High-Throughput Multimodal Single-Cell Targeted DNA and Surface Protein Analysis Using the Mission Bio Tapestri Platform.

An important aspect of understanding cancer biology is to connect the diverse repertoire of genotype-to-phenotype displays in individual specimens and ultimately resolve disease course outcome through informative datasets. A focus of cancer genomics has strived to provide predictive capabilities using genomic information to further inform therapeutic strategies. The advent of single-cell sequencing and analysis now provides a route to decipher high-resolution genomic diversity in individual samples and facilitate detailed understanding of clonal evolution in clinical research settings. In addition to generating high-throughput single-cell genomic SNV and CNV data, this protocol describes a new analytical ability that adds a second dimension which provides for interrogation of surface protein marker expression. The first immediate application of this technology is quite suitable to heme cancer cell studies. This multimodal approach allows for correlation of diverse genomic signatures to key phenotypic biomarkers such as immunophenotypes in leukemic diseases.

Simultaneous DNA, RNA, and Protein Analysis from Single Cells Using a High-Throughput Microfluidic Workflow for Resolution of Genotype-to-Phenotype Modalities.

Understanding the genomic landscape of cancer in single cells can be valuable for the characterization of molecular events that drive evolution of tumorigenesis and fostering progress in identifying druggable regimens for patient treatment scenarios. We report a new approach to measure multiple modalities simultaneously from up to 10,000 individual cells using microfluidics paired with next-generation sequencing. Our procedure determines targeted protein levels, mRNA transcript levels, and somatic gDNA sequence variations including copy number variants. This approach can resolve over 20 proteins, 100s of targeted transcripts, and DNA amplicons.

Technical Insights into Highly Sensitive Isolation and Molecular Characterization of Fixed and Live Circulating Tumor Cells for Early Detection of Tumor Invasion.

Circulating Tumor Cells (CTC) and Circulating Tumor Microemboli (CTM) are Circulating Rare Cells (CRC) which herald tumor invasion and are expected to provide an opportunity to improve the management of cancer patients. An unsolved technical issue in the CTC field is how to obtain highly sensitive and unbiased collection of these fragile and heterogeneous cells, in both live and fixed form, for their molecular study when they are extremely rare, particularly at the beginning of the invasion process. We report on a new protocol to enrich from blood live CTC using ISET® (Isolation by SizE of Tumor/Trophoblastic Cells), an open system originally developed for marker-independent isolation of fixed tumor cells. We have assessed the impact of our new enrichment method on live tumor cells antigen expression, cytoskeleton structure, cell viability and ability to expand in culture. We have also explored the ISET® in vitro performance to collect intact fixed and live cancer cells by using spiking analyses with extremely low number of fluorescent cultured cells. We describe results consistently showing the feasibility of isolating fixed and live tumor cells with a Lower Limit of Detection (LLOD) of one cancer cell per 10 mL of blood and a sensitivity at LLOD ranging from 83 to 100%. This very high sensitivity threshold can be maintained when plasma is collected before tumor cells isolation. Finally, we have performed a comparative next generation sequencing (NGS) analysis of tumor cells before and after isolation from blood and culture. We established the feasibility of NGS analysis of single live and fixed tumor cells enriched from blood by our system. This study provides new protocols for detection and characterization of CTC collected from blood at the very early steps of tumor invasion.

A single-molecule barcoding system using nanoslits for DNA analysis.

Molecular confinement offers new routes for arraying large DNA molecules, enabling single-molecule schemes aimed at the acquisition of sequence information. Such schemes can rapidly advance to become platforms capable of genome analysis if elements of a nascent system can be integrated at an early stage of development. Integrated strategies are needed for surmounting the stringent experimental requirements of nanoscale devices regarding fabrication, sample loading, biochemical labeling, and detection. We demonstrate that disposable devices featuring both micro- and nanoscale features can greatly elongate DNA molecules when buffer conditions are controlled to alter DNA stiffness. Furthermore, we present analytical calculations that describe this elongation. We also developed a complementary enzymatic labeling scheme that tags specific sequences on elongated molecules within described nanoslit devices that are imaged via fluorescence resonance energy transfer. Collectively, these developments enable scaleable molecular confinement approaches for genome analysis.