Radioluminescence microscopy of FLT uptake in single cells

364 Objectives 39-deoxy-39-[18F]fluorothymidine (FLT) is a radiotracer that is commonly used to study tumor cell proliferation in vivo. However, positron emission tomography measurements aggregate the FLT uptake from millions of cells and thus true measurements of the fraction of cells that are actively dividing may be lost. We have developed a technique called radioluminescence microscopy that can image the uptake of FLT and other radiotracers on a single cell level. Our objective is to characterize the single-cell distribution of FLT and compare it against a standard fluorescence-based measure of cell proliferation. Methods We image the proliferation of live human MDA-MB-231 cells, grown under different serum conditions (0% and 20%), using radioluminescence microscopy with FLT. Absolute uptake of FLT is quantified in single cells from the radioluminescence micrographs. As a reference, we also image incorporation of EdU (5-Ethynyl-29-deoxyuridine) in fixed cells using fluorescence microscopy. Results We demonstrate that it is possible to image human breast cancer cells with single cell precision. To our knowledge, this is the first reported instance of single cell FLT analysis. Our data are comparable to single cell measurements of EdU incorporation in fixed cells. EdU and similar compounds are the gold standard for measuring cell proliferation in vitro. The side-by-side comparison of FLT and EdU therefore shows that the two tracers measure comparable proliferation processes in cells and allows comparisons to be made between clinical uses of FLT, and in vitro data using either FLT or EdU. FLT can be used to image live cells directly, while EdU data typically requires the fixation of cells. Notably, we are able to obtain radioluminescence data using a picomolar concentration of FLT, as opposed to EdU, where the recommended concentration is in the millimolar range. Based on EdU staining data, few cells (1%) actively cycle under serum deprivation while a majority of them (71%) do under 20% serum. The distribution of FLT reflects this dynamic. At 0% serum, uptake of FLT is heterogeneous but relatively low. At 20% serum, a subpopulation of FLT-avid cells appears to emerge, representing 61% of the total population. This dichotomous distribution of FLT uptake is not typically observed with other radiotracers such as 2-deoxy-2-[18F]fluoro-D-glucose (FDG). Conclusions Our results confirm that increased uptake of FLT by proliferating tumors is due to a greater fraction of FLT-avid cells rather than a change in FLT uptake by individual cells. This finding is consistent with the fact that FLT uptake is mediated by expression of thymidine kinase 1 (TK1), which is only expressed in actively dividing cells. . While a baseline level of uptake is observed even in serum-starved cells, FLT uptake by arrested cells is nearly 5-fold lower than that by proliferating cells. The level of FLT uptake in arrested cells is not significantly different between the various serum conditions. This suggests that, within the same patient, changes in FLT uptake are reflective of changes in the number of actively dividing cells provided other parameters remain the same.