Circulating tumor cells, enumeration and beyond

Abstract

The detection and enumeration of circulating tumor cells (CTCs) has shown significant clinical utility with respect to prognosis in breast, colorectal and prostate cancers. Emerging studies show that CTCs can provide pharmacodynamic information to aid therapy decision making. CTCs as a 'virtual and real-time biopsy' have clear potential to facilitate exploration of tumor biology, and in particular, the process of metastasis. The challenge of profiling CTC molecular characteristics and generating CTC signatures using current technologies is that they enrich rather than purify CTCs from whole blood; we face the problem of looking for the proverbial 'needle in the haystack'. This review summarizes the current methods for CTC detection and enumeration, focuses on molecular characterization of CTCs, unveils some aspects of CTC heterogeneity, describes attempts to purify CTCs and scans the horizon for approaches leading to comprehensive dissection of CTC biology.

Figure 1

Characterization of CTCs. CTCs isolated from SCLC patients by either the Veridex CellSearch Platform or isolated by ScreenCell ISET size exclusion. Panel A shows a single CTC, isolated by ISET and stained by H&E, with high nuclear to cytoplasmic ratio, cell size >8 µm (the pore size of membrane) and irregular nuclear shape. Panel B shows a SCLC CTM (black arrow) isolated by ISET alongside a leukocyte positively stained brown for CD45 (white arrow). Panel C demonstrates that the DAPI nuclear staining can identify apoptosis (left panel) and nuclear morphology consistent with mitosis (right panel) of CTCs detected by the CellSearch. Panel D SCLC associated diagnostic biomarkers, NSE and TTF-1 were immunohistochemically stained with white blood cells used as internal negative control to further confirm the identity of ISET isolated CTM and CTCs Panel E shows FISH analysis to detect bcl-2 genetic abnormalities such as amplification and triploidy from SCLC CTCs. Blood samples were collected from SCLC patients undertaking chemotherapy at the Christie Hospital, Manchester UK according to ethically approved protocols.

Figure 2

Feasibility evaluation of Tandem CellSearch-FACS for CTC purification. Spiked SCLC H146 cells were processed by the CellSearch system using CellSearch Profile kit. Panel A The recovered cells were stained with Alexa Fluor 488 conjugated CD56 antibody and 7-AAD before the sorting of viable CD56 positive cells by FACS. Panel B A quality check on extracted RNA from FACS sorted cells in panel A showed good quality of RNA with high RNA integrity number (RIN).