Introduction

Single-cell sequencing (SCS) is a powerful new tool for investigating evolution and diversity in cancer and understanding the role of rare cells in tumor progression. These methods have begun to unravel key questions in cancer biology that have been difficult to address with bulk tumor measurements.

Role of sequencing

A single cell is the ultimate denomination of a multicellular organism. The human body is composed of approximately 37.2 trillion cells that live harmoniously among their neighbors. However, in cancer, a single cell can lead to the downfall of an entire organism. As a single cell begins its journey to evolve into a malignant mass of tumor cells, the lineages diverge and form distinct subpopulations resulting in intratumor heterogeneity. Clonal diversity is a salient feature of many human and provides fuel for evolution to select upon. A tumor is analogous to an ecosystem, and many principles from ecology and population genetics can help us understand how tumor cell populations respond to selective pressures.

Applications of single cell sequencing

While single-cell genomic methods such as SNS are feasible in a research setting, they will not be useful in the clinic until advances are made in reducing the cost and time of sequencing. Fortunately, the cost of DNA sequencing is falling precipitously as a direct result of industry competition and technological innovation. Sequencing has an additional benefit over microarrays in the potential for massive multiplexing of samples using barcoding strategies. Barcoding involves adding a specific 4 to 6 base oligonucleotide sequence to each library as it is amplified, so that samples can be pooled together in a single sequencing reaction. After sequencing, the reads are deconvoluted by their unique barcodes for downstream analysis. With the current throughput of the Illumina HiSeq2000, it is possible to sequence up to 25 single cells on a single-flow cell lane, thus allowing 200 single cells to be profiled in a single run. Moreover, by decreasing the genomic resolution of each single-cell copy number profile (for example from 50 kb to 500 kb) it is possible to profile hundreds of cells in parallel on a single lane, or thousands on a run, making single-cell profiling economically feasible for clinical applications.

A major application of single-cell sequencing will be in the detection of rare tumor cells in clinical samples, where fewer than a hundred cells are typically available. These samples include body fluids such as lymph, blood, sputum, urine, or vaginal or prostate fluid, as well clinical biopsy samples such as fine-needle aspirates (Figure 1a) or core biopsy specimens. In breast cancer, patients often undergo fine-needle aspirates, nipple aspiration, ductal lavages or core biopsies; however, genomic analysis is rarely applied to these samples because of limited DNA or RNA. Early stage breast cancers, such as low-grade ductal carcinoma in situ (DCIS) or lobular carcinoma in situ, which are detected by these methods, present a formidable challenge to oncologists, because only 5% to 10% of patients with DCIS typically progress to invasive carcinomas.

Conclusion

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