The mice were then anaesthetized with 2% isoflurane gas and 1?L/min oxygen and whole-body SPECT images obtained using a NanoSPECT/CT four-head camera (Bioscan, Inc., Washington DC, USA) fitted with 1.4?mm pinhole collimators in helical scanning mode (24 projections, 40?min acquisition). patients with solid tumors. To accelerate progress and rapidly characterize emerging toxicities, systems that permit the repeated and non-invasive assessment of CAR T-cell bio-distribution would be invaluable. An ideal solution would entail the use of a non-immunogenic reporter that mediates specific uptake of an inexpensive, non-toxic and clinically established imaging tracer by CAR T cells. Here we show the utility of the human sodium iodide symporter (hNIS) for the temporal and spatial monitoring of CAR T-cell behavior in a cancer-bearing host. This system provides a clinically compliant toolkit for high-resolution serial imaging of CAR T cells in vivo, addressing a fundamental unmet need for future clinical development Zoledronic acid monohydrate in the field. Introduction Chimeric antigen receptors are genetically delivered fusion molecules that couple the binding of a native tumor-associated cell surface target to delivery of a bespoke T-cell activating signal1,2. Efficient disease control by CAR T cells has been exhibited in pre-clinical models representative of a broad range of cancer types1,3C9. Unprecedented clinical impact has been achieved in patients with refractory B-cell malignancies10C14, with complete remissions in heavily pre-treated patients highlighting the truly groundbreaking nature of this advance. Some patients do not gain benefit from CD19-targeted CAR T-cell therapy, exhibiting primary resistance. Appreciation of the frequency of disease relapse, either with CD19-positive or CD19-unfavorable disease, is growing SLC3A2 as more clinical experience is usually gained in CD19 expressing hematological malignancies13. Moreover, patients may endure severe side effects due to cytokine release syndrome (CRS), neurotoxicity, or on-target off-tumor toxicity that is frequently unanticipated prior to first in man evaluation15,16. The next anticipated breakthrough is the demonstration of meaningful efficacy of CAR T-cell immunotherapy in patients with solid tumors. This will require that CAR T cells can migrate to, penetrate and then persist in an active state within a tumor microenvironment that is profoundly immunosuppressive8,11,12,14,17C21. Given these considerations, pre-clinical and early clinical development of novel CAR T-cell therapies would be greatly facilitated if we could undertake repeated and reliable tracking of these cells after their infusion in animal studies and patients. An ideal approach would be noninvasive, cost-efficient and equally compatible with both small animal and Zoledronic acid monohydrate clinical imaging modalities22. Single-photon emission-computed tomography (SPECT/CT) tracking of indium-111 (111In) labeled CAR T cells provides a brief snapshot of the fate of adoptively transferred cells in vivo23,24. This approach gives good image resolution but is usually hampered by key limitations. First, due to isotope decay, signal is lost within 96?h. Second, labeling is usually agnostic to CAR expression by T cells. Thirdly, passive labeling does not report on cellular proliferation following adoptive transfer as signal is not maintained in daughter cells and, finally, activity may be mis-registered through phagocytosis of dying labeled cells. Co-expression of a CAR and a reporter gene within the same cell can overcome these limitations. Proof of concept was first exhibited using Zoledronic acid monohydrate the herpes simplex virus thymidine kinase 1 (HSV1tk) reporter, co-expressed with a CAR and luciferase reporter. This system enabled the serial imaging of CAR T cells using both positron emission tomography (PET) and bioluminescence imaging (BLI)25. More recently, PET imaging of HSV1tk+ CAR T cells has been achieved in patients with high-grade glioma26. However, HSV1tk is usually a viral protein which is usually immunogenic in man, favoring immune-mediated recognition and elimination of HSV1tk transduced T cells27. Use of a human reporter gene, such as norepinephrine receptor or hNIS, would overcome this concern28,29. As yet however, neither has been successfully adapted to achieve real-time imaging of CAR T cells. The somatostatin receptor type 2 (SSTR2) has been recently co-expressed with an intracellular adhesion molecule-1 directed CAR and imaged by PET-CT with gallium-68-labeled octreotide analog (68Ga-DOTATOC)30,31. However, this approach has two limitations. Firstly, the SSTR2 receptor internalizes on conversation with its substrates, risking poor sensitivity, especially at lower reporter gene expressing cell density32. Secondly, SSTR2 is usually expressed on T cells and other immune cells33, accounting for the ability of octreotide analogs and their radiolabeled derivatives to inhibit T-cell function34. This is clearly undesirable for a broadly applicable strategy to image therapeutic T-cell products in cancer patients. The hNIS gene is usually localized on.