Whole-body tracking of single cells via positron emission tomography

Abstract

In vivo molecular imaging can measure the average kinetics and movement routes of injected cells through the body. However, owing to non-specific accumulation of the contrast agent and its efflux from the cells, most of these imaging methods inaccurately estimate the distribution of the cells. Here, we show that single human breast cancer cells loaded with mesoporous silica nanoparticles concentrating the ⁶⁸Ga radioisotope and injected into immunodeficient mice can be tracked in real time from the pattern of annihilation photons detected using positron emission tomography, with respect to anatomical landmarks derived from X-ray computed tomography. The cells travelled at an average velocity of 50 mm s^-1 and arrested in the lungs 2-3 s after tail-vein injection into the mice, which is consistent with the blood-flow rate. Single-cell tracking could be used to determine the kinetics of cell trafficking and arrest during the earliest phase of the metastatic cascade, the trafficking of immune cells during cancer immunotherapy and the distribution of cells after transplantation.

Fig. 1 |. Overview of the CellGPS workflow.

a. Mesoporous silica nanoparticles (MSNs) are used to concentrate ⁶⁸Ga from a clinical PET generator. b. The nanoparticles are loaded to cells into achieve up to 100 Bq / cell (less than 1 millionth of a standard PET dose). c. Isolated single cells are administered into mice. Gamma rays emitted from single cells are captured by a small-animal PET scanner and processed by an algorithm to estimate the location of the moving cell in real time. In this example, single human breast cancer cells administered IV arrested in the lung, as confirmed by ex vivo dissection.

Fig. 2 |. ⁶⁸Ga-MSN labelling method for sensitive cell tracking.

a. Transmission electron microscopy (TEM), showing MSN pore structure. b. Energy dispersive spectroscopy and elemental mapping of single MSN labelled with stable ⁶⁹Ga. c. Radiochemical purity of ⁶⁸Ga-MSN quantified using iTLC. d. TEM showing uptake of lipid-coated MSNs by MDA-MB-231 cell. e. Cellular uptake of FITC-labelled MSNs (green). Red highlights the cell membrane and blue the nucleus. f. Cell uptake of FITC-labelled MSN analysed by flow cytometry. g. Fluorescence (Hoechst 33342) and radioluminescence microscopy (RLM) of MDA-MB-231 cells following uptake of ⁶⁸Ga-MSN. Colour bar shown as counts per pixel. h. Cell viability assay following treatment with non-radioactive MSN and ⁶⁸Ga-labelled MSN. Experiment performed in triplicate. The P-values (two-sided) were smaller than 0.001. NS, not significant. i. DNA damage measured as γH2AX staining (40 min after the end of the labelling procedure). j. Cell enumeration in Terasaki plates by fluorescence microscopy. k. PET imaging of various numbers of ⁶⁸Ga-MSN-labelled cells (top: coronal view, bottom: sagittal view; colour scale shown as kBq/cc). l. Cell uptake (in vitro gamma counting) for different numbers of cells.

Fig. 3 |. Static PET imaging of small cell populations in mice and ex vivo validation.

a. MDA-MB-231 cells arrested in the lungs following IV injection of 100 radiolabelled cells. b. Visualization of a cluster of 10 cells after subcutaneous injection in the right thigh. c. Single radiolabelled cell detected after injection in the right forepaw. d. Single radiolabelled cell imaged after arrest in the left lung, following IV injection (experiment repeated three times). e. Photograph and PET/CT images of ex vivo organs from the same mouse (from top to bottom: brain, heart, lung, liver, spleen, stomach, kidney), and f. magnified view of the lungs. g. Gamma counting quantification of radioactivity in ex vivo organs. h. Brightfield and fluorescence imaging of ex vivo lung tissue highlights single locus with high DiI fluorescence. i. Confocal microscopy demonstrates dual staining for nucleus (Hoechst 33342, blue) and membrane (DiI, red) but no green fluorescence (negative control), confirming the localization in the lung of the injected cancer cell.

Fig. 4 |. Dynamic PET tracking of single cells.

a. A single radiolabelled cell is flowed through a length of tubing coiled around a 3D-printed cylinder. b. Cell trajectory reconstructed from list-mode PET data (black line) and closest helical fit (red line). c. Estimated cell velocity as a function of time (black line), and average velocity of the cell medium (dashed red line). d. Butterfly catheter inserted into mouse tail vein for simultaneous cell injection and PET imaging. e. Coincidence event rate recorded by the scanner. Events measured before cell arrival are due to background noise. f. Dynamic tracking of single cell in vivo, shown at three different time points. The blue lines represent coincidence events detected by the scanner. The red dot is the estimated location of the cell. The yellow line is the reconstructed trajectory. Coronal CT views are shown for anatomical reference. g. Reconstructed trajectory shown as a transaxial view, including cross-sectional CT slice through cell arrest location. h. Reconstructed trajectory shown with respect to 3D surface-rendered bony anatomy.