76 Chapter 4 FUTURE PERSPECTIVES The prospect of ICI treatment strategies lies in our ability to translate our increasing understanding of the human immune responses in the tumor microenvironment to more effective treatments. PD-L1 assessment on tumor biopsies using IHC is unlikely to play a role in this respect2,3. PD-1/ PD-L1 imaging has shown to visualize whole-body PD-1/PD-L1 expression and accessibility. However, the presence of single immune checkpoints is likely an oversimplification of factors determining ICI therapy response. Therefore, PD-1/PD-L1 imaging may give a more accurate representation of whole-body PD-1/PD-L1 expression compared with IHC PD-L1, but its role as predictor of response remains to be determined. Next to pretreatment imaging, it would be very relevant to explore the feasibility of (repetitive) on-treatment imaging to assess the dynamics of PD-L1/PD-1 on anticancer treatment. Different types of radiotracers have already demonstrated their potential in the (pre)clinical setting to image PD-1/PD-L1, including intact monoclonal antibodies, nanobodies, and adnectins. Which one is most suitable depends on the research question. For example, smaller proteins, such as nanobodies and peptides, can be of growing interest because they are rapidly eliminated from the bloodstream and can reach the core of tumors, whereas antibodies may be more limited in their tissue-penetrating capacity because of their size48. Therefore, these molecules may be more suitable to answer fundamental research questions about the expression levels of immune checkpoints in tumors. On the other hand, intact therapeutic antibodies provide us with important information of the whole-body distribution of ICI and the accessibility of tumors for monoclonal antibodies in the clinical setting. In addition to PD-1/PD-L1 imaging, further insights into the fundamental mechanisms that regulate early aspects of T-cell activation will provide key information to better understand the variable ICI response rates. In this respect, some promising molecular imaging approaches have been reported recently16,60-62. Imaging CD8+ T-cells using antibody fragments allows for direct assessment of the location, density, and proximity of CD8+ T-cells, which is relevant because ICI invigorates CD8+ T-cell responses to specific tumor antigens63,64. Several clinical studies in lymphoma and solid malignancies (NCT03802123, NCT03610061) are ongoing; however, a tolerogenic tumor microenvironment can inhibit the activity of cytotoxic T-cells. Granzyme B is a serine-protease released from CD8+ T-cells and natural killer cells and is one of the key markers for immune cell activation. Granzyme B PET imaging using a targeted peptide, GZP, can visualize and quantify granzyme B; therefore, it allows for the identification of active tumor infiltrating lymphocytes to monitor the therapeutic efficacy of ICI treatment65. Currently, the in vivo visualization of active TILs is being evaluated using [18F]FAraG PET/MR in patients with urothelial carcinoma receiving neo-adjuvant anti-PD-1/PD-L1 ICI treatment (NCT03007719).