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EJNMMI Research

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Open Access 01-12-2014 | Original research

Camelid reporter gene imaging: a generic method for in vivo cell tracking

Authors: Lode RY Goethals, Tomas J Bos, Luc Baeyens, Frank De Geeter, Nick Devoogdt, Tony Lahoutte

Published in: EJNMMI Research | Issue 1/2014

Abstract

Background

To combine the sensitivity of bioluminescent imaging (BLI) with the 3D and quantitative properties of pinhole single-photon emission computed tomography (SPECT)/micro-computed tomography (CT) (phSPECT/micro-CT), we generated stable cell lines that express a yellow-fluorescent protein (YFP) and Gaussia luciferase (GLuc) fusion protein (YFP/GLuc). For in vivo phSPECT detection of this YFP/GLuc protein, a nanobody, targeted against yellow and green fluorescent proteins (anti-YFP-Nb), was site specifically labelled with ^99mTc.

Methods

Human embryonic kidney cells (HEK293T) were cultured and passaged every 3 days. 10E5 cells were transduced with YFP/GLuc-containing vector: both membrane-targeted (MT-YFP/GLuc) and non-targeted (YFP/GLuc) fusion proteins were developed. These vectors were compared against a SKOV-3 cell line stably expressing green fluorescent-firefly luciferase (GFP/Fluc) and HEK293T cells expressing red fluorescent protein in combination with a Gaussia luciferase (Red/GLuc). Transduction efficiencies were scored by fluorescence microscopy, and transduced cells were enriched by fluorescence-activated cell sorting (FACS). GLuc and FLuc functionality was tested in vitro by list-mode BLI. Subsequently, cells were transplanted subcutaneously in athymic (nu/nu) mice (MT-YFP/GLuc: n = 4, YFP/GLuc: n = 6, GFP/FLuc: n = 6, Red/GLuc: n = 4). Labelling efficiency of anti-YFP-Nb was measured using instant thin layer chromatography. One week after transplantation, ^99mTc-labelled anti-YFP-Nb was injected intravenously and pinhole (ph) SPECT/micro-CT was performed, followed by in vivo BLI.

Results

Cells showed high levels of fluorescence after transduction. The cells containing the MT-YFP/GLuc were positive on fluorescence microscopy, with the fluorescent signal confined to the cell membrane. After cell sorting, transduced cells were assayed by BLI and showed a significantly higher light output both in vitro and in vivo compared with non-transduced HEK293T cells. The anti-YFP-Nb labelling efficiency was 98%, and subsequent phSPECT/micro-CT demonstrated visible cell binding and significantly higher transplant-to-muscle ratio for both the MT-YFP/GLuc and YFP/GLuc transplanted cells, compared with the GFP/FLuc and Red/GLuc group.

Conclusion

This study provides a proof of principle for a nanobody-based cell tracking method, using a YFP/GLuc fusion protein and anti-YFP-Nb in a model of subcutaneously transplanted transduced HEK293T cells.

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Title: Camelid reporter gene imaging: a generic method for in vivo cell tracking
Authors: Lode RY Goethals
Tomas J Bos
Luc Baeyens
Frank De Geeter
Nick Devoogdt
Tony Lahoutte
Publication date: 01-12-2014
Publisher: Springer Berlin Heidelberg
Published in: EJNMMI Research / Issue 1/2014
Electronic ISSN: 2191-219X
DOI: https://doi.org/10.1186/s13550-014-0032-8

At a glance: The ONWARDS insulin icodec trials

Springer Medicine

Camelid reporter gene imaging: a generic method for in vivo cell tracking

Abstract

Background

Methods

Results

Conclusion

At a glance: The ONWARDS insulin icodec trials

Springer Medicine

Abstract

Background

Methods

Results

Conclusion

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