Most cancer deaths are caused by secondary tumors formed through metastasis, yet due to our limited understanding of this process, prevention remains a major challenge. Recently, cancer stem cells (CSCs) have been proposed as the source of metastases, but only little is known about their migratory behavior. Oxygen gradients in the tumor have been linked to directional migration of breast cancer cells. Here, we present a method to study the effect of oxygen gradients on the migratory behavior of breast CSCs using a microfluidic device. Our chip contains a chamber in which an oxygen gradient can be generated between hypoxic (<1%) and ambient (21%) conditions. We tracked the migration of CSCs obtained from MDA-MB-231 breast cancer cells, and found that their migration patterns do not differ from the average MDA-MB-231 population. Surprisingly, we found that the cells migrate towards low oxygen levels, in contrast with an earlier study. We hypothesize that in our device, migration is exclusively due to the pure oxygen gradient, whereas the effects of oxygen in earlier work were obscured by additional cues from the tumor microenvironment (e.g., nutrients and metabolites). These results open new research directions into the role of oxygen in directing cancer and CSC migration.

Cytotoxic T-cells (CTLs) exhibit strong effector functions to leverage antigen-specific anti-tumoral and anti-viral immunity. When naïve CTLs are activated by antigen-presenting cells (APCs) they display various levels of functional heterogeneity. To investigate this, we developed a single-cell droplet microfluidics platform that allows for deciphering single CTL activation profiles by multi-parameter analysis. We identified and correlated functional heterogeneity based on secretion profiles of IFNγ, TNFα, IL-2, and CD69 and CD25 surface marker expression levels. Furthermore, we strengthened our approach by incorporating low-melting agarose to encapsulate pairs of single CTLs and artificial APCs in hydrogel droplets, thereby preserving spatial information over cell pairs. This approach provides a robust tool for high-throughput and single-cell analysis of CTLs compatible with flow cytometry for subsequent analysis and sorting. The ability to score CTL quality, combined with various potential downstream analyses, could pave the way for the selection of potent CTLs for cell-based therapeutic strategies.