Fluorometric Quantification of Single-Cell Velocities to Investigate Cancer Metastasis.

Hematogenous metastasis is a multistep, selectin-regulated process whose mechanisms remain poorly understood. To investigate this biological pathway of cancer dissemination and better understand circulating cancer cells, we developed a high-throughput methodology that integrates organ-on-chip-like microfluidic and photoconvertible protein technologies. Our approach can ascribe single-cell velocity as a traceable cell property for off-chip analysis of the direct relationships between cell molecular profiles and adhesive phenotypes in the context of physiologically relevant fluid flow. We interrogate how natively expressed selectin ligands relate to colon cancer cell rolling frequencies and velocities and provide context for previously reported disparities in in vitro and in vivo models of selectin-mediated adhesion and metastasis. This integrated methodology represents a versatile approach for the development of anti-metastatic therapeutics as well as to generate and test mechanistic hypotheses regarding spatiotemporal processes that occur over timescales of seconds to hours with single-cell resolution.

P-, but not E- or L-, selectin-mediated rolling adhesion persistence in hemodynamic flow diverges between metastatic and leukocytic cells.

The ability of leukocytic cells to engage selectins via rolling adhesion is critical to inflammation, but selectins are also implicated in mediating metastatic dissemination. Using a microfluidic- and flow-based cell adhesion chromatography experimental and analytical technique, we interrogated the cell-subtype differences in engagement and sustainment of rolling adhesion on P-, E-, and L-selectin-functionalized surfaces in physiological flow. Our results indicate that, particularly at low concentrations of P-selectin, metastatic but not leukocytic cells exhibit reduced rolling adhesion persistence, whereas both cell subtypes exhibited reduced persistence on L-selectin and high persistence on E-selectin, differences not revealed by flow cytometry analysis or reflected in the extent or velocity of rolling adhesion. Conditions under which adhesion persistence was found to be significantly reduced corresponded to those exhibiting the greatest sensitivity to a selectin-antagonist. Our results suggest that potentially therapeutically exploitable differences in metastatic and leukocytic cell subtype interactions with selectins in physiological flow are identifiable through implementation of functional assays of adhesion persistence in hemodynamic flow utilizing this integrated, flow-based cell adhesion chromatography analytical technique.