To get a clearer picture of the relevance of CTC biology including EMT it is possible to take advantage of the simplicity of blood sampling and establish multiple longitudinal blood samples to build a picture of CTC biology during the course of disease progression. (CK-19) mRNA in peripheral blood of patients with breast malignancy (37-39). Similarly, in CRC a panel of genes including carcinoembryonic antigen (CEA), CK-19, CK-20 and CD133 has been used to verify the presence of colorectal tumour cells in enriched blood samples (40). However, any study carried out on enriched CTC populations will inevitably suffer from reduced sensitivity Rabbit polyclonal to AFF3 through the effective dilution caused by varying levels of contaminating WBCs. Over the last decade, advances in both CTC enrichment protocols and molecular methodologies has culminated in a range of successful strategies for genome wide amplification and analysis of single CTCs which have extended many of the observations made with enriched CTCs. There Isocorynoxeine are several advantages of single CTC analysis including: (I) single CTC analysis is 100% real (assuming a CTC was isolated) and avoids any complications or dilution of transmission due to contaminating cells; (II) since a cell has on average only two DNA copies of each gene and around 100,000 mRNA molecules (41) the degree of next generation sequencing (NGS) required for generating a representative single cell snapshot is usually relatively low; (III) single cell genomic analysis can unambiguously identify co-existing genetic changes; (IV) the integration of RNA and DNA analysis from your same cell can establish linkage between the genetic alterations and alterations in RNA pathways (42); (V) single Isocorynoxeine cell data provides an ideal means of establishing the degree of heterogeneity and estimating tumour development (43,44). For single CTC molecular analysis the main problems encountered are largely technical, financial and logistical. Since the amplification process necessary for single CTC analysis is extremely sensitive it is critical that actions are taken to avoid or identify potential contamination such as defining individual clean areas and use of unfavorable controls. Given the vanishingly small amounts of DNA (6.6 pg) present in a single Isocorynoxeine cell, whole genome amplification (WGA) is a pre-requisite for extensive genomic analysis of CTCs. The common use of CTC WGA was pioneered by Christoph Klein and co-workers through the establishment of a simple single tube amplification system (available as the AMPLI1? WGA kit, Silicon Biosystems) based on restriction enzyme digestion, ligation of primer and subsequent PCR amplification (45). There are now many commercially available single cell WGA systems to choose from generally based on PCR (46,47) or linear amplification (48-50). There are obvious pros and cons for each WGA system (51) and the choice of which WGA system to use needs to be based on the clinical/scientific aim of the study, whether the WGA system is compatible Isocorynoxeine with the CTC enrichment process used and the resources available. Following WGA of CTCs one of the most widely used approach for CTC molecular analysis has been establishing genome copy number alterations (CNA) which provides a simple snapshot of each CTC which will allow comparisons between CTCs and, when available, the matching tumour [provides examples of non-small cell lung malignancy (NSCLC) CTC CNA profiles]. Initial single cell studies compared targeted mutation profiles and copy number profiles of CTCs with the primary tumours and metastases of the corresponding patients. One such study of circulating melanoma cells using single cell comparative genomic hybridisation (single-cell CGH) revealed multiple CNA consistent with melanoma which was in sharp contrast to the normal cells that revealed balanced CGH profiles (52). In addition, the use of hierarchical clustering provided a means of establishing linkages between each cell and suggested a clonal origin of the melanoma cells examined (52). Open in a separate window Physique 1 Determining CTC copy number alterations (CNA). (A) Shows a schematic outline of the CTC NGS analysis workflow used. (B) Presents genome wide CNA profiles and accompanying images of single CTCs and WBCs isolated by epitope dependent and independent technologies from two patients with NSCLC. For all four cells the CNA pattern is presented in a linear format Isocorynoxeine with marked copy number increases shown in reddish and losses in blue alongside the fluorescent stained images of the starting cells. The top panel presents the CNA patterns and images of a NSCLC cytokeratin (CK) positive, CD45 unfavorable CTC and a CK unfavorable, CD45 positive WBC obtained following CellSearch? EpCAM enrichment.