Extracellular Vesicles Provide a Real-Time Recording of Intracellular Signaling: EGFR receptor presence and implications for personalized therapy

Abstract

The epidermal growth factor receptor (EGFR) family, particularly EGFR and HER2, plays a critical role in cell signaling related to proliferation, survival, and progression of breast cancer. To better understand the causes of cancer aggressiveness, the extracellular signaling activity of EGFR was investigated in triple-negative breast cancer (TNBC) 4T1 cells, which, despite their classification, display wild-type EGFR and HER2 receptors similar to double-positive BT-474 breast cancer cells. Phosphorylation of cytoplasmic regions of EGFR and HER2 strongly correlate with patient susceptibility to anti-HER therapy. HER2 and EGFR signaling at the cell surface is followed by movement of the receptor complex into endosomes. It is unclear whether these endosomes become extracellular vesicles (EVs) that reflect the internal signaling events of the cells that release them. EVs may offer a way to eavesdrop on the ongoing internal signaling activity of the HER family receptors and could potentially be used to individualize therapy. 4T1 cells were treated with EGF, then EV isolation was performed through centrifugation, and remaining cells were lysed. EVs were purified using IZON columns before Western Blot analysis. Phosphorylation of EGFR (Y1068) and HER2 (Y877) was measured at different times after ligand stimulation and compared to the EVs released by the same cells. p-EGFR was clearly elevated in the EVs in a dose- and time-dependent manner. These results suggest that the phosphorylated state of HER family receptors in EVs can reflect internal signaling. This finding has clinical implications and may help guide future personalized therapies using EVs from tumor biopsies or blood samples.