared to visible fluorescence. SNAPf is a fast-labeling variant of SNAP-tag that reacts with a fluorescent dye-conjugated benzylguanine (BG) substrate, leading to covalent attachment of the fluorescent dye to the SNAPf. This property makes SNAPf a valuable tool for fluorescence imaging. The NIR fluorescent substrate BG-800, a conjugate between BG and IRDye 800CW, was synthesized and characterized in this study. HEK293, MDA-MB-231 and SK-OV-3 cells stably expressing SNAPf-Beta-2 adrenergic receptor (SNAPf-ADRβ2) fusion protein were created. The ADRβ2 portion of the protein directs the localization of the protein to the cell membrane. The expression of SNAPf-ADRβ2 in the stable cell lines was confirmed by the reaction between BG-800 substrate and cell lysates. Microscopic examination confirmed that SNAPf-ADRβ2 was localized on the cell membrane. The signal intensity of the labeled cells was dependent on the BG-800 concentration. In vivo imaging study showed that BG-800 could be used to visualize xenograph tumors expressing SNAPf-ADRβ2. However, the background signal was relatively high, which may be a reflection of non-specific accumulation of BG-800 in the skin. To address the background issue, quenched substrates that only fluoresce upon reaction with SNAP-tag were synthesized and characterized. Although the fluorescence was successfully quenched, in vivo imaging with the quenched substrate CBG-800-PEG-QC1 failed to visualize the SNAPf-ADRβ2 expressing tumor, possibly due to the reduced reaction rate. Further improvement is needed to apply this system for in vivo imaging.

Alkaline phosphatase assay using a near-infrared fluorescent substrate merocyanine 700 phosphate

Alkaline phosphatase (ALP) is a phosphomonoester hydrolase that is commonly used as a conjugating enzyme in biological research. A wide variety of substrates have been developed to assay its activity. In this study, we developed an ALP assay method utilizing merocyanine 700 (MC700) based substrate MC700 phosphate (MC700p). MC700 is a near-infrared fluorescent merocyanine dye, and has excitation/emission maxima at 686 nm/722 nm in ALP assay buffer. Upon hydrolysis by ALP, MC700p is converted to MC700. The fluorescence of MC700 is dependent on the pH and detergent concentration in the buffer. The fluorescence signal produced by MC700p hydrolysis is linearly related to the ALP amount and substrate concentration. A stop solution containing EDTA could be used to stop the ALP/MC700p reaction. It was also demonstrated that MC700p could substitute pNpp as the ALP substrate in a commercial 17β-Estradiol enzyme immunoassay kit.

A comparative study of affibody, panitumumab, and EGF for near-infrared fluorescence imaging of EGFR- and EGFRvIII-expressing tumors

Aberrant overexpression and/or activation of epidermal growth factor receptor (EGFR) is associated with many types of cancers. EGFR variant III (EGFRvIII) is a common in-frame deletion mutant, which lacks a large part of the extracellular portion (exons 2–7), including components of the ligand-binding domain. Although EGFR has been extensively studied as a molecular imaging target, information about EGFRvIII-targeted molecular imaging is lacking. In this study, the EGFR-specific affibody, therapeutic antibody panitumumab, and ligand EGF were labeled with IRDye 800CW (Ex/Em: 774/789 nm), yielding Aff800, Pan800, and EGF800, respectively. The binding affinities of the labeled agents were compared in cell-based assays using a rat glioma cell line F98 parental (F98-p) lacking EGFR expression, and 2 F98-derived transgenic cell lines expressing EGFR or EGFRvIII (designated as F98-EGFR and F98-vIII, respectively). Results showed that all agents could bind to F98-EGFR, with Pan800 having the highest binding affinity, followed by Aff800 and EGF800. Pan800 and Aff800, but not EGF800, also bound to F98-vIII. In vivo animal imaging demonstrated that compared with F98-p tumors, F98-EGFR tumors generated higher signals with all three agents. However, in the case of F98-vIII, only Pan800 and Aff800 signals were higher. Analysis of tissue lysates showed that a large portion of Pan800 was degraded into small fragments in F98-EGFR and F98-vIII tumors, possibly due to proteolytic digestion after its specific binding and internalization. In conclusion, Pan800 and Aff800 could be used as imaging agents for both wild-type EGFR and EGFRvIII, whereas EGF800 only targets wild-type EGFR.

A homogeneous fluorescence-based method to measure antibody internalization in tumor cells

We have developed a simple fluorescence-based method to monitor antibody internalization. Panitumumab was dual-labeled with the fluorophore IRDye 800CW and quencher IRDye QC-1 to yield the biomolecular probe Pan800QC. The fluorescence of IRDye 800CW is quenched by IRDye QC-1 on the same intact antibody. After incubation with epidermal growth factor receptor (EGFR)-expressing cells, internalization of Pan800QC was detected by an increase in fluorescence signal due to enzymatic digestion of the antibody and separation of IRDye 800CW and IRDye QC-1. By optimizing reaction conditions, a signal-to-background ratio of 8.5 was obtained. This homogeneous assay can be applied in the characterization and screening of internalizing antibodies.

Development of a near-infrared fluorescence ELISA method using tyramide signal amplification

In this study, we applied tyramide signal amplification (TSA) to fluorescence enzyme-linked immunosorbent assay (ELISA) employing horseradish peroxidase (HRP) as the detection enzyme. When used with a human epidermal growth factor ELISA kit, the TSA method led to a >100-fold increase in fluorescence signal intensity in comparison to an unamplified method. It also showed wider dynamic range and better sensitivity compared to a conventional method using tetramethylbenzidine as the HRP substrate.

Targeting EGFR and HER2 for Molecular Imaging of Cancer

The epidermal growth factor (EGF) receptor (EGFR, HER1, ErbB1) and human epidermal growth factor receptor type 2 (HER2, ErbB2) belong to the ErbB family of type I tyrosine kinases (TKs). This family of receptor TKs also includes another two closely related members HER3/ErbB3 and HER4/ErbB4. The general structure of these cell surface receptor proteins contains an extracellular ligand-binding domain, a hydrophobic transmembrane domain, an intracellular tyrosine kinase domain and a non-catalytic carboxyl terminal tail (Ferguson et al., 2000; Kari et al., 2003; Mitsudomi & Yatabe, 2010; Pines et al., 2010). Various ligands for EGFR, HER3 and HER4 have been identified, with EGF the most extensively characterized for its binding and activation of EGFR. These ErbB proteins are present in the plasma membrane as monomers. Upon ligand binding, the ErbB receptors can associate with each other to form different receptor dimers, which may be homodimers (e.g., EGFR-EGFR) or heterodimers (e.g., EGFR-HER2). The dimerization results in the activation of kinase activity and downstream signaling pathways, which often leads to cell proliferation and malignant tumor growth. HER2 is an exception, with no compatible ligand identified. However, it is the preferred heterodimerization partner for all other ErbB members (Mishani & Hagooly, 2009; Niu et al., 2008; Tzahar et al., 1996; Yarden & Sliwkowski, 2001).

Abstract 3922: A comparative study of affibody, antibody and EGF for near-infrared fluorescence imaging of EGFR- and EGFRvIII-expressing tumors.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Aberrant overexpression and/or activation of epidermal growth factor receptor (EGFR) is associated with many types of cancers. EGFR variant III (EGFRvIII) is a common in-frame deletion mutant, which lacks a large part of the extracellular portion (exons 2-7), including components of the ligand-binding domain. Although EGFR has been extensively studied as a molecular imaging target, information about EGFRvIII-targeted molecular imaging is lacking. In this study, an EGFR-specific affibody, a therapeutic antibody (panitumumab) and the ligand EGF were labeled with IRDye 800CW (Ex/Em: 774/789 nm), and named as Aff800, Pan800 and EGF800 respectively. The binding affinities of the labeled agents were compared in cell-based assays using a rat glioma cell line F98 parental (F98-p) lacking EGFR, and two F98-derived transgenic cell lines expressing EGFR or EGFRvIII (designated as F98-EGFR and F98-vIII respectively). Results showed that all agents could bind to F98-EGFR, with Pan800 having the highest binding affinity, followed by Aff800 and EGF800. Pan800 and Aff800, but not EGF800, bound to F98-vIII with similar affinities. In vivo animal imaging studies demonstrated that compared to F98-p tumor signals, F98-EGFR tumor generated higher signals for all three agents. However, in the case of F98-vIII, only Pan800 and Aff800 signals were higher. Analysis of tissue lysates showed that a large portion of Pan800 was degraded into small fragments in F98-EGFR and F98-vIII tumors, possibly due to proteolytic digestion after its specific binding and internalization. In conclusion, Pan800 and Aff800 could be used as imaging agents for both wild type EGFR and EGFRvIII, whereas EGF800 only targets wild type EGFR. Citation Format: Haibiao Gong, Joy L. Kovar, Lael Cheung, Eben L. Rosenthal, David M. Olive. A comparative study of affibody, antibody and EGF for near-infrared fluorescence imaging of EGFR- and EGFRvIII-expressing tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3922. doi:10.1158/1538-7445.AM2013-3922

Abstract 5295: Near-infrared fluorescence in vivo tumor imaging using SNAP-tag technology

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The SNAP-tag is a small protein derived from the human DNA repair protein O6-alkylguanine-DNA-alkyltransferase (hAGT). It reacts with fluorescent dye conjugated benzylguanine (BG) substrate, leading to covalent labeling of the fluorescent dye on the SNAP-tag®. This property makes the SNAP-tag a valuable tool for fluorescence imaging. In this study, we extended the SNAP-tag technology to near-infrared (NIR) fluorescence imaging using IRDye® 800CW conjugated BG substrate (BG-800). Compared to the visible spectrum, fluorescence in the NIR spectrum has the advantage of low light absorption and reduced autofluorescence, which is highly desirable for in vivo imaging. We created HEK293, MDA-MB-231, SK-OV-3 and SK-BR-3 cells stably expressing SNAP-ADRB2 fusion protein. The ADRB2 potion of the protein directs the localization of the protein to the cell membrane. The expression of SNAP-ADRB2 was confirmed by the reaction between BG-800 substrate and the cell lysate. Microscopic examination confirmed that SNAP-ADRB2 was localized on the cell membrane. The signal intensity of the stable cells after reaction with the substrate BG-800 was dependent on the BG-800 concentration. In vivo imaging study showed that BG-800 could visualize xenograft tumors established with SNAP-ADRB2 overexpressing SK-OV-3 cells. These results demonstrated that the application of SNAP-tag technology could be extended to in vivo imaging when combined with NIR fluorescent fluorophores. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5295. doi:10.1158/1538-7445.AM2011-5295

Abstract 4952: Study of mouse tumor models with an IRDye 800CW SNAP-tag imaging probe

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Non-invasive optical analysis of molecular targets inside living animals has become an important tool for disease progression and treatment assessment. One of the essential elements of molecular imaging is the development of specific, sensitive imaging contrast agents to investigate these biological processes. The use of longer wavelength dyes, i.e. near-infrared (NIR) dyes, presents distinct advantages over inherently fluorescent protein tags, including reduced autofluorescence background and increased sensitivity in imaging small animals. In addition, the adoption of efficient and specific labeling techniques is a key step in the generation of protein-based fluorescent imaging agents. In the present study, we explore the versatility of a self-labeling protein termed SNAP-tag, derived from human O6-alkylguanine-DNA alkyltransferase, in tagging polypeptides with a near-infrared dye, IRDye® 800CW. In cell-based assays, an IRDye 800CW conjugated SNAP-tag successfully labeled cells transiently transfected with a pSNAP-ADRB2 plasmid. For in vivo experiments, a synthetic epidermal growth factor receptor (EGFR)-binding ligand was used to construct an IRDye 800CW-EGFR-SNAP-tag. Following injection of the agent into nude mice bearing EGFR-overexpressing A431 xenografts, tumors were clearly visualized with a Pearl® Imaging system. Furthermore, the specificity of binding was demonstrated in competition experiments with unlabeled EGF. Similar probes labeled with visible fluorophores were used to image EGFR-expressing cultured cells by confocal microscopy. This fluorescent imaging system using a self-labeling protein tag and highly sensitive organic dyes provides versatile tools for cancer research, drug discovery and small animal imaging. 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 4952.