title: Camelid reporter gene imaging: a generic method for in vivo cell tracking.
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 (99m)Tc.
  
  
  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, (99m)Tc-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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: In Vivo Cellular and Molecular Imaging Laboratory, Vrije Universiteit Brussel, Laarbeeklaan 103, Jette 1090, Belgium ; Department of Radiology, Universitair Ziekenhuis Brussel, Laarbeeklaan 101, Jette 1090, Belgium.
authors.forename: Lode Ry
authors.initials: LR
authors.lastname: Goethals
authors.affiliation: Department of Cellular and Molecular Medicine, UC San Diego, 9500 Gilman Drive, La Jolla 92093, CA, USA.
authors.forename: Tomas J
authors.initials: TJ
authors.lastname: Bos
authors.affiliation: Beta Cell Neogenesis, Vrije Universiteit Brussel, Laarbeeklaan103, Jette 1090, Belgium.
authors.forename: Luc
authors.initials: L
authors.lastname: Baeyens
authors.affiliation: In Vivo Cellular and Molecular Imaging Laboratory, Vrije Universiteit Brussel, Laarbeeklaan 103, Jette 1090, Belgium.
authors.forename: Frank
authors.initials: F
authors.lastname: De Geeter
authors.affiliation: In Vivo Cellular and Molecular Imaging Laboratory, Vrije Universiteit Brussel, Laarbeeklaan 103, Jette 1090, Belgium.
authors.forename: Nick
authors.initials: N
authors.lastname: Devoogdt
authors.affiliation: In Vivo Cellular and Molecular Imaging Laboratory, Vrije Universiteit Brussel, Laarbeeklaan 103, Jette 1090, Belgium ; Department of Nuclear Medicine, Universitair Ziekenhuis Brussel, Laarbeeklaan 101, Jette 1090, Belgium.
authors.forename: Tony
authors.initials: T
authors.lastname: Lahoutte
keywords: Bioluminescence; Cell tracking; Fluorescence; Nanobody; SPECT/CT; YFP