Ultrasensitive and multiplexed tracking of single cells using whole-body PET/CT

Abstract

In vivo molecular imaging tools are crucially important for elucidating how cells move through complex biological systems; however, achieving single-cell sensitivity over the entire body remains challenging. Here, we report a highly sensitive and multiplexed approach for tracking upward of 20 single cells simultaneously in the same subject using positron emission tomography (PET). The method relies on a statistical tracking algorithm (PEPT-EM) to achieve a sensitivity of 4 becquerel per cell and a streamlined workflow to reliably label single cells with over 50 becquerel per cell of ¹⁸F-fluorodeoxyglucose (FDG). To demonstrate the potential of the method, we tracked the fate of more than 70 melanoma cells after intracardiac injection and found they primarily arrested in the small capillaries of the pulmonary, musculoskeletal, and digestive organ systems. This study bolsters the evolving potential of PET in offering unmatched insights into the earliest phases of cell trafficking in physiological and pathological processes and in cell-based therapies.

Fig. 1.. Workflow for tracking single radiolabeled cells in vivo with PET.

Cancer cells (B16) were radiolabeled with FDG (1) and then sorted and dispensed as single cells using a microfluidic device (2). As a proof of concept, the labeled cells were injected into a murine model via either intravenous (IV) (3a) or intracardiac (3b) routes. Cells injected intravenously were trapped in the lungs, whereas those injected intracardially were widely disseminated throughout the entire body. We used PET/CT and a custom algorithm to estimate the 3D locations of these single cells (4). Figure 1 was created in part with BioRender.com.

Fig. 2.. In vitro characterization of FDG cell labeling.

(A and B) Single FDG-labeled cells (B16) were placed in vials and imaged with PET/CT before and after adding lysis buffer, showing that focal PET signal is a characteristic feature of live cells. PET images are fused with CT and displayed as maximum intensity projections, with a slice thickness of 6 mm to capture the contents of the entire vial. (C) The radioactivity of single cells was measured with a gamma counter, revealing that B16 cells could be labeled with FDG more effectively than MDA-MB-231 and 4T1 cells. (D) Viability of FDG-labeled cells was >88%, as assessed by calcein-AM staining. Inset picture: Fluorescence micrograph confirming that dispensing of exactly one cell by the device. (E) CCK-8 assay, demonstrating a 75% cell metabolic rate 54 hours after labeling, relative to control cells (the number of biological replicates in each group is three). (F) Annexin V assay, showing comparable levels of cell apoptosis in labeled and control cells 3 hours after labeling and substantially less than the positive control (cisplatin). (G and H) DNA damage characterized using γH2AX staining for control, x-ray irradiated, and FDG-labeled cells, measured 10 min (G) and 24 hours after labeling (H). (I) FDG efflux from FDG-labeled cells in the presence of different concentrations of d-glucose (0, 1.5, and 4.5 g/liter) (the number of biological replicates in each group is three). CTR, control; A.U., arbitrary units; MeOH, methanol; ns, not significant.

Fig. 3.. In vivo PET imaging of FDG-labeled single cells.

(A and C) PET/CT images of radiolabeled single cells, which were introduced into mice via intravenous (A) or intracardiac injection (C). PET images were reconstructed using the conventional OSEM method. The focal signals seen in the PET images represent single labeled cells (green arrows). High-frequency reconstruction noise is also visible in the images near the edge of the field of view (blue arrows). (B and D) ROI quantification of the PET images, showing the change in radioactivity in single cells and bladder over time. PET and CT images are fused together and displayed as maximum intensity projections, with a slice thickness of 21.2 mm.

Fig. 4.. PEPT-EM improves the detection of single cells.

(A) Schematic representation of OSEM, the standard algorithm for PET reconstruction. (B) PET image obtained by OSEM reconstruction, showing five FDG-labeled cells imaged in vials. The lowest detectable cell had 12 Bq. PET images are displayed as maximum intensity projections, with a slice thickness of 7.1 mm, and fused to CT. (C) Schematic representation of PEPT-EM, a tracking algorithm based on a Gaussian mixture model that estimates the 3D positions of radioactive sources directly from the recorded coincident annihilation photons. (D) The 3D positions of the discrete sources were reconstructed by PEPT-EM from the same PET dataset, shown here as red asterisks over the CT image of the vials. (E) The PEPT-EM algorithm was initialized by generating random cell locations. As the algorithm iterates, it progressively converges toward the maximum-likelihood location of the cells, leading to a reduction in the SD of the estimated position (represented by the radius of the circle).

Fig. 5.. In vivo tracking single cells using PEPT-EM.

(A) The PEPT-EM algorithm was applied to a PET dataset acquired after intravenously injecting 10 to 20 single cells into Foxn1^nu mice. The resulting cell positions (red asterisks; right) are compared to the conventional OSEM reconstruction of the same dataset (left). (B and C) Both algorithms were also applied to a PET dataset obtained by injecting FDG-labeled cells into the left ventricle of a mouse. The results are shown for 10-min (B) and 1-min acquisitions (C). Fused PET/CT images are shown as maximum intensity projections, with a slice thickness of 21.2 mm (coronal view) and 20.2 mm (sagittal view).

Fig. 6.. In vivo tracking of the fate of single cells after intracardiac injection.

(A) PET/CT image (OSEM reconstruction; maximum intensity projection) of a mouse right after intracardiac (left ventricle) injection, with labels indicating the anatomical locations where cancer cells were arrested. Fused PET/CT images are displayed as maximum intensity projections, with a slice thickness of 21.2 mm. (B) A comprehensive summary of the results, showing the sites and organ systems in which 74 labeled cells arrested (n = 6 mice). (C) 3D rendering showing single-cell distribution (OSEM reconstruction) relative to the segmented bony and cardiovascular anatomy.