Human CXCR2 Stable Cell Line-HEK293

Interleukin-8 receptor β (CXCR2) is a highly promising target for molecular imaging of inflammation and inflammatory diseases. This is because it is expressed almost exclusively on neutrophils. Here, researchers designed modified fluorinated ligands based on a squaramide template and explored various modification sites and synthetic strategies. These promising candidates were then tested for their affinity for CXCR2 using a nano-BRET competition assay, ultimately yielding the tracer candidate 16b. Because direct labeling with 18F using established tosyl chemistry failed to yield the desired radiotracer, an indirect labeling approach was developed. Using tert-butyl (S)-3-(tosyloxy)pyrrolidinecarboxylate and pentafluorophenol ester, the radiotracer [18F]16b was obtained in 15% radiochemical yield. The specificity of the radiotracer was subsequently confirmed by time-dependent uptake of [18F]16b in CXCR2-negative and CXCR2-overexpressing human embryonic kidney cells. Further studies using human neutrophils demonstrated the potential of this tracer for diagnostic imaging of neutrophil function.

To assess CXCR2 specificity, researchers used recombinant human embryonic kidney (HEK293) cells stably overexpressing hCXCR2 (HEK-CXCR2). [18F]16b uptake by hCXCR2-overexpressing HEK293 cells was measured over time in the presence and absence of the reference ligand 10 and the commonly used CXCR2-specific inhibitor AZD5069 (Figure 1A). Cells were pretreated with vehicle, AZD5069, or 10 for 30 minutes before the addition of [18F]16b. For hCXCR2-overexpressing HEK293 cells, vehicle-treated cells showed significant [18F]16b uptake, while both AZD5069- and 10-treated cells inhibited uptake of the developed radiotracer. No uptake was observed in CXCR2-negative HEK293 cells (Figure 1A). Therefore, compound [18F]16b can be considered a specific ligand for CXCR2. To further validate the potential application of [18F]16b as a neutrophil marker, it was incubated with human and mouse peripheral blood cells. Similar to the HEK-CXCR2 experiments, AZD5069 was used as a CXCR2-specific blocker to examine the specific uptake of [18F]16b. In human neutrophils, [18F]16b was rapidly taken up in a time-dependent manner. This uptake could be blocked by AZD5069, demonstrating the CXCR2 specificity of [18F]16b in human neutrophils. In contrast, in mouse neutrophils, no significant difference in uptake was observed between the control and AZD5069-blocked groups. Furthermore, the level of uptake was significantly lower than that in human neutrophils (Figure 1B), strongly suggesting species differences.

Figure 1. Uptake of [18F]16b in different cell types. (Spatz P, et al., 2024)