Preprocessing method for fluorescence image diagnosis

Abstract

The purpose of the present invention is to provide a method for causing a target cell to emit light, said target cell being present inside a cell mass and being capable of causing a fluorescent probe to emit light. The present invention provides a method for causing a target cell to fluorescently emit light, comprising: a step for removing calcium ions from a cell mass or tissue containing the target cell; and a step of bringing a fluorescent probe into contact with the cell mass or tissue, the fluorescent probe being capable of emitting fluorescence by being brought into contact with or incorporated into the target cell. Thus, since the target cells present in the interior of the cell mass and the tissue can be made to emit light, the present invention can be used to detect the target cells in a sample, such as a clinical sample, in which the target cells may not necessarily be present on the surface.

Description

[0001] This case is a divisional application of a case with an application date of July 12, 2019, an invention name of "Preprocessing Method for Fluorescence Image Diagnosis" and an application number of 201980035559.9. Technical Field

[0002] The present invention relates to a method for pre-treating tissue when performing fluorescence imaging diagnosis using a fluorescent diagnostic agent that reacts with a tumor marker or the like and emits fluorescence. Background Art

[0003] The success of cancer surgery depends on the early and accurate determination of the location of cancer cells and their complete removal. Therefore, visualization of the tumor area during surgery is required, and fluorescent probes that react specifically to tumors have been developed (non-patent literature 1 to 8). However, simply applying the fluorescent probe directly to the tumor area has problems such as insufficient fluorescence intensity in the early stage, difficulty in distinguishing from the cell's inherent spontaneous light emission, and rapid disappearance of fluorescence. Therefore, methods for determining the cancer area using kinetic maps are being studied (non-patent literature 9); cutting, compression and cleaning treatments (non-patent literature 10); multispectral open-air methods (non-patent literature 11) and fluorescent probes that cross-link with intracellular proteins (non-patent literature 12).

[0004] Prior art literature

[0005] Non-patent literature

[0006] Non-patent document 1: Yasuteru Urano et al., Science Translational Medicine; 3(110): 110-119 (2011)

[0007] Non-patent document 2: Yoichi Miyata et al., Scientific Reports; 7: 36542 (2017)

[0008] Non-patent document 3: Takeshi Mizushima et al., BMC Cancer; 16; 411 (2016)

[0009] Non-patent document 4: Yasuteru Urano, Current Opinion in Chemical Biology; 16: 602-608 (2012)

[0010] Non-patent document 5: Makoto Mitsunaga et al., Gut; 62: 1179-1186 (2013)

[0011] Non-patent document 6: Hiroaki Ueo et al., Scientific Reports; 5: 12080 (2015)

[0012] Non-patent document 7: Yoshiaki Shinden et al., Scientific Reports; 6: 27525 (2016)

[0013] Non-patent document 8: Haruaki Hino et al., Translational Oncology; 9: 203-210 (2016) Non-patent document 9: Yuko Nakamura et al., Oncotarget; 7 (32): 51124-51137 (2016)

[0014] Non-patent literature 10: Toshiko Harada et al., Contrast Media Mo. Imaging (2016) DOI: 10.1002 / cmmi.1775

[0015] Non-patent document 11: Ali Behrooz et al., Optics Letters; 42(15): 2964-2967 (2017) Non-patent document 12: Yuko Nakamura et al., Oncotarget; 8(24): 39512-39521 (2017) Summary of the invention

[0016] Problems to be solved by the invention

[0017] When the inventors of the present invention studied the staining of cell clusters or tissues using fluorescent probes, they found that although the tissues and lymph nodes removed by surgery in the clinic were obviously cancerous, even if the fluorescent probes were brought into contact, there were cases where no fluorescence that could be distinguished from non-cancer areas was observed. As a result of further research, the inventors of the present invention found that when the fluorescent probes were brought into contact with cancer cell clusters, the cancer cells on the surface quickly fluoresced, but on the other hand, the cancer cells inside took a long time to glow. As a result, the inventors of the present invention discovered a new problem: by using the previous method of detecting cancer cells using fluorescent probes, it is difficult to detect cancer cells that are not present on the surface of cell clusters. Therefore, the purpose of the present invention is to provide a method for rapidly emitting light to target cells, which are present inside cell clusters and can be made to emit light by fluorescent probes.

[0018] Methods used to solve problems

[0019] The inventors of the present invention have studied various treatments for improving luminescence of cell aggregates containing target cells that are made to emit fluorescence by fluorescent probes, and found that by removing calcium ions in the cell aggregates containing target cells, the entire interior of the cell aggregates can be rapidly luminesced, thereby completing the present invention. The treatment method of the present invention is a general method that can utilize any adherent cells and any fluorescent probes regardless of the type of cells and the type of fluorescent probes.

[0020] Therefore, the present invention relates to the following:

[0021] (1) A method for causing a target cell to emit fluorescence, comprising:

[0022] a step of removing calcium ions from a cell mass or tissue containing the target cells; and

[0023] A step of bringing a fluorescent probe into contact with the cell mass or tissue, wherein the fluorescent probe is capable of emitting fluorescence by contacting with the target cell or being incorporated into the target cell.

[0024] (2) The method according to (1), which is a method of causing the interior of a target cell mass to emit fluorescence, or a method of causing target cells present in a cell mass or tissue to emit fluorescence.

[0025] (3) A method for determining a region where a target cell exists, comprising:

[0026] a step of removing calcium ions from cell aggregates or tissues where the target cells are believed to be present;

[0027] a step of bringing a fluorescent probe into contact with a cell mass or tissue believed to contain the target cell, wherein the fluorescent probe is capable of emitting fluorescence by contacting with the target cell or being incorporated into the target cell; and

[0028] A step of determining that the region where the fluorescence emission caused by the fluorescent probe is observed is the region where the target cell exists.

[0029] (4) The method according to any one of (1) to (3), wherein

[0030] The fluorescent probe that emits fluorescence by contacting with or being incorporated into the target cell is a fluorescent probe that emits fluorescence by reacting with a substance contained in the target cell, by physical properties such as pH of the target cell, or by a hypoxic state of the target cell.

[0031] (5) The method according to any one of (1) to (4), wherein the substance contained in the target cells is an enzyme, an ion, nitric oxide or a reactive oxygen species.

[0032] (6) The method according to (5), wherein the enzyme is a glycosidase, a protease or a peptidase.

[0033] (7) The method according to (5) or (6), wherein the enzyme is an enzyme present on the surface of a cell membrane.

[0034] (8) The method according to (5), wherein the ions are calcium ions or metal ions.

[0035] (9) The method according to any one of (1) to (8), wherein the removal of calcium ions is performed by immersing the cell mass or tissue containing the target cells in a buffer containing a metal ion chelator or a buffer not containing calcium.

[0036] (10) The method according to (9), wherein the immersion is performed for 30 seconds to 30 minutes.

[0037] (11) The method according to any one of (1) to (10), wherein the contacting step with the fluorescent probe is performed at pH 6 to 8.

[0038] (12) The method according to any one of (1) to (11), wherein the target cell is a cancer cell or a tumor cell.

[0039] (13) The method according to (12), wherein the cancer is a solid cancer.

[0040] (14) The method according to any one of (1) to (13), wherein the target cells are caused to emit fluorescence having an intensity capable of distinguishing the target cells from non-target cells.

[0041] (15) The method according to any one of (1) to (14), wherein the fluorescent probe is gGlu-HMRG, Leu-HMRG, EP-HMRG or FeRhoNox-1.

[0042] (16) The method according to any one of (1) to (15), wherein the method is performed in vitro.

[0043] (17) The method according to any one of (1) to (16), wherein the method is performed on a target cell in a living body.

[0044] (18) A method for distinguishing cancer cells or tumor cells from non-cancer cells or non-tumor cells, comprising:

[0045] By the method described in (12), a step of causing cancer cells or tumor cells to emit fluorescence; and

[0046] The step of distinguishing cancer cells or tumor cells from non-cancer cells or non-tumor cells by determining that cells that emit fluorescence are cancer cells or tumor cells and determining that cells that do not emit fluorescence are non-cancer cells or non-tumor cells.

[0047] (19) A method for diagnosing cancer or tumor, comprising:

[0048] A step of removing calcium ions from a cell mass sample or a tissue sample from a subject;

[0049] a step of bringing a fluorescent probe into contact with the sample, wherein the fluorescent probe is capable of emitting fluorescence by contacting with cancer cells or tumor cells or being incorporated into cancer cells or tumor cells; and

[0050] When cells emitting fluorescence are present in the sample, the subject is diagnosed as having cancer or tumor.

[0051] Effects of the Invention

[0052] The method of causing target cells to emit fluorescence of the present invention can cause target cells existing inside cell clusters and tissues to emit fluorescence. Therefore, the present invention can be used to detect target cells in samples such as clinical samples where target cells may not necessarily exist on the surface. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 : Effect of PBS(-) pretreatment in clinical cancer samples. Lymph node samples removed during breast cancer surgery were pretreated by immersion in PBS(-) for 0 minutes, 1 minute, 5 minutes, 10 minutes or during the progression period. Fluorescence increment after immersion in 50μM gGlu-HMRG solution for 10 minutes. In the pathological diagnosis of the sample, those diagnosed with cancer metastasis are indicated by black circles, and those without cancer metastasis are indicated by white triangles.

[0054] Figure 2 : Effect of PBS(-) pretreatment in cancer-derived cultured cell aggregates (spheroids). Changes in fluorescence intensity over time when HeLa cell spheroids were washed with HBSS and then immersed in a 1 μM gGlu-HMRG solution dissolved in HBSS (top), and after pretreatment in PBS(-) for 10 minutes, immersed in a 1 μM gGlu-HMRG solution dissolved in PBS(-) (bottom).

[0055] Figure 3 : Effect of PBS(-) on cell adhesion. Transmitted light images of spheroids without PBS(-) pretreatment (left) and spheroids pretreated with PBS(-) for 10 minutes after immersion in 1 μM gGlu-HMRG solution. The upper image shows the whole image, and the lower image shows the enlarged image of the edge of the cell mass.

[0056] Figure 4 : Schematic representation of the method to analyze the penetration and response of reagents to spheroids. Figure 5-Figure 10 When the depth (Depth) shown in the figure is, for example, 200μm, the fluorescence intensity of the white area is calculated from the fluorescence intensity of the image obtained by fluorescence microscopy. As shown in this figure, the white area has a circumference of 200μm from the outer periphery as the inner side and a circumference of 100μm from the inner side to the outer side as the outer side.

[0057] Figure 5 : Changes in the penetration and response of reagents to spheroids pretreated with PBS(-). Fluorescence intensity distribution of HeLa cell spheroids after immersion in a 50μM gGlu-HMRG solution dissolved in HBSS for 5 minutes without pretreatment (black triangles) and after immersion in PBS(-) for 10 minutes and then in a 50μM gGlu-HMRG solution dissolved in PBS(-) for 5 minutes (black circles).

[0058] Figure 6 : Differences in pretreatment effects due to the composition of the pretreatment solution. Fluorescence intensity distribution of A549 cell spheroids after immersion in PBS- (black circles), PBS+ (black diamonds), PBS+Ca(*), and PBS+Mg (black squares) for 10 minutes and then in 50 μM gGlu-HMRG solutions dissolved in each solution for 5 minutes.

[0059] Figure 7 : Differences in pretreatment effects due to the composition of the pretreatment solution. Fluorescence intensity distribution after A549 cell spheroids were immersed in PBS- (black circles), PBS+ (black diamonds), and saline (white squares, *) for 10 minutes, and then immersed in 50 μM gGlu-HMRG solutions dissolved in PBS- (black circles, *), PBS+ (black diamonds), and saline (white squares) for 5 minutes.

[0060] Figure 8 : Differences in the penetration and reaction of reagents due to the presence or absence of pretreatment and the composition of the pretreatment solution. Fluorescence intensity distribution of HeLa cell spheroids without pretreatment (black triangles), immersed in PBS (black circles), PBS+ (black diamonds), and PBS-EDTA (white diamonds) for 10 minutes, and then immersed in 50μM gGlu-HMRG solutions dissolved in PBS (black triangles, black circles), PBS+ (black diamonds), and PB-EDTA (white diamonds) for 5 minutes.

[0061] Fig. 9 : Differences in the penetration and reaction of reagents due to the presence or absence of pretreatment and the composition of the pretreatment solution. Fluorescence intensity distribution of A549 cell spheroids after being immersed in PBS (black circle), PBS+ (black diamond), and PBS-EDTA (white diamond) solutions for 10 minutes without pretreatment (black triangle), and then immersed in 50μM gGlu-HMRG solutions dissolved in PBS (black triangle, black circle), PBS+ (black diamond), and PB-EDTA (white diamond) for 5 minutes.

[0062] Fig.10 : Differences in pretreatment effects due to the composition of the pretreatment solution. Fluorescence intensity distribution of A549 cell spheroids after immersion in PBS- (black circles), PBS+ (black diamonds), PBS+Ca(*), and PB-EDTA (white circles) for 10 minutes and then in 50 μM FeRhoNoxg-1 solutions dissolved in each solution for 5 minutes. DETAILED DESCRIPTION

[0063] In one aspect, the present invention is a method for causing a target cell to emit fluorescence, comprising:

[0064] a step of removing calcium ions from a cell mass or tissue containing the target cells; and

[0065] The step of contacting the cell mass or tissue with a fluorescent probe, wherein the fluorescent probe can emit fluorescence by contacting the target cell or being incorporated into the target cell. Preferably, the present invention is a method for causing the inside of a target cell mass to emit fluorescence, or a method for causing target cells present in a cell mass or tissue to emit fluorescence, comprising the above steps.

[0066] In another aspect, the present invention relates to a method for determining an area where a target cell is present, comprising:

[0067] a step of removing calcium ions from cell aggregates or tissues where the target cells are believed to be present;

[0068] a step of bringing a fluorescent probe into contact with a cell mass or tissue believed to contain the target cell, wherein the fluorescent probe is capable of emitting fluorescence by contacting with the target cell or being incorporated into the target cell; and

[0069] The step of determining that the region where the fluorescence emission caused by the fluorescent probe is observed is the region where the target cell exists.

[0070] In another aspect, the present invention relates to a method for determining whether a target cell is present, comprising:

[0071] A step of removing calcium ions from the cell mass or tissue being examined;

[0072] a step of bringing a fluorescent probe into contact with the target cell mass or tissue, wherein the fluorescent probe is capable of emitting fluorescence by contacting with the target cell or being incorporated into the target cell; and

[0073] A step of determining the presence of the target cells when fluorescence emission from the fluorescent probe is observed.

[0074] In the present invention, "target cells" refer to cells that can emit light by contacting with the adopted fluorescent probe or incorporating the fluorescent probe. Therefore, the target cells and the adopted fluorescent probe can be appropriately selected according to the purpose. For example, when the fluorescent probe is gGlu-HMRG, the target cells are cancer cells, especially solid cancer cells. "Cancer" in this specification can be listed as but not limited to: lung cancer, non-small cell lung cancer, small cell lung cancer, non-Hodgkin's lymphoma, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, cerebellar astrocytoma in children, pediatric brain astrocytoma, basal cell carcinoma, skin cancer (non-melanoma), biliary cancer, extrahepatic bile duct cancer, intrahepatic bile duct cancer, bladder cancer, bone and joint cancer, osteosarcoma and malignant fibrous histiocytoma, brain cancer, brain tumor, brain stem glioma, cerebellar astrocytoma, glioma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial Neuroectodermal tumors, optic pathway and hypothalamic gliomas, head and neck cancer, metastatic squamous cell carcinoma, bronchial adenoma / carcinoid tumor, adrenal tumors, nervous system lymphoma, cancer of the central nervous system, central nervous system lymphoma, neuroblastoma, childhood cancer, Seiary syndrome, extracranial germ cell tumors, extragonadal germ cell tumors, extrahepatic bile duct cancer, eye cancer, intraocular melanoma, retinoblastoma, salivary gland cancer, oral cancer, oral cavity cancer, oropharyngeal cancer, oral cancer, tongue cancer, esophageal cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), small intestine cancer, Colon cancer, colorectal cancer, rectal cancer, anorectal cancer, anal cancer, germ cell tumors, hepatocellular (liver) cancer, Hodgkin's lymphoma, laryngeal cancer, pharyngeal cancer, hypopharyngeal cancer, nasopharyngeal cancer, paranasal and nasal cancer, pharyngeal cancer, islet cell tumors (endocrine pancreas), pancreatic cancer, parathyroid cancer, liver cancer, gallbladder cancer, appendix cancer, kidney cancer, urethra cancer, renal pelvis and ureteral transitional cell cancer and other urinary cancers, squamous cell carcinoma, penile cancer, testicular cancer, thymoma, thymoma and thymic cancer, thyroid cancer, prostate cancer, ovarian cancer, ovarian epithelial cancer, ovarian low malignant potential tumor, ovarian germ cell tumor, gestational trophoblastic tumor Tumor, breast cancer, uterine cancer, uterine sarcoma, cervical cancer, endometrial cancer, uterine corpus cancer, vaginal cancer, vulvar cancer, Wilms' tumor, skin cancer (non-melanoma), skin cancer (melanoma), pheochromocytoma, Merkel cell skin cancer, mesothelioma, malignant mesothelioma, multiple endocrine neoplasia syndrome, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorders, pineoblastoma and pituitary tumor, plasma cell tumor, pleuropulmonary blastoma, retinoblastoma, rhabdomyosarcoma, sarcoma, Ewing's sarcoma, soft tissue sarcoma, soft tissue sarcoma, mycosis fungoides, Kaposi's sarcoma.

[0075] In this specification, "cell mass" refers to a mass formed by a plurality of cells of the same or different species, including tissue. In this specification, "tissue" refers to a biological structure formed by a three-dimensional arrangement of cells of the same or different species, for example, including epithelial tissue, connective tissue, muscle tissue and nervous tissue. "Cell mass or tissue containing target cells" can include target cells, and can also include cells or tissues other than other target cells. In addition, "cell mass or tissue in which target cells are believed to be present" does not necessarily have to have target cells, as long as it is a cell mass or tissue in which target cells may be present, and can have cells or tissues other than target cells. "Target cell mass" refers to a cell mass mainly composed of target cells.

[0076] The fluorescence luminescence brought by the target cell is carried out by generating a fluorescent pigment by the environment of the cell surface or the cell by contacting the target cell surface with a fluorescent probe, or by being incorporated into the target cell interior. The environment in which the fluorescent pigment is generated from the fluorescent probe can be an environment such as hypoxia or acidic / alkaline, or an environment in which a specific substance is present. That is, "a fluorescent probe that emits fluorescence by contacting with a target cell or being incorporated into its interior" refers to a fluorescent probe that emits fluorescence by reacting with a substance contained in the target cell, or a fluorescent probe that emits fluorescence by hypoxia, acidity or alkalinity in the target cell or its surroundings. Wherein, acidity or alkalinity refers to the height of the pH relative to the organism, for example, acidity can be below pH6, and can be above pH8.

[0077] In addition, as examples of the above-mentioned specific substances, enzymes, ions, nitric oxide or reactive oxygen species can be listed. As long as the enzyme is a protein that can catalyze a specific reaction, it is not particularly limited, for example, glycosidase, protease or peptidase (γ-glutamine transpeptidase, etc.) can be listed. In addition, the above-mentioned enzyme can be an enzyme present on the cell membrane surface, or it can be an enzyme present in the cell. The ions in this manual can list calcium ions or metal ions (such as iron ions, copper ions, zinc ions). As reactive oxygen species, hypochlorous acid, hydrogen peroxide, reactive oxygen, peroxynitrite (peroxynitrite) and hydroxyl radical can be listed.

[0078] For example, the fluorescent probe may be a substance that is not incorporated into cells by being made hydrophilic, etc. Specifically, the fluorescent probe may be gGlu-HMRG, Leu-HMRG, EP-HMRG, or FeRhoNox-1. In addition, the fluorescent probe may be DDS-formed by being embedded in a medium such as a ribosome.

[0079] If the removal of calcium ions is a method for removing calcium ions in cell masses and tissues, it is not necessary to be particularly limited. For example, it can be carried out by immersing the cell masses or tissues containing the target cells in a buffer containing a metal ion chelator or a buffer containing no calcium ions. The metal ion chelator can use a divalent metal ion chelator such as ethylenediaminetetraacetic acid (EDTA), ethylene glycol ether diaminetetraacetic acid (EGTA) or citric acid. In addition, the buffer can include phosphate buffered saline (PBS), phosphate buffer, sodium citrate buffer, citric acid-phosphate buffer, Tris hydrochloric acid buffer (Tris-HCl), MES, PIPES, HEPES, ADA, ACES, MOPSO, BES, MOPS, TES, DIPSO, TAPSO, POPSO, HEPPSO, EPPS, Bicine, TAPS, Tricine. The immersion can be carried out for 30 seconds to 1 hour, 30 seconds to 30 minutes, 1 to 20 minutes, 3 to 10 minutes, 5 to 15 minutes or 10 to 20 minutes. In addition, the immersion can be performed at pH 7 to 8. Removal of calcium ions does not mean 100% removal, and it is sufficient as long as the adhesion between cells is loosened and the fluorescent probe can reach the target site in the cell mass or tissue. In particular, when the fluorescent probe reacts with a substance on the cell surface to produce a fluorescent substance, it is desirable to remove calcium ions without washing out the substance contained on the surface of the cell mass and tissue and the fluorescent substance generated by the reaction.

[0080] The contact of the fluorescent probe with the cell mass or tissue can be carried out by immersing the cell mass or tissue in a liquid containing the fluorescent probe, or by dripping the liquid containing the fluorescent probe onto or spreading it to the cell mass or tissue or the position containing the two by spraying, etc. The liquid containing the fluorescent probe is preferably a buffer, more preferably a buffer that can remove calcium ions from the cell mass or tissue. For example, the fluorescent probe can be used by containing a buffer containing a chelating agent or a calcium-free PBS.

[0081] Measuring and observing the fluorescence emission from the target cells can be performed by appropriately using devices and methods known to those skilled in the art to detect the emission at a wavelength corresponding to the fluorescent probe used. Preferably, the fluorescence emission from the target cells is a fluorescence with an intensity that can distinguish the target cells from non-target cells. In addition, the fluorescence emission from the target cells can also be an intensity that can distinguish the target cells from non-target cells by visual observation.

[0082] The method of the present invention is particularly useful when tumor cells and cancer cells are present inside a cell mass, but the tumor cells and cancer cells are not detected by contacting the fluorescent probe alone. By using the method of the present invention, tumor cells and cancer cells inside a cell mass can be made to emit light, thereby reliably detecting the presence of tumors and cancer cells inside a cell mass and tissue.

[0083] Therefore, in one aspect, the target cells are cancer cells or tumor cells. The present invention relates to a method for distinguishing cancer cells or tumor cells from non-cancer cells or non-tumor cells, comprising: a step of causing cancer cells or tumor cells to emit fluorescence by the above method; and a step of determining cells that emit fluorescence as cancer cells or tumor cells, and determining cells that do not emit fluorescence as non-cancer cells or non-tumor cells, thereby distinguishing cancer cells or tumor cells from non-cancer cells or non-tumor cells. This distinction between cancer cells or tumor cells and non-cancer cells or non-tumor cells can, for example, accurately remove cancer cells or tumor cells during surgery, and can also improve diagnostic accuracy when diagnosing cancer or tumors.

[0084] In another aspect, the present invention relates to a method for diagnosing or monitoring cancer or tumor, or a method for providing information therefor, comprising: a step of removing calcium ions from a cell mass sample or a tissue sample from a subject; a step of contacting a fluorescent probe with the sample, wherein the fluorescent probe can emit fluorescence by contacting with cancer cells or tumor cells or being incorporated into cancer cells or tumor cells; and, when there are cells emitting fluorescence in the sample, a step of diagnosing the subject as having cancer or tumor or determining that the subject has cancer or tumor (or has a poor follow-up or prognosis), or a step of providing the above information. In the present method, as a fluorescent probe that emits fluorescence by contacting with cancer cells or tumor cells or by being incorporated into cancer cells or tumor cells, for example, gGlu-HMRG can be cited. In the present specification, "monitoring method" includes: monitoring whether a patient who may have cancer or tumor has cancer or tumor; monitoring whether the follow-up of a cancer or tumor patient after surgery or treatment is good, whether the cancer or tumor patient is good, or whether the prognosis is good.

[0085] The method of the present invention can be performed in vitro, ex vivo or in vivo. For example, when the method of the present invention is performed in order to determine the area where cancer cells are present during cancer surgery, the method of the present invention is performed on target cells in a living body. In this case, the method of the present invention may further include the step of removing cells determined to be cancer cells or tumor cells by the above method.

[0086] Hereinafter, the present invention will be described in more detail based on examples. However, the present invention is not limited to these examples. In addition, all documents cited in the entire application are directly incorporated into the present application by reference.

[0087] Example

[0088] (Example 1) Clinical Samples

[0089] During breast cancer surgery, the lymph nodes including the sentinel lymph nodes removed for pathological diagnosis are separated and carried from the operating room to the examination room wrapped in gauze moistened with PBS. The reagents and PBS (140 mM NaCl, 2.7 mM KCl, 10 mM PO4 3- , pH = 7.4). The PBS-immersed samples were immersed in a container containing PBS for 1 minute, 5 minutes, 10 minutes, or a progression time of 1 minute to 30 minutes. The non-PBS-immersed samples were stored in a state of being wrapped in gauze moistened with PBS, instead of PBS immersion, for fluorescence measurement. The lymph node was carefully removed with tweezers, placed on a plate, and a visible image of autofluorescence was captured using a fluorescence measurement machine. 50μMgGlu-HMRG dissolved in PBS was added to the plate containing the lymph node, and the fluorescence measurement was continued until the lymph node was completely submerged. In addition, the samples used in this experiment were pathologically diagnosed and used as a control as the final result of whether the cancer cells had metastasized to each lymph node.

[0090] Figure 1 The fluorescence intensity increase in the lymph node 10 minutes after the start of the measurement is shown. The fluorescence intensity is expressed as a ratio to the fluorescence intensity of a fluorescence intensity standard plate having a certain fluorescence intensity (fluorescence intensity obtained by measurement / fluorescence intensity of the fluorescence intensity standard plate).

[0091] Among all 25 samples, almost no increase in fluorescence was observed in the five samples in which no cancer metastasis was found in pathological diagnosis. In the 20 cases where cancer was found, strong fluorescence was observed in the samples immersed in PBS regardless of the immersion time. On the other hand, only weak fluorescence was observed in the samples not immersed in PBS, and examples were observed that were difficult to distinguish from samples without cancer cells. Based on the present results, it is shown that the diagnostic accuracy of gGlu-HMRG can be improved by immersing the samples in PBS before using a fluorescent reagent to detect cancer.

[0092] (Example 2) Changes in the fluorescence intensity of gGlu-HMRG in spheroids prepared from cultured cancer cells

[0093] Take 10 5HeLa cells were inoculated into cells / well and cultured for 4-6 days. The obtained spheroids were placed on a glass-bottomed culture dish, cultured for about 3 hours, the culture medium was removed, and the spheroids were immersed in PBS(-) for 10 minutes. The control group was not immersed in PBS(-) and washed with HBSS. A 1μM gGlu-HMRG solution dissolved in PBS(-) or HBSS was added to the spheroids immersed in PBS(-) or the control group spheroids, respectively. The spheroids were photographed using a fluorescence microscope (Leica DMI 6000CS, lens: 10×, filterLeica L5, excitation wavelength 460-500nm, fluorescence wavelength 512-542nm) when the gGlu-HMRG solution was just added (0 minutes later), 10 minutes later, 20 minutes later, 30 minutes later, and 60 minutes later. In addition, a transmitted light image of the spheroid was taken 10 minutes after immersion in PBS(-).

[0094] Figure 2 The results of fluorescence observation are shown. Compared with PBS (-), it was observed that the fluorescence in the central part of the spheroid rose slowly in HBSS (a physiological buffer closer to blood). That is, it was shown that the solution composition and pre-immersion treatment are important for the reaction and penetration speed of the reagent. In addition, the transmitted light image ( Figure 3 ), it was observed that the adhesion between cells became weaker due to PBS(-) immersion.

[0095] (Example 3) Quantification of gGlu-HMRG response and permeability using spheroids from cultured cancer cells.

[0096] 1×10 HeLa cells (human cervical cancer cell line) or A549 cells (adenocarcinoma human alveolar basal epithelial cell line) were seeded on Nunclon sphera. 4 Cells were cultured for 7-14 days. The obtained spheroids were adhered to a poly-L-lysine coated glass-bottomed culture dish for about 24 hours. The culture medium was replaced with each buffer and incubated at room temperature for 10 minutes (immersion, rinse). The buffer in the culture dish was replaced with the buffer obtained by dissolving gGlu-HMRG to a final concentration of 0.1μM (A549 cells) or 1μM (Hela cells), and observed with a fluorescence microscope (Leica DMI 6000CS, lens: 10×, filter Leica L5, ex460-500nm, em.512-542nm). Acquired by ImageJ Figure 4The region (ROI) at a certain distance from the surface was analyzed for the increase in average brightness at the start of the measurement and after 5 minutes. The fluorescence was also observed when the cells were immersed in physiological saline and Glu-HMRG dissolved in PBS (-) was added.

[0097] The buffers used and their compositions are as follows:

[0098] PBS(-): 140mM NaCl, 2.7mM KCl, 10mM PO4 3- (pH = 7.4)

[0099] PBS(+): In addition to the above PBS(-), 0.90 mM CaCl2 and 0.33 mM MgCl2 were added (pH = 7.4)

[0100] PBS-Ca: In addition to the above PBS(-), 0.90 mM CaCl2 (pH = 7.4) was added

[0101] PBS-Mg: In addition to the above PBS(-), 0.33 mM MgCl2 (pH = 7.4) was added

[0102] PBS-EDTA: In addition to the above PBS (-), 0.2 mM EDTA (pH = 7.4) was added to the physiological saline: 154 mM NaCl

[0103] HBSS(+): HBSS-Hank's balanced salt solution (Thermo Fisher Scientific,

[0104] #14025076).

[0105] Figure 5 The results of PBS(-) and HBSS(+) using Hela cells are shown. When the spheroids formed by Hela cells react with gGlu-HMRG dissolved in HBSS close to body fluids without washing with buffer, the rise of fluorescence becomes slower as they leave the surface. In contrast, the fluorescence rises faster when the spheroids are immersed in PBS(-) for 10 minutes and then immersed in gGlu-HMRG dissolved in PBS(-).

[0106] Figure 6 The results of using PBS(-), PBS(+), PBS-Ca and PBS-Mg for A549 cells are shown. The fluorescence intensity rises faster when treated with PBS(-) than with PBS(+). 2+The same results as those of PBS(-) were obtained in PBS-Mg. 2+ PBS-Ca, the fluorescence hardly rises like PBS(+). From this result, the presence or absence of calcium ions is considered to be important.

[0107] Figure 7 The results of using A549 cells and adding gGlu-HMRG dissolved in PBS (-), PBS (+), saline, and saline + PBS (-) are shown. In saline, the fluorescence intensity rises slowly. The pH stabilization effect produced by the phosphate buffer contained in PBS is also considered to be important.

[0108] (Example 4) Evaluation of the effects of chelators on gGlu-HMRG response and permeability using cultured cancer-derived cell spheroids.

[0109] In order to evaluate the effect of the adhesion dependent on divalent ions such as calcium, fluorescence was observed under the same conditions as in Example 3 except that the following buffer was used.

[0110] (1) Without pretreatment (no rinse), immersed in gGlu-HMRG solution dissolved in PBS (-)

[0111] (2) Pretreatment with PBS(-) immersion

[0112] (3) Pretreatment by PBS(+) immersion

[0113] (4) Pretreatment with PBS(-)+0.2mM EDTA

[0114] Figure 8 Results using HeLa cells are shown. 2+ Mg 2+ Compared with the case where the samples were immersed in PBS(-) containing divalent ions or PBS(+) containing divalent ions, the fluorescence rise was faster at a distance from the surface. In addition, the fluorescence rise was faster by adding a divalent ion chelator (EDTA).

[0115] Fig. 9 The results using A549 cells are shown. A549 cells showed the same tendency as HeLa cells. 2+ ,Mg 2+ ) had a fluorescence rise rate that was approximately the same as that of the case without immersion. On the other hand, when 0.2 mM EDTA, which chelates divalent ions, was added, the fluorescence intensity increased significantly at a depth deeper than that of PBS(-).

[0116] (Example 5) Effect of pretreatment when using different fluorescent probes

[0117] The fluorescence was measured in the same manner as in Example 3 except that 5 μM of FeRhoNox-1 probe was used instead of Glu-HMRG as the fluorescent probe. Only A549 cells were used as cells.

[0118] Fig.10 The results are shown in Figure 2. The same tendency as when gGlu-HMRG was used as a fluorescent probe was also observed for the FeRhoNox-1 probe. This suggests that calcium removal also promotes the penetration of other fluorescent probes into the cell mass.

[0119] As described above, the fluorescence rise of gGlu-HMRG in the center of the spheroids was accelerated by pre-immersion in PBS(-). 2+ This effect was hardly observed with solutions containing calcium such as PBS-EDTA. On the contrary, this effect was further enhanced if calcium ions were actively removed using PBS-EDTA. Judging from the cell images, PBS(-) immersion hindered calcium-dependent adhesion, formed gaps between cells, and facilitated the penetration of reagents. No obvious effect was observed with physiological saline, which also did not contain calcium. This phenomenon was observed not only in HeLa cells but also in A549 cells, and is considered to be independent of specific cell tumors. In addition, this phenomenon was observed not only on gGlu-HMRG but also on FeRhoNox-1, indicating that it can be applied to situations where attempts are made to detect cell characteristics using functional fluorescent probes.

[0120] (Example 6) Effect of pH on pretreatment using different fluorescent probes

[0121] gGlu-HMRG was reacted in cultured cells cultured on a glass surface, and the fluorescence intensity was compared. Hela cells were seeded at the same density in a 4-divided glass-bottomed culture dish. They were cultured in DMEM + 8% FBS, PS at 37°C and 5% CO2 for about 8 hours. After washing three times with PBS (-) adjusted to pH 6.5, 7.3, and 7.8, the solution was replaced with a solution in which 2 μM gGlu-HMRG was added to each solution. The staining was performed at 37°C and 5% CO2 for 20 minutes, and the solution was not replaced and observed under a fluorescence microscope under the same conditions. 100ms exposure, gain 5int 5, ND1. While referring to the image overlapped with DIC, the fluorescence intensity of five cells in the fluorescence image and the fluorescence intensity of the background were measured, and the average value and standard deviation were plotted. In addition, the fluorescence intensity of gGlu-HMRG and the fluorescence intensity of HMRG (fluorescent substance after reaction with GGT) were measured at each pH.

[0122] The fluorescence intensity is pH-dependent. When the pH decreases, the signal decreases and the background increases (not shown). At pH 7.3 or higher, the signal intensity increases. In addition, the results of measuring the fluorescence intensity of gGlu-HMRG and the fluorescence intensity of HMRG (fluorescent substance after reaction with GGT) at various pH values ​​show that the fluorescence intensity decreases as the pH increases, preferably under conditions of pH 6 to 8, and more preferably pH 6 to 7.5 (not shown). These results indicate that pH has an effect on the determination of fluorescence intensity.

Claims

1. A method for emitting fluorescence capable of distinguishing target cells from non-target cells, comprising: removing calcium ions from a cell mass or tissue containing the target cells, contacting the cell mass or tissue with a fluorescent probe, wherein the fluorescent probe fluoresces when in contact with or incorporated into the target cell, and enabling the cell mass or tissue from which calcium ions have been removed and which has been in contact with the fluorescent probe to emit fluorescence, wherein the fluorescent probe that emits fluorescence when in contact with the target cell or incorporated into the target cell is a fluorescent probe that emits fluorescence when reacting with an enzyme or iron ion contained in the target cell, The removal of calcium ions is carried out by immersing the cell mass or tissue containing the target cells in a buffer containing a metal ion chelator or a buffer not containing calcium. Wherein, the method is performed in vitro. 2 . The method according to claim 1 , which is a method of emitting fluorescence from the inside of the cell mass, or a method of causing target cells present inside the cell mass or tissue to emit fluorescence.

3. A method for determining an area where a target cell is present, comprising: removing calcium ions from a cell mass or tissue predicted to contain the target cells, contacting the cell mass or tissue predicted to contain the target cell with a fluorescent probe, wherein the fluorescent probe emits fluorescence when in contact with or incorporated into the target cell, and determining that the region where the fluorescence emission from the fluorescent probe is observed is the region where the target cell exists, wherein the fluorescent probe that emits fluorescence when in contact with the target cell or incorporated into the target cell is a fluorescent probe that emits fluorescence when reacting with an enzyme or iron ion contained in the target cell, The removal of calcium ions is performed by immersing the cell mass or tissue containing the target cells in a buffer containing a metal ion chelator or a buffer not containing calcium.

4. The method of claim 1, wherein: The enzyme is a glycosidase, a protease or a peptidase.

5. The method of claim 4, wherein: The enzyme is an enzyme present on the surface of the cell membrane.

6. A method as claimed in any one of the preceding claims, wherein: The immersion is performed for 30 seconds to 30 minutes.

7. The method of claim 1, wherein: The fluorescent probe emits fluorescence when in contact with the target cell or incorporated into the target cell, Wherein, the target cell is a cancer cell or a tumor cell.

8. The method of claim 7, wherein: The cancer is a solid cancer.

9. A method as claimed in any one of the preceding claims, wherein: The fluorescent probe is gGlu-HMRG, Leu-HMRG, EP-HMRG or FeRhoNox-1.

10. A method for diagnosing cancer or tumor, comprising: removing calcium ions from a cell mass sample or a tissue sample obtained from a subject, contacting the sample with a fluorescent probe, wherein the fluorescent probe fluoresces when contacted with or incorporated into a cancer cell or a tumor cell, and When cells that fluoresce are present in the sample, diagnosing the subject as having cancer or a tumor, The fluorescent probe that emits fluorescence when in contact with a target cell or incorporated into a target cell is a fluorescent probe that emits fluorescence when reacting with an enzyme or iron ion contained in the target cell. The removal of calcium ions is carried out by immersing the cell mass or tissue containing the target cells in a buffer containing a metal ion chelator or a buffer not containing calcium. Wherein, the method is performed in vitro.

11. The method of claim 10, wherein: The method includes providing image data for the sample from which the calcium ions have been removed and which has been contacted with the fluorescent probe.

12. The method according to claim 10 or 11, wherein: The immersion is performed for 30 seconds to 30 minutes.

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