Abstract 3793: Discovery and isolation of enormous number of circulating tumor cells in breast cancer patient’s blood based upon cell attachment property

Circulating tumor cells (CTCs) are detached from primary tumors into the bloodstream and known to play a role in tumor metastasis. CTCs are known to be rare, estimated at 1-10 CTCs among 1010 blood cells in a milliliter of blood. Most of existing technologies for CTC enrichment are based on marker expression or size. However, recent studies have suggested that CTCs may have a wide range of marker expression and sizes. The number of CTCs that is isolated from the patient’s blood is fewer than expected. This infrequent and unreliable detection impedes the clinical use of CTCs. We adopted a concept that presumptive CTC would have differential attachment properties compared to WBC in tissue culture plate because it originated from carcinoma where tumor cells attach each other. To prove the concept, we spiked breast cancer cell lines into 7.5ml of healthy blood. RBC was removed by Ficoll. Diluted peripheral blood mononuclear cells were incubated with 5% CO2 in cell culture incubator for 2h at 37°C. After a thorough rinse with 1×PBS, the plates were treated with 10mM EDTA to detach cells from the bottom. The cells collected were followed by lysis and DNA extraction, or placed on glass slide and stained with anti-CD45, -CK, -HER2, and DAPI. Typically, slides were analyzed by confocal microscope or Metafer slide scanning platform for enumeration of cells. 7.5 mL of peripheral blood was collected from breast cancer patient, and handled as described above. Our method showed higher detection sensitivities for 6 breast cancer cell lines. We verified that breast cancer cell lines were collected more than 80% of spiked cell number in 2 hours. This technique is applied to isolation of CTCs in actual breast cancer patients, and unexpectedly considerable numbers of CTCs were observed. We found in a range of CTCs from 0.5 × 105 to 1 × 106 per ml of blood. These numbers are in striking contrast to previous studies. CTCs were observed in 61 of 114 samples (53.51%) from metastatic breast cancer patients and 15 of 48 samples (31.25%) from early breast cancer patients. We also discovered that there are various sized CTCs, and categorized these CTCs according to their diameter; small (10 μM) CTCs. All three types of CTCs appeared in large numbers. HER2 and EpCAM expression of CTC vary by patients and didn’t always coincide with expression in primary tumor. In summary, we have developed a promising strategy for CTC isolation by using the prefe