The use of mass cytometry (CyTOF) to evaluate the cellular uptake of stable radiopharmaceutical surrogates in single cells: a proof-of-concept study

Abstract Background Current methods for assessing radiopharmaceutical uptake are based on scintillation counting (e.g., γ-counting) or imaging techniques, such as positron emission tomography (PET) and single photon emission computed tomography (SPECT), which evaluate the average radiopharmaceutical uptake in biological samples. Single-cell analysis of radiopharmaceutical uptake and biodistribution would provide a better understanding of the possible heterogeneous uptake in cellular populations and organs, with the potential to improve current dosimetry and toxicity assessments. In this proof-of-concept study, we evaluate the use of mass cytometry (CyTOF) as an analytical tool to study the uptake of non-radioactive radiopharmaceutical surrogates at the single-cell level. Results The preclinical immunoconjugate DOTA-cAC10, targeting CD30 (a receptor overexpressed in lymphomas), was labeled either with radioactive lutetium-177 ( 177 Lu) resulting in the [ 177 Lu]Lu-DOTA-cAC10 radioimmunoconjugate, or stable lutetium-175 ( 175 Lu), yielding the surrogate [ 175 Lu]Lu-DOTA-cAC10. For CyTOF experiments, cells were incubated with the stable surrogate and an iridium DNA intercalator (Cell-ID™) to enable concurrent determination of immunoconjugate uptake and cell identification. CyTOF analysis of the surrogate was performed in three T-cell lymphoma cell lines with varying CD30 expression (Karpas 299, Myla and Jurkat) and compared to γ-counting data obtained for the radioimmunoconjugate. Radioimmunoconjugate [ 177 Lu]Lu-DOTA-cAC10 cellular uptake studies by γ-counting showed receptor-dependent accumulation, with Karpas 299 cells exhibiting the highest uptake levels (32.5 ± 1.6% of total added activity), followed by Myla (20.0 ± 1.0%) and Jurkat cells (15.7 ± 1.2%). Mass cytometry enabled analysis of the stable surrogate [ 175 Lu]Lu-DOTA-cAC10 uptake at single-cell resolution, revealing median signal intensities of 175 Lu of 32.2, 14.0, and 2.94 a.u. for Karpas 299, Myla, and Jurkat cells, respectively. Conclusions The sensitivity of CyTOF enabled discrimination of uptake between cell models even at low metal-to-antibody stoichiometric ratios. It also revealed intercellular variability in uptake that can only be captured with single-cell methods. Overall, we showed that CyTOF is a robust, high-throughput, multiplexed approach for characterizing the cellular uptake of stable radiopharmaceutical surrogates at single-cell resolution, paving the way for future studies.