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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.

The ability of cancers to grow indefinitely has fueled the idea that cancer and stem cells may have common underlying mechanisms. Detailed gene expression maps have now shown the diversity and distinctiveness in gene expression programs associated with stemness in embryonic and adult stem cells. These maps have further revealed a shared transcriptional program in embryonic stem cells (ESC) and cancer stem cells. Surprisingly, forced activation of an ESC-like gene expression program in adult epithelial cells can reprogram them into human cancer stem cells and achieve pathologic self-renewal. The ability to create induced cancer stem cells (iCSC) may provide opportunities to better define the biology of cancer stem cells in order to trace or eliminate them in human patients.

CpG island methylation plays an important role in normal cellular processes, such as genomic imprinting and X-chromosome inactivation, as well as in abnormal processes, such as neoplasia. However, the dynamics of de novo CpG island methylation, during which a CpG island is converted from an unmethylated, active state to a densely methylated, inactive state, are largely unknown. It is unclear whether the development of de novo CpG island methylation is a progressive process, in which a subset of CpG sites are initially methylated with a subsequent increase in methylation density, or a single event, in which the initial methylation event encompasses the entire CpG island. The tumor suppressor gene p16/CDKN2a/INK4a (p16) is inactivated by CpG island methylation during neoplastic progression in a variety of human cancers. We investigated the development of methylation in the p16 CpG island in primary human mammary epithelial cell strains during escape from mortality stage 0 (M(0)) growth arrest. The methylation status of 47 CpG sites in the p16 CpG island on individual DNA molecules was determined by sequencing PCR clones of bisulfite-treated genomic DNA. The p16 CpG island was initially methylated at a subset of sites in three discrete regions in association with p16 transcriptional repression and escape from M(0) growth arrest. With continued passage, methylation gradually increased in density and methylation expanded to sites in adjacent regions. Thus, de novo methylation in the p16 CpG island is a progressive process that is neither site specific nor completely random but instead is region specific. Our results suggest that early detection of methylation in the CpG island of the p16 gene will require methylation analysis of the three regions and that the identification of region-specific methylation patterns in other genes may be essential for an accurate assessment of methylation-mediated transcriptional silencing.

Proliferation of human mammary epithelial cells (HMEC) is limited to a few passages in culture due to an arrest in G1 termed selection or mortality stage 0, M0. A small number of cells spontaneously escape M0, continue to proliferate in culture, and then enter a second mortality stage, M1, at which they senesce. Evidence that M0 involves the Rb pathway comes from the observation that expression of human papillomavirus type 16 E7 alleviates the M0 proliferation block, and we further show that the Rb-binding region of E7 is required to allow cells to bypass M0. In contrast, E6 does not prevent HMEC from entering M0 but, rather, is involved in M1 bypass. Here we show that inactivation of the D-type cyclin-dependent kinase inhibitor p16INK4A is associated with escape from the M0 proliferation block. Early-passage HMEC express readily detectable amounts of p16 protein, whereas normal or E6-expressing HMEC that escaped M0 expressed markedly reduced amounts of p16 mRNA and protein. This initial reduction of p16 expression was associated with limited methylation of the p16 promoter region CpG island. At later passages, a further reduction in p16 expression occurred, accompanied by increased CpG island methylation. In contrast, reduction of p16 expression did not occur in E7-expressing HMEC that bypassed M0, due to inactivation of Rb. These observations in the E6-expressing HMEC correlate well with the finding that CpG island methylation is a mechanism of p16 inactivation in the development of human tumors, including breast cancer.