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Evaluation of two internalizing carcinoembryonic antigen reporter genes for molecular imaging.

Barat B, Kenanova VE, Olafsen T, Wu AM - Mol Imaging Biol (2011)

Bottom Line: Surface expression of TR(1-99)-NA3 was lower than that of NA3-CD5.Specific targeting of TR(1-99)-NA3 or NA3-CD5 was shown by PET in xenografted mice.The in vivo imaging studies suggest a potential application of the internalizing form of CEA (N-A3) as a PET reporter gene.

View Article: PubMed Central - PubMed

Affiliation: Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095-1770, USA.

ABSTRACT

Purpose: The objective of this article is to develop internalizing positron emission tomography (PET) reporter genes for tracking genetically modified T cells in vivo.

Procedures: The transmembrane and cytoplasmic domains of the human transferrin receptor (TfR) and CD5 were each fused to the carcinoembryonic (CEA) minigene N-A3 and expressed in Jurkat T cells. Internalization was evaluated by confocal microscopy or by intracellular uptake of ¹²⁵I-labeled anti-CEA scFv-Fc. Reporter gene-transfected Jurkat xenografts in mice were analyzed by immunohistochemistry (IHC) and imaged by PET using ¹²⁴I- or ⁶⁴Cu-scFv-Fc as tracers.

Results: Surface expression of TR(1-99)-NA3 was lower than that of NA3-CD5. Both reporter genes were internalized following binding of the anti-CEA antibody fragment. IHC of tumors showed strong staining of NA3-CD5, whereas TR(1-99)-NA3 stained weakly. Specific targeting of TR(1-99)-NA3 or NA3-CD5 was shown by PET in xenografted mice.

Conclusions: The in vivo imaging studies suggest a potential application of the internalizing form of CEA (N-A3) as a PET reporter gene.

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Flow cytometry analysis of recombinant CEA reporter protein expression in Jurkat T cells. Nontransfected Jurkat (solid gray) and transfected Jurkat (black line).
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Fig2: Flow cytometry analysis of recombinant CEA reporter protein expression in Jurkat T cells. Nontransfected Jurkat (solid gray) and transfected Jurkat (black line).

Mentions: In order to evaluate the expression of recombinant CEA reporters on Jurkat cells, TR(1–99)-NA3 and NA3-CD5 transfected Jurkat cells were incubated with the anti-CEA antibody and examined by flow cytometry. Nontransfected Jurkat cells were used as a negative control. Although the fluorescence intensity shifted both cell types to the right (Fig. 2), the MFI values differed almost four fold. The MFI value for TR(1–99)-NA3 transfected cells was 26 versus 92 for NA3-CD5 transfected cells. When cells were passaged and examined for expression over time, we noted that the cell surface expression of NA3-CD5 remained relatively stable, whereas TR(1–99)-NA3 expression of recombinant protein consistently dropped over time. Western blot analyses of the recombinant proteins (TR(1–99)-NA3 and NA3-CD5) showed that they migrated consistent with their predicted molecular weights (approximately 42 kDa for both) under reducing conditions (data not shown).Fig. 2


Evaluation of two internalizing carcinoembryonic antigen reporter genes for molecular imaging.

Barat B, Kenanova VE, Olafsen T, Wu AM - Mol Imaging Biol (2011)

Flow cytometry analysis of recombinant CEA reporter protein expression in Jurkat T cells. Nontransfected Jurkat (solid gray) and transfected Jurkat (black line).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2992604&req=5

Fig2: Flow cytometry analysis of recombinant CEA reporter protein expression in Jurkat T cells. Nontransfected Jurkat (solid gray) and transfected Jurkat (black line).
Mentions: In order to evaluate the expression of recombinant CEA reporters on Jurkat cells, TR(1–99)-NA3 and NA3-CD5 transfected Jurkat cells were incubated with the anti-CEA antibody and examined by flow cytometry. Nontransfected Jurkat cells were used as a negative control. Although the fluorescence intensity shifted both cell types to the right (Fig. 2), the MFI values differed almost four fold. The MFI value for TR(1–99)-NA3 transfected cells was 26 versus 92 for NA3-CD5 transfected cells. When cells were passaged and examined for expression over time, we noted that the cell surface expression of NA3-CD5 remained relatively stable, whereas TR(1–99)-NA3 expression of recombinant protein consistently dropped over time. Western blot analyses of the recombinant proteins (TR(1–99)-NA3 and NA3-CD5) showed that they migrated consistent with their predicted molecular weights (approximately 42 kDa for both) under reducing conditions (data not shown).Fig. 2

Bottom Line: Surface expression of TR(1-99)-NA3 was lower than that of NA3-CD5.Specific targeting of TR(1-99)-NA3 or NA3-CD5 was shown by PET in xenografted mice.The in vivo imaging studies suggest a potential application of the internalizing form of CEA (N-A3) as a PET reporter gene.

View Article: PubMed Central - PubMed

Affiliation: Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095-1770, USA.

ABSTRACT

Purpose: The objective of this article is to develop internalizing positron emission tomography (PET) reporter genes for tracking genetically modified T cells in vivo.

Procedures: The transmembrane and cytoplasmic domains of the human transferrin receptor (TfR) and CD5 were each fused to the carcinoembryonic (CEA) minigene N-A3 and expressed in Jurkat T cells. Internalization was evaluated by confocal microscopy or by intracellular uptake of ¹²⁵I-labeled anti-CEA scFv-Fc. Reporter gene-transfected Jurkat xenografts in mice were analyzed by immunohistochemistry (IHC) and imaged by PET using ¹²⁴I- or ⁶⁴Cu-scFv-Fc as tracers.

Results: Surface expression of TR(1-99)-NA3 was lower than that of NA3-CD5. Both reporter genes were internalized following binding of the anti-CEA antibody fragment. IHC of tumors showed strong staining of NA3-CD5, whereas TR(1-99)-NA3 stained weakly. Specific targeting of TR(1-99)-NA3 or NA3-CD5 was shown by PET in xenografted mice.

Conclusions: The in vivo imaging studies suggest a potential application of the internalizing form of CEA (N-A3) as a PET reporter gene.

Show MeSH
Related in: MedlinePlus