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

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. 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

Imaging characteristics of heterotopic mesenteric ossification on FDG PET and Tc-99m bone SPECT

A CT scan of a 69-year-old male patient, performed for staging of suspected lung carcinoma, incidentally showed an irregular lesion of 10 cm in the upper abdomen. Further investigation using FDG-PET showed only moderately increased glucose metabolism, whereas Tc-99m MDP SPECT revealed intense osteoblastic activity inside the lesion. A CT-guided biopsy was performed and histologic analysis established the diagnosis of heterotopic mesenteric ossification. This pathology is rare and mostly diagnosed when it is complicated by small bowel obstruction

Preclinical evaluation of TriMix and antigen mRNA-based anti-tumor therapy

The use of tumor-associated antigen (TAA) mRNA for therapeutic purposes is under active investigation. To be effective, mRNA vaccines need to deliver activation stimuli in addition to TAAs to dendritic cells (DC). In this study, we evaluated whether intranodal delivery of TAA mRNA together with TriMix, a mix of mRNA encoding CD40 ligand, constitutive active Toll-like receptor 4 and CD70, results in the in situ modification and maturation of DCs, hence, priming of TAA-specific T cells. We showed selective uptake and translation of mRNA in vivo by lymph node resident CD11c(+) cells. This process was hampered by codelivery of classical maturation stimuli but not by TriMix mRNA. Importantly, TriMix mRNA induced a T-cell-attracting and stimulatory environment, including recruitment of antigen-specific CD4(+) and CD8(+) T cells and CTLs against various TAAs. In several mouse tumor models, mRNA vaccination was as efficient in CTL induction and therapy response as vaccination with mRNA-electroporated DCs. Together, our findings suggest that intranodal administration of TAA mRNA together with mRNA encoding immunomodulating molecules is a promising vaccination strategy