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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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Immunohistochemical staining of N-A3/CEA expression in tumors (×20 magnification). a Paraffin-embedded sections of nontransfected and TR(1–99)-NA3 transfected Jurkat tumors stained with cT84.66 anti-CEA antibody. b Frozen sections of nontransfected and NA3-CD5 transfected Jurkat tumors stained with anti-CEA scFv-Fc H310A antibody fragment.
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Fig4: Immunohistochemical staining of N-A3/CEA expression in tumors (×20 magnification). a Paraffin-embedded sections of nontransfected and TR(1–99)-NA3 transfected Jurkat tumors stained with cT84.66 anti-CEA antibody. b Frozen sections of nontransfected and NA3-CD5 transfected Jurkat tumors stained with anti-CEA scFv-Fc H310A antibody fragment.

Mentions: Subcutaneous tumors were established in athymic nude mice by coinjecting transfected or nontransfected Jurkat cells with irradiated HT1080 human sarcoma feeder cells, respectively. Tumors were excised, frozen, and analyzed for expression of CEA by IHC. Immunohistochemical staining of tumors produced by TR(1–99)-NA3 transfected Jurkat cells showed that the expression of protein was very low (Fig. 4a), whereas the expression of NA3-CD5 transfected Jurkat tumors was more prominent (Fig. 4b).Fig. 4


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

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

Immunohistochemical staining of N-A3/CEA expression in tumors (×20 magnification). a Paraffin-embedded sections of nontransfected and TR(1–99)-NA3 transfected Jurkat tumors stained with cT84.66 anti-CEA antibody. b Frozen sections of nontransfected and NA3-CD5 transfected Jurkat tumors stained with anti-CEA scFv-Fc H310A antibody fragment.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2992604&req=5

Fig4: Immunohistochemical staining of N-A3/CEA expression in tumors (×20 magnification). a Paraffin-embedded sections of nontransfected and TR(1–99)-NA3 transfected Jurkat tumors stained with cT84.66 anti-CEA antibody. b Frozen sections of nontransfected and NA3-CD5 transfected Jurkat tumors stained with anti-CEA scFv-Fc H310A antibody fragment.
Mentions: Subcutaneous tumors were established in athymic nude mice by coinjecting transfected or nontransfected Jurkat cells with irradiated HT1080 human sarcoma feeder cells, respectively. Tumors were excised, frozen, and analyzed for expression of CEA by IHC. Immunohistochemical staining of tumors produced by TR(1–99)-NA3 transfected Jurkat cells showed that the expression of protein was very low (Fig. 4a), whereas the expression of NA3-CD5 transfected Jurkat tumors was more prominent (Fig. 4b).Fig. 4

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