JP2006003293A - Method for measuring ability to inhibit carcinoma metastasis and measuring instrument thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for measuring and retrieving ingredients, having the ability to inhibit carcinoma metastasis and measuring the degree of their metastasis inhibition, in a carcinoma metastasis model system reflecting in-vivo kinetics in an initial phase-surrounding carcinoma cells and vascular endothelial cells from a view point on the side of host cells for the purpose of screening drugs having a metastasis inhibiting action for prognostic improvements, regarding patients who have experienced carcinoma detection and extirpative surgery, and to provide a measuring instrument. <P>SOLUTION: In this method, to coexistently culture both the vascular endothelial cells (5) and the carcinoma cells without direct contact, supply ingredients involved in a cleavage degree part (9) of the vascular endothelial cells considered as secreting from carcinoma cells, and measure the degree of its cleavage by measuring the electrical resistance level between an upper layer part (7) and a lower layer part (8) of a culture compartment. The carcinoma metastasis inhibition ability measuring instrument uses this measuring method. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method and a measuring instrument for searching (screening) a component having cancer metastasis inhibiting ability and measuring the inhibiting ability.

Conventionally, screening of test reagents for cancer metastasis inhibitory action, which is generally performed widely, has been performed by animal experiments using mice. (Non-Patent Document 1)
Japanese Patent Laid-Open No. 5-21893

Moreover, as a method for determining the effect of a metastasis inhibitor in a test tube, an evaluation method in accordance with each step of invasion, exercise, and metastasis formation of angiogenesis has been devised and widely used. (Non-Patent Document 1)
Cancer Metastasis Study Group “Cancer Invasion and Metastasis Research Manual” Kinyoshido 1994

  However, in animal experiments in which cancer cells are administered to experimental animals and the number of nodules metastasized to metastasis-forming organs is measured, it takes 3 to 4 weeks to form metastases, which is inconvenient. Including the method of measuring angiogenesis using a rabbit cornea, it is permissible to handle a large amount of test reagents that need to process a large amount of samples for primary screening from the viewpoint of animal welfare. Not.

  Therefore, as shown in the above known technique, it is desired to establish a measurement method capable of quickly and accurately grasping the kinetics of cancer transfer in a test tube without using an experimental animal. .

Furthermore, as a typical method for measuring the infiltration ability, an extracellular matrix component is applied on a filter attached to a chemotaxis chamber, and further, endothelial cells are cultured on the filter until saturation, The cancer cells are cultivated in layers for a certain period of time, and the invasion capacity of the cancer cells is measured by counting the number of cancer cells that pass through the endothelial cell interval and the filter pores and fall into the lower well. There is. (For example, Patent Document 1)
Furthermore, the method described in Patent Document 1 is a system for evaluating the ability of cancer cells to infiltrate endothelial cells and motility from the viewpoint of cancer cells.

However, according to these patented technologies, cancer metastasis from the viewpoint of pharmacokinetics in which cancer cells are removed clinically because the cancer cells and the endothelial cells of normal cells corresponding to the host are in direct contact with each other. Is a step,
1) Proliferation of cancer cells at the primary lesion 2) Infiltration of surrounding tissues,
3) Invasion into blood vessels 4) Intravascular movement 5) Establishment of metastatic organs in blood vessels 6) After evacuation
In the step of proliferating in metastasis organ tissue and reaching metastasis formation, it corresponds to invasion into blood vessels, and the stage is advanced, and metastasis suppression and metastasis prevention are too late and become a stage.

In addition, a system has been reported in which the level of endothelial cell cleavage is evaluated by measuring endothelial cells whose morphology has been changed by a confocal laser microscope. (Non-Patent Document 2)
International Journal of Oncology 21: 541-546 2002

  However, in addition to the need for an expensive microscope called a confocal laser microscope, the above-mentioned conventional system that measures the number of cells under the microscope does not completely eliminate the possibility that an artificial factor may be introduced by the measurer to determine the cells. Met.

  Therefore, the present invention provides a measurement procedure by a simple operation that is excellent in sensitivity that can detect even a slight change in the cleavage state of endothelial cells, can be used for long-time measurement, and does not enter the individual differences of the practitioner. In addition, cancer cells and vascular endothelial cells from the viewpoint of host cells are used for the purpose of screening for drugs that have metastasis-suppressing effects aimed at improving the prognosis of patients after cancer is found and undergoes resection surgery. To provide a component and a method for measuring a metastasis suppression process by searching for a component having cancer metastasis suppression ability, which is a cancer metastasis model system reflecting the in vivo dynamics in the early stage surrounding the above.

  In order to solve the above-mentioned problems, the first invention is a method for measuring cancer metastasis, that is, a method invention, wherein a substance that is a candidate for an antimetastatic agent is selected from the viewpoint of host endothelial cells. The objective is to evaluate the degree of cell spacing between vascular endothelial cells produced by co-culturing vascular endothelial cells and cancer cells by the amount of electrical resistance across the endothelial cells.

  In addition, the second invention is an invention of a resistance measuring instrument, and a polycarbonate filter and a polystyrene cylinder having pores which are chemotaxis chambers using an existing device designed for analyzing chemotaxis. Is placed so as not to contact the bottom of the 24-well cell culture plate, and vascular endothelial cells are saturated in the small compartment and placed in the 24-well cell culture plate. This is a resistance measuring instrument in which electrodes are inserted at a fixed shortest distance between vascular endothelial cells produced by co-culturing cancer cells.

  According to this invention, first, according to the first invention, that is, the invention according to claim 1, the cancer cells and the vascular endothelial cells are not directly contacted, that is, can be co-cultured in a non-contact state. It is possible to supply components involved in the vascular endothelial cell cleavage phenomenon, which are thought to be secreted from cancer cells, over a long period of time. By focusing on the vascular endothelial cell cleavage phenomenon, a cancer metastasis model was constructed. The metastatic ability can be evaluated.

  In addition, in order to evaluate the cleavage level of vascular endothelial cells cultured in the chemotaxis chamber according to the invention of claim 2, which is the second invention, fixation is performed at the shortest distance by inserting a layer of cultured endothelial cells. Since the electrode to be used is used, there are extremely beneficial effects such that the cleavage level of the endothelial cells can be measured with high sensitivity.

  Hereinafter, the best mode for carrying out the invention described in claim 1 of the present invention will be described. A non-contact co-culture system of vascular endothelial cells and cancer cells will be described.

  FIG. 1 shows an example of a non-contact co-culture system of vascular endothelial cells and cancer cells according to the present invention. This culture system is a polypropylene to which a polycarbonate filter (1) having pores having a pore diameter of 0.4 to 8 micrometers is attached. The cylindrical chemotaxis chamber (2) is suspended from the polypropylene 24-well culture plate (3).

  The extracellular filter component (4) of the polycarbonate filter (1) is coated, on which human umbilical cord-derived vascular endothelial cells are seeded according to a conventional method, and after 3 to 5 days, the vascular endothelial cells are in the chamber (2). A layer (5) of vascular endothelial cells is formed on one side of the filter (1).

  If the polycarbonate filter (1) can be seen through, the cell spacing can be observed under a microscope, but the electrical resistance between the culture compartment upper layer (7) and lower layer (8), which will be described in detail later. Can be judged by level. Depending on the purpose, the sample is added to the medium of the upper layer part (7) and the lower layer part (8) and pre-cultured.

  On the bottom of the polypropylene 24-well culture plate (3) described above, cancer cells established as cell lines and cancer cells (6) obtained by culturing cancer tissues isolated from the human body are seeded. Depending on the purpose, a sample is added to the medium and precultured. The type of cancer cell used in the present invention is not particularly limited, but the timing of measuring the electrical resistance described later differs slightly depending on the type of cancer cell.

  A chemotaxis chamber (2) having a vascular endothelial cell layer (5) formed on one side of the filter (1) is cultured in a well of a 24-well culture plate (3), and cancer cells (6) are contained in the culture solution. In vitro modeling of the process by which cancer cells cleave vascular endothelial cells, infiltrate the surrounding tissues, and metastasize by culturing for a certain period of time together with a culture plate in which components that cleave endothelial cells are secreted from To build.

  From the above, according to the best mode for carrying out the present invention, vascular endothelial cells that are considered to be secreted from cancer cells by co-culturing without direct contact between vascular endothelial cells and cancer cells. It becomes possible to supply the components involved in the cleavage for a long time.

  Next, the best mode of the cancer metastasis inhibitory capacity measuring apparatus according to the second invention will be described. As shown in FIG. 2, a 24-well culture plate (3) and a resistance measuring compartment container (10) similar to a well are used. In order to measure the electrical resistance level between the upper layer part (7) and the lower layer part (8) of the culture compartment, the upper part (7) is prepared. The electrode (11) of the resistor (not shown) is placed as close as possible to the endothelial cells (5), and the other electrode (12) is fitted into the bottom of the container (10). To do. By fixing the electrode (11) installed in the upper layer part (7) to the lid (13) of the container (10) used at the time of measurement, the distance from the bottom surface (1) of the chemotaxis chamber (2) can be measured. It can always be constant. In addition, (14) shown in FIG. 3 is a resistance measuring device EVOM (manufactured by WPI), and (15) is a resistance measuring compartment Endohm (manufactured by WPI).

  From the above, according to the best mode for carrying out the present invention, the degree of cleavage of vascular endothelial cells is evaluated by a numerical value called resistance value, and the personal will of the measurer called cell measurement under a microscope is introduced. It is possible to reduce the room.

  The present invention has industrial applicability by searching for a component having cancer metastasis-inhibiting ability and establishing a technique and a measuring instrument for measuring metastasis-inhibiting ability.

It is explanatory drawing which shows one Example of this invention. It is explanatory drawing which shows the other Example of this invention. It is explanatory drawing which shows one Example of the resistance measuring device used for this invention. It is explanatory drawing which shows the other Example of this invention. It is the graph which measured the cleavage degree of the endothelial cell by the cancer cell by this invention, and a non-cancer cell.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Filter 2 Chemotaxis chamber 3 24-well culture plate 4 Extracellular matrix component 5 Vascular endothelial cell 6 Cancer cell 7 Upper part of culture compartment 8 Lower part of culture compartment 9 Decomposition part of vascular endothelial cell 10 Resistance measurement compartment container 11 Compartment Upper layer electrode 12 Compartment lower layer electrode 13 Resistance measurement container lid 14 Resistance measurement container EVOM (WPI)
15 Resistance measurement compartment Endohm (WP1 company)

Claims (2)

  For the purpose of selecting substances that are candidates for anti-metastatic agents from the viewpoint of host-side endothelial cells, the cell spacing of vascular endothelial cells produced by non-contact co-culture of vascular endothelial cells and cancer cells is determined to be endothelium. A method for measuring the ability to suppress cancer metastasis, characterized by evaluating the amount of electrical resistance across cells.   A 24-well cell culture plate having an opening at the top and cancer cells on the bottom, a chemotaxis chamber developed for analyzing chemotaxis and provided with a polycarbonate filter having pores at the bottom, and the 24-well cell The bottom surface of the culture plate is suspended by a suspension means so as not to contact the chemotaxis chamber, a small compartment is formed, vascular endothelial cells are saturated in the small compartment, and cancer is contained in the 24-well cell culture plate. A cancer metastasis inhibitory capacity measuring instrument, wherein an electrode is inserted at a certain shortest distance through the polycarbonate filter at a cell interval of vascular endothelial cells produced by co-culturing cells.