Detection and characterization of circulating tumor cells (CTCs) may reveal insights into the diagnosis and treatment of malignant disease. Technologies for isolating CTCs developed thus far suffer from one or more limitations, such as low throughput, inability to release captured cells, and reliance on expensive instrumentation for enrichment or subsequent characterization. We report a continuing development of a magnetic separation device, the magnetic sifter, which is a miniature microfluidic chip with a dense array of magnetic pores. It offers high efficiency capture of tumor cells, labeled with magnetic nanoparticles, from whole blood with high throughput and efficient release of captured cells. For subsequent characterization of CTCs, an assay, using a protein chip with giant magnetoresistive nanosensors, has been implemented for mutational analysis of CTCs enriched with the magnetic sifter. The use of these magnetic technologies, which are separate devices, may lead the way to routine preparation and characterization of “liquid biopsies” from cancer patients.

Circulating tumor cells (CTCs) are established cancer biomarkers for the “liquid biopsy” of tumors. Molecular analysis of single CTCs, which recapitulate primary and metastatic tumor biology, remains challenging because current platforms have limited throughput, are expensive, and are not easily translatable to the clinic. Here, we report a massively parallel, multigene-profiling nanoplatform to compartmentalize and analyze hundreds of single CTCs. After high-efficiency magnetic collection of CTC from blood, a single-cell nanowell array performs CTC mutation profiling using modular gene panels. Using this approach, we demonstrated multigene expression profiling of individual CTCs from non–small-cell lung cancer (NSCLC) patients with remarkable sensitivity. Thus, we report a high-throughput, multiplexed strategy for single-cell mutation profiling of individual lung cancer CTCs toward minimally invasive cancer therapy prediction and disease monitoring.

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 107 per mL. In this work, we present a high-throughput full-length mRNA-seq protocol incorporating a magnetic sifter and magnetic nanoparticle-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 cell lines (NCI-H1650 and NCI-H1975) are spiked into whole blood, before being enriched for single-cell 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. In addition, while mRNA-seq data is typically only used for gene expression analysis of transcriptomic data, we demonstrate the use of full-length mRNA-seq chemistries like Smart-seq2 to facilitate variant analysis of expressed genes. This enables the use of mRNA-seq data for differentiating cells in a heterogeneous population by both their phenotypic and variant profile. In a simulated heterogeneous mixture of circulating tumor cells in whole blood, we utilize this high-throughput protocol to differentiate these heterogeneous cells by both their phenotype (lung cancer versus white blood cells), and mutational profile (H1650 versus H1975 cells), in a single sequencing run. This high-throughput method can help facilitate single-cell analysis of rare cell populations, such as circulating tumor or endothelial cells, with demonstrably high-quality transcriptomic data.

Numerous techniques for isolating circulating tumor cells (CTCs) have been developed. Concurrently, single-cell techniques that can reveal molecular components of CTCs have become widely available. We discuss how the combination of isolation and multigene profiling of single CTCs in our platform can facilitate eventual translation to the clinic.

Circulating tumor cells (CTCs) are currently widely studied for their potential application as part of a liquid biopsy. These cells are shed from the primary tumor into the circulation, and are postulated to provide insight into the molecular makeup of the actual tumor in a minimally invasive manner. However, they are extremely rare in blood, with typical concentrations of 1-100 in a milliliter of blood; hence, a need exists for a rapid and high-purity method for isolating CTCs from whole blood. Here, we describe the application of a microfabricated magnetic sifter toward isolation of CTCs from whole blood at volumetric flow rates of 10 mL/h, along with the use of a PDMS-based nanowell system for single-cell gene expression profiling. This method allows rapid isolation of CTCs and subsequent integration with downstream genetic profiling methods for clinical applications such as targeted therapy, therapy monitoring, or further biological studies.

Background: Circulating tumor cells (CTCs), defined as epithelial cells shed from a primary tumor into the bloodstream, are valuable prognostic, and possibly diagnostic, biomarkers that contain actionable genetic information for cancer treatment. Unfortunately, the rarity of CTCs in comparison to other blood components necessitates high-throughput separation technologies for efficient enrichment and practical downstream analysis. Moreover, genetic data extraction from CTCs currently suffers from a dearth of reliable analytical methods capable of handling low cell numbers. Technological innovations are urgently required to developing platforms that can help to optimize cancer management. Aim: To measure gene expression profiles of individual CTCs for cancer management via an integrated nanoscale platform. Methods: We have developed a protocol to effectively enrich rare cells via a magnetic sifting technology, whose methodology is based on using magnetic nanoparticles to tag CTCs in conjunction with magnetic filtration to enable high-throughput enrichment with release capability. This magnetic sifter offers 1) high capture efficiency at fast flow rates due to extreme field gradients at the pore edges, 2) high throughput due to the density of pores (∼200 pores/mm2), 3) scalability via standard lithographic fabrication, and 4) harvesting of viable cells. For subsequent characterization, a robust nanowell-based assay was designed to circumvent experimental errors associated with ensemble measurements through detection of mRNA transcripts directly from single CTCs (using one-step RT-PCR). Using standard photolithography, 25,600 nanowells are positioned on top of polydimethyl-siloxane (PDMS) and designed for subsequent RT-PCR reaction from a single CTC. These massive single-cell arrays are able to isolate up to thousands of individual NSCLC cells to measure gene expression. Also, this device is easily interrogated by conventional fluorescence microscopy to detect a candidate panel of genes on CTCs that are relevant for cancer detection or therapy monitoring. Nanowell is innovative as a low-cost (PDMS-based), easily scalable (from currently 25k to more than 100k nanowells), adjunctive solution to existing diagnostic methods. Results and Discussion: To date, we have assayed 23 NSCLC patients using the MagSifter & Nanowell and detected CTCs with valid biomarkers (hTERT and cMET). Each 4-mL whole blood sample was processed within one working day using the current workflow. From these samples, individual CTCs were assessed for hTERT and cMet expression. Single CTCs displaying hTERT only, cMet only, and both were evident upon fluorescent imaging. Direct comparison with CTC enumeration confirms better sensitivity by Nanowell assay, since the Nanowell utilizes PCR amplification in fluorescence as a signal generator. We believe this is the first demonstration of ex vivo visualization of gene expression from individual lung cancer CTCs. Citation Format: Seung-min Park, Dawson J. Wong, Chin Chun Ooi, Viswam S. Nair, Ophir Vermesh, Sang Hun Lee, Susie Suh, Luke P. Lee, Shan X. Wang, Sanjiv S. Gambhir. Gene expression profiling of individual circulating tumor cells from non-small cell lung cancer (NSCLC) patients via integrated nanotechnologies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-280. doi:10.1158/1538-7445.AM2015-LB-280

Microfluidic devices are widely used for applications such as cell isolation. Currently, the most common method to improve throughput for microfluidic devices involves fabrication of multiple, identical channels in parallel. However, this “numbering up” only occurs in one dimension, thereby limiting gains in volumetric throughput. In contrast, macrofluidic devices permit high volumetric flow rates but lack the finer control of microfluidics. Here, it is demonstrated how a micropore array design enables flow homogenization across a magnetic cell capture device, thus creating a massively parallel series of microscale flow channels with consistent fluidic and magnetic properties, regardless of spatial location. This design enables scaling in two dimensions, allowing flow rates exceeding 100 mL h−1 while maintaining >90% capture efficiencies of spiked lung cancer cells from blood in a simulated circulating tumor cell system. Additionally, this design facilitates modularity in operation, which is demonstrated by combining two different devices in tandem for multiplexed cell separation in a single pass with no additional cell losses from processing.