An integrated enrichment system to facilitate isolation and molecular characterization of single cancer cells from whole blood

Abstract

Circulating tumor cells (CTCs) carry valuable biological information. While enumeration of CTCs in peripheral blood is an FDA-approved prognostic indicator of survival in metastatic prostate and other cancers, analysis of CTC phenotypic and genomic markers is needed to identify cancer origin and elucidate pathways that can guide therapeutic selection for personalized medicine. Given the emergence of single-cell mRNA sequencing technologies, a method is needed to isolate CTCs with high sensitivity and specificity as well as compatibility with downstream genomic analysis. Flow cytometry is a powerful tool to analyze and sort single cells, but pre-enrichment is required prior to flow sorting for efficient isolation of CTCs due to the extreme low frequency of CTCs in blood (one in billions of blood cells). While current enrichment technologies often require many steps and result in poor recovery, we demonstrate a magnetic separator and acoustic microfluidic focusing chip integrated system that enriches rare cells in-line with FACS™ (fluorescent activated cell sorting) and single-cell sequencing. This system analyzes, isolates, and index sorts single cells directly into 96-well plates containing reagents for Molecular Indexing (MI) and transcriptional profiling of single cells. With an optimized workflow using the integrated enrichment-FACS system, we performed a proof-of-concept experiment with spiked prostate cancer cells in peripheral blood and achieved: (i) a rapid one-step process to isolate rare cancer cells from lysed whole blood; (ii) an average of 92% post-enrichment cancer cell recovery (R² = 0.9998) as compared with 55% recovery for a traditional benchtop workflow; and (iii) detection of differentially expressed genes at a single cell level that are consistent with reported cell-type dependent expression signatures for prostate cancer cells. These model system results lay the groundwork for applying our approach to human blood samples from prostate and other cancer patients, and support the enrichment-FACS system as a flexible solution for isolation and characterization of CTCs for cancer diagnosis. © 2018 International Society for Advancement of Cytometry.

Keywords: CTC; cell sorting; in-line enrichment; single cell RNA sequencing.

Figure 1.

In-line enrichment system. (A) Image of the in-line enrichment system connected directly to the BD FACSMelody™ sorter. The instrument located at the left is a BD FACSMelody™ sorter and the smaller instrument on the right is the enrichment system. (B) Schematic drawing of the in-line enrichment system connected to the flow cytometer. Key parts of FACS sorter are represented in the dashed box. Outside the box, key elements of the enrichment system are highlighted. The entire fluidics system is driven by air so there are three air sources in the system–positive pressure to the sample tube, wash tube, or waste tube. Sample is pressurized and pushed through the magnetic separator and AF chip in sequential order. If negative depletion is not needed, then an alternative configuration could bypass the magnet and process the sample directly in the AF chip. The outlet of the AF chip is connected to the flow cell of the sorter. Inserted images: 1) Magnetic separation tubing containing magnet-labeled cells (brownish color) in the process of being magnetically pulled to the sides of the tubing. The tubing allows continuous separation of WBCs in 50 ml of whole blood without dropping separation efficiency (data not shown). The tubing can be moved in and out from the magnetic separator to allow capture of magnetically labeled cells and back flushing of the captured cells. 2) Channel of AF chip showing acoustic washing. Polystyrene beads suspended in a tracking blue dye solution are focused into the wash buffer in the center of the channel and as a consequence of washing, the blue dye solution flows to the side channel and ends up going to the waste). Green bars in the fluidic paths are flow meters that read the volumetric flow rate in μl/min real time. Valves are indicated by opposing triangles.

Figure 2.

Performance characterization of the in-line enrichment system. (A) Recovery of spiked cancer cells and percentage of debris removal with the sample running through AF chip only. 22RV1 prostate cancer cells were stained and 100 cells sorted into lysed whole blood (n = 3 replicates). No error bars are shown for Cell Recovery since all three replicates at each of the 3 flow rates had the same value. (B) Recovery of spiked cancer cells, percentage of debris removal, and percentage of CD45+ cells depleted, with the sample running through AF chip and magnetic depletion. The spiked-in samples went through the enrichment system at a sample flow rate of 96, 120, or 160 μl/min and data was recorded for 2 min (three replicates for each sample flow rate; see volumetric calculation in Materials and Methods). (C) Spiked tumor cell recovery after processing cells through the in-line pre-enrichment system and BD FACSMelody™ sorter. Samples representing 1, 2, 10, and 100 VPD^pos/CD45^neg/EpCAM^pos 22RV1 cells were sorted into 5 ml lysed whole blood. After adding 2 ml PBS to each spiked-in 5 ml lysed whole blood sample, the samples were processed through the enrichment system at 160 μl/min and immediately analyzed by the BD FACSMelody™ sorter (n = 3). (D) Schematic illustration of the spiking experiments to compare a traditional benchtop magnet-based enrichment work flow with the integrated enrichment-FACS system for detecting spiked cancer cells in whole blood. The bar chart shows the enrichment recovery of 100 22Rv1 cells spiked in 5 ml of lysed blood from both the integrated enrichment system and the traditional magnetic depletion, wash, and centrifugation workflow, and the integrated system showed significantly higher recovery than the traditional workflow (P-value = 0.0027).

Figure 3.

Characterization of PC3 and 22Rv1 cells with BD Precise™ assay. (A) Heat map of the top differentially expressed genes in PC3 and 22Rv1 cells as measured by log mean number of molecules per cell at P-value threshold of 1E-10. (B) Box plots of the gene expression levels measured as log number of molecules from replicate plates of PC3 and 22Rv1 cells in a subset of representative genes. (C) Correlation between the log-transformed number of EPCAM transcripts per cell measured by the BD Precise assay and the log-transformed fluorescence intensity of EPCAM protein of each cell measured by flow cytometry. A “jitter” function built in the BD DataView software was applied to introduce slight irregular variation in log-transformed signals for visualization of the data.