Immunomagnetic Beads
Immunomagnetic beads are magnetic beads which are coated with antibodies against antigens expressed on target cells. Examples include MACS (Miltenyi Biotech, Germany) and Dynabeads (Life Technologies, Carlsbad, CA, USA). In this procedure, the beads are incubated with blood samples which may or may not have undergone prior enrichment strategies. Two common purification methods are used: positive selection, where the target cells are bound by the beads and magnetically enriched; or negative selection, where non-target cells are bound by the beads and removed magnetically, reducing the amount of 'contaminating' cells and increases the relative fraction of the target cell population.[23,108,109] CTCs isolated by immunomagnetic methods could be further studied for more detailed characterization.
MACS
Using MACS, CTC was identified from the CD45-negative cell population in the peripheral blood in 37% of renal cell carcinoma patients. The presence of CTC was found to be associated with advanced tumor stage and more aggressive tumors. Furthermore, detailed analyses indicated that half of patients with CTC developed distant metastases or died of renal cell carcinoma within 2 years.[109] Besides showing a potential for clinical utility, MACS has also been used for elucidation of cellular pathways important for survival of disseminated tumor cells (DTCs). In a recent report, DTCs isolated from the bone marrow of gastric cancer patients by MACS was found to have significantly decreased expression of microRNA-144 (miR-144). In gastric cancer patients, downregulation of miR-144 expression in cancer tissues was associated with poor prognosis. It is believed that disseminated cancer cells in bone marrow survive by downregulation of miR-144, resulting in upregulation of Z finger X-chromosomal protein (ZFX) expression and implicating a pivotal role for the miR-144-ZFX axis in gastric cancer progression.[110]
Dynabeads
Previously, we have developed a refined assay to identify CTC from patients with CRC by staining for intestinal-specific markers including CK20 and CDX2 following CTC capture with BerEP4-coated Dynabeads (Figure 3).[110,111] Using this assay, we showed that CTCs could be detected in 62% (CK20) and 81% (CDX2) of CRC patients, in 6% (CK20) and 7.5% (CDX2) of patients with colorectal adenoma, 0% (CK20) and 2.5% (CDX2) of patients with other cancers but not in any patients with benign colorectal disease or in normal subjects. Using both methods, CTC number was found to be significantly associated with tumor-node-metastasis (TNM) stage, lymph node status, presence of recurrent or metastatic CRC and overall survival. Furthermore, we have also showed that chromosome 17 aneusomy detected in CK20-positive CTCs FISH were consistent with those from their respective primary tumor tissues in 90% of cases. This development has opened up new possibilities for characterization of CTCs isolated by immunomagnetic enrichment techniques.
Figure 3.
Refined assay for identification of cytokeratin-positive circulating tumor cells using Dynabead®.
Peripheral blood mononuclear cells are incubated with Dynabeads coated with anti-BerEP4 antibodies. BerEP4-positive CTCs in the sample are bound by the beads and are magnetically enriched. The isolated cells are then incubated with goat-anti-mouse antibodies to block antigenic sites present on the mouse antibodies on the Dynabeads. Finally, the captured cells are then incubated with mouse anti-CK20 antibodies for specific identification of CK20-positive CTCs in the sample.
Immunomagnetic Enrichment: Fluorescence-activated Cell Sorting
Recently, Magbanua and Park have demonstrated that it is possible to isolate CTCs with high purity using an immunomagnetic enrichment (IE) followed by fluorescence-activated cell sorting (FACS) approach. In this method, samples are incubated with magnetic beads conjugated to monoclonal antibodies against EpCAM to enrich for tumor cells followed by purification using FACS to remove hematopoietic mononuclear cells.[112] Up to 20 ml of blood sample can be analyzed in about 30 min with recoveries of approximately 80% for cells with high EpCAM expression levels and approximately 30% for those with low EpCAM levels. By use of a trypan blue exclusion assay, approximately 73% of cells were found to be viable after cell sorting. Application of this approach allowed them to identify frequent androgen receptor gene amplification in CTCs from castration-resistant prostate cancer patients.[113] Moreover, CTCs from breast cancer patients isolated using this method were amenable to genome-wide copy number analysis via array comparative genomic hybridization (aCGH), which revealed copy number alterations consistent with those reported for breast cancer. Also, a comparison of the isolated CTCs with matched archival primary tumors confirmed both common and divergent lineages at the genome level.[114] It would be interesting to see if this method could be used in conjunction with whole genome sequencing, which would allow more detailed investigations of CTCs at the genome and transcriptome level and open new horizons to further our understanding of tumor progression.
Expert Rev Proteomics. 2013;10(6):579-589. © 2013 Expert Reviews Ltd.
