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


Cytokeratins (CK) constitute the major component of the epithelial cytoskeleton where they serve to protect cells from mechanical and non-mechanical injuries.[25,26] They are resistant to degradation and are highly antigenic. Their expression is regulated according to differentiation and developmental stage. Among the different types of cytokeratins, CK8, 18 and 19 are mainly expressed in simple epithelial cells and are most commonly associated with epithelial malignancies.[27–29] Some mesenchymal cells may also express CK, including smooth muscle cells of the myometrium, vascular endothelial cells and in some mesenchymal neoplasms such as synovial sarcomas, epithelioid sarcomas and chordomas.[30–35] On the other hand, squamous cell carcinomas mainly express CK5, CK6, CK14, CK16 and CK17.[36] These markers are also downregulated during EMT and thus also suffer from reduced sensitivity in such cases.

Mucin 1

Mucin 1 (MUC1) is a transmembrane glycoprotein normally expressed at low levels in glandular epithelial cells. During tumorigenesis, upregulated expression of MUC1 is detected in a variety of cancers including colon, lung, pancreas, breast, ovarian, prostate kidney, stomach and head and neck.[37–40] In cancer cells, MUC1 promotes tumorigenicity by enhancing cell signaling through growth factor receptors, inhibiting apoptosis and increasing motility and invasiveness.[40] In colon cancer and lung cancer, upregulated expression of MUC1 was shown to be significantly associated with poor prognosis.[38,41,42] The extracellular domain of MUC1 can be detected in peripheral blood and is known as the CA15.3 antigen, which is associated with poor prognosis in various cancers and is used as a diagnostic and prognostic marker for breast cancer patients.[43,44] This marker is often used for identification of CTCs in breast cancer patients.[45]

Human Epidermal Growth Factor Receptor 2

Human epidermal growth factor receptor 2 (HER2) is overexpressed in 20% of breast cancers and predicts poor prognosis. Use of HER2 targeted agents such as trastuzumab, lapatinib and pertuzumab in patients with HER2-positive breast cancers has been found to significantly improve treatment outcomes.[46,47] It would therefore be of great benefit to the patient if sampling of breast cancer cells could be obtained from CTC in blood rather than through a biopsy. However, results on the evaluations of the concordance of HER2 status between CTCs and primary tumors varied among different studies. Discordances between CTCs and primary tumor tissues have been reported by different groups which may be due the differences in methods used for HER2 quantification.[5,48–52] Nevertheless, irrespective of the HER2 status of the primary tumor, the detection of HER2-positive CTCs has been found to be associated with worse disease-free survival and overall survival.[53,54] Interestingly, a recent report found that CTCs with enhanced HER2 activity but without HER2 overexpression were detected in 18% (3/17) of relapsed breast cancer patients with HER2-negative tumors. It was suggested that a 'HER2 status conversion' may have occurred in those patients who could significantly impact our understanding of cancer progression in breast cancer patients.[55]


CD45 is a hematopoietic cell type specific tyrosine phosphatase which is involved in T- and B-cell antigen receptor signaling.[56,57] Multiple forms of CD45 exist where expression is dependent on cell type, differentiation status and activation state of cells.[58] Besides its phosphatase role in B and T cells, CD45 modulates cytokine production in NK cells, mast cells and neutrophils.[59,60] In addition, its expression was also found to be critical for lymphocyte survival, as CD45−/− mice display increased apoptosis.[61]

Since CD45 expressed in all hematopoietic cells, its main use in CTC studies is for targeting of peripheral blood mononuclear cells during negative immunomagnetic selection. This is performed to minimize contamination of CTCs with blood cells which may decrease CTC purity and negatively affect accuracy of results.


CD133 (prominin-1) is a transmembrane glycoprotein which has been identified as a specific marker for hematopoietic stem cells.[62,63] Currently, it is one of the most commonly used markers for isolation of cancer stem cells from various organs such as brain,[64,65] prostate,[66] liver,[67,68] pancreas,[69] lung,[70,71] colon[72] and ovary.[73]

A recent study by Nadal et al. found CD133-positive CTCs in 65% of breast cancer patients. Moreover, they also observed preferential enrichment of CD133-positive CTCs in patients with non-luminal breast cancer subtype after chemotherapy, suggesting that CD133 may be a potential marker for chemoresistance in these patients.[74] On the other hand, CD133 has also been reported to be expressed in 15–60% human lung cancers[75–77] and has been successfully used for isolation of lung cancer cells with stem cell properties. Such cells were found to be capable of forming tumor spheres, were highly tumorigenic in mice and resistant to chemotherapeutic drugs.[70,78] However, whether the detection of CD133-positive cells in lung cancer patients has prognostic significance is still inconclusive and requires further investigations.[76,79]

In spite of the studies presented above, recent investigations have provided data showing that CD133 expression can actually be found on both undifferentiated and differentiated cells and that CD133-negative cells were also capable of undergoing self-renewal and establishment of tumors in immunocompromised mice, casting doubts as to whether CD133 is a specific stem cell marker.[44,80] It has been proposed that glycosylation status of CD133, rather than its expression, may be a better marker for identification of stem cells.[81]

Chemokine Receptors

Human chemokines are secreted ligands which bind to G protein-coupled chemokine receptors on cell surfaces and affect cellular migratory behavior.[82] The expression of specific chemokine receptor pattern on metastatic cells has been proposed to mediate metastasis by promoting their re-population on specific organs at distant sites. Examples include CXCR4, CCR6, CCR7 and CCR9.[83–85] The expression of these chemokine receptors have either been detected in CTCs or associated with metastatic disease in several tumor types including multiple myeloma,[83] gastric cancer,[86] non-small cell lung cancer,[87,88] renal cancer,[89] colorectal cancer (CRC)[90] and breast cancer.[91] The detection of chemokine receptors in such a wide variety of cancers has led to clinical trials evaluating their use as a therapeutic target for prevention or treatment of metastasis.[84,85]

Adhesion Molecules

The expression of adhesion molecules including VCAM-1 and ICAM-1 have been found to be crucial for promoting physical interactions between CTCs and hematopoietic cells which promote CTC survival, extravasation and metastasis.[92]

VCAM-1 is normally expressed by endothelial cells in response to stimulation by TNF-α and IL-1.[93] Recently, VCAM-1 expression on breast cancer CTCs was found to promote their binding with macrophages and protect CTCs from proapoptotic cytokines such as TNF-related apoptosis-inducing ligand (TRAIL).[94]

ICAM-1 is a glycoprotein also expressed on endothelial cells in response to TNF-α and IL-1 during inflammation. Its expression on CTCs has been found to encourage their transendothelial migration in melanoma,[95] pancreatic[96] and breast cancers[97] by promoting their interaction with neutrophils.[98,99]