A High-Density Microchamber Array for the Analysis of Extracellular Vesicles Derived from Single Cells under Drug Treatment

Extracellular vesicles (EVs) are key players in cancer development and drug resistance. For example, heat shock protein 90 (HSP90) carried via EVs from secreting cancer cells to distant recipient cells mediates apoptosis and metastasis. Here, we study EV secretion from individual breast cancer cells and the changes under treatment with the HSP90-inhibiting cancer drug tanespimycin (17AAG). We introduce a two-layer microfluidic platform with an array of microchambers that coencapsulate single cancer cells with functionalized beads for the capturing and immunostaining of EVs. Microchambers are created by pressurizing a microfluidic layer with densely packed, round openings, which are aligned with cell- and bead-traps. This new design facilitates the isolation of cells in over 5100 microchambers and efficient EV capture. We characterize the EV secretion of two breast cancer cell lines: triple-negative MDA-MB-231 cells and HER2-positive SkBr3 cells secrete EVs that carry distinguishable levels of tetraspanins (CD9, CD63, and CD81) and HSPs (-90 and -70). Upon drug treatment, the signals for HSP90- and HSP70-positive EVs increase for both cell lines. However, analysis of protein colocalization on the EV surface revealed a significant difference in EV subpopulations: while MDA-MB-231 cells have no HSP90 on CD63-positive EVs, these two markers are colocalized on SkBr3-derived EVs, indicating different intracellular biogenesis pathways for HSP90-loaded EVs. Moreover, our results emphasize that using CD63 as the sole EV capture protein may hide important EV subpopulations. Overall, our platform may support future choices of EV biomarkers for diagnostic and biomedical purposes and help in understanding the heterogeneous drug response of cancer cells.