Marrying Nanomagnetics with RNA Sequencing of Single Cancer Cells

Single-cell characterization techniques, such as mRNA-seq, have been applied to a diverse range of applications in cancer biology, yielding great insight into mechanisms leading to therapy resistance and tumor clonality. While single-cell techniques can yield a wealth of information, a common bottleneck is the lack of throughput, with many current processing methods being limited to the analysis of small volumes of single cell suspensions with cell densities on the order of 10
⁷
per mL. In this work [1], we present a highthroughput full-length mRNA-seq protocol incorporating a magnetic sifter and magnetic nanoparticle (MNP)-antibody conjugates for rare cell enrichment, and Smart-seq2 chemistry for sequencing. We evaluate the efficiency and quality of this protocol with a simulated circulating tumor cell system, whereby non-small-cell lung cancer (NSCLC) cell lines (NCI-H1650 and NCI-H1975) are spiked into whole blood, before being enriched for singlecell mRNA-seq by EpCAM-functionalized magnetic nanoparticles and the magnetic sifter. We obtain high efficiency (>90%) capture and release of these simulated rare cells via the magnetic sifter, with reproducible transcriptome data, enabling highly predictive cancer diagnostics and treatment. We demonstrate the first integration of a high-throughput immunomagnetic cell separation platform (magnetic sifter), with a 96-well microplate-based single-cell full-length mRNA-seq technique (Smart-seq2 [2]), enabling rapid enrichment and sequencing of rare cells. This speed allows Smart-seq2 sequencing of a rare subset of cells (~10
²
) from a background of ~10
⁹
cells to start in under 4 hours. In contrast, recent notable papers reporting singlecell sequencing of circulating tumor cells (a notable rare cell of biological and clinical interest) have required longer overall processing times, separately due to incubation times [3], slower volumetric processing [4], or manual cell picking [5]. Our validation studies also demonstrate that our mRNA-seq data is of comparable quality to previous single-cell studies [6]. Additionally, we describe variant analysis on mRNA-seq data, capitalizing on the full-length reads from Smart-seq2, and illustrate how a combination of expression and variant analysis on single cells can help differentiate heterogeneous cells, in contrast to the current paradigm of thinking solely in terms of either variant (DNA-based) or expression (RNA-based) analysis. This is of interest to the single-cell sequencing community as it illustrates the strength of full-length mRNA-seq methods relative to 3'-biased methods, and might be a key consideration for future single-cell mRNA-seq studies. In summary, we present a unique MNP-based method of broad interest to the multidisciplinary research communities of cell separation and single-cell sequencing. We demonstrate a cell separation platform (magnetic sifter) that enables rapid single-cell sequencing of rare cells in complex biological systems, and envision this method would facilitate time-sensitive studies in areas such as immune or cancer biology, and liquid biopsies in diagnostics and immuno-therapy. This work was supported by US NIH through the Center for Cancer Nanotechnology Excellence (U54CA151459) and the Innovative Molecular Analysis Technologies (R33CA138330). CCO was supported by an A*STAR fellowship (Singapore).