In Vivo Fluorescent Labeling of Tumor Cells with the HaloTag® Technology

Tseng, Jen-Chieh; Benink, Hélène A; McDougall, Mark G.; Chico‐Calero, Isabel; Kung, Andrew L.

doi:10.2174/1875397301206010048

Cited by 11 publications

(11 citation statements)

References 13 publications

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“…The availability of HaloTag ligands with fluorescent dyes ranging from blue to far red and near-infrared permits multiplexed detection of this proteasome reporter in combination with markers for specific cell types or proteins by microscopy and flow cytometry. 54 Labeling with far red or near-infrared dyes will also improve the detection of cancer cells with low proteasome activity in animal models by intravital microscopy and/or whole animal fluorescence imaging because longer wavelengths of light transmit more efficiently through tissues and avoid endogenous autofluorescence. 55 This reporter system can also be coupled with ligands for other imaging modalities, such as positron emission tomography (PET), with improved sensitivity for whole animal studies of tumor-initiating cells during tumor progression and therapy.…”

Section: Discussionmentioning

confidence: 99%

Imaging Reporters for Proteasome Activity Identify Tumor- and Metastasis-Initiating Cells

et al. 2015

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Tumor-initiating cells, also designated as cancer stem cells, are proposed to constitute a subpopulation of malignant cells central to tumorigenesis, metastasis, and treatment resistance. We analyzed activity of the proteasome, the primary organelle for targeted protein degradation, as a marker of tumor- and metastasis-initiating cells. Using human and mouse breast cancer cells expressing a validated fluorescent reporter, we found a small subpopulation of cells with low proteasome activity that divided asymmetrically to produce daughter cells with low or high proteasome activity. Breast cancer cells with low proteasome activity had greater local tumor formation and metastasis in immunocompromised and immunocompetent mice. To allow flexible labeling of cells, we also developed a new proteasome substrate based on HaloTag technology. Patient derived glioblastoma cells with low proteasome activity measured by the HaloTag reporter show key phenotypes associated with tumor-initiating cells, including expression of a stem cell transcription factor, reconstitution of the original starting population, and enhanced neurosphere formation. We also show that patient-derived glioblastoma cells with low proteasome activity have higher frequency of tumor formation in mouse xenografts. These studies support proteasome function as a tool to investigate tumor-and metastasis-initiating cancer cells and potential biomarker for outcomes in patients with several different cancers.

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Section: Discussionmentioning

confidence: 99%

Imaging Reporters for Proteasome Activity Identify Tumor- and Metastasis-Initiating Cells

et al. 2015

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“…For example, Tseng et al employed a HCT116 xenograft model expressing HaloTag protein for whole animal fluorescence imaging. 115 The HaloTag ligand displayed enhanced uptake in the HaloTag-expressing tumor, with minimal uptake in the control tumor. Similarly, tumor nodules from cancer cells highly expressing HaloTag receptors were conjugated with four different fluorophore ligands to evaluate cellular growth at specific time points.…”

Section: In Vivo Molecular Imagingmentioning

confidence: 99%

HaloTag Technology: A Versatile Platform for Biomedical Applications

2015

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Exploration of protein function and interaction is critical for discovering links between genomics, proteomics, and disease state; yet the immense complexity of proteomics found in biological systems currently limit our investigational capacities. While affinity and autofluorescent tags are widely employed for protein analysis, these methods have limited success as they lack specificity and require multiple fusion tags and genetic constructs. As an alternative approach, the innovative HaloTag protein fusion platform allows for comprehensive analysis of protein function and interaction using a single genetic construct with multiple capabilities. This is accomplished using a simplified process, in which a variable HaloTag ligand binds rapidly to the HaloTag protein (usually linked to the protein of interest) with high affinity and specificity. In this review article, we examine all current applications of the HaloTag technology platform for biomedical applications such as the study of protein isolation and purification, protein function, protein-protein and protein-DNA interactions, biological assays, in vitro cellular imaging, and in vivo molecular imaging. In addition, novel uses of the HaloTag platform are briefly discussed with potential future applications.

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“…We chose the HaloTag technology, 7 which is particularly well suited for in vivo labeling. 8 Based on the use of a modified haloalkane dehalogenase protein tag (HaloTag), this strategy allows the covalent and site-specific conjugation of synthetic haloalkane ligands (Halo-ligands) onto the protein of interest (fused with the HaloTag). This irreversible and specific coupling has recently drawn much attention for the purification of proteins with high yield and efficiency, 9 for the grafting of proteins onto various surfaces, 7a,10 and for protein labeling with small-molecule ligands.…”

Section: Introductionmentioning

confidence: 99%

Advanced Fluorescent Polymer Probes for the Site-Specific Labeling of Proteins in Live Cells Using the HaloTag Technology

et al. 2019

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We report the site-specific and covalent bioconjugation of fluorescent polymer chains to proteins in live cells using the HaloTag technology. Polymer chains bearing a Halo-ligand precisely located at their α-chain-end were synthesized in a controlled manner owing to the RAFT polymerization process. They were labeled in lateral position by several organic fluorophores such as AlexaFluor 647. The resulting Halo-ligand polymer probe was finally shown to selectively recognize and label HaloTag proteins present at the membrane of live cells using confocal fluorescence microscopy. Such a polymer bioconjugation approach holds great promises for various applications ranging from cell imaging to cell surface functionalization.

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In Vivo Fluorescent Labeling of Tumor Cells with the HaloTag® Technology

Cited by 11 publications

References 13 publications

Imaging Reporters for Proteasome Activity Identify Tumor- and Metastasis-Initiating Cells

Imaging Reporters for Proteasome Activity Identify Tumor- and Metastasis-Initiating Cells

HaloTag Technology: A Versatile Platform for Biomedical Applications

Advanced Fluorescent Polymer Probes for the Site-Specific Labeling of Proteins in Live Cells Using the HaloTag Technology

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