Head-to-head comparison of nuclear imaging approaches to quantify tumor CD8+T-cell infiltration
Authors: Sandker Gerwin G.W. , Raavé René , Antunes Inês F. , Boswinkel Milou , Cornelissen Lenneke , Franssen Gerben M. , Molkenboer-Kuenen Janneke , Wierstra Peter J. , Hagemans Iris M. , de Vries Erik F.J. , Bussink Johan , Adema Gosse , Verdoes Martijn , Aarntzen Erik H.J.G. , Heskamp Sandra 
Published: October 2024

AbstractMany immunotherapies focus on (re)invigorating CD8+T cell anti-cancer responses and different nuclear imaging techniques have been developed to measure CD8+T cell distributions.In vivolabeling approaches using radiotracers primarily show CD8+T cell distributions, whileex vivolabeled CD8+T cells can show CD8+T cell migration patterns, homing, and tumor infiltration. Currently, a comprehensive head-to-head comparison ofin vivoandex-vivocell labeling with respect to their tumor and normal tissue targeting properties and correlation to the presence of CD8+T cells is lacking, yet essential for correct interpretation of clinical CD8+imaging applications. Therefore, we performed a head-to-head comparison of three different CD8+T cell imaging approaches: 1)89Zr-labeled DFO-conjugated Fc-silent anti-CD8 antibody ([89Zr]Zr-anti-CD8-IgG2asilent), 2)ex vivo89Zr-oxine labeled ovalbumin-specific CD8+T cells ([89Zr]Zr-OT-I cells), and 3)18F-labeled IL2 ([18F]AlF-RESCA-IL2).MethodsB16F10/OVA tumor-bearing C57BL/6 mice (n=10/group) received intravenously one of the three radiopharmaceuticals. PET/CT images were acquired starting 72 h ([89Zr]Zr-anti-CD8-IgG2asilent), 24 and 48 h ([89Zr]Zr-OT-I cells), and 10 min ([18F]AlF-RESCA-IL2) post injection. Subsequently,ex vivobiodistribution analysis of the radiopharmaceuticals was performed followed by flow cytometric analysis to evaluate the number of intratumoral CD8+T cells. Additionally, the intratumoral radiolabel distributions was assessed by autoradiography and immunohistochemistry (IHC) on tumor slices.Results[89Zr]Zr-anti-CD8-IgG2asilent,[89Zr]Zr-OT-I cells, and [18F]AlF-RESCA-IL2 showed uptake in CD8-rich tissues, with preferential targeting to the spleen. Biodistribution analysis showed tumor uptake above blood level for all radiopharmaceuticals, except [18F]AlF-RESCA-IL2. For all three approaches, the uptake in the tumor-draining lymph node was significantly higher compared with the contralateral axial lymph node, suggesting that all approaches allow evaluation of immune responses involving CD8+T cells. Tumor uptake of [89Zr]Zr-anti-CD8-IgG2asilent(R2=0.65, p<0.01) and [89Zr]Zr-OT-I cells (R2=0.74, p<0.01) correlated to the number of intratumoral CD8+T cells (flow cytometry). The intratumoral distribution pattern of the radiosignal was different forex vivoandin vivoradiolabeling techniques. The short half-life of18F precluded autoradiography assessment of [18F]AlF-RESCA-IL2.ConclusionWe show that [89Zr]Zr-anti-CD8-IgG2asilentand [89Zr]Zr-OT-I cells PET/CT imaging can be used to evaluate intratumoral CD8+T cells, even though their normal tissues and intratumoral distribution patterns are significantly different. Based on their characteristics, [89Zr]Zr-anti-CD8-IgG2asilentmight be most useful to immunophenotyping the TME, while theex vivocell labeling approach visualizes CD8+T cell migrations patterns and the permissiveness of tumors for invasion, whereas [18F]AlF-RESCA-IL2 allows for rapid recurrent imaging and might prove useful for tracking rapid changes in CD8+T cell distributions. In conclusion, our head-to-head comparison of the three prototype CD8+T cell labeling approaches provides new insights which can aid in correct interpretation of clinical CD8 imaging and may guide in the selection of the optimal imaging approach for the research question of interest.