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Method for detecting the degree of malignancy of each of the circulating tumor cells, and a kit for the same

a technology for circulating tumor cells and kits, which is applied in the field of methods for detecting the degree of malignancy of each circulating tumor cell, and the kit for detecting the degree of malignancy of the circulating tumor cell, can solve the problems of serious wrong diagnosis, and achieve the effect of accurate diagnosis

Inactive Publication Date: 2015-03-05
ON CHIP BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention describes a method for detecting the degree of malignancy of circulating tumor cells (CTCs) in a patient. This can help in accurately diagnosing the cancer and predicting its metastasis after surgery or monitoring its progression. The method can also be used to evaluate the effectiveness of anticancer drugs and measure the degree of epithelial-to-mesenchymal transition (EMT) in the CTCs.

Problems solved by technology

Thus, a count loss of CTCs or a contamination between samples of patients leads to a seriously wrong diagnosis.

Method used

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  • Method for detecting the degree of malignancy of each of the circulating tumor cells, and a kit for the same
  • Method for detecting the degree of malignancy of each of the circulating tumor cells, and a kit for the same
  • Method for detecting the degree of malignancy of each of the circulating tumor cells, and a kit for the same

Examples

Experimental program
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Effect test

example 1

[0109]In this Example, in order to establish the detection method of the present invention, A549 cell line derived from lung cancer was mixed with the peripheral blood of the volunteer, as a substitute for CTC. Then, cytokeratin and vimentin were detected and the EMT index was calculated.

[0110]Specifically, 45 mL of lysing buffer was added to 4 mL of peripheral blood in which A549 cells (100 cells) were mixed, and the whole was mixed and allowed to stand on ice so as to lyse erythrocytes. 0.1 mL of solution wherein ImmunoTOKUI (On-chip Biotechnologies Co., Ltd) was diluted by a factor of 20 with PBS buffer containing 0.5% BSA and 2 mM EDTA (hereinafter referred to as T-buffer) was added thereto. The mixture was centrifuged, and the supernatant was aspirated so as to obtain a cell pellet. The resulting cell pellet was resuspended in 200 μL of T-buffer, and 100 μL of Fc Blocking Reagent was added thereto. The whole was incubated at 4° C. for 15 minutes. After the incubation, 200 μL of...

example 2

[0117]In this example, a method for determining the apparatus constants A and B of the flow cytometer in the formula i.e. EMT index=[A(FL2 / (FL1+FL2))+B] used in Example 1 is explained.

[0118]In order to obtain a standard linear of fluorescence signals of a “100% CK (FITC)” condition wherein cytokeratin only expresses and vimentin does not express, particles bound with FITC were measured by the flow cytometer. Further, in order to obtain a standard linear of fluorescence signals of a “100% Vimentin (PE)” condition wherein vimentin only expresses and cytokeratin does not express, particles bound with PE were measured by the flow cytometer.

[0119]In particular, the apparatus constants A and B are calculated as follows. In order to obtain a standard linear of fluorescence signals of a “100% CK (FITC)” condition wherein cytokeratin only expresses and vimentin does not express, the FITC-bound particles having a diameter of 3 μm were measured by the flow cytometer so as to obtain a standard ...

example 3

[0123]In this Example, recovery rates in the detection method of the present invention were examined by using A549 cells or cancer cells other than A549 cells.

[0124]The procedures described in Example 1 were repeated using A549 cells. Further, the procedures described in Example 1 were repeated except that KATO-III cells, PC-9 cells, or PC-14 cells were used instead of A549 cells. Then, EMT indexes thereof were calculated, and further detection rates of these cells were calculated simultaneously.

[0125]As shown in FIG. 4, the detection rates of KATO-III cells, A549 cells, PC-9 cells, and PC-14 cells were approximately 100%, 89%, 75%, and 100%, respectively.

[0126]The detection rate of PC-14 cells in which EpCAM was not expressed, was 100%. Therefore, it was revealed that the detection method of the present invention can effectively detect CTCs of metastatic cancer in which the EMT was induced.

[0127]Finally, a characteristic feature of the present invention is shown from the other poin...

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Abstract

The object of the present invention is to provide an evaluation method capable of accurately determining a metastasis of cancer, the stage of cancer progression, or the malignancy of cancer.The object can be solved by a method for detecting the degree of malignancy of each of the circulating tumor cells, characterized by the following steps: (a) bringing an epithelial cell-binding component, which specifically binds to a marker molecule expressed on epithelial cells and is fluorescently-labeled or luminescent enzyme-labeled, and a mesenchymal cell-binding component, which specifically binds to a marker molecule expressed on mesenchymal cells and is fluorescently-labeled or luminescent enzyme-labeled, into contact with a sample that possibly contains circulating tumor cells, (b) detecting a fluorescence signal or a luminescence signal of the epithelial cell-binding component and a fluorescence signal or a luminescence signal of the mesenchymal cell-binding component of each of the cells, and (c) determining the degree of epithelial-mesenchymal transition of circulating tumor cells based on the signal amount of the epithelial cell-binding component (E) and the signal amount of the mesenchymal cell-binding component (M).

Description

TECHNICAL FIELD[0001]The present invention relates to a method for detecting the degree of malignancy of each of the circulating tumor cells, a kit for detecting the degree of malignancy of the circulating tumor cells, and an apparatus for detecting the degree of malignancy of the circulating tumor cells.BACKGROUND ART[0002]Recently, it has become important to select an effective anticancer drug for each cancer patient by removing cancer cells from the cancer patient, and diagnosing the cancer cells thoroughly, for example, a molecular biological analysis such as gene variation analysis. It is known that extremely-low concentrated cancer cells can be detected in the blood of the cancer patients. These cancer cells are referred to as circulating tumor cell (hereinafter sometimes referred to as CTC). The number of the detected CTC is correlated to a prognosis of the patient. Therefore, a measurement of CTC begins to be performed, so as to evaluate a stage of cancer progression.[0003]W...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/574
CPCG01N33/57492G01N33/574C12N5/0693G01N33/58G01N33/582
Inventor TAKEDA, KAZUOYAMASHITA, NAMIKOFUJIMURA, YUUNISHIO, KAORIKOH, YASUHIROWATANABE, MASARUKOIZUMI, FUMIAKIUEHARA, YURI
Owner ON CHIP BIOTECH
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