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

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.

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

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 upwards of 20 single cells simultaneously in the same subject using positron emission tomography (PET). The method relies on a new tracking algorithm (PEPT-EM) to push the cellular detection threshold to below 4 Bq/cell, and a streamlined workflow to reliably label single cells with over 50 Bq/cell of 18F-fluorodeoxyglucose (FDG). To demonstrate the potential of method, we tracked the fate of over 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.

Radioluminescence from polymer dots based on thermally activated delayed fluorescence.

We demonstrate that polymer dots doped with thermally activated delayed fluorescence (TADF) molecules clearly exhibit blue radio-luminescence upon hard X-ray and electron beam irradiation, which is a new design for nano-sized scintillators.

Red, green, and blue radio-luminescent polymer dots doped with heteroleptic tris-cyclometalated iridium complexes.

In this study, we synthesized radioexcitable luminescent polymer dots (P-dots) doped with heteroleptic tris-cyclometalated iridium complexes that emit red, green, and blue light. We investigated the luminescence properties of these P-dots under X-ray and electron beam irradiation, revealing their potential as new organic scintillators.

Preclinical Evaluation of 89Zr-Panitumumab for Biology-Guided Radiation Therapy.

PURPOSE: Biology-guided radiation therapy (BgRT) uses real-time line-of-response data from on-board positron emission tomography (PET) detectors to guide beamlet delivery during therapeutic radiation. The current workflow requires 18F-fluorodeoxyglucose (FDG) administration daily before each treatment fraction. However, there are advantages to reducing the number of tracer injections by using a PET tracer with a longer decay time. In this context, we investigated 89Zr-panitumumab (89Zr-Pan), an antibody PET tracer with a half-life of 78 hours that can be imaged for up to 9 days using PET. METHODS AND MATERIALS: The BgRT workflow was evaluated preclinically in mouse colorectal cancer xenografts (HCT116) using small-animal positron emission tomography/computed tomography (PET/CT) for imaging and image-guided kilovoltage conformal irradiation for therapy. Mice (n = 5 per group) received 7 MBq of 89Zr-Pan as a single dose 2 weeks after tumor induction, with or without fractionated radiation therapy (RT; 6 × 6.6 Gy) to the tumor region. The mice were imaged longitudinally to assess the kinetics of the tracer over 9 days. PET images were then analyzed to determine the stability of the PET signal in irradiated tumors over time. RESULTS: Mice in the treatment group experienced complete tumor regression, whereas those in the control group were killed because of tumor burden. PET imaging of 89Zr-Pan showed well-delineated tumors with minimal background in both groups. On day 9 postinjection, tumor uptake of 89Zr-Pan was 7.2 ± 1.7 in the control group versus 5.2 ± 0.5 in the treatment group (mean percentage of injected dose per gram of tissue [%ID/g] ± SD; P = .07), both significantly higher than FDG uptake (1.1 ± 0.5 %ID/g) 1 hour postinjection. To assess BgRT feasibility, the clinical eligibility criteria was computed using human-equivalent uptake values that were extrapolated from preclinical PET data. Based on this semiquantitative analysis, BgRT may be feasible for 5 consecutive days after a single 740-MBq injection of 89Zr-Pan. CONCLUSIONS: This study indicates the potential of long-lived antibody-based PET tracers for guiding clinical BgRT.

Development of a Lensless Radiomicroscope for Cellular-Resolution Radionuclide Imaging.

The action of radiopharmaceuticals takes place at the level of cells. However, existing radionuclide assays can only measure uptake in bulk or in small populations of single cells. This potentially hinders the development of effective radiopharmaceuticals for disease detection, staging, and treatment. Methods: We have developed a new imaging modality, the lensless radiomicroscope (LRM), for in vitro, cellular-resolution imaging of ß- and α-emitting radionuclides. The palm-sized instrument is constructed from off-the-shelf parts for a total cost of less than $100, about 500 times less than the radioluminescence microscope, its closest equivalent. The instrument images radiopharmaceuticals by direct detection of ionizing charged particles via a consumer-grade complementary metal-oxide semiconductor detector. Results: The LRM can simultaneously image more than 5,000 cells within its 1 cm2 field of view, a 100-times increase over state-of-the-art technology. It has spatial resolution of 5 µm for brightfield imaging and 30 µm for 18F positron imaging. We used the LRM to quantify 18F-FDG uptake in MDA-MB-231 breast cancer cells 72 h after radiation treatment. Cells receiving 3 Gy were 3 times larger (mean = 3,116 µm2) than their untreated counterparts (mean = 940 µm2) but had 2 times less 18F-FDG per area (mean = 217 Bq/mm2), a finding in agreement with the clinical use of this tracer to monitor response. Additionally, the LRM was used to dynamically image the uptake of 18F-FDG by live cancer cells, and thus measure their avidity for glucose. Conclusion: The LRM is a high-resolution, large-field-of-view, and cost-effective approach to image radiotracer uptake with single-cell resolution in vitro.

Real-time optical oximetry during FLASH radiotherapy using a phosphorescent nanoprobe.

The rapid depletion of oxygen during irradiation at ultra-high dose rate calls for tissue oximeters capable of high temporal resolution. This study demonstrates a water-soluble phosphorescent nanoprobe and fiber-coupled instrument, which together are used to measure the kinetics of oxygen depletion at 200 Hz during irradiation of in vitro solutions.

Fluorinated diselenide nanoparticles for radiosensitizing therapy of cancer.

Radiation resistance of cancer cells represents one of the major challenges in cancer treatment. The novel self-assembled fluoralkylated diselenide nanoparticles (fluorosomes) based on seleno-l-cystine (17FSe2) possess redox-active properties that autocatalytically decompose hydrogen peroxide (H2O2) and oxidize the intracellular glutathione (GSH) that results in regulation of cellular oxidative stress. Alkylfluorinated diselenide nanoparticles showed a significant cytotoxic and radiosensitizing effect on cancer cells. The EL-4 tumor-bearing C56BL/6 mice treated with 17FSe2 followed by fractionated radiation treatment (4 × 2Gy) completely suppressed tumor growth. Our results suggest that described diselenide system behaves as a potent radiosensitizer agent targeting tumor growth and preventing tumor recurrence.

3D computational model of oxygen depletion kinetics in brain vasculature during FLASH RT and its implications for in vivo oximetry experiments.

PURPOSE: Ultra-high-dose-rate irradiation, also known as FLASH, has been shown to improve the therapeutic ratio of radiation therapy (RT). The mechanism behind this effect has been partially explained by the radiochemical oxygen depletion (ROD) hypothesis, which attributes the protection of the normal tissue to the induction of transient hypoxia by ROD. To better understand the contribution of oxygen to the FLASH effect, it is necessary to measure oxygen (O2 ) in vivo during FLASH irradiation. This study's goal is to determine the temporal resolution required to accurately measure the rapidly changing oxygen concentration immediately after FLASH irradiation. METHODS: We conducted a computational simulation of oxygen dynamics using a real vascular model that was constructed from a public fluorescence microscopy dataset. The dynamic distribution of oxygen tension (po2 ) during and after FLASH RT was modeled by a partial differential equation (PDE) considering oxygen diffusion, metabolism, and ROD. The underestimation of ROD due to oxygen recovery was evaluated assuming either complete or partial depletion, and a range of possible values for parameters such as oxygen diffusion, consumption, vascular po2 and vessel density. RESULT: The O2 concentration recovers rapidly after FLASH RT. Assuming a temporal resolution of 0.5 s, the estimated ROD is only 50.7% and 36.7% of its actual value in cases of partial and complete depletion, respectively. Additionally, the underestimation of ROD is highly dependent on the vascular density. To estimate ROD rate with 90% accuracy, temporal resolution on the order of milliseconds is required considering the uncertainty in parameters involved, especially, the diverse vascular density of the tissue. CONCLUSION: The rapid recovery of O2 poses a great challenge for in vivo ROD measurements during FLASH RT. Temporal resolution on the order of milliseconds is recommended for ROD measurements in the normal tissue. Further work is warranted to investigate whether the same requirements apply to tumors, given their irregular vasculature.

Mechanoporation enables rapid and efficient radiolabeling of stem cells for PET imaging.

Regenerative medicine uses the patient own stem cells to regenerate damaged tissues. Molecular imaging techniques are commonly used to image the transplanted cells, either right after surgery or at a later time. However, few techniques are fast or straightforward enough to label cells intraoperatively. Adipose tissue-derived stem cells (ADSCs) were harvested from knee joints of minipigs. The cells were labeled with PET contrast agent by flowing mechanoporation using a microfluidic device. While flowing through a series of microchannels, cells are compressed repeatedly by micro-ridges, which open transient pores in their membranes and induce convective transport, intended to facilitate the transport of 68Ga-labeled and lipid-coated mesoporous nanoparticles (MSNs) into the cells. This process enables cells to be labeled in a matter of seconds. Cells labeled with this approach were then implanted into cartilage defects, and the implant was imaged using positron emission tomography (PET) post-surgery. The microfluidic device can efficiently label millions of cells with 68Ga-labeled MSNs in as little as 15 min. The method achieved labeling efficiency greater than 5 Bq/cell on average, comparable to 30 min-long passive co-incubation with 68Ga-MSNs, but with improved biocompatibility due to the reduced exposure to ionizing radiation. Labeling time could also be accelerated by increasing throughput through more parallel channels. Finally, as a proof of concept, ADSCs were labeled with 68Ga-MSNs and quantitatively assessed using clinical PET/MR in a mock transplant operation in pig knee joints. MSN-assisted mechanoporation is a rapid, effective and straightforward approach to label cells with 68Ga. Given its high efficiency, this labeling method can be used to track small cells populations without significant effects on viability. The system is applicable to a variety of cell tracking studies for cancer therapy, regenerative therapy, and immunotherapy.

Simultaneous dose and dose rate optimization (SDDRO) of the FLASH effect for pencil-beam-scanning proton therapy.

PURPOSE: Compared to CONV-RT (with conventional dose rate), FLASH-RT (with ultra-high dose rate) can provide biological dose sparing for organs-at-risk (OARs) via the so-called FLASH effect, in addition to physical dose sparing. However, the FLASH effect only occurs, when both dose and dose rate meet certain minimum thresholds. This work will develop a simultaneous dose and dose rate optimization (SDDRO) method accounting for both FLASH dose and dose rate constraints during treatment planning for pencil-beam-scanning proton therapy. METHODS: SDDRO optimizes the FLASH effect (specific to FLASH-RT) as well as the dose distribution (similar to CONV-RT). The nonlinear dose rate constraint is linearized, and the reformulated optimization problem is efficiently solved via iterative convex relaxation powered by alternating direction method of multipliers. To resolve and quantify the generic tradeoff of FLASH-RT between FLASH and dose optimization, we propose the use of FLASH effective dose based on dose modifying factor (DMF) owing to the FLASH effect. RESULTS: FLASH-RT via transmission beams (TB) (IMPT-TB or SDDRO) and CONV-RT via Bragg peaks (BP) (IMPT-BP) were evaluated for clinical prostate, lung, head-and-neck (HN), and brain cases. Despite the use of TB, which is generally suboptimal to BP for normal tissue sparing, FLASH-RT via SDDRO considerably reduced FLASH effective dose for high-dose OAR adjacent to the target. For example, in the lung SBRT case, the max esophageal dose constraint 27 Gy was only met by SDDRO (24.8 Gy), compared to IMPT-BP (35.3 Gy) or IMPT-TB (36.6 Gy); in the brain SRS case, the brain constraint V12Gy≤15cc was also only met by SDDRO (13.7cc), compared to IMPT-BP (43.9cc) or IMPT-TB (18.4cc). In addition, SDDRO substantially improved the FLASH coverage from IMPT-TB, e.g., an increase from 37.2% to 67.1% for lung, from 39.1% to 58.3% for prostate, from 65.4% to 82.1% for HN, from 50.8% to 73.3% for the brain. CONCLUSIONS: Both FLASH dose and dose rate constraints are incorporated into SDDRO for FLASH-RT that jointly optimizes the FLASH effect and physical dose distribution. FLASH effective dose via FLASH DMF is introduced to reconcile the tradeoff between physical dose sparing and FLASH sparing, and quantify the net effective gain from CONV-RT to FLASH-RT.

Noninvasive and Highly Multiplexed Five-Color Tumor Imaging of Multicore Near-Infrared Resonant Surface-Enhanced Raman Nanoparticles In Vivo.

In vivo multiplexed imaging aims for noninvasive monitoring of tumors with multiple channels without excision of the tissue. While most of the preclinical imaging has provided a number of multiplexing channels up to three, Raman imaging with surface-enhanced Raman scattering (SERS) nanoparticles was suggested to offer higher multiplexing capability originating from their narrow spectral width. However, in vivo multiplexed SERS imaging is still in its infancy for multichannel visualization of tumors, which require both sufficient multiplicity and high sensitivity concurrently. Here we create multispectral palettes of gold multicore-near-infrared (NIR) resonant Raman dyes-silica shell SERS (NIR-SERRS) nanoparticle oligomers and demonstrate noninvasive and five-plex SERS imaging of the nanoparticle accumulation in tumors of living mice. We perform the five-plex ratiometric imaging of tumors by varying the administered ratio of the nanoparticles, which simulates the detection of multiple biomarkers with different expression levels in the tumor environment. Furthermore, since this method does not require the excision of tumor tissues at the imaging condition, we perform noninvasive and longitudinal imaging of the five-color nanoparticles in the tumors, which is not feasible with current ex vivo multiplexed tissue analysis platforms. Our work surpasses the multiplicity limit of previous preclinical tumor imaging methods while keeping enough sensitivity for tumor-targeted in vivo imaging and could enable the noninvasive assessment of multiple biological targets within the tumor microenvironment in living subjects.

High-resolution positron emission microscopy of patient-derived tumor organoids.

Tumor organoids offer new opportunities for translational cancer research, but unlike animal models, their broader use is hindered by the lack of clinically relevant imaging endpoints. Here, we present a positron-emission microscopy method for imaging clinical radiotracers in patient-derived tumor organoids with spatial resolution 100-fold better than clinical positron emission tomography (PET). Using this method, we quantify 18F-fluorodeoxyglucose influx to show that patient-derived tumor organoids recapitulate the glycolytic activity of the tumor of origin, and thus, could be used to predict therapeutic response in vitro. Similarly, we measure sodium-iodine symporter activity using 99mTc- pertechnetate and find that the iodine uptake pathway is functionally conserved in organoids derived from thyroid carcinomas. In conclusion, organoids can be imaged using clinical radiotracers, which opens new possibilities for identifying promising drug candidates and radiotracers, personalizing treatment regimens, and incorporating clinical imaging biomarkers in organoid-based co-clinical trials.

Flow radiocytometry using droplet optofluidics.

Flow-based cytometry methods are widely used to analyze heterogeneous cell populations. However, their use for small molecule studies remains limited due to bulky fluorescent labels that often interfere with biochemical activity in cells. In contrast, radiotracers require minimal modification of their target molecules and can track biochemical processes with negligible interference and high specificity. Here, we introduce flow radiocytometry (FRCM) that broadens the scope of current cytometry methods to include beta-emitting radiotracers as probes for single cell studies. FRCM uses droplet microfluidics and radiofluorogenesis to translate the radioactivity of single cells into a fluorescent signal that is then read out using a high-throughput optofluidic device. As a proof of concept, we quantitated [18F]fluorodeoxyglucose radiotracer uptake in single human breast cancer cells and successfully assessed the metabolic flux of glucose and its heterogeneity at the cellular level. We believe FRCM has potential applications ranging from analytical assays for cancer and other diseases to development of small-molecule drugs.

High-resolution radioluminescence microscopy of FDG uptake in an engineered 3D tumor-stoma model.

PURPOSE: The increased glucose metabolism of cancer cells is the basis for 18F-fluorodeoxyglucose positron emission tomography (FDG-PET). However, due to its coarse image resolution, PET is unable to resolve the metabolic role of cancer-associated stroma, which often influences the metabolic reprogramming of a tumor. This study investigates the use of radioluminescence microscopy for imaging FDG uptake in engineered 3D tumor models with high resolution. METHOD: Multicellular tumor spheroids (A549 lung adenocarcinoma) were co-cultured with GFP-expressing human umbilical vein endothelial cells (HUVECs) within an artificial extracellular matrix to mimic a tumor and its surrounding stroma. The tumor model was constructed as a 200-µm-thin 3D layer over a transparent CdWO4 scintillator plate to allow high-resolution imaging of the cultured cells. After incubation with FDG, the radioluminescence signal was collected by a highly sensitive widefield microscope. Fluorescence microscopy was performed using the same instrument to localize endothelial and tumor cells. RESULTS: Simultaneous and co-localized brightfield, fluorescence, and radioluminescence imaging provided high-resolution information on the distribution of FDG in the engineered tissue. The microvascular stromal compartment as a whole took up a large fraction of the FDG, comparable to the uptake of the tumor spheroids. In vitro gamma counting confirmed that A549 and HUVEC cells were both highly glycolytic with rapid FDG uptake kinetics. Despite the relative thickness of the tissue constructs, an average spatial resolution of 64 ± 4 µm was achieved for imaging FDG. CONCLUSION: Our study demonstrates the feasibility of imaging the distribution of FDG uptake in engineered in vitro tumor models. With its high spatial resolution, the method can separately resolve tumor and stromal components. The approach could be extended to more advanced engineered cancer models but also to surgical tissue slices and tumor biopsies.

Microfluidics-Coupled Radioluminescence Microscopy for In Vitro Radiotracer Kinetic Studies.

Integrated bioassay systems that combine microfluidics and radiation detectors can deliver medical radiopharmaceuticals to live cells with precise timing, while minimizing radiation dose and sample volume. However, the spatial resolution of many radiation imaging systems is limited to bulk cell populations. Here, we demonstrate microfluidics-coupled radioluminescence microscopy (µF-RLM), a new integrated system that can image radiotracer uptake in live adherent cells growing inside microincubators with spatial resolution better than 30 µm. Our method enables on-chip radionuclide imaging by incorporating an inorganic scintillator plate (CdWO4) into a microfluidic chip. We apply this approach to investigate the factors that influence the dynamic uptake of [18F]fluorodeoxyglucose (FDG) by cancer cells. In the first experiment, we measured the effect of flow on FDG uptake of cells and found that a continuous flow of the radiotracer led to fourfold higher uptake than static incubation, suggesting that convective replenishment enhances molecular radiotracer transport into cells. In the second set of experiments, we applied pharmacokinetic modeling to show that lactic acidosis inhibits FDG uptake by cancer cells in vitro and that this decrease is primarily due to downregulation of FDG transport into the cells. The other two rate constants, which represent FDG export and FDG metabolism, were relatively unaffected by lactic acidosis. Lactic acidosis is common in solid tumors because of the dysregulated metabolism and inefficient vasculature. In conclusion, µF-RLM is a simple and practical approach for integrating high-resolution radionuclide imaging within standard microfluidics devices, thus potentially opening venues for investigating the efficacy of radiopharmaceuticals in in vitro cancer models.

Multicellular Spheroids as In Vitro Models of Oxygen Depletion During FLASH Irradiation.

PURPOSE: The differential response of normal and tumor tissues to ultrahigh-dose-rate radiation (FLASH) has raised new hope for treating solid tumors but, to date, the mechanism remains elusive. One leading hypothesis is that FLASH radiochemically depletes oxygen from irradiated tissues faster than it is replenished through diffusion. The purpose of this study was to investigate these effects within hypoxic multicellular tumor spheroids through simulations and experiments. METHODS AND MATERIALS: Physicobiological equations were derived to model (1) the diffusion and metabolism of oxygen within spheroids; (2) its depletion through reactions involving radiation-induced radicals; and (3) the increase in radioresistance of spheroids, modeled according to the classical oxygen enhancement ratio and linear-quadratic response. These predictions were then tested experimentally in A549 spheroids exposed to electron irradiation at conventional (0.075 Gy/s) or FLASH (90 Gy/s) dose rates. Clonogenic survival, cell viability, and spheroid growth were scored postradiation. Clonogenic survival of 2 other cell lines was also investigated. RESULTS: The existence of a hypoxic core in unirradiated tumor spheroids is predicted by simulations and visualized by fluorescence microscopy. Upon FLASH irradiation, this hypoxic core transiently expands, engulfing a large number of well-oxygenated cells. In contrast, oxygen is steadily replenished during slower conventional irradiation. Experimentally, clonogenic survival was around 3-fold higher in FLASH-irradiated spheroids compared with conventional irradiation, but no significant difference was observed for well-oxygenated 2-dimensional cultured cells. This differential survival is consistent with the predictions of the computational model. FLASH irradiation of spheroids resulted in a dose-modifying factor of around 1.3 for doses above 10 Gy. CONCLUSIONS: Tumor spheroids can be used as a model to study FLASH irradiation in vitro. The improved survival of tumor spheroids receiving FLASH radiation confirms that ultrafast radiochemical oxygen depletion and its slow replenishment are critical components of the FLASH effect.

Tb-Doped core-shell-shell nanophosphors for enhanced X-ray induced luminescence and sensitization of radiodynamic therapy.

The development of radiation responsive materials, such as nanoscintillators, enables a variety of exciting new theranostic applications. In particular, the ability of nanophosphors to serve as molecular imaging agents in novel modalities, such as X-ray luminescence computed tomography (XLCT), has gained significant interest recently. Here, we present a radioluminescent nanoplatform consisting of Tb-doped nanophosphors with an unique core/shell/shell (CSS) architecture for improved optical emission under X-ray excitation. Owing to the spatial confinement and separation of luminescent activators, these CSS nanophosphors exhibited bright optical luminescence upon irradiation. In addition to standard physiochemical characterization, these CSS nanophosphors were evaluated for their ability to serve as energy mediators in X-ray stimulated photodynamic therapy, also known as radiodynamic therapy (RDT), through attachment of a photosensitizer, rose bengal (RB). Furthermore, cRGD peptide was used as a model targeting agent against U87 MG glioblastoma cells. In vitro RDT efficacy studies suggested the RGD-CSS-RB in combination with X-ray irradiation could induce enhanced DNA damage and increased cell killing, while the nanoparticles alone are well tolerated. These studies support the utility of CSS nanophosphors and warrants their further development for theranostic applications.

High-Z Metal-Organic Frameworks for X-ray Radiation-Based Cancer Theranostics.

X-ray radiation is commonly employed in clinical practice for diagnostic and therapeutic applications. Over the past decade, developments in nanotechnology have led to the use of high-Z elements as the basis for innovative new treatment platforms that enhance the clinical efficacy of X-ray radiation. Nanoscale metal-frameworks (nMOFs) are coordination networks containing organic ligands that have attracted attention as therapeutic platforms in oncology and other areas of medicine. In cancer therapy, X-ray activated, high-Z nMOFs have demonstrated potential as radiosensitizers that increase local radiation dose deposition and generation of reactive oxygen species (ROS). This minireview summarizes current research on high-Z nMOFs in cancer theranostics and discusses factors that may influence future clinical application.

Identification of Lymphatic and Hematogenous Routes of Rapidly Labeled Radioactive and Fluorescent Exosomes through Highly Sensitive Multimodal Imaging.

There has been considerable interest in the clinical use of exosomes as delivery vehicles for treatments as well as for promising diagnostic biomarkers, but the physiological distribution of exosomes must be further elucidated to validate their efficacy and safety. Here, we aimed to develop novel methods to monitor exosome biodistribution in vivo using positron emission tomography (PET) and optical imaging. Exosomes were isolated from cultured mouse breast cancer cells and labeled for PET and optical imaging. In mice, radiolabeled and fluorescently labeled exosomes were injected both via lymphatic and hematogenous metastatic routes. PET and fluorescence images were obtained and quantified. Radioactivity and fluorescence intensity of ex vivo organs were measured. PET signals from exosomes in the lymphatic metastatic route were observed in the draining sentinel lymph nodes. Immunohistochemistry revealed greater exosome uptake in brachial and axillary versus inguinal lymph nodes. Following administration through the hematogenous metastasis pathway, accumulation of exosomes was clearly observed in the lungs, liver, and spleen. Exosomes from tumor cells were successfully labeled with 64Cu (or 68Ga) and fluorescence and were visualized via PET and optical imaging, suggesting that this simultaneous and rapid labeling method could provide valuable information for further exosome translational research and clinical applications.