(487c) Nanoscale Hydrogel Coatings for Rapid Sorting of Circulating Tumor Cells: Relating Marker Expression to Isolation Yield

A link between the presence of tumor cells in systemic circulation and metastatic cancer progression has long been established. Observations of these circulating tumor cells (CTCs) has prompted efforts to develop a fluid biopsy, with the goal of detecting these rare cells in patient peripheral blood as surrogate markers of metastatic disease for partial replacement or supplement to invasive tissue biopsies. While such an assay could potentially provide valuable prognostic information among numerous other clinical advantages, the implementation of fluid biopsies has been critically hindered by current available technologies for rare cell isolation. Numerous platforms have been designed; most commonly magnetically-activated cell sorting (MACS), fluorescent-activated cell sorting (FACS), and a myriad of antigen-based microfluidic capture technologies. Yet, these have generally failed to support a reliable fluid biopsy assay, due to poor performance parameters such as low throughput, low purity of enriched antigen positive cells, and insufficiently low detection thresholds to detect poor expressed surface markers of target cell  populations. In this work, we build on a recently developed rapid cell sorting technique called Antigen Specific Lysis (ASL) based on photo-crosslinked polymer film encapsulation to isolate tumor cells in suspension. Briefly, antibodies direct the specific binding of streptavidin-eosin-5-isothiocyanate (SA-EITC, type II photoinitiator) to surface markers of intact cells, then upon immersion in an aqueous monomer solution, visible light initiates surface-mediated radical crosslinking of nanoscale PEG diacrylate hydrogel films solely around the antigen positive population. Finally, anionic surfactants lyses virtually 100% of uncoated cells while fully encapsulated cells remain protected, and are then easily collected by centrifugation. This sorting process has shown >90% viability of sorted cells, and can be performed in 1-2 hours with inexpensive and disposable labware. Further, because this technique is not dependent on microfluidics or spatial mobility of target cells, we expect it is fundamentally capable of accommodating very large batch sized (10¹¹ cells) with only marginal changes in processing time. Here, we focus on determining the response of process yield on the level of photoinitiator loading on target cell. The surface-mediated gelation response has been shown to be dependent on the density of photoinitiator at the initiating surface – therefore, we posit this is a critical parameter to assess for attaining high processing yield, particularly during the lysis-based negative depletion. Further, a lower initiator density threshold is seen in these surface initiated PEG diacrylate films, so that a lower threshold of marker expression for SA-EITC loading will determine successful coating and, ultimately, isolation of target populations. Particularly for CTC isolation, recent evidence suggests that the most highly potent and aggressive CTCs often downregulate epithelial markers that are commonly targeted for cell isolation, further highlighting the need to determine the effects of expression level on performance of this system. To methodically simulate lower expression levels of epithelial markers, we immunolabel Epithelial Cell Adhesion molecule (EpCAM) on a non-small cell lung cancer line H358 with biotin antibodies, then competitively bind incrementally varied ratios of streptavidin and streptavidin-photointiators and evaluate the percent yield of isolated cells versus relative photoinitiator loading determined by flow cytometry. We expect these data will inform robust targeting strategies with optimal yield performance to allow the development of ASL as reliable tumor cell isolation platform for clinical use.