Abstract 14903: SPECT-CT and Bioluminescence Imaging Reveal the Important Contribution of Cellular Bioenergetics on the Molecular Imaging Signal from Stem Cells Expressing Luciferase and The Sodium-Iodide Symporter

Abstract

Molecular imaging is useful for longitudinal assessment of engraftment. However, it is not known whether cellular bioenergetics can influence the molecular imaging signal. We investigated the effects of dissociation and suspension on cellular bioenergetics and the molecular imaging signal obtained by human Na-Iodide symporter (hNIS) and firefly luciferase (fluc) labeling of rat cardiosphere-derived cells (rCDCs). CDCs were lentivirally transduced to express fluc or hNIS. In vitro ¹⁸FDG uptake, ATP levels, cellular metabolism (oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)), ^99mTc-pertechnetate/NIS uptake and bioluminescence (BLI) were measured in rCDCs which were adherent for 24hrs, and following rCDC dissociation and suspension for 1hr. In vivo dual isotope SPECT-CT imaging (following intravenous injection of ^99mTc and ²⁰¹Tl), and BLI imaging were performed at 1hr & 24hrs, (which corresponded to the in vitro assay time points) following transplantation in a rat model. CDC retention was confirmed by qPCR and ex vivo luciferase assay. Cell dissociation followed by suspension for 1hr resulted in decreased glucose uptake, cellular ATP, ^99mTc uptake, BLI signal, OCR and ECAR by 82%, 43%, 42%, 44%, 52% and 60% (p<0.05) respectively, compared to adherent cells. ADP/ATP ratio increased from 0.27 in adherent rCDCs, to 1.1 and 1.8 following suspension for 1hr and 3hrs respectively, indicating progressive bioenergetic deficits. In vitro ^99mTc uptake was similar following dissociation with trypsin or non-enzymatic dissociation solution. In vivo cell loss (∼3 fold) was observed at 24hrs in both fluc⁺ and hNIS⁺ rCDCs. The in vivo ^99mTc uptake was significantly lower at 1hr compared to 24hrs following transplantation (71.6±17.2 vs. 185.7±54 kBq at 1&24hrs, n=7, p<0.01), in contrast to the in vivo BLI signal which was ∼3 fold higher at 1hr than at 24hrs (n=3, p<0.01). The BLI signal was 2.5 and 3.5 fold higher at 1hr and 24hr, respectively when CDCs were suspended in medium compared to saline. Cell dissociation and lack of substrates in the suspension medium impair CDC bioenergetics, ^99mTc uptake and BLI signal. BLI and NIS imaging may be useful for in vivo optimization of bioenergetics in transplanted cells.