Capturing early-stage single-cell ADCC response using a microfluidic lab-in-a-trench platform

Conventional antibody-dependent cell-mediated cytotoxicity (ADCC) assays based on measurement of Chromium-51 and lactate dehydrogenase yield target cell population-averaged ADCC outcomes after many hours and provide only one endpoint measure of cell death based on membrane integrity. These gold standard techniques do not adequately reflect single-cell response dynamics over short periods of time. To address these limitations, we present a simple ADCC assay that utilises a microfluidic Lab-in-a-Trench (LiaT) platform to dynamically capture peripheral blood mononuclear cell (PBMC)-mediated ADCC responses at a single-cell resolution at early timepoints (<60-min), while simultaneously providing analysis of two separate measurements of cell state (apoptosis and viability) via fluorescence-based measurements. The study demonstrates active immune-mediated therapeutic effects induced by the ADCC-capable anti-HER2 therapeutic monoclonal antibody trastuzumab against a HER2-positive breast cancer cell line (SKBR3) at short exposure times, highlighting a rapid onset ADCC response in target cell subpopulations. The impact of tyrosine kinase inhibitor (TKI)-pretreatment on the trastuzumab ADCC response was also studied. At assay commencement, the LiaT platform showed the irreversible pan-HER TKI neratinib induced a more widely distributed population of apoptotic and non-viable target cells compared to the reversible dual EGFR/HER2 TKI lapatinib, parameters that are not measured in standard ADCC assays. The PBMC-mediated ADCC response to trastuzumab was greater in lapatinib pre-treated target cells than neratinib-pre-treated cells. Together, these features demonstrate the LiaT platform’s ability to offer more comprehensive, high-throughput screening of novel ADCC-capable agents, and more specific real-time analysis of individual target cell phenotype in future studies.