Evaluation of single-cell heterogeneity and invasive potential in cancer cells via secreted protease activity assay.

BACKGROUND: This paper presents an assay for the secreted protease activity of single cancer cells isolated within chambers of a microfluidic array. The methodology builds on the dielectrophoresis and bipolar electrode (DEP-BPE) platform, enabling live-cell secretion analysis following selective, label-free DEP-based cell isolation. While many existing assays rely on population-level measurements or labeling strategies, there remains a critical need for label-free, functional tools that capture heterogeneity in live-cell secretion behavior at the single-cell level. RESULTS: The reported workflow allows for quantification of protease secretion behavior among individual cells, acquisition of temporal secretion profiles, and identification of highly invasive cells. Specifically, we assess the matrix metalloprotease 9 (MMP9) secretion of isolated cancer cells and report substantial heterogeneity in both secreted MMP9 concentration and secretion dynamics at the single-cell level. A linear fluorescence response (R2 = 0.98) enables quantification down to 3.31 nM MMP9 (5.4 × 105 molecules) in 280 pL volumes. In a mixed population, 34 of 132 cells (26 %) exhibited MMP9 secretion above background, and 14 cells secreted more than any untreated control cell, revealing a highly invasive subpopulation obscured in ensemble measurements. SIGNIFICANCE: This platform enables functional single-cell analysis of invasive potential, providing insights into cellular heterogeneity that are critical for evaluating disease progression and treatment response. The novelty of this work lies in its integration of single-cell isolation by DEP, which is selective for cell type, with a functional assay for a secreted enzyme. The approach is generalizable to selective isolation and molecular assay of many cell types.