Advanced Technologies for Studying Circulating Tumor Cells at the Protein Level

Charles Ming Lok Chan; Thomas Chi Chuen Au; Anthony Tak Cheung Chan; Brigette Buig Yue Ma; Nancy Bo Yin Tsui; Simon Siu Man Ng; Edwin Pun Hui; Lawrence Wing Chi Chan; Wing Shan Ho; Benjamin Yat Ming Yung; Sze Chuen Cesar Wong


Expert Rev Proteomics. 2013;10(6):579-589. 

In This Article

Technologies for CTC Detection Using Protein Markers


Currently, CellSearch (Janssen Diagnostics, Raritan, NJ, USA) is the only US FDA-approved method for the clinical detection of CTC from cancer patients. Its use has been approved for prognostication and disease monitoring in patients with breast, colon and prostate cancer.[7,100–102] In this method, blood is collected into blood tubes containing a cell preservative. The blood tubes are then placed into an automated sample preparation system which positively enriches for EpCAM-positive cells using immunomagnetic ferrofluids. Capture efficiency was found to be dependent on level of EpCAM expression as cell line spike-in experiments revealed approximately 75% efficiency for high EpCAM expressing cells whereas only approximately 42% recovery was achieved for cells with low EpCAM expression.[103] The enriched cells are then incubated with antibodies against CD45, CK8, 18, 19 and stained with DAPI. During analysis, appropriately sized cells with correct morphological characteristics which are DAPI-, EpCAM- and CK-positive, but negative for CD45 staining are identified as CTCs and enumerated (Figure 1).[104]

Figure 1.

Circulating tumor cell identification by CellSearch®.
Tubes containing blood are centrifuged to separate blood into plasma, buffy coat and red blood cell layer. The blood tube is then placed into the CellTrack® Autoprep system where blood cells are incubated with antibodies against EpCAM, CK, CD45 and are stained with DAPI. In this step, EpCAM-positive CTCs are labeled with anti-CK-PE antibodies while leukocytes are labeled with anti-CD45-APC antibodies. The labeled cells are then analyzed and enumerated in CellTracks® Analyzer. CTCs are identified as CK-positive/DAPI-positive/CD45-negative cells while leukocytes are identified as CD45-positive/DAPI-positive/CK-negative cells.

Using a threshold of 3 CTC/7.5 ml blood for patients with metastatic colorectal carcinoma and 5 CTC/7.5 ml blood for patients with metastatic breast cancer and metastatic prostate cancer, CTC counts were significantly associated with overall survival at any time during treatment and thus, could be used for treatment monitoring. Moreover, that study also revealed that in all three cancer types, patients who developed CTC during the course of treatment and those with persistent CTC counts performed unfavorably. By contrast, improved prognosis was observed in patients whose CTC count dropped from a value above threshold to one below threshold.[7,101,102,105] On the other hand, the Southwest Oncology Group (SWOG) 0500 has initiated a randomized Phase III study to investigate whether it is beneficial to change therapy in metastatic breast cancer patients receiving chemotherapy based on elevated CTC count by CellSearch at first follow-up.[201] Results from this study may form the cornerstone for the use of CTC count as a basis for treatment decisions.

CellSearch could also be used to identify HER2-positive CTCs in patients with breast cancer. Results were found to be highly concordant with those obtained by HER2 FISH analysis of CTC.[48] However, as mentioned above, several studies have reported on the discrepancies in HER2 status between CTC and primary tumor tissue[5,48–52] and more studies would have to be performed to clarify this issue.


The CTC-chip consists of an array of 78,000 microposts coated with anti-EpCAM antibodies within a 970 mm2 surface for CTC capture. The blood sample is pumped continuously through the chip by a pneumatic pressure regulated pump at a flow rate of 1–2 ml/h. This slow flow rate ensures optimal duration for CTC-micropost contact and facilitates CTC-micropost attachment (Figure 2). In fact, cells with both high and low levels of EpCAM expression were found to be captured with similar efficiencies. Recovery rates of >60% could be achieved with approximately 50% purity. Using blood samples from cancer patients and healthy subjects, CTCs were identified in 115 out of 116 patient samples, including those from lung, prostate, pancreatic, breast and colon cancer patients, but not in any of the 20 healthy controls, giving a sensitivity of 99.1% and a specificity of 100%. CTC numbers were found to correlate well with treatment response. Increases in number of CTCs found were associated with clinical progression and shows close concordance with radiographic assessment of tumor volume. The isolated CTCs are also amenable to further characterization by immunostaining, RT-PCR and mutation detection.[106,107] In fact, the use of CTC-chip correctly identified EGFR mutations with a sensitivity of 92% and allowed monitoring of changes in CTC genotype during treatment.[107] These findings indicate that use of the CTC-chip may find application in disease monitoring and provide clinicians with valuable information regarding tumor characteristics during treatment.

Figure 2.

Circulating tumor cell identification by circulating tumor cell-chip.Unprocessed whole blood is pumped into the CTC-chip by a pneumatic pressure-regulated pump at a flow rate of 1–2 ml/h. EpCAM-positive CTCs are bound to anti-EpCAM antibody-coated microposts on the CTC-chip while most of the contaminating blood cells flow through. The captured cells could then be analyzed by immunostaining (as shown) or by other methods such as RT-PCR.