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 (R 2 = 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.

CTC Technologies and Tools

“…A description of several of these EpCAM independent isolation methods can be found in this issue (39)(40)(41)(42)(43)(44)(45). A description of several of these EpCAM independent isolation methods can be found in this issue (39)(40)(41)(42)(43)(44)(45).…”

“…Addition of fluorescently labeled antibodies recognizing treatment targets to the staining cocktail is relatively easy, although, one is limited by the number of fluorochromes that can be determined simultaneously. Tools to isolate the single cells from CTC enriched cell suspensions are available (39,41,42,56,57). Probing for gene aberrations of CTC by fluorescence in situ hybridization has also been demonstrated and again reiterated their extensive intra-and inter-patient heterogeneity (52)(53)(54)(55).…”

“…With the introduction of new methods for CTC isolation many have argued that the lack of detection of CTC in cancer patients with the CellSearch system may be contributed to the CTC definition used as it detects only cells expressing both EpCAM and Cytokeratin. A description of several of these EpCAM independent isolation methods can be found in this issue (39)(40)(41)(42)(43)(44)(45). However, for these different CTC phenotypes, clinical confirmation of the correlation of CTC to patient survival will be necessary.…”

“…For extensive interrogation of the CTC the individual cells will have to be isolated and for molecular characterization the nucleic acids will have to be amplified. Tools to isolate the single cells from CTC enriched cell suspensions are available (39,41,42,56,57). The steps needed to get from blood to single CTC surely will be associated with cell losses and the different steps will have to be optimized to minimize these losses.…”

Self-Seeding Microwells to Isolate and Assess the Viability of Single Circulating Tumor Cells

“…Other systems are available to assess single cells, for example a self-sufficient micro-droplet generation system that facilitates encapsulation, chemical stimulation, and microscopic analysis of viable cells inside droplets [35]. However, many cells are used and it has not been shown that such systems work for rare cell populations like CTC single cell isolation technologies suitable for CTC analysis, including viable cells do exist like DEPArray™ [36], ALS cellselector™ [37] or FACS sorting [38] but no successful CTC cultures have been published yet. A workflow to successfully obtain viable CTC using self-sorting microwells is presented here.…”