Development of novel chimeric transmembrane proteins for multimodality imaging of cancer cells

Abstract

Tracking the migration of cancer cells is essential to understanding the metastatic process. In order to facilitate the tracking of metastatic progression, we have generated transgenic cancer cell lines that express novel chimeric proteins composed of truncated human type II transmembrane proteins fused in-frame to a red fluorescence protein. These chimeric proteins have been engineered to display the fluorescence domain on the surface of cells. The three novel chimeric proteins exhibited high transient expression in several cancer cell lines. Membrane expression was well characterized in MCF-7 cell lines that stably express the chimeric proteins. Indirect immunocytochemistry of non-premeablized cells demonstrated co-localization of endogenous red and green fluorescence labeled secondary antibody bound on the cell surfaces. Immunoblots of total protein prepared from membrane, cytosolic and nuclear fractions indicated that the chimeric proteins were mainly associated with the membrane fraction. Further evidence of the membrane expression of these proteins was confirmed by confocal microscopy. Moreover, the chimeric protein was detected on the cell surface by T2-weighted magnetic resonance imaging using anti-red fluorescence protein antibody and superparamagnetic iron oxide particles in vitro and by optical imaging in vivo. The development of this non-mammalian cell surface marker, that can be detected by multi-modality imaging, will find utility in non-invasive longitudinal tracking of biological processes including metastatic progression, solid tumor treatment regimes and the fate of cells used in cell therapies such as islet or stem cells.

Multimodality Imaging Probes: Design and Challenges

“…Recent examples include transduction of MCF-7 breast cancer cells to express a red fluorescent protein–human type II transmembrane protein chimera that localized to the plasma membrane of the cells. Superparamagnetic iron oxide nanoparticles (SPIO) were then bound to the cells by associating biotinylated anti-RFP antibodies to the cells and then allowing these to couple to avidin-modified SPIO 311. In another example, human endothelial progenitor cells (HEPCs) (from umbilical cord) were transfected with vectors carrying human sodium iodide symporter (NIS) and were found to stably express the NIS protein on their surfaces.…”

The in Vivo TRPV6 Protein Starts at a Non-AUG Triplet, Decoded as Methionine, Upstream of Canonical Initiation at AUG

“…Confocal Microscopy-For confocal microscopy HEK293 cells were co-transfected with the TRPV6-S or the TRPV6-XL cDNAs (with or without the pore mutation, D542A) fused to the eGFP cDNA and the pCherryPicker cDNA (Clontech), a vector that constitutively expresses the cDNA of the red fluorescent protein mCherry (14) fused to the cDNA of the transferrin receptor membrane-anchor domain (15); the fusion protein was used as a plasma membrane marker. The cDNA of tmem 16a cloned from human placenta and fused to the GFP cDNA was expressed as independent control.…”

Antibody-Free Magnetic Cell Sorting of Genetically Modified Primary Human CD4+ T Cells by One-Step Streptavidin Affinity Purification

“…The truncated LNGFR, which lacks a cytoplasmic domain, has been previously used as a non-functional cell surface marker for antibody-based cell selection, including in vitro and in vivo for purification of transduced human lymphocytes in the setting of allogeneic bone marrow transplantation [21], [22]. The level of cell surface streptavidin-binding peptide expression achieved was critically dependent on the fusion protein chosen, since preliminary experiments using the 38 amino acid SBP fused to the HLA-A2 heavy chain, or the streptavidin-binding Nano-tag peptide fused to a membrane-targeted red fluorescent protein construct [9], [23], showed poor staining at the surface of transfected cells.…”