Detection and sorting of Circulating Tumor Cells (CTCs)

[Summary]
After removal of the blood cells in 4ml of peripheral blood, sample were dyed with CTC marker (cytokeratin (CK-FITC) and EpCAM(CD326-PE)) and leukocyte common antigen(CD45-Alexa700). Detection and sorting of the cancer cell(CTCs) was carried out by On-chip Sort.  As a result, the detection and sorting of various CTCs that are independent of the expression of EpCAM are attained.

[Introduction]
Recently, it comes to be considered that circulating tumor cell (CTC) is suitable biomarker for selection of therapy and monitoring of curative effects.
 
On-chip Biotechnlogies Inc. takes part in New Energy and Industrial Technology Development Organization(NEDO) project and developed of the detection of CTC independent of the expression level of EpCAM (CD326), and realization of sorting of rare CTC in peripheral blood.

Cell sorting by disposable chip can realize closed system sorting. The CTCs without the cross contamination between blood samples can be evaluated certainly, and the chip after measurement can be processed like other medical wastes.

[Object]
Recently, a diagnostic technique which uses circulating tumor cells (CTCs) attracts a great deal of attention for one of a monitoring marker which shows choice of a better treatment with collecting blood and a curative effect.  Sorting of rare CTC in the peripheral blood which is not dependent on the expression level of EpCAM (CD326) using On-chip Sort was investigated. The stability and reliability of diagnosis were checked with healthy peripheral blood using the verification model which added the cultured cancer cell.

[Method (1) ; Blind test between two laboratories]
We spike several number of cancer cells (0 cell, 10 cells, 100 cells, 500 cells and 1000cells) into 4 mL of peripheral blood. After spiked cancer cells, white blood cells in the sample were removed at each laboratory. Then, the sample was dyed with common CTC markers (cytokeratine (CK)-FITC, EpCAM(CD326)-PE) and common leukocyte antigen (CD45-Alexa700). Finally, Sample was detected with On-chip Sort and compared.

CTC(Circulating Tumor Cell):Cytokeratin & Vimentin positive, CD45 Negative 2

Figure 1. Gateing for CTC detection

Circulating Tumor Cell Cell Line kato3 A549 PC-14  mcf-7

Figure 2. The result of CTCs recovery test between two laboratories.
It was revealed that there was no difference about detection between laboratories.

[Method (2); Detection and sorting of CTC from peripheral blood of patients with lung cancer]
After hemolyzing red blood cells from sample (peripheral blood 4mL) of patients with lung cancer, white blood cells were removed by anti-CD45 antibody immobilized bead, and the cell fraction of cytokeratin positivity and CD45 negativity was sorted.

[Result(2) ; Detection and sorting of CTCs in peripheral blood of patients with lung cancer]
It became clear that the expression of EpCAM in patients with lung cancer was low.
As a result, regarding measurement of CTC, On-chip method which is not dependent on EpiCAM rather than the result of CellSearch method depending on EpiCAM shows a high positive ratio.
The detection of CTCs (CD45(-)/cyto keratin(+)) in peripheral blood of cancer patient’s were carried out (Fig. 3).

Fluorescence micrograph of captured Circulating Tumor Cell Cytokeratin Vimentin

Figure 3 : The photograph which observed the cells after sorting under the microscope.
The CTC in blood was lebelled with anti-Cytokeratin and anti-Vimentin antibodies, and sorted.