*2.4. Selection of CCK-2R-Expressing Human Cancer Cell Lines*

To identify the most suitable cell line for the in vitro evaluation of the radiolabeled ligand, CCK-2R protein expression was investigated in human cancer cell lines derived from tumors of different origins (non-small cell lung cancer, skin cancer, prostate cancer, breast cancer, and colon cancer). Western blot analysis revealed that CCK-2R was expressed to a certain extent by all the tested human cancer cell lines, with A549, PC3, and SK-BR-3 exhibiting the highest expression levels (Figure 1, panel A, B). Eventually, the A549 cell line was selected for the following experiments since, upon equal CCK-2R expression, it has the major advantage of ease of cultivation and efficiently forms tumors in nude mice xenograft models. Importantly, to determine whether CCK-2R was also expressed on the A549 cell surface, flow cytometry studies were performed on live cells. Cells were incubated with two different fluorescently-labelled anti-CCK-2R antibodies and, alternatively, with the fluorescent endogenous octapeptide CCK-8 (FAM-CCK-8), which binds CCK-2R with a subnanomolar affinity. Expression was confirmed, to some extent, using both antibodies and the natural ligand (Figure 1, panel C).

**Figure 1.** (**A**) Western blot and (**B**) quantification of the total cholecystokinin-2 receptor (CCK-2R) expression on different cell lines. GADPH served as the loading control for total proteins. (**C**) Levels of CCK-2R expression on cell surface by flow cytometry. Dead cells were removed from the analysis using LIVE/DEAD staining. The fluorescence intensity of control cells stained only with the LIVE/DEAD was subtracted. Mean fluorescence intensity plus SEM of three independent experiments is shown.
