Detection of radiopharmaceuticals and their cold surrogates by Imaging Mass Cytometry enables assessment of single-cell functional response, therapeutic biodistribution, and modulation of the immune microenvironment

Radiopharmaceuticals (RPT) can improve therapeutic responses while decreasing adverse reactions through targeted radiation delivery to the tumor. While localized radionuclide uptake can currently be measured, these techniques lack the resolution to examine biodistribution at the single-cell level or capacity to simultaneously assess multiple measures of response. Here, we show that Imaging Mass Cytometry (IMC), a technique combining immunostaining with laser ablation-enabled inductively coupled plasma mass spectrometry, can measure the spatial distribution of RPT metal, as well as the therapeutic response within multiplexed measurements. Testing the abilities and limitations of this approach, we benchmarked IMC and autoradiography readouts, compared isotope-labelling approaches and dosing necessary for IMC detection, and provide examples of a variety of unique single cell readouts of RPT distribution and cellular response. Using both hot and cold labelling strategies, we measured the distribution of either carrier-added metal or cold surrogate and matched target for two tumor-directed RPTs, seeing clear differences in on- and off-target distributions within the tumor. Metal from carrier added [ 177 Lu]Lu-DOTA-RW03, a fully humanized CD133 antibody, co-localized with CD133 expression in certain regions of the tumor, with CD133 + RPT metal - regions interspersed. A cold analog of an engineered antibody fragment against the prostate stem cell antigen ( 169 Tm-PSCA A2DM) was observed in both the tumor mass and peripheral areas of collagen-rich stroma in a syngeneic model using human PSCA expressing cells. Over a 28-day time course, on- versus off-target ratios improved with time, and varying therapeutic dose was shown to change single cell RPT amounts with analog doses as low as 10 µg detected by IMC, highlighting its sensitivity. Diving deeper, IMC enabled functional assessments of response showing higher single-cell positivity for markers of DNA damage and apoptosis in regions with higher RPT exposure. Finally, we show the ability of IMC to assess the RPT-initiated immune response and impact of radioisotope selection by comparing lymphocyte and myeloid cell infiltration in response to alpha or beta emitters conjugated to the same targeting reagent. These findings show the potential of IMC quantification of RPT dose, distribution, and response, which will expand our understanding of localized absorbed dose, how radioisotope selection impacts response, and inform future pre-clinical therapeutic design.