Circulating Tumor Cell Analysis: Technical and Statistical Considerations for Application to the Clinic
Figure 1
Specific molecular features of CTCs can be analyzed via secondary phenotyping using the CellSearch system (Veridex) (a)â(c). Breast cancer CTCs in 7.5âmL of blood were processed on the CellTracks AutoPrep system using the CellSearch CTC kit and additional characterization antibodies against either (a) EGFR (Veridex), (b) CD44 (4âg/mL; BD BioSciences), or (c) M30 (3.7âg/mL; Alexis Biochemicals). Samples were then analyzed by the CellTracks Analyzer II. CTCs were identified and enumerated via positive staining for CK and DAPI (respective red and blue staining in left panels), negative staining for CD45 (not shown), and size and morphological characteristics. The additional FITC channel (green staining in right panels) was exploited for identification of molecular characteristics in individual CTCs. Expression of markers such as (a) EGFR and (b) the cancer stem cell marker CD44 may provide information regarding disease aggressiveness and/or indicate patient suitability for specific targeted therapies. Apoptosis markers such as (c) M30 (caspase-cleaved CK18) and the corresponding morphological characteristic of apoptotic membrane blebbing could provide information in patients on active treatment regarding efficacy of therapy and antitumor effects. The sample shown in (c) was exposed to palitaxel chemotherapy prior to CTC analysis.
