JP2016048232A - Method and device for detecting cancer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for easily and rapidly detecting the presence of cancer in tissues collected from a living body.SOLUTION: The method for detecting cancer sequentially includes the steps of: applying markers to a tissue sample collected from a living body, the markers having a site specifically bonded and adsorbed or coordinated to cancer cells and a site detectable by an optical technique; performing cleaning for removing a marker not bonded and adsorbed or coordinated to the cancer cells from the tissue sample; and detecting the markers remaining in the tissue sample by the optical technique.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a rapid and simple cancer detection method and detection apparatus.

  In diagnosis of gastrointestinal cancer such as laryngeal cancer, esophageal cancer, stomach cancer, colon cancer, etc., a site suspected of cancer is collected using forceps by endoscopy or the like, and pathological diagnosis (for example, see Patent Document 1) The presence or absence of cancer and the degree of progression are determined by diagnosis using immunostaining (see, for example, Patent Document 2). However, the conventional determination method requires complicated processing such as preparation and staining of a microscope sample, requires a long time until diagnosis, and requires a skill in determination. A determination method is required.

JP 2010-160018 A International Publication No. 2010/098435

  An object of the present invention is to provide a detection method in which the presence or absence of cancer cells in a tissue sample collected from a living body can be quickly determined with a simple technique.

The present inventors have reached the present invention as a result of intensive studies in view of the above. That is, the present invention provides a coating step in which a tissue sample collected from a living body is coated with a marker having a site that specifically binds, adsorbs or coordinates with cancer cells and a site that can be detected by an optical technique;
A washing step of removing a marker that is not bound, adsorbed or coordinated to cancer cells from a tissue sample coated with the marker;
A method for detecting cancer, comprising a detection step of detecting a marker remaining in a tissue specimen after removal of a marker that is not bound, adsorbed or coordinated with a cancer cell, by an optical technique.

  An object of the present invention is to provide a detection method in which the presence or absence of cancer cells can be quickly and easily determined by a simple technique.

It is a flowchart which shows the detection method of the cancer of this invention. It is the schematic of the detection method of the cancer of this invention at the time of using the marker which emits fluorescence or phosphorescence as a marker of this invention. The tissue sample 1 is a cross-sectional view of the tissue sample. It is a conceptual diagram which shows the detection means of the apparatus of this invention. 2 is a fluorescence micrograph of cancer tissue piece 1. 2 is a fluorescence micrograph of a normal tissue piece 1. It is an image divided into gradations 0 to V by image processing of the cancer tissue piece 1. It is an image divided into gradations 0 to V by image processing of the normal tissue piece 1.

  Hereinafter, the present invention will be described, but will be described using the flowchart of FIG. 1 and the schematic diagram of FIG. 2 as necessary.

[Coating process]
First, a tissue sample collected from a living body has a marker that has a site that specifically binds, adsorbs or coordinates to cancer cells and a site that can be detected by an optical technique (hereinafter referred to as “the marker of the present invention”). Will be described. In the applying step, the marker 4 of the present invention is applied to the tissue specimen 1 of FIG. 2A, and the marker 4 is attached to the normal cell portion 2 and the cancer cell portion 3 as shown in FIG. The purpose of the applying step is to bind, adsorb or coordinate the marker of the present invention to cancer cells by applying the marker of the present invention to the collected tissue specimen.

  The tissue specimen to which the cancer detection method of the present invention can be applied is not particularly limited as long as it is derived from a living tissue in which cancer occurs. Examples of such biological tissues include digestive tracts such as the oral cavity, esophagus, stomach, small intestine, large intestine, and anus; digestive glands such as the liver, gallbladder, and pancreas; Organs; urinary organs such as kidney, ureter, bladder, urethra; circulatory organs such as heart, spleen, bone marrow; skin, muscle, subcutaneous, cartilage, bone (pelvis / vertebra), lymph node, breast, thyroid, prostate, testis ( Epididymis), peripheral nerve, uterine vagina, endometrium and the like. Among these, since the detection of cancer is easy, the gastrointestinal tissue is preferable, the stomach and large intestine tissues are more preferable, and the large intestine tissue is most preferable.

  The marker of the present invention is a marker having a site that specifically binds, adsorbs or coordinates to cancer cells and a site that can be detected by an optical technique. The site of the marker of the present invention that specifically binds, adsorbs or coordinates to cancer cells is preferably a site having a residue to which a protein that specifically binds, adsorbs or coordinates to cancer cells is bound. As a protein that specifically binds, adsorbs or coordinates to cells, a lectin or a monoclonal antibody is preferable, and a lectin is more preferable because it is easily available. Various lectins that specifically bind to, adsorb or coordinate to cancer cells are known, and lectins excellent in binding property to cancer cell Thomsen-Friedenreich antigen are preferable, and peanut lectins are more preferable.

  The site | part detectable by the optical method of the marker of this invention is a site | part which the compound detectable by an optical method couple | bonds, adsorb | sucks, or coordinates. Compounds that can be detected by an optical method include compounds that reflect or absorb visible light and develop color (for example, dyes, dyes, pigments, etc.), and compounds that absorb excitation light such as ultraviolet rays and emit fluorescence or phosphorescence. A compound that absorbs excitation light and emits fluorescence is preferable because the contrast ratio between the portion where the marker is present and the portion where the marker is absent can be increased. Examples of compounds that absorb excitation light and emit fluorescence include fluorescein compounds, rhodamine compounds, coumarin compounds, BODIPY compounds, bimane compounds, 5-dimethylaminonaphthalenesulfonyl compounds, dapoxyl compounds, MANT compounds, toluidinylnaphthalenesulfonyl compounds, bimane compounds , Pyrene compounds, cyanine compounds, NBD compounds and the like.

  In the marker of the present invention, a compound that specifically binds, adsorbs or coordinates to a cancer cell and a compound that can be detected by an optical technique may bind directly or indirectly through a spacer. In addition, a compound that specifically binds, adsorbs or coordinates to cancer cells, or a compound that specifically binds, adsorbs or coordinates to cancer cells is detected by an optical technique. Possible compounds may adsorb or coordinate.

  As a marker in which a compound that specifically binds, adsorbs or coordinates to a cancer cell and a compound that can be detected by an optical technique are bound directly or indirectly through a spacer, for example, a marker such as FITC Examples include lectins that are fluorescently labeled with a fluorescent labeling reagent, and compounds that specifically bind to, adsorb, or coordinate to cancer cells bind to, adsorb, or coordinate with compounds that can be detected by optical techniques. Examples of the marker include those disclosed in International Publication No. 2007/097318.

  Examples of the application method when applying the marker of the present invention to a tissue specimen include dipping, dropping, spraying, spraying, brushing, and the like. The tissue specimen coated with the marker of the present invention is subjected to the next washing step after a sufficient time has elapsed for the marker of the present invention to bind to, adsorb or coordinate with cancer cells.

  The marker of the present invention may be applied to a tissue specimen as it is, but it is easy to apply and the marker that is not bound, adsorbed or coordinated to cancer cells in the next washing step can be easily removed. It is preferably applied by dissolving or dispersing in an aqueous medium. In the present invention, the aqueous solvent solution or dispersion of the marker of the present invention may be referred to as “the marker liquid of the present invention”. In the present invention, the aqueous medium refers to a liquid that contains at least 50% of water and is liquid at 25 ° C. Examples of the aqueous medium include water, physiological saline, buffer solution, polyol compound aqueous solution and the like. Examples of the buffer solution include acetate buffer solution, phosphate buffer solution, citrate buffer solution, borate buffer solution, tartaric acid buffer solution, Tris buffer solution, HEPES buffer solution, MES buffer solution and the like. Examples of the polyol compound in the polyol compound aqueous solution include propylene glycol, 1,3-butanediol, glycerin, dipropylene glycol, diglycerin, erythrose, sorbitol and the like.

[Washing process]
Next, a washing process for removing a marker that has not been bound, adsorbed or coordinated to cancer cells from a tissue specimen coated with a marker will be described. In the application step, the marker of the present invention is applied not only to cancer cells but also to normal cells. In order to detect cancer cells, the marker of the present invention needs to be present only in the cancer cell portion. For this reason, in the washing step, in order to facilitate the detection of cancer cells in the next detection step, as shown in FIG. 2 (c), the cancer cell portion 3 of the present invention bound, adsorbed or coordinated is used. Only the marker 4 is left and the other markers are removed.

  The removal of the applied marker has less adverse effect on the tissue specimen, so that an aqueous medium is preferably used, and more preferably the same aqueous medium as that used for the marker of the present invention. Examples of the washing method include (1) a method of immersing a tissue specimen in an aqueous medium, and (2) a method of spraying or dropping an aqueous medium on the tissue specimen. When washing by dipping, it is sufficient to leave the tissue specimen in an aqueous medium, but in order to increase the efficiency of washing, the tissue specimen or the aqueous medium is vibrated, or the aqueous medium is agitated to remove water. A flow may occur in the medium. Moreover, when washing | cleaning by immersion, you may replace | exchange an aqueous medium and may perform immersion several times.

[Detection process]
Next, a detection process for detecting a marker remaining in a tissue specimen after removing a marker that is not bound, adsorbed or coordinated with a cancer cell by an optical technique will be described. By detecting a marker remaining in the tissue specimen by an optical technique, it can be confirmed that cancer cells are present in the tissue specimen. Detection of the marker of the present invention by an optical technique is a compound in which a compound that is bound, adsorbed or coordinated to a detectable site and is detectable by an optical technique reflects or absorbs visible light and is colored. In the case of a compound that emits visible light and in the case of a compound that absorbs excitation light and emits fluorescence or phosphorescence, the compound is irradiated with the excitation light, and the coloration or fluorescence or phosphorescence derived from the marker of the present invention is detected. Is called.

  When the marker of the present invention is a marker that emits fluorescence or phosphorescence, the excitation light 5 irradiated to the tissue specimen 1 is absorbed as it is in the normal cell portion 2 as shown in FIG. In the cancer cell portion 3, the presence of the marker 4 of the present invention emits fluorescence or phosphorescence 6. By detecting this fluorescence or phosphorescence 6, it can be confirmed that the cancer cell portion 3 exists.

  The detection of the coloration or fluorescence or phosphorescence derived from the marker of the present invention may be carried out visually, but it is possible to reduce the error by the detector and handle a large number of samples, so that a detection device can be used. preferable. When the marker of the present invention is a marker that can be detected by fluorescence, a fluorescence detector is used. Data obtained from the fluorescence detector may be imaged or digitized. When the data obtained from the fluorescence detector is digitized, a threshold is set in consideration of the background (fluorescence or phosphorescence derived from normal cells), and when the measured value exceeds the threshold, cancer cells It shall be.

[Detection device]
This invention is invention of the detection apparatus which implement | achieves the detection method of the above-mentioned cancer. Specifically,
Application means for applying a marker having a site that specifically binds, adsorbs or coordinates to cancer cells and a site detectable by an optical technique to a tissue sample collected from a living body;
A washing means for removing a marker that is not bound, adsorbed or coordinated to cancer cells from a tissue specimen coated with the marker;
A cancer detection apparatus having a detection means for detecting a marker remaining in a tissue specimen after removal of a marker that is not bound, adsorbed or coordinated to a cancer cell by an optical technique.

[Applying means]
An application means for applying a marker that has a site that specifically binds, adsorbs or coordinates to cancer cells and a site that can be detected by an optical technique to a tissue sample collected from a living body will be described. The coating unit of the present invention is a unit that can perform the above-described coating process. As a method for applying a tissue sample collected from a living body to the marker of the present invention, since it is simple and can be uniformly applied, the method of immersing the tissue sample in the marker solution of the present invention, the present invention is applied to the tissue sample. The method of dripping or spraying the marker solution is preferable, and the method of dropping or spraying the marker solution of the present invention on the tissue specimen is preferable because automation is easy. In the case of the immersion method, for example, the marker solution of the present invention may be placed in a petri dish or a test tube, and a tissue specimen may be further immersed therein. Moreover, in the case of the method of dripping or spraying, for example, a tissue specimen may be placed on a belt conveyor-like transfer means, and the marker solution of the present invention may be dripped or sprayed from above.

[Washing means]
A cleaning means for removing a marker that is not bound, adsorbed or coordinated to a cancer cell from a tissue sample coated with the marker will be described. The cleaning unit is a unit that can perform the above-described cleaning step. As a method for washing, a method of immersing a tissue specimen in an aqueous medium and a method of dropping or spraying the aqueous medium on a tissue specimen are preferable because they are simple, and since automation is easy, an aqueous medium is applied to the tissue specimen. The method of dripping or spraying is preferable. In the case of the immersion method, for example, a tissue sample may be placed in a petri dish or a test tube, an aqueous medium may be added, and shaken lightly. In the case of a small tissue sample, a microtube may be used. Moreover, in the case of the method of dripping or spraying, for example, a tissue sample may be placed on a belt conveyor-like transfer means, and the aqueous medium may be dripped or sprayed from the top.

[Detection means]
Next, detection means for detecting a marker remaining in a tissue specimen after removing a marker that is not bound, adsorbed or coordinated to a cancer cell by an optical technique will be described. The detection means of the present invention is a means capable of performing the detection process described above. The detection means of the present invention requires an irradiator that irradiates the washed tissue specimen with visible light or excitation light, and a detector that detects coloration or fluorescence or phosphorescence derived from the marker of the present invention. That is, as shown in FIG. 3, the tissue sample 7 after the washing treatment is irradiated with visible light or excitation light 10 from an irradiator 8, and the coloration or fluorescence or phosphorescence derived from the marker of the present invention generated thereby. 11 is detected by the detector 9. Although the presence or absence of cancer is determined based on the data obtained by the detector, cancer cells are not necessarily present on the entire surface of the tissue sample, and detection is performed in the portion of the tissue sample where cancer cells are present and in the portion that does not exist. The data obtained by the vessel will vary greatly. For this reason, in order to improve the accuracy of cancer detection, it is necessary to measure the portion where cancer cells are present, that is, the portion where the data obtained by the detector has a higher measurement value. Preferably, the data is sent to the computer 12 and imaged and image-processed to determine the presence or absence of cancer based on the measurement value and the size of the portion where the measurement value of the detector is high.

  Hereinafter, the present invention will be specifically described by way of examples.

[Marker solution]
A marker A was synthesized by the same method as in Example 2 of WO 2007/097318 except that coumarin 6 was used instead of fluoresceinated cholesterol, and a davelcolic acid buffer solution containing calcium ions and magnesium ions ( A marker solution was prepared by dispersing the marker A to 1% by mass in Sigma-Aldrich Japan, trade name: hereinafter abbreviated as DPBS (+)). Marker A is a fine particle having coumarin 6 as a fluorescent compound and peanut lectin as a site that binds, adsorbs or coordinates with cancer, and the average particle size measured by a dynamic light scattering method is 400 nm.

[Body tissue used for evaluation]
Human colon cancer cell line (HT-29) was transplanted under the serosa of the rectum of nude mice, and colon cancer tissues that had been bred for 28 days to establish and grow cancer cells and normal tissues of the colon of nude mice were tested. Using.

〔Test method〕
Tissue pieces (three pieces of cancer tissue pieces and two normal tissue pieces) cut into small pieces were immersed in the marker solution for 2 minutes, and then the excess detection solution was removed using DPBS (+). The tissue piece treated with the detection solution was photographed with a fluorescence microscope (Olympus, model: BX63) (observation magnification 40 times, exposure time 1/60, using FITC filter), and the photographed image was image software (Mitani Corporation). , Product name WinROOF) was subjected to image processing by the following method, and the area% ratio of gradations III to V was determined by fluorescence intensity by the following method. The results are shown in Table 1. 4 and 5 show fluorescence micrographs of cancer tissue pieces or normal tissue pieces, and FIGS. 6 and 7 show images divided into gradations 0 to V by image processing of cancer tissue pieces or normal tissue pieces.
(1) The image (color image) of the fluorescence microscope is converted into a black and white image.
(2) A black and white image of a cancer tissue piece is divided into 256 gradations according to shading.
(3) The gradation without the texture piece is divided into gradation 0 and the remaining gradation is divided into five gradations with the gradation width shown in Table 1, and gradations I to V are set in ascending order of emission intensity.
(4) The area% ratio of each of the gradations I to V is obtained.
(5) For the image of the normal tissue piece, the gradation percentage of the same scale as that of the image of the cancer tissue piece is applied to obtain the area% ratio of the gradations I to V (therefore, the gradation IV or V is 0). % Is also possible).

  It is difficult to discriminate between a cancer tissue piece and a normal tissue only with the value of gradation IV or V having the highest fluorescence intensity, but with the total value of gradations III to V, all of the cancer tissue pieces are 5%. However, the normal tissue piece is less than 1%, and the cancer tissue piece and the normal tissue can be easily distinguished. 4 and 5 are fluorescence micrographs of the cancer tissue piece 1 and the normal tissue piece 1, and FIGS. 6 and 7 are images that are divided into gradations I to V after image processing. Although it is difficult to discriminate between a cancer tissue fragment and a normal tissue only with a fluorescent micrograph, it is easy to discriminate by image processing, but it becomes easier to discriminate by digitizing as described above. Therefore, it can be said that the method of the present invention is a detection method in which the presence or absence of cancer cells in a tissue sample collected from a living body can be quickly determined with a simple technique.

DESCRIPTION OF SYMBOLS 1 Tissue sample 2 Normal cell part 3 Cancer cell part 4 Marker 5 Excitation light 6 Fluorescence or phosphorescence 7 Tissue sample 8 after washing processing Irradiator 9 Detector 10 Visible light or excitation light 11 Coloration or color derived from the marker of the present invention Fluorescent or phosphorescent 12 Computer

Claims (11)

An application step of applying a marker having a site that specifically binds, adsorbs or coordinates to cancer cells and a site detectable by an optical technique to a tissue sample collected from a living body;
A washing step of removing a marker that is not bound, adsorbed or coordinated to cancer cells from a tissue sample coated with the marker;
A detection step of detecting a marker remaining in a tissue specimen after removal of a marker that is not bound, adsorbed or coordinated to a cancer cell by an optical technique;
A method for detecting cancer.   The method for detecting cancer according to claim 1, wherein the site that specifically binds, adsorbs or coordinates to the cancer cell is a site that has a binding property to a sugar chain antigen.   The method for detecting cancer according to claim 1 or 2, wherein the site detectable by an optical technique is a site that absorbs excitation light and emits fluorescence and becomes optically detectable.   The method for detecting cancer according to claim 3, wherein the detection by an optical technique in the detection step is measurement of fluorescence using a fluorescence detector.   The detection step of detecting by an optical method is a step of detecting a marker remaining in a tissue specimen by imaging and image-processing data obtained by an optical method. The method for detecting cancer according to 1.   A tissue sample collected from a living body is coated with a marker that has a site that specifically binds, adsorbs or coordinates to cancer cells and a site that can be detected by optical techniques, and binds, adsorbs or coordinates to cancer cells. A cancer detection apparatus comprising detection means for detecting a marker remaining in a tissue specimen obtained by removing unmarked markers by an optical technique. Application means for applying a marker having a site that specifically binds, adsorbs or coordinates to cancer cells and a site detectable by an optical technique to a tissue sample collected from a living body;
A washing means for removing a marker that is not bound, adsorbed or coordinated to cancer cells from a tissue specimen coated with the marker;
A cancer detection apparatus comprising detection means for detecting a marker remaining in a tissue specimen after removal of a marker that is not bound, adsorbed or coordinated to a cancer cell by an optical technique.   The cancer detection apparatus according to claim 6 or 7, wherein the site that specifically binds, adsorbs, or coordinates to a cancer cell is a site that has a binding property to a sugar chain antigen.   The cancer detection apparatus according to any one of claims 6 to 8, wherein the site that can be detected by an optical technique is a site that absorbs excitation light and emits fluorescence and becomes optically detectable.   The cancer detection apparatus according to any one of claims 6 to 9, wherein the detection by the optical means in the detection means is measurement of fluorescence using a fluorescence detector.   The detection means for detecting by an optical technique is means for detecting a marker remaining in a tissue specimen by imaging and processing data obtained by an optical technique. The cancer detection apparatus according to 1.

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