Human KISS1R Stable Cell Line-CHO-K1

Kisspeptins (KPs) and their receptor (KISS1R) promote metastasis and tumor progression in various cancers, such as triple-negative breast cancer (TNBC). Targeting KISS1R holds great promise for the molecular imaging and targeted radionuclide therapy of aggressive, metastatic cancers. Here, researchers synthesized high-purity N-terminally functionalized conjugates of KP-10, KP-54, and the hybrid peptide KiSS-34 (AMBA-2-Nal-Gly-Leu-Arg-Trp-NH2)-including scrambled sequence controls-for comparative study. Conjugation of these peptides with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and Alexa-Fluor-488 (AF-488) preserved their biological activity; this was confirmed by calcium mobilization assays in CHO-KISS1R-transfected cells, which yielded EC50 values ​​in the sub-nanomolar to nanomolar range (0.05–0.85 nM). Conventional target detection methods using antibodies (Abs) and AF-488-KPs failed to successfully visualize KISS1R in model cell lines (CHO-KISS1R) and native cancer cell lines, likely due to non-specific antibody binding and rapid KISS1R internalization following agonist stimulation. However, live-cell imaging using AF-488-KP-10 and the novel analog AF-488-KiSS-34 successfully captured the rapid internalization process of KISS1R. Furthermore, internalization assays were conducted following efficient radiolabeling of DOTA-KPs with Lu-177; the results demonstrated that [177Lu]Lu-DOTA-KiSS-34 exhibited the highest uptake and internalization rates in CHO-KISS1R cells compared to the KP-10 analogs.

The researchers hypothesized that agonist stimulation triggers rapid endocytic dynamics in KISS1R, posing methodological and experimental challenges. Consequently, they employed live-cell imaging to study KISS1R and successfully observed the rapid endocytic dynamics of CHO-KISS1R-transfected cells following treatment with AF-488-KP-10. The results showed that AF-488-KP-10 rapidly accumulated at the plasma membrane of CHO-KISS1R cells and was subsequently internalized into vesicular structures within 60 minutes (Figure 1). This process was accompanied by distinct co-localization with wheat germ agglutinin (WGA).

Figure 1. Binding and endocytosis of AF-488-KP-10 via the KISS1R receptor in CHO-KISS1R-transfected cells; cells were treated with 100 nM AF-488-KP-10 (green) and analyzed by live-cell microscopy over 60 minutes. The merged image shows the plasma membrane and endocytic vesicles, WGA (yellow), and nuclei (blue). (Taş H, et al., 2026)