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

TL;DR

This paper introduces a new microfluidic platform to study extracellular vesicles from individual breast cancer cells and how they change under drug treatment.

Contribution

The study introduces a high-density microchamber array platform for analyzing EVs from single cells under drug treatment.

Findings

Two breast cancer cell lines secrete EVs with distinct levels of tetraspanins and HSPs.

Drug treatment increases HSP90- and HSP70-positive EVs in both cell lines.

EV subpopulations differ between cell lines, suggesting different biogenesis pathways for HSP90-loaded EVs.

Abstract

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.

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