Lode Ry Goethals MD, Presenter: Nothing to Disclose

Tomas Bos PhD, Abstract Co-Author: Nothing to Disclose

Veere De Grove, Abstract Co-Author: Nothing to Disclose

Luc Baeyens PhD, Abstract Co-Author: Nothing to Disclose

Inneke Willekens MD, Abstract Co-Author: Nothing to Disclose

Frank De Geeter MD, PhD, Abstract Co-Author: Nothing to Disclose

Johan De Mey, Abstract Co-Author: Research grant, General Electric Company

Tony Lahoutte, Abstract Co-Author: Nothing to Disclose

To combine the sensitivity of bioluminescent imaging (BLI) with the 3D and quantitative properties of SPECT/CT, we developed a lentiviral vector expressing a membrane bound Yellow-Fluorescent Protein - Gaussia Luciferase fusion protein (MB-YFP/GLuc). For SPECT detection of YFP/GLuc, a nanobody, targeted against YFP (anti-YFP-Nb), was developed and site specifically labelled with 99mTc. After intravenous injection of anti-YFP-Nb, SPECT/micro-CT was performed to determine feasibility of tracking YFP+ cells.

Human embryonic kidney cells (HEK 293T) were cultured and passaged every 3 days. 10E5 cells were transduced with the MB-YFP/GLuc vector or a negative control vector containing red fluorescent protein. Transduction efficiencies were scored by fluorescence microscopy. Positive YFP or Red fluorescent cells were subcloned by FACS. GLuc functionality was tested in vitro by using list mode BLI. 2 x 10E6 sorted HEK 293T cells were transplanted subcutaneously in 6 athymic (nu/nu) mice and in vivo BLI was performed. Labelling efficiency of anti-YFP-Nb was measured using instant thin layer chromatography. Specific binding af anti-YFP-Nb was determined in vitro using competitive binding studies. 1 week upon transplanation, 99mTc labelled anti-YFP-Nb was injected intraveneously and triple pinhole SPECT/micro-CT was performed. Image were analysed with AMIDE.

After transduction with the MB-YFP/GLuc vector, cells were positive on fluorescent microscopy, confirmed to be confined to the cell membrane on confocal microscopy. After FACS, 100% YFP+ cells were further cultured. In vitro BLI showed a significantly higher signal compared with the red fluorescent control group. Anti-YFP-Nb labelling efficiency was 98%, YFP specific binding was shown, with blocking after an excess of cold nanobody. In vivo BLI showed significantly increased light output in the MB-YFP/GLuc cells. SPECT/Micro-CT demonstrated visible cell binding and significantly higher target to muscle ratio with the MB-YFP/GLuc cells. (Figure: in vivo SPECT/CT)

We provided proof op principle for a nanobody based cell tracking method, using a MB-YFP/Gluc protein and anti-YFP-Nb's in a model of subcutaneously transplanted HEK293T cells.

This novel method for cell tracking will be applied in several pre-clinical models of cell transplantation, yielding insights in cell survival/proliferation in different disease models.

Goethals, L, Bos, T, De Grove, V, Baeyens, L, Willekens, I, De Geeter, F, De Mey, J, Lahoutte, T, Development and Validation of a Nanobody-based Tri-Modality Cell Tracking Method Combining Fluorescence, Bioluminescence and SPECT/micro-CT.  Radiological Society of North America 2012 Scientific Assembly and Annual Meeting, November 25 - November 30, 2012 ,Chicago IL. http://archive.rsna.org/2012/12021157.html