The arrival of cell-based therapies is a revolution in medicine. However, its safe clinical application in a rational manner depends on reliable, clinically applicable methods for determining the fate and trafficking of therapeutic cells in vivo using medical imaging techniques─known as in vivo cell tracking. Radionuclide imaging using single photon emission computed tomography (SPECT) or positron emission tomography (PET) has several advantages over other imaging modalities for cell tracking because of its high sensitivity (requiring low amounts of probe per cell for imaging) and whole-body quantitative imaging capability using clinically available scanners. For cell tracking with radionuclides, ex vivo direct cell radiolabeling, that is, radiolabeling cells before their administration, is the simplest and most robust method, allowing labeling of any cell type without the need for genetic modification. This Review covers the development and application of direct cell radiolabeling probes utilizing a variety of chemical approaches: organic and inorganic/coordination (radio)chemistry, nanomaterials, and biochemistry. We describe the key early developments and the most recent advances in the field, identifying advantages and disadvantages of the different approaches and informing future development and choice of methods for clinical and preclinical application.

The integration of nuclear imaging with nanomedicine is a powerful tool for efficient development and clinical translation of liposomal drug delivery systems. Furthermore, it may allow highly efficient imaging-guided personalised treatments. In this article, we critically review methods available for radiolabelling liposomes. We discuss the influence that the radiolabelling methods can have on their biodistribution and highlight the often-overlooked possibility of misinterpretation of results due to decomposition in vivo. We stress the need for knowing the biodistribution/pharmacokinetics of both the radiolabelled liposomal components and free radionuclides in order to confidently evaluate the images, as they often share excretion pathways with intact liposomes (e.g. phospholipids, metallic radionuclides) and even show significant tumour uptake by themselves (e.g. some radionuclides). Finally, we describe preclinical and clinical studies using radiolabelled liposomes and discuss their impact in supporting liposomal drug development and clinical translation in several diseases, including personalised nanomedicine approaches.

Imaging plays a key role in the preclinical evaluation of nanomedicine-based drug delivery systems and it has provided important insights into their mechanism of action and therapeutic effect. Its role in supporting the clinical development of nanomedicine products, however, has been less explored. In this review, we summarize clinical studies in which imaging has provided valuable information on the pharmacokinetics, biodistribution, and target site accumulation of nanomedicine-based drug delivery systems. Importantly, these studies provide convincing evidence on the uptake of nanomedicines in tumors, confirming that the enhanced permeability and retention (EPR) effect is a real phenomenon in patients, albeit with fairly high levels of inter- and intraindividual variability. It is gradually becoming clear that imaging is critically important to help address this high heterogeneity. In support of this notion, a decent correlation between nanomedicine uptake in tumors and antitumor efficacy has recently been obtained in two independent studies in patients, exemplifying that image-guided drug delivery can help to pave the way towards individualized and improved nanomedicine therapies.

Gammadelta T (γδ-T) cells are strong candidates for adoptive immunotherapy in oncology due to their cytotoxicity, ease of expansion, and favorable safety profile. The development of γδ-T cell therapies would benefit from non-invasive cell-tracking methods and increased targeting to tumor sites. Here we report the use of [89Zr]Zr(oxinate)4 to track Vγ9Vδ2 T cells in vivo by positron emission tomography (PET). In vitro, we showed that 89Zr-labeled Vγ9Vδ2 T cells retained their viability, proliferative capacity, and anti-cancer cytotoxicity with minimal DNA damage for amounts of 89Zr ≤20 mBq/cell. Using a mouse xenograft model of human breast cancer, 89Zr-labeled γδ-T cells were tracked by PET imaging over 1 week. To increase tumor antigen expression, the mice were pre-treated with PEGylated liposomal alendronate. Liposomal alendronate, but not placebo liposomes or non-liposomal alendronate, significantly increased the 89Zr signal in the tumors, suggesting increased homing of γδ-T cells to the tumors. γδ-T cell trafficking to tumors occurred within 48 hr of administration. The presence of γδ-T cells in tumors, liver, and spleen was confirmed by histology. Our results demonstrate the suitability of [89Zr]Zr(oxinate)4 as a cell-labeling agent for therapeutic T cells and the potential benefits of liposomal bisphosphonate treatment before γδ-T cell administration.

Chimeric antigen receptor T cell therapy (CAR-T) has been rolled out as a new treatment for hematological malignancies. For solid tumor treatment, CAR-T has been disappointing so far. Challenges include the quantification of CAR-T trafficking, expansion and retention in tumors, activity at target sites, toxicities, and long-term CAR-T survival. Non-invasive serial in vivo imaging of CAR-T using reporter genes can address several of these challenges. For clinical use, a non-immunogenic reporter that is detectable with exquisite sensitivity by positron emission tomography (PET) using a clinically available non-toxic radiotracer would be beneficial. Here, we employed the human sodium iodide symporter to non-invasively quantify tumor retention of pan-ErbB family targeted CAR-T by PET. We generated and characterized traceable CAR T cells and examined potential negative effects of radionuclide reporter use. We applied our platform to two different triple-negative breast cancer (TNBC) models and unexpectedly observed pronounced differences in CAR-T tumor retention by PET/CT (computed tomography) and confirmed data ex vivo. CAR-T tumor retention inversely correlated with immune checkpoint expression in the TNBC models. Our platform enables highly sensitive non-invasive PET tracking of CAR-T thereby addressing a fundamental unmet need in CAR-T development and offering to provide missing information needed for future clinical CAR-T imaging.

The ionophore 8-hydroxyquinoline (oxine) has been used to radiolabel cells and liposomal medicines with ¹¹¹In and, more recently, ⁸⁹Zr, for medical nuclear imaging applications. Oxine has also shown promising ionophore activity for the positron-emitting radionuclide ⁵²Mn that should allow imaging of labelled cells and nanomedicines for long periods of time (>14 days). However, to date, the radiometal complex formed and its full labelling capabilities have not been fully characterised. Here, we provide supporting evidence of the formation of [⁵²Mn]Mn(oxinate)₂ as the metastable complex responsible for its ionophore activity. The cell labelling properties of [⁵²Mn]Mn(oxinate)₂ were investigated with various cell lines. The liposomal nanomedicine, DOXIL® (Caelyx) was also labelled with [⁵²Mn]Mn(oxinate)₂ and imaged in vivo using PET imaging. [⁵²Mn]Mn(oxinate)₂ was able to label various cell lines with moderate efficiency (15–53%), however low cellular retention of ⁵²Mn (21–25% after 24 h) was observed which was shown not to be due to cell death. PET imaging of [⁵²Mn]Mn-DOXIL at 1 h and 24 h post-injection showed the expected pharmacokinetics and biodistribution of this stealth liposome, but at 72 h post-injection showed a profile matching that of free ⁵²Mn, consistent with drug release. We conclude that oxine is an effective ionophore for ⁵²Mn, but high cellular efflux of the isotope limits its use for prolonged cell tracking. [⁵²Mn]Mn(oxinate)₂ is effective for labelling and tracking DOXIL in vivo. The release of free radionuclide after liposome extravasation could provide a non-invasive method to monitor drug release in vivo.