Assessment of in vitro interactions between eosinophils and cancer cells by imaging-enhanced flow cytometry and...

Eosinophils are a rare immune cell subset with important roles in Th2 immunity and cancer. Interleukin IL-33 (IL-33) plays important functions in the survival and activation of eosinophils. Recent evidence unraveled the role of IL-33 in activating the anti-tumor activities of eosinophils enhancing their degranulation and tumor cell killing in a contact-dependent manner. We propose a dual approach methodology to extrapolate the physical interactions of eosinophils with tumor cells as a result of eosinophil stimulation. Human eosinophils isolated from the blood of healthy donors by dextran sedimentation followed by magnetic sorting are exposed to either IL-33 (Eos33) or IL-5 (Eos5) for 18 h. These pre-conditioned cells are then co-cultured with A375P melanoma cells to monitor cell-cell interactions. Acoustic focusing flow cytometry analysis is employed to evaluate the presence of eosinophil-tumor cell conjugates after 1 h incubation of human eosinophils and A375P melanoma cells. Moreover, a 24 h time-lapse video recording approach is employed to obtain single cell tracking eosinophil profiles. This allows to quantitatively determine the interaction extent of Eos33, as opposed to Eos5, with tumor cells. In conclusion, our protocols easily and quickly allow the extrapolation of relevant kinematic and biologically relevant parameters for tumor reactive eosinophils. Furthermore, these methods are adaptable to various models with other types of immune cell subsets and cancer cells and can be implemented on different video microscopy platforms and advanced flow cytometry systems.