Recently, we demonstrated a robust increase of CCL2 in the brain tissue of canine patients bearing high-grade glioma.<h4>Methods</h4>We performed a series of in vitro experiments using canine Tregs and patient-derived canine glioma cell lines (GSC 1110, GSC 0514, J3T-Bg, G06A) to interrogate the CCL2-CCR4 signaling axis in the canine.<h4>Results</h4>We established a flow cytometry gating strategy for identification and isolation of FOXP3<sup>+</sup> Tregs in dogs. Here, CCL2 is linked to glioma.