Dynamic SPECT Imaging Kinetic Modelling Characterises Tracer Influx and Efflux in Tumour-Infiltrating CAR T Cells

Chimeric Antigen Receptor (CAR) T cell therapies for solid tumours face challenges regarding inefficient trafficking to the tumour site, limited extravasation and impaired persistence within an immunosuppressive microenvironment. Non-invasive imaging approaches capable of capturing the dynamic behaviour and biodistribution of CAR T cells are therefore needed to better understand these barriers. This study explores the use of compartmental modelling to interpret technetium-99 m pertechnetate ([99mTc]TcO4−) uptake by CAR T cells co-expressing the sodium iodide symporter (NIS) reporter gene in a prostate cancer NSG mouse model using single-photon emission computed tomography computerised tomography (SPECT/CT). Co-expression of hNIS with a prostate-specific membrane antigen (PSMA) targeting CAR did not impair CAR T cell function and enabled non-invasive visualisation of CAR-hNIS T cell infiltration in vivo. Dynamic SPECT/CT imaging revealed clear differences in tumour uptake between CAR-hNIS treated mice and control groups. Compartmental modelling using a reversible two-compartment framework demonstrated increased tracer influx and reduced tracer efflux in CAR-hNIS treated tumours. These kinetic changes indicate that tumour signal arises from NIS-mediated tracer uptake and delayed tracer washout in tumours containing infiltrating CAR T cells, rather than from passive perfusion or non-specific delivery alone. Immunohistochemistry confirmed CAR T cell infiltration in the tumours. This work establishes a proof-of-concept for applying kinetic modelling to interpret CAR T cell infiltration in vivo using dynamic hNIS-based SPECT imaging. By distinguishing tracer delivery from tracer washout kinetics and correcting for variability in tracer bioavailability, this approach provides a more sophisticated biological interpretation of tumour uptake than static imaging alone. These findings highlight the potential of integrating kinetic modelling into future quantitative cell-tracking strategies to improve the evaluation and optimisation of CAR T cell therapies for solid tumours.