Fluorescent dye agent and method for detecting tumor cells

Abstract

The present invention relates to providing a new method for easily detecting tumor cells in tissue from a living body. The present invention includes a fluorescent dye containing a compound that exhibits solvatochromism for detecting tumor cells in tissue. The present invention also includes a method for staining tissue derived from a living body or applying to a living body the fluorescent dye to detect tumor cells or specify the range of tumor removal.

Description

[Technical Field]

[0001] The present invention relates to a fluorescent dye agent used for detecting tumor cells and a method for detecting tumor cells. [Background technology]

[0002] Various staining methods are used to detect tumor cells. For example, hematoxylin-eosin (HE) staining is the most widely used stain in pathological diagnosis and is considered the gold standard in many diagnostic methods. However, HE staining stains normal tissue as well as tumor cells, resulting in poor optical transparency, necessitating thin sectioning of tissue specimens. Because thin sections only provide two-dimensional information, determining the distribution of tumor cells requires the preparation and observation of many sections. Furthermore, preparing tissue specimens requires advanced skills, is tedious, and takes a long time.

[0003] Furthermore, HE staining can sometimes result in low contrast and unclear boundaries between tumor cells and normal tissue.

[0004] Another method is to stain specific tissues with antibodies conjugated with fluorescent dyes, radionuclides, or metal particles to obtain high-contrast images of tumor cells. However, because the antibody moiety does not pass through the cell membrane, treatment to increase membrane permeability is required, making the procedure complicated. In addition, the reagents are expensive.

[0005] On the other hand, a method for detecting skin diseases has also been reported in which light is irradiated without using dyes, and the difference in Raman scattering between abnormal and normal tissue is utilized (for example, Patent Document 1). However, it is thought that it would be difficult to identify tumor areas at the cellular level using the Raman scattering method.

[0006] On the other hand, some compounds are known to exhibit a phenomenon known as solvatochromism, in which their absorption maximum wavelength, fluorescence wavelength, or both change depending on the polarity of surrounding molecules such as solvents. Compounds exhibiting solvatochromism typically undergo polarization via intramolecular charge transfer (ICT) upon photoexcitation. The stability of the excited state of the polarized molecule changes depending on the degree of polarity of the solvent molecules, so the energy difference between the excited state and the ground state of the molecule changes based on the polarity of the solvent.

[0007] Non-Patent Document 1 describes 1-acetyl-6-piperidylpyrene (PK) and its aldehyde analog (PA) as compounds exhibiting such solvatochromism, and also describes the use of PK to stain normal tissues or HeLa cells. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Japanese Patent Application Publication No. 2018-201678 [Non-patent literature]

[0009] [Non-Patent Document 1] Analytical Chemistry, 2020, Vol.92, Issue 9, p.6512-6520 Summary of the Invention [Problem to be solved by the invention]

[0010] However, Non-Patent Document 1 does not describe an example in which tissue containing both normal cells and tumor cells is stained with a compound exhibiting solvatochromism, nor does it describe that normal cells and tumor cells can be distinguished from each other based on the difference in fluorescence wavelength caused by solvatochromism.

[0011] In view of the above circumstances, an object of the present invention is to provide a new method for simply detecting tumor cells in tissue derived from a living body. [Means for solving the problem]

[0012] The present inventors have discovered that tumor cells in tissue can be easily detected by applying a fluorescent dye agent containing a compound exhibiting solvatochromism to staining tissue derived from a living body, and have completed the present invention.

[0013] That is, the present invention provides the following fluorescent dye agents. [1] A fluorescent dye agent for detecting tumor cells in tissue derived from a living organism, which contains a compound exhibiting solvatochromism. [2] The fluorescent dye agent according to [1], wherein the compound contains a condensed polycyclic π-conjugated structure having 2 to 6 rings. [3] The fluorescent dye agent according to [2], wherein the compound further comprises one or more hydrophilic substituents containing one or more atoms selected from the group consisting of boron, nitrogen, oxygen, phosphorus, sulfur, and halogen. [4] The compound is a compound represented by the following chemical formula (II), 1-acetyl-6-piperidylpyrene, Nile Red, POLARIC TM The fluorescent dye agent according to any one of [1] to [3], which is selected from the group consisting of , Laurdan, di-4-ANEPPDHQ, Prodan and derivatives thereof. [ka] [5] The fluorescent dye agent according to any one of [1] to [4], wherein the compound has an absorption maximum wavelength of 300 to 600 nm in a 20 mM phosphate buffer solution (pH 7.4) at 25°C. [6] The maximum fluorescence wavelength in dimethyl sulfoxide (DMSO) at 25°C is λ DMSO , the fluorescence maximum wavelength in toluene at 25 °C is λ TolIn this case, the λ DMSO and λ Tol The fluorescent dye agent according to any one of [1] to [5], wherein the difference is 20 to 200 nm. [7] A kit comprising the fluorescent dye agent according to any one of [1] to [6].

[0014] Furthermore, the present invention provides the following method. [8] A method for detecting tumor cells, comprising staining tissue derived from a living organism with a fluorescent dye agent containing a compound exhibiting solvatochromism. [9] A method for identifying a tumor removal area in tissue in a living organism, comprising staining the living organism with a fluorescent dye agent containing a compound exhibiting solvatochromism.

[10] Use of a compound exhibiting solvatochromism for the detection of tumor cells in tissue derived from a living organism.

[11] Use of compounds exhibiting solvatochromism to identify the extent of tumor removal.

[12] Use of a compound exhibiting solvatochromism for the manufacture of a composition for treating or diagnosing a tumor.

[13] Compounds exhibiting solvatochromism for use in the treatment or diagnosis of tumors. [Effects of the Invention]

[0015] Tumor cells stained with the fluorescent dye of the present invention can be easily distinguished from normal cells and detected. Furthermore, the fluorescent dye of the present invention can stain biological tissues simply and quickly. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 shows charts of (left) absorption spectrum measurement and (right) fluorescence spectrum measurement (normalized) of PC (compound A) in an organic solvent in an example. [Figure 2A]1A is a two-photon microscope image of the tissue at the lesion site of Paget's disease in Test Example 1. The area corresponding to one tumor cell (Paget's cell) is indicated by an arrowhead. [Figure 2B] Figure 2B is a two-photon microscopy image of healthy skin tissue. [Figure 3A] Figure 3A is a two-photon microscope image of the lesional tissue of Paget's disease in Test Example 2. The arrowheads represent Paget cells that have proliferated in the epidermis, the asterisks attached to the arrowheads represent Paget cells that have infiltrated and proliferated in hair follicles, and the arrows represent Paget cells that have infiltrated and proliferated in the dermis. [Figure 3B] Figure 3B is a two-photon microscope image of the lesion tissue of Paget's disease in Test Example 2. The arrowheads indicate Paget's cells infiltrating around the hair follicle. [Figure 3C] Figure 4C is a two-photon microscope image of melanoma lesion tissue in Test Example 2. The arrow indicates melanoma cells that have proliferated in the epidermal basal cell layer, and the arrowhead indicates melanoma cells that have proliferated in the dermis and formed tumor honeycombs. [Figure 4A] FIG. 4A is a two-photon microscope image of healthy skin tissue in Test Example 3. [Figure 4B] Figure 4B is a two-photon microscope image of the lesion tissue of Paget's disease in Test Example 3. The arrowheads indicate Paget's cells proliferating in the epidermis. [Figure 4C] Figure 4C is a two-photon microscope image of melanoma lesion tissue in Test Example 3. The arrowheads indicate melanoma cells proliferating within the epidermis. [Figure 5] 5 shows two-photon microscopic images of the rectal mucosal epithelial tissue in Test Example 4. Panels A and B show healthy rectums, and panels C and D show well-differentiated adenocarcinoma portions of the rectum. DETAILED DESCRIPTION OF THE INVENTION

[0017] As used herein, a "compound exhibiting solvatochromism" (abbreviated as an "SC compound" or "solvatochromic compound" in this specification) refers to a compound whose maximum absorption wavelength, maximum fluorescence wavelength, or both change depending on the polarity (hydrophobicity) of the surroundings of the compound.

[0018] Although not limited to a specific mechanism of action, it is presumed that the SC compounds of the present invention have different cell membrane environments (e.g., polarity, orientation, membrane fluidity, etc.) between normal cells and tumor cells, resulting in differences in the stability of the excited state of the SC compounds in the cell membrane, as described above, and causing tumor cells and normal cells to emit fluorescence of different wavelengths.

[0019] [Fluorescent dye] The present invention includes a fluorescent dye agent containing an SC compound, which is used for detecting tumor cells in tissue derived from a living body.

[0020] (detection target) The living body is not particularly limited as long as it is a multicellular animal, and is preferably a mammal (including humans and non-human mammals), more preferably a human.

[0021] The tissue is not particularly limited as long as it contains a plurality of cells. Examples of tissues include skin, brain, spinal cord, esophagus, stomach, small intestine, large intestine, duodenum, rectum, liver, pancreas, gallbladder, bladder, kidney, heart, spleen, thymus, prostate, uterus, ovaries, testes, breast, lung, bronchi, eyeball, nose, paranasal cavity, oral cavity, pharynx, salivary gland, thyroid, parathyroid, adrenal gland, muscle, bone marrow, blood vessel, nerve, lymph node, peritoneum, diaphragm, and blood. In one embodiment, the tissue is skin, e.g., epidermis or dermis, or a combination of both. In another embodiment, the tissue is rectum.

[0022] Furthermore, the fluorescent dye agent of the present invention can be applied to tissues isolated from a living body by surgical procedures such as excision, resection, puncture, blood sampling, etc., or to the above tissues obtained from feces, urine, sweat, and other body fluids.

[0023] In one embodiment, the form of the tissue can be appropriately selected depending on the detection method, and may be, for example, an organ or tissue itself, or a thin section or three-dimensional fragment thereof.

[0024] Depending on the form of the tissue, the tissue may have been subjected to treatments such as fixation with formalin or the like, paraffin embedding, deparaffinization, dehydration, and clearing.

[0025] The tumors detected by the fluorescent dye agent of the present invention may be benign or malignant (such as cancer or sarcoma cells), but are preferably malignant tumors. The type of tumor cells is not particularly limited, and examples include tumors occurring in the above tissues.

[0026] In one embodiment, the tumor is a skin tumor. Examples of such tumors include sweat gland tumors (e.g., extramammary Paget's disease, Paget's disease of the breast, eccrine porocarcinoma, microcystic adnexal carcinoma, and cutaneous mucinous carcinoma), malignant melanoma, epidermal and hair follicle tumors (e.g., basal cell carcinoma, squamous cell carcinoma, actinic keratosis, Bowen's disease, leukoplakia, and keratoacanthoma), nervous system tumors (e.g., Merkel cell carcinoma and malignant peripheral nerve sheath tumor), and mesenchymal tumors (e.g., dermatofibrosarcoma protuberans, solitary fibrous tumor, muscle tumors, liposarcoma, angiosarcoma, Kaposi's sarcoma, spindle cell hemangioendothelioma, heterogeneous fibroxanthomatosis, epithelioid sarcoma, synovial sarcoma, and undifferentiated pleomorphic cell sarcoma). In a specific embodiment, the tumor is extramammary Paget's disease or malignant melanoma.

[0027] (Solvatochromic compounds (SC compounds)) In the fluorescent dye preparation of the present invention, the SC compound is not particularly limited as long as it is applicable to tissues derived from living organisms. The SC compound may be used alone or in combination of two or more kinds, but usually, a clear stained image can be obtained by using only one kind.

[0028] The smaller the molecular size of the SC compound, the easier it is to translocate to the cell membrane and the better its staining ability, making it preferable. The molecular weight of the SC compound can be, for example, 800 or less, 700 or less, 600 or less, 500 or less, or 450 or less. The molecular weight of the SC compound can also be, for example, 200 or more.

[0029] In one embodiment, the SC compound is capable of distributing to the cell membrane and intracellularly, and in a more specific embodiment, the SC compound is capable of inserting into the cell membrane. In one embodiment, the SC compounds exhibit strong fluorescence anisotropy at the cell membrane. In one embodiment, the SC compound has the property of exhibiting lower fluorescence intensity outside the cell compared to the cell membrane and / or inside the cell. In a specific embodiment, the SC compound exhibits substantially no fluorescence outside the cell. For example, the fluorescence intensity outside the cell of the SC compound may be, but is not limited to, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, or 1% or less of the fluorescence intensity inside the cell. The comparison of intracellular and extracellular fluorescence intensities is performed by irradiating cells derived from the tissue of interest of the present invention with light of a wavelength capable of exciting the SC compound, acquiring fluorescence microscopy images under conditions that allow detection of light of the maximum fluorescence wavelength, and comparing the average signal intensities of intracellular and extracellular fluorescence.

[0030] In one embodiment, the SC compound includes a fused polycyclic π-conjugated structure having 2, 3, 4, 5, or 6 rings. The fused polycyclic π-conjugated structure is a polycyclic structure having delocalized π-electrons and formed by condensing two or more rings, and may include one or more heteroatoms (nitrogen, oxygen, sulfur, etc.), such as a benzophenoxazine ring. In a specific embodiment, the fused polycyclic π-conjugated structure is a fused polycyclic aromatic hydrocarbon structure (e.g., naphthalene, azulene, anthracene, phenanthrene, pyrene, etc.). In a specific embodiment, the SC compound includes a pyrene skeleton (four rings) as the fused polycyclic π-conjugated structure. SC compounds including a pyrene skeleton are particularly suitable for use in the present invention because they have a high fluorescence quantum yield even when their molecular size is reduced.

[0031] In one embodiment, the SC compound includes a structure in which 2, 3, 4, 5, or 6 conjugated rings are conjugated to one another. The conjugated rings may be one or a combination of two or more selected from the group consisting of a benzene ring, a naphthalene ring, an anthracene ring, a phenanthrene ring, a fluorene ring, a pyridine ring, a thiophene ring, a pyrrole ring, a furan ring, a benzothiophene ring, a benzofuran ring, a benzopyrrole ring, an imidazole ring, a quinoline ring, an isoquinoline ring, a carbazole ring, a thiazole ring, and a dibenzothiophene ring.

[0032] From the viewpoint of improving water solubility and promoting dispersion into cell membranes and cells, the SC compound preferably further contains one or more hydrophilic substituents containing one or more atoms selected from the group consisting of boron, nitrogen, oxygen, phosphorus, sulfur, and halogen. In particular, from the viewpoint of suppressing reaction with biomolecules, the hydrophilic substituent contained in the SC compound preferably contains one or more types selected from the group consisting of a tertiary amino group, a quaternary ammonium group, and a carbonyl group (excluding an aldehyde group).

[0033] In a specific embodiment, from the viewpoint of staining ability, the SC compound is a compound represented by the following chemical formula (II): PC, 1-acetyl-6-piperidylpyrene (PK), Nile Red, POLARIC TM , Laurdan, di-4-ANEPPDHQ, Prodan or derivatives thereof. In a more particular embodiment, the SC compound is selected from PC, PK, Nile Red and POLARIC, preferably PC or PK, more preferably PC.

[0034] 1-Acetyl-6-piperidylpyrene (PK) is an SC compound represented by the following chemical formula (I). As shown in Table S1 in Analytical Chemistry, 2020, Vol. 92, Issue 9, pp. 6512-6520, PK exhibits high fluorescence quantum yields in various solvents. [ka]

[0035] PC, also known as (E)-1-(6-(piperidin-1-yl)pyren-1-yl)hex-1-en-3-one, is an SC compound represented by the following chemical formula (II). As shown in the examples, it exhibits high fluorescence quantum yields in various solvents. [ka]

[0036] Nile Red is a compound with CAS number 7385-67-3. POLARIC is a compound and its derivatives described in Chem. Lett., 2011, Vol. 40, pp. 989-991, including, for example, the POLARIC series sold by Goryo Chemical Co., Ltd. Laurdan is a compound with CAS number 74515-25-6. Details of di-4-ANEPPDHQ are described, for example, in Biophys. J., 2006, Vol. 90, Issue 7, pp. 2563-2575. Prodan is a compound with CAS number 70504-01-7.

[0037] Among the above SC compounds, PC, PK, and Nile Red are preferred because they have good photostability and high fluorescence quantum yield. Furthermore, PK and PC are more preferable than Nile Red because they are less likely to nonspecifically adsorb to tissues, and the difference in fluorescence wavelength between tumor and normal tissues tends to be greater, thereby suppressing background fluorescence.

[0038] In one embodiment, the absorption maximum wavelength of the SC compound is, for example, between 300 and 600 nm in 20 mM phosphate buffer (pH 7.4) at 25° C. The absorption maximum wavelength of the SC compound may be, for example, within a range between any two values ​​selected from the group consisting of 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, 350 nm, 360 nm, 370 nm, 380 nm, 390 nm, 400 nm, 410 nm, 420 nm, 430 nm, 440 nm, 450 nm, 460 nm, 470 nm, 480 nm, 490 nm, 500 nm, 510 nm, 520 nm, 530 nm, 540 nm, 550 nm, 560 nm, 570 nm, 580 nm, 590 nm, and 600 nm.

[0039] The absorption maximum wavelength of PK is 407 nm in 20 mM phosphate buffer (pH 7.4) at room temperature, as shown in Table S1 of the Supporting Information in Analytical Chemistry, 2020, Vol. 92, Issue 9, pp. 6512-6520.

[0040] In one embodiment, the fluorescence maximum wavelength in methanol at 25° C. is λ Met , the fluorescence maximum wavelength in n-heptane at 25 °C is λ Hep In this case, λ of the SC compound Met and λ Hep The difference between λ and λ is, for example, 50 nm or more, 60 nm or more, 70 nm or more, 80 nm or more, 90 nm or more, 100 nm or more, or 110 nm or more. Met and λ Hep The larger the difference between the λ and λ peaks, the easier it is to detect tumor tissue with high contrast compared to healthy tissue, which is preferable. Met and λ Hep The difference may be 300 nm or less, 200 nm or less, or 150 nm or less.

[0041] In one embodiment, the fluorescence maximum wavelength in dimethyl sulfoxide (DMSO) at 25° C. is λ DMSO , the fluorescence maximum wavelength in toluene at 25 °C is λ Tol In this case, λ of the SC compoundDMSO and λ Tol The difference between λ and λ is, for example, 20 nm or more, 30 nm or more, 40 nm or more, 50 nm or more, 60 nm or more, 70 nm or more, 80 nm or more, 90 nm or more, or 100 nm or more. DMSO and λ Tol The larger the difference between the λ and λ peaks, the easier it is to detect tumor tissue with high contrast compared to healthy tissue, which is preferable. DMSO and λ Tol The difference may be 200 nm or less, 180 nm or less, 160 nm or less, 140 nm or less, or 120 nm or less.

[0042] The fluorescence maximum wavelength of PK is λ , as shown in Table S1 of the Supporting Information in Analytical Chemistry, 2020, Vol. 92, Issue 9, pp. 6512-6520. Met = 593 nm, λ Hep = 474 nm, λ Tol = 503 nm, λ DMSO = 558 nm.

[0043] In one embodiment, the two-photon absorption maximum wavelength of the SC compound is preferably between 600 and 1200 nm in 20 mM phosphate buffer (pH 7.4) at 25° C. Such SC compounds can be excited with light of a wavelength that is not easily absorbed by biological materials in tissue, and are therefore suitable for use in two-photon microscopy.

[0044] (Other compositions) In one embodiment, the fluorescent dye agent consists solely of the SC compound.

[0045] In one embodiment, the fluorescent dye agent may further contain one or more of a pH buffer, a surfactant, a salt, a solvent, a dye composition other than the SC compound, etc.

[0046] Examples of pH buffers include one or more selected from the group consisting of tris(hydroxymethyl)aminomethane; Good's buffers (HEPES, MOPS, etc.); and pH buffers containing citric acid, acetic acid, lactic acid, oxalic acid, phthalic acid, imidazole, triethanolamine, diethanolamine, glycine, boric acid, phosphoric acid, or carbonate.

[0047] Examples of the solvent include one or more selected from the group consisting of water, ethanol, methanol, 2-propanol, dimethyl sulfoxide (DMSO), N,N-dimethylformamide, acetonitrile, acetone, ethyl acetate, tetrahydrofuran, and 1,2-dichloroethane.

[0048] The dye composition other than the SC compound is not particularly limited as long as it does not interfere with the detection of tumor cells by the SC compound. For example, nuclear staining dyes such as propidium iodide (PI), ethidium bromide, acridine orange, DAPI, and Hoechst are suitable because they do not interfere with the staining of the SC compound. Furthermore, dye compositions used in various tissue staining procedures, such as hematoxylin-eosin (HE) staining, Azan staining, Masson-Trichrome staining, Elastica-van Gieson staining, silver impregnation staining, Victoria blue staining, PAM staining, PTAH staining, Sudan III staining, Oil Red O staining, PAS staining, Alcian blue staining, toluidine blue staining, colloidal iron staining, mucicarmine staining, Congo red staining, Dylon staining, Grimelius staining, Fontana-Masson staining, Kossa staining, Berlin blue staining, Bodian staining, Klüver-Barrera staining, and Giemsa staining, can be used. These other dye compositions may be used singly or in combination of two or more.

[0049] (Dosage form) The dosage form of the fluorescent dye agent of the present invention is not particularly limited, and examples thereof include solids such as powders and liquids.

[0050] (Application) When tissue is stained with the fluorescent dye agent of the present invention, the SC compound may be distributed in both tumor cells and normal tissue cells, but the fluorescence wavelength of the SC compound in tumor cells is shifted from the fluorescence wavelength in normal tissue cells. Therefore, by appropriately selecting the wavelength of the fluorescence to be detected, tumor cells can be detected with high contrast from normal tissue. Thus, the fluorescent dye agent of the present invention can be used for the examination or diagnosis of tumors, particularly malignant tumors.

[0051] The fluorescent dye agent of the present invention may also be applied to the living body itself or to tissue that is part of the living body and not separated from the living body.

[0052] When applied to a living body, the fluorescent dye agent of the present invention can be used for diagnosing tumors, particularly malignant tumors. Furthermore, in tumor treatment, the fluorescent dye agent of the present invention may be applied to a living body before, during, or after tumor removal treatment as part of the treatment to identify the extent of tumor removal, particularly cancer removal, or to check whether any tumor remains.

[0053] The fluorescent dye agent of the present invention may be, for example, a reagent used in clinical tests, basic research, etc., as well as a drug or quasi-drug.

[0054] (kit) The fluorescent dye agent of the present invention can also be combined with, for example, reagents and instruments for staining or tissue specimen preparation to form a kit. In certain embodiments, the kit includes a reagent for preparing a staining solution. In further specific embodiments, the reagent for preparing a staining solution may include, for example, one or a mixture of two or more selected from the group consisting of the above-mentioned pH buffer, surfactant, salt, solvent, and other dye composition.

[0055] [Method for detecting tumor cells in tissue] The present invention includes a method for detecting tumor cells, which comprises staining tissue derived from a living organism with a fluorescent dye agent containing a compound that exhibits solvatochromism.

[0056] Examples of fluorescent dye agents containing compounds exhibiting solvatochromism include those described above under [Fluorescent dye agents].

[0057] Examples of tissues derived from living organisms include those described above in the section [Fluorescent dye agent]. The method of the present invention may include a step of preparing such tissues.

[0058] In one embodiment, the method of the present invention is applied to tissues that are organs or organ systems themselves or three-dimensional fragments thereof. In this embodiment, a step of clearing the tissue prior to staining is preferably included. Examples of tissue clearing methods include the TDE method, LUCID method, CLARITY method, PACT / PARS method, CUBIC method, 3DISCO method, Scale method, ScaleS method, SeeDB method, FocusClear method, Clear method, BABB method, iDISCO method, and uDISCO method. These clearing methods are described, for example, in Cell Chem. Biol. 2016, Vol. 23, pp. 137-157 and Laser Photonics Rev. 2019, Vol. 13, pp. 1800292.

[0059] In one embodiment, the method of the present invention is applied to a living organism or to tissue that is part of a living organism and not separated from the living organism.

[0060] In one embodiment, the method of the present invention is applied to thin sections, which may optionally be subjected to treatments commonly used in clinical examinations, such as fixation, dehydration, dealcoholization, paraffin infiltration, paraffin embedding, deparaffinization, immersion, and staining using the various tissue staining methods described above.

[0061] Staining is usually performed by contacting a staining solution containing an SC compound with the tissue. The concentration of the SC compound in the staining solution is adjusted to, for example, 0.001 mg / mL or more, 0.01 mg / mL or more, 0.1 mg / mL or more, 0.2 mg / mL or more, 0.3 mg / mL or more, 0.4 mg / mL or more, 0.5 mg / mL or more, 0.6 mg / mL or more, 0.7 mg / mL or more, 0.8 mg / mL or more, 0.9 mg / mL or more, or 1 mg / mL or more, relative to the total volume of the staining solution. The concentration of the SC compound in the staining solution is adjusted to, for example, 500 mg / mL or less, 200 mg / mL or less, 100 mg / mL or less, 50 mg / mL or less, 20 mg / mL or less, 10 mg / mL or less, 5 mg / mL or less, or 2 mg / mL or less, relative to the total volume of the staining solution.

[0062] The temperature during dyeing is not particularly limited, but is, for example, 0 to 80°C, 4 to 50°C, or 20 to 45°C, and preferably 35 to 42°C.

[0063] The time for which the staining solution is brought into contact with the tissue is, for example, 1 minute or more, 10 minutes or more, 20 minutes or more, 1 hour or more, 2 hours or more, 1 day or more, or 2 days or more, at 20 to 40°C, for example, 14 days or less or 7 days or less. In one embodiment, the time for which the staining solution is contacted with the tissue is at 35 to 40°C, for example, 12 hours or less, 6 hours or less, preferably 2 hours or less, more preferably 1 hour or less, even more preferably 30 minutes or less, and even more preferably 10 minutes or less, and may be, for example, 1 minute or more, 2 minutes or more, 5 minutes or more, or 10 minutes or more.

[0064] Tissue stained with a staining solution containing an SC compound can be used directly to detect tumor cells, but may optionally be subjected to further processing such as staining with other dye compositions before detection.

[0065] The method of the present invention may further include a step of detecting tumor cells. Tumor cells can be detected, for example, by exciting the SC compound with light of an appropriate wavelength and detecting the emitted fluorescence. Detection can be performed using, for example, a confocal laser scanning microscope, or, depending on the thickness of the section, a microscope capable of multiphoton excitation, such as a two-photon microscope, can be used. For example, when the compound of chemical formula (II) (PC) or 1-acetyl-6-piperidylpyrene (PK) is used as the SC compound, it is suitable for measurement by two-photon microscopy, as shown in the examples. PC, in particular, has a fluorescence maximum wavelength even longer than PK, making it particularly suitable for detecting tumors located deeper within the tumor.

[0066] In one embodiment, tumor cells are detected by selecting a fluorescent light containing one particular wavelength that provides contrast between the tumor cells and cells of normal tissue, and measuring the fluorescent intensity. In another embodiment, tumor cells are detected by multi-wavelength measurement, i.e., tumor cells are detected by detecting fluorescence containing two or more different specific wavelengths and integrating the intensity of each fluorescence.

[0067] In one embodiment, when PC or PK is used as the SC compound, the detected fluorescence comprises one or more wavelengths selected from the ranges of 450 to 550 nm, 480 to 520 nm, or 490 to 500 nm, for example. In a specific embodiment, the detected fluorescence comprises light at 495 nm.

[0068] As described above in the section [Fluorescent dye agent], the detection method of the present invention can be used to examine tumors, identify the extent of tumor removal, diagnose tumors, or treat tumors.

[0069] [Preferred embodiment of the present invention] The present invention includes the following preferred embodiments. <1> A fluorescent dye agent for detecting tumor cells in tissue derived from a living organism, which contains a compound exhibiting solvatochromism. <2> <1> A kit comprising the fluorescent dye agent described in . <3> A method for detecting tumor cells, comprising staining tissue derived from a living organism with a fluorescent dye agent containing a compound exhibiting solvatochromism. <4> A method for identifying a tumor removal area in tissue in a living organism, comprising staining the living organism with a fluorescent dye agent containing a compound exhibiting solvatochromism. <5> Staining a tissue or a living organism derived from a living body using a fluorescent dye agent containing a compound exhibiting solvatochromism; and Detecting tumors based on staining images; A method for treating or diagnosing a tumor, comprising: <6> Use of a compound exhibiting solvatochromism for the detection of tumor cells in tissue derived from a living organism. <7> Use of compounds exhibiting solvatochromism to identify the extent of tumor removal. <8> Use of a compound exhibiting solvatochromism for the manufacture of a composition for treating or diagnosing a tumor. <9> Compounds exhibiting solvatochromism for use in the treatment or diagnosis of tumors. <10> The aforementioned <1> ~ <9> In the formula (I), the compound contains a fused polycyclic π-conjugated structure having 2 to 6 rings, and preferably contains a pyrene skeleton. <11> The aforementioned <1> ~ <10> wherein the compound further comprises one or more hydrophilic substituents containing one or more atoms selected from the group consisting of boron, nitrogen, oxygen, phosphorus, sulfur, and halogen. <12> The aforementioned <1> ~ <11> wherein the molecular weight of the compound is 800 or less, 700 or less, 600 or less, 500 or less, or 450 or less, and is 200 or more. <13> The aforementioned <1> ~ <12> In the above, the compound is a compound represented by the following chemical formula (II), 1-acetyl-6-piperidylpyrene, Nile Red, POLARIC TM , Laurdan, di-4-ANEPPDHQ, Prodan and derivatives thereof, preferably selected from the compound represented by chemical formula (II), 1-acetyl-6-piperidylpyrene, Nile Red and POLARIC, more preferably the compound represented by chemical formula (II) or 1-acetyl-6-piperidylpyrene, and even more preferably the compound represented by chemical formula (II). [ka] <14> The aforementioned <1> ~ <13> wherein the absorption maximum wavelength of the compound in 20 mM phosphate buffer (pH 7.4) at 25°C is 300 to 600 nm, and is within a range between any two values ​​selected from the group consisting of, for example, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, 350 nm, 360 nm, 370 nm, 380 nm, 390 nm, 400 nm, 410 nm, 420 nm, 430 nm, 440 nm, 450 nm, 460 nm, 470 nm, 480 nm, 490 nm, 500 nm, 510 nm, 520 nm, 530 nm, 540 nm, 550 nm, 560 nm, 570 nm, 580 nm, 590 nm, and 600 nm. <15> The aforementioned <1> ~ <14> The maximum fluorescence wavelength in methanol at 25°C is λ Met , the fluorescence maximum wavelength in n-heptane at 25 °C is λ Hep In this case, λ of the SC compound Met and λ Hep The difference is 50 nm or more, 60 nm or more, 70 nm or more, 80 nm or more, 90 nm or more, 100 nm or more, or 110 nm or more, and is 300 nm or less, 200 nm or less, or 150 nm or less. <16> The aforementioned <1> ~ <15> The maximum fluorescence wavelength in dimethyl sulfoxide (DMSO) at 25°C is λ DMSO , the fluorescence maximum wavelength in toluene at 25 °C is λTol In this case, λ of the SC compound DMSO and λ Tol The difference is 20 nm or more, 30 nm or more, 40 nm or more, 50 nm or more, 60 nm or more, 70 nm or more, 80 nm or more, 90 nm or more, or 100 nm or more, and is 200 nm or less, 180 nm or less, 160 nm or less, 140 nm or less, or 120 nm or less. <17> The aforementioned <1> ~ <16> wherein the living body is a human or non-human mammal. <18> The aforementioned <1> ~ <17> wherein the tissue in which the tumor or tumor cells are present is selected from skin, brain, spinal cord, esophagus, stomach, small intestine, large intestine, duodenum, rectum, liver, pancreas, gallbladder, urinary bladder, kidney, heart, spleen, thymus, prostate, uterus, ovary, testis, breast, lung, bronchus, eyeball, nose, paranasal cavity, oral cavity, pharynx, salivary gland, thyroid gland, parathyroid gland, adrenal gland, muscle, bone marrow, blood vessel, nerve, lymph node, peritoneum, diaphragm, and blood. <19> The aforementioned <1> ~ <18> wherein the tumor is a malignant tumor. <20> The aforementioned <1> ~ <19> wherein the tissue in which the tumor or tumor cells are present is skin; The tumor is selected from sweat gland tumors, malignant melanoma, epidermal-hair follicle tumors, nervous system tumors, and mesenchymal tumors, and is preferably extramammary Paget's disease or malignant melanoma. [Example]

[0070] [1. Materials] [1-1. Synthesis and analysis of PC] PC was synthesized according to the following scheme: [ka]

[0071] To a known pyrene derivative, Compound 1 (100 mg, 0.32 mmol), and 2-pentanone (47 μL, 0.48 mmol), 1 M aqueous sodium hydroxide solution (1 mL) and dehydrated ethanol (6 mL) were added, and the resulting solution was heated and stirred at 60°C for 4 hours under an argon atmosphere.

[0072] Next, water was added to the above solution, and the resulting precipitate was collected by filtration.

[0073] The resulting precipitate was purified by silica gel column chromatography (dichloromethane:hexane=2:1) ​​and further recrystallized from acetonitrile to obtain the target compound A (PC) (yield: 20 mg, 16%).

[0074] ( 1 H-NMR analysis, and 13 C-NMR analysis) 1 H-NMR, and 13 The C-NMR analysis was performed using a nuclear magnetic resonance spectrometer (JMN-LA500 manufactured by JEOL Ltd.) The results obtained for Compound A are shown below. · 1 H NMR (500MHz, CDCl3): δ(ppm)=8.71(d,J=15.6Hz,1H),8.46(d,J=9.2Hz,1H),8.33(d,J=9.2H z,1H),8.25(d,J=9.2Hz,1H),8.15(d,J=8.1Hz,1H),8.09(d,J=8.1Hz,1H),8.08(d,J=9.2Hz, 1H),8.03(d,J=9.2Hz,1H),7.74(d,J=8.1Hz,1H),7.01(d,J=15.6Hz,1H),3.22(s,4H),2.78 (t,J=7.4Hz,2H),1.93-1.95(m,4H),1.77-1.85(m,2H),1.73(s,2H),1.06(t,J=7.33Hz,1H). · 13C NMR(CDCl3,TMS)δ(ppm)=14.11,18.12,24.67,26.84,43.49,55.21,117.53,120.49,124.26,124.48,124.71 ,125.09,125.68,126.02,126.31,126.51,126.57,127.66,128.75,130.80,133.21,139.25,150.33,200.49.

[0075] (High resolution mass spectrometry) High-resolution mass spectrometry was performed using a high-resolution mass spectrometer (JMS-700 manufactured by JEOL Ltd.) The results obtained for Compound A are shown below. HRMS (ESI + ),calcd for C 25 H 23 NO[M+Na] + 404.1985,found 404.1981.

[0076] (Optical properties measurement of PC in organic solvents) The absorption and fluorescence spectra of the above PC in each organic solvent were measured.

[0077] Absorption and fluorescence spectra were measured using a UV-visible-near-infrared spectrophotometer (JASCO Corporation: V-670) and a spectrofluorometer (JASCO Corporation: FP6600), respectively. Fluorescence quantum yield was measured using an absolute PL quantum yield measurement system (Hamamatsu Photonics K.K.: C9920-02V). The concentration of Compound A was 5 μM in each solvent. The solvents used were toluene, dichloromethane (DCM), DMSO, and ethanol. The results are shown in Figure 1 and Table 1. The absorption maximum wavelength of Compound A in 20 mM phosphate buffer (pH 7.4) at 25°C was 421 nm, and the fluorescence maximum wavelength was 621 nm.

[0078] [Table 1]

[0079] As shown in Figure 1 and Table 1, the absorption maximum wavelength was almost the same for all solvents, but the fluorescence maximum wavelength differed depending on the solvent. More specifically, the fluorescence wavelength was longer in polar solvents, demonstrating fluorescent solvatochromism that is sensitive to solvent polarity.

[0080] [1-2. Preparation of SC compounds other than PC] PK was synthesized by the method described in Analytical Chemistry, 2020, Vol. 92, Issue 9, pp. 6512-6520. Nile Red was manufactured by Tokyo Chemical Industry Co., Ltd. POLARIC was used. TM 500BCS (manufactured by Goryo Chemical Co., Ltd.) was used.

[0081] [1-3. Preparation of staining solution] The staining solutions in Test Examples 1 and 2 were prepared by dissolving PK in DMSO at 1 mg / mL. The staining solutions in Test Examples 3 and 4 were both DMSO solutions containing 1 mM of SC compound, and were diluted 100-fold before use.

[0082] [1-4.Tissue used] The healthy skin tissue, extramammary Paget's disease lesion tissue, and melanoma lesion tissue used in the test examples were obtained by resection and then sectioned. A dermatologist had confirmed in advance that each tissue was appropriate. Healthy rectal tissue and tumor tissue were also prepared by sectioning tissue obtained by resection, and a specialist had confirmed that each tissue was appropriate. The collection and use of tumor tissue was approved by the Ehime University Hospital Clinical Research Ethics Review Committee (No. 1802009) and was carried out in accordance with the study protocol.

[0083] [2. Test Example 1: Observation of tumor cells by PK staining of thin sections] Extramammary Paget's disease lesion tissue and healthy tissue were embedded in paraffin and then cut to prepare tissue sections. Sections (5 μm thick) were prepared using the procedure normally used in histopathological examinations. Staining with PK staining solution was performed by deparaffinizing the tissue using conventional methods, followed by immersion in PK staining solution at room temperature for several days to a week. In practice, sufficient staining was achieved by immersion in PK staining solution at room temperature for 2 to 3 days.

[0084] A two-photon microscope, A1R MP+ (NIKON), was used for microscopic observation. A 960 nm laser light source and a 495 nm filter were used for measurements of PK, and a 561 nm filter was used for measurements of PI. Scans were performed to a depth of 100 μm into the sample. Three-dimensional reconstruction images were obtained by processing the data using the accompanying software.

[0085] Figures 2A and 2B show images of 10-μm-thick sections of the 3D reconstructions of each tissue. As shown in Figure 2A, the fluorescent wavelength shifted and the signal level decreased in areas where tumor cells (Paget cells) were present. As a result, cells behind the tumor cells were visible, creating apparent gaps. Meanwhile, the cell membrane outlines of the surrounding normal cells were stained with PK. However, as shown in Figure 2B, no such areas of low signal level were present in the healthy tissue.

[0086] These results demonstrate that tumor cells can be distinguished from normal cells by staining with PK and selecting appropriate excitation and fluorescence wavelengths. Furthermore, PK is shown to be inserted into the cell membrane.

[0087] [3. Test Example 2: Observation of tumor cells by PK staining of cleared three-dimensional tissue] 500 μm-thick extramammary Paget's disease lesion tissue and 100 μm-thick malignant melanoma lesion tissue were cleared without fixation or sectioning, and then stained with PK and propidium iodide (PI). Using the LUCID method, clearing was performed according to the procedure described in Sawada K, Kawakami R, Shigemoto R, and Nemoto T. Eur. J. Neurosci., 2018, Vol. 47, No. 9, 1033-1042. The cleared tissue was stained with PK staining solution as in Test Example 1. PI staining was performed using a method commonly used for tissue staining. Furthermore, the same microscopic observation conditions as in Test Example 1 were used for detecting PK signals.

[0088] Figures 3A and 3B show images of a 10-μm-thick section of a 3D reconstruction of extramammary Paget's disease tissue. Using two-photon microscopy, we were able to obtain 3D PK-stained images of tissue as thick as 500 μm without slicing. Tumor cells (Paget's cells) could be detected even when they were scattered alone. Furthermore, in areas corresponding to tumor cells with low levels of 495 nm fluorescence signal, only the nuclei were observed stained with PI. This confirmed the actual presence of cells in those areas.

[0089] Furthermore, a three-dimensional reconstruction image of melanoma lesion tissue is shown in Figure 3C. As with Paget's disease lesions, no fluorescent signal was observed in melanoma cells with PK staining, and only the nuclei were observed with PI staining. Thus, even if the tumor cell types are different, the fluorescent dye agent of the present invention can specifically detect tumor cells.

[0090] The tissues observed included normal cells of the epidermis, dermis, and hair follicles, and all of these cells showed significant PK staining signals. Therefore, it is understood that PK staining can be used to detect tumor cells and distinguish them from these various normal cells.

[0091] [4. Test Example 3: Comparison of staining of normal tissue and tumor tissue by various SC compounds] Healthy skin tissue and extramammary Paget's disease lesion tissue, which had been made transparent by the LUCID method in the same manner as in Test Example 2, were stained with a staining solution containing PK, PC, Nile Red, or POLARIC. The stained tissue sections were observed using the two-photon microscope observation method in Test Example 1. Fluorescent wavelengths of 492 nm or less were colored cyan, 500 to 550 nm green, 560 to 593 nm yellow, and 593 nm or greater red. The images shown have been appropriately corrected for brightness and contrast.

[0092] In healthy tissue, as shown in Figure 4A, regardless of which staining solution was used, a planar structure filled with cells without any gaps was observed.

[0093] On the other hand, in the extramammary Paget's disease lesion tissue, as shown in Figure 4B, the fluorescence wavelength in the region corresponding to the tumor cells shifted, resulting in the observation of void-like areas similar to those in Test Example 1. Comparing the imaging power during fluorescence observation, PC had the best imaging power, followed by PK, Nile Red, and POLARIC. The higher the fluorescence intensity and staining contrast, the higher the imaging power. The fluorescence intensity of Nile Red and POLARIC in the original image was lower than that of PK and PC, and adjustment of brightness and contrast was required to identify the tumor.

[0094] Next, melanoma skin tissue was cleared in the same way and stained in the same way, and observed under a two-photon microscope. As shown in Figure 4C, the tumor cells were observed to have gaps, similar to those in Paget's disease lesions.

[0095] Comparing the drawing power during fluorescence observation, PC had the best drawing power, followed by PK, POLARIC, and Nile Red. Nile Red and POLARIC showed a relatively small shift in fluorescence wavelength due to solvatochromism, indicating that the degree of the shift in fluorescence wavelength is related to drawing power.

[0096] It was also found that POLARIC tends to emit strong light in the observed wavelength range when adsorbed to normal tissue. POLARIC tends to produce background light when staining tumor tissue, and in order to obtain good images of the lesion, it was necessary to reduce the overall fluorescence intensity of the observed image by, for example, lowering the laser power to 1 / 4 (2.5%) of that of PK and PC. On the other hand, with the other SC compounds, images with high imaging power were obtained without any special adjustment of the background intensity.

[0097] [5. Test Example 4: Comparison of staining of normal and tumor tissues in the rectum] Healthy rectal tissue and well-differentiated adenocarcinoma lesion tissue that had been made transparent by the LUCID method were stained with PK and Hoechst in the same manner as in Test Example 2. The stained tissue sections were observed using a two-photon microscope in accordance with the method used in Test Example 1.

[0098] The results are shown in Figure 5. In normal rectal mucosal epithelium (columnar epithelium), the cell membrane was positive for PK staining, the cytoplasm was poorly stained, and only Hoechst-stained nuclei were observed. On the other hand, in tumor lesions, cytoplasmic staining by PK staining was high, and the distribution of cells was observed. In other words, contrary to the cases of Test Examples 1 to 3, the fluorescence of the rectal tumor tissue became observable, and a stained image that was clearly distinguishable from normal tissue was obtained.

Claims

1. A fluorescent dye agent for detecting tumor cells in tissue derived from a living organism, which contains a compound exhibiting solvatochromism that can be inserted into cell membranes.

2. 2. The fluorescent dye agent according to claim 1, wherein the compound contains a condensed polycyclic π-conjugated structure having 2 to 6 rings.

3. 3. The fluorescent dye agent according to claim 2, wherein the compound further comprises one or more hydrophilic substituents selected from the group consisting of a tertiary amino group, a quaternary ammonium group, and a carbonyl group (excluding an aldehyde group).

4. The fluorescent dye agent according to any one of claims 1 to 3, wherein the compound is selected from the group consisting of a compound represented by the following chemical formula (II), 1-acetyl-6-piperidylpyrene, Nile Red, POLARIC (registered trademark)-500BCS, Laurdan, di-4-ANEPPDHQ, and Prodan. 【Chemistry 1】

5. 5. The fluorescent dye agent according to claim 1, wherein the compound has an absorption maximum wavelength of 300 to 600 nm in 20 mM phosphate buffer (pH 7.4) at 25°C.

6. A kit comprising the fluorescent dye agent according to any one of claims 1 to 5.

7. A method for detecting tumor cells, comprising staining tissue derived from a living organism with a fluorescent dye agent containing a compound exhibiting solvatochromism that can be inserted into a cell membrane.

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