Methods. We used lentivirus to transduce HEK293T cells to express a cancer-relevant antigen (PD-L1), with and without a GFP tag. We used image and flow cytometry to characterize the resulting cell cultures, and single vesicle flow cytometry (vFC) to measure vesicles released upon treatment with ionophore (A23187). The instrument (Northern Lights, Cytek) was calibrated to estimate molecules of GFP and antibody binding capacity (ABC) of cells and EVs, as well as vesicle size.
Results. We found that both the fraction of cells expressing detectable GFP (MOI-1: 20%; MOI-5: 85%; MOI-10: 95%), increased with the multiplicity of infection. We generated several single cell clones and characterized these with respect to their GFP and PDL1 surface expression. Individual clones expressed GFP as distributions with median intensities ranging from ~100,000-450,000 GFP molecules per cell, and PDL1-PE from ~40,000-450,000 ABC per cell, both of which increased with MOI.
We selected one high expressing clone from each cell line and expanded these for EV production. Cells were cultured in vesicle-free media, induced to release vesicles, and the media was processed by ultrafiltration (UF-100 kDa, Amicon) and the vesicles measured by vFC. Our instrument detected EVs with a size limit of detection (LOD) of ~50 nm, and the generation of ~100 EVs per cell after ionophore treatment, of which 50% express PD-L1 above the limit of detection of ~50 PE-MESF.
Conclusions. We used quantitative cytometry to characterize engineered cell lines expressing high amounts of PDL1, with and without a GFP tag, and the EVs released by the cells. Our current efforts are aimed at optimizing vesicle production in terms of uniformity and level of expression and production at commercial scale.
Supported by NIH CA272098-01.