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

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).

1 . 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).   
     
     
         2 . The method for detecting the degree of malignancy of each of the circulating tumor cells according to  claim 1 , further comprising the step of (d) removing erythrocytes and/or leucocytes from the sample, before or after step (a). 
     
     
         3 . The method for detecting the degree of malignancy of each of the circulating tumor cells according to  claim 1 , wherein a leucocyte-binding component, which specifically binds to a marker molecule expressed on leucocytes and is fluorescently-labeled or luminescent enzyme-labeled, is brought into contact with a sample that possibly contains circulating tumor cells in the step (a). 
     
     
         4 . The method for detecting the degree of malignancy of each of the circulating tumor cells according to  claim 1 , wherein the fluorescence signal or the luminescence signal is detected by a flow cytometer or an image analyzer in the detection step (b). 
     
     
         5 . The method for detecting the degree of malignancy of each of the circulating tumor cells according to  claim 1 , wherein the degree of epithelial-mesenchymal transition is represented by a formula selected from a group consisting of formula (1)
     P=M /( E+M )  (1),
     formula (2)       P=E /( E+M )  (2),
     formula (3)       P =Log [ M /( E+M )]  (3), and
     formula (4)       P =Log [ E /( E+M )]  (4)
   wherein P is the degree, E is the signal amount of the epithelial cell-binding component, and M is the signal amount of the mesenchymal cell-binding component, in step (c).   
     
     
         6 . The method for detecting the degree of malignancy of each of the circulating tumor cells according to  claim 1 , further comprising the step of (e) setting a reference of state before an onset of epithelial-mesenchymal transition (0) and a reference of state after a termination of epithelial-mesenchymal transition (1) by measuring particles to which a fluorescence substance labeling the epithelial cell-binding component is bound, and particles to which a fluorescence substance labeling the mesenchymal cell-binding component is bound. 
     
     
         7 . The method for detecting the degree of malignancy of each of the circulating tumor cells according to  claim 6 , step (e) is carried out together with steps (b) and (c). 
     
     
         8 . The method for detecting the degree of malignancy of each of the circulating tumor cells according to  claim 1 , wherein the epithelial cell-binding component is an antibody or aptamer specifically binding to cytokeratin, EpCAM, or E-cadherin, and the mesenchymal cell-binding component is an antibody or aptamer specifically binding to vimentin, or N-cadherin. 
     
     
         9 . A kit for detecting the degree of malignancy of the circulating tumor cells, comprising
 (a) an epithelial cell binding antibody which specifically binds to a marker molecule expressed on epithelial cells and is fluorescently-labeled or luminescent enzyme-labeled, and   (b) a mesenchymal cell-binding antibody which specifically binds to a marker molecule expressed on mesenchymal cells and is fluorescently-labeled or luminescent enzyme-labeled.   
     
     
         10 . The kit for detecting the degree of malignancy of the circulating tumor cells according to  claim 9 , further comprising:
 (c) a leucocyte-binding antibody which specifically binds to a marker molecule expressed on leucocytes and is fluorescently-labeled or luminescent enzyme-labeled, and/or   (d) particles to which a fluorescence substance or luminescent enzyme labeling the epithelial cell-binding antibody is bound, and (e) particles to which a fluorescence substance or luminescent enzyme labeling the mesenchymal cell-binding antibody is bound.   
     
     
         11 . The kit for detecting the degree of malignancy of the circulating tumor cells according to  claim 9 , wherein the epithelial cell-binding antibody is an antibody specifically binding to cytokeratin, EpCAM, or E-cadherin, and the mesenchymal cell-binding antibody is an antibody specifically binding to vimentin, or N-cadherin. 
     
     
         12 . A use for preparing a kit for detecting the degree of malignancy of the circulating tumor cells, of an epithelial cell binding antibody specifically binding to a marker molecule expressed on epithelial cells, and/or a mesenchymal cell-binding antibody specifically binding to a marker molecule expressed on mesenchymal cells. 
     
     
         13 . The use for preparing a kit for detecting the degree of malignancy of the circulating tumor cells according to  claim 12 , wherein the epithelial cell-binding antibody is an antibody specifically binding to cytokeratin, EpCAM, or E-cadherin, and the mesenchymal cell-binding antibody is an antibody specifically binding to vimentin, or N-cadherin. 
     
     
         14 . An apparatus for detecting the degree of malignancy of the circulating tumor cells, characterized by comprising:
 (a) a means of receiving an amount of fluorescence signal or the luminescence signal of epithelial cell-binding component bound to each of the circulating tumor cells and an amount of fluorescence signal or the luminescence signal of mesenchymal cell-binding component bound to each of circulating tumor cells, and   (b) a means of determining the degree of epithelial-mesenchymal transition of each of the circulating tumor cells based on the received amount of signal of the epithelial cell-binding component (E) and the received amount of signal of the mesenchymal cell-binding component (M).   
     
     
         15 . The apparatus for detecting the degree of malignancy of the circulating tumor cells according to  claim 14 , wherein the degree of epithelial-mesenchymal transition in the means (b) is represented by a formula selected from a group consisting of formula (1)
     P=M /( E+M )  (1),
     formula (2)       P=E /( E+M )  (2),
     formula (3)       P =Log [ M /( E+M )]  (3), and
     formula (4)       P =Log [ E /( E+M )]  (4)
   wherein P is the degree, E is a signal amount of the epithelial cell-binding component, and M is a signal amount of the mesenchymal cell-binding component.   
     
     
         16 . The apparatus for detecting the degree of malignancy of the circulating tumor cells according to  claim 14 , wherein the epithelial cell-binding component is an antibody specifically binding to cytokeratin, EpCAM, or E-cadherin, and the mesenchymal cell-binding component is an antibody specifically binding to vimentin, or N-cadherin. 
     
     
         17 . A method for evaluating cells, characterized in that the amounts of multiple molecules expressed on each of the cells are analyzed in each cell, and the characteristic feature of each cell is quantified and indicated by using the amounts of multiple molecules, in procedures for measuring cells. 
     
     
         18 . The method for evaluating cells according to  claim 17 , wherein the amounts of multiple molecules analyzed in each cell are an amount of marker molecules expressed on epithelial cells (E) and an amount of marker molecules expressed on mesenchymal cells (M), the characteristic feature of each cell is a degree of epithelial-mesenchymal transition which is obtained by a formula calculated from E and M. 
     
     
         19 . An apparatus for evaluating cells, characterized in that the apparatus can measure cells, and the amounts of multiple molecules expressed on each of the cells are analyzed in each cell, and the characteristic feature of each cell is quantified and indicated by using the amounts of multiple molecules, in procedures for measuring cells. 
     
     
         20 . The apparatus for evaluating cells according to  claim 19 , wherein the amounts of multiple molecules analyzed in each cell are an amount of marker molecules expressed on epithelial cells (E) and an amount of marker molecules expressed on mesenchymal cells (M), the characteristic feature of each cell is a degree of epithelial-mesenchymal transition obtained by a formula calculated from E and M.