WO2009116322A1 - センチネルリンパ節内転移癌細胞検出キット - Google Patents
センチネルリンパ節内転移癌細胞検出キット Download PDFInfo
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- WO2009116322A1 WO2009116322A1 PCT/JP2009/051385 JP2009051385W WO2009116322A1 WO 2009116322 A1 WO2009116322 A1 WO 2009116322A1 JP 2009051385 W JP2009051385 W JP 2009051385W WO 2009116322 A1 WO2009116322 A1 WO 2009116322A1
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57492—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds localized on the membrane of tumor or cancer cells
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- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
- A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
- A61K47/6913—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome the liposome being modified on its surface by an antibody
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- A61K49/00—Preparations for testing in vivo
- A61K49/04—X-ray contrast preparations
- A61K49/0433—X-ray contrast preparations containing an organic halogenated X-ray contrast-enhancing agent
- A61K49/0447—Physical forms of mixtures of two different X-ray contrast-enhancing agents, containing at least one X-ray contrast-enhancing agent which is a halogenated organic compound
- A61K49/0461—Dispersions, colloids, emulsions or suspensions
- A61K49/0466—Liposomes, lipoprotein vesicles, e.g. HDL or LDL lipoproteins, phospholipidic or polymeric micelles
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- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
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- A61K49/1806—Suspensions, emulsions, colloids, dispersions
- A61K49/1812—Suspensions, emulsions, colloids, dispersions liposomes, polymersomes, e.g. immunoliposomes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C07—ORGANIC CHEMISTRY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
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- C07—ORGANIC CHEMISTRY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2821—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against ICAM molecules, e.g. CD50, CD54, CD102
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
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- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
Definitions
- the present invention relates to a kit for detecting cancer cells that have undergone lymphatic metastasis from a cancer primary lesion to a lymph node, particularly a sentinel lymph node, and a drug transporter that selectively reaches the sentinel lymph node.
- Cancer cell metastasis is mainly caused by lymphatics.
- surgery is done to dissect the primary tumor or lymph nodes that may have metastasized.
- lymph node dissection has a complicated surgical procedure and a great physical burden on the patient.
- lymph node metastasis was performed in order to examine postoperatively whether the lymphatic system that had metastasized could be completely dissected, and whether there was a possibility of re-metastasis of cancer cells to the lymphatic system. Laboratory tests should be performed as indicators.
- This sentinel lymph node is the site where lymphatic micrometastases of cancer cells first occur.
- the clinical importance of such sentinel lymph node testing has been demonstrated in many breast cancer patients.
- the biological and histological characteristics of the lymphatic endothelial cells (LEC) in the sentinel lymph nodes that act on micrometastatic cancer cells and the closest import lymph nodes are still clear. Not.
- Patent Document 1 the present inventors have established a human lymphatic endothelial cell line derived from imported lymphatic vessels of sentinel lymph nodes of breast cancer patients using protease.
- the present inventors investigated the effect of cancer cell culture supernatant on the expression of adhesion molecules on human lymphatic endothelial cells using human breast cancer cells MDA-MB-231 or MCF-7, and expressed the adhesion molecules We investigated whether it promotes cancer cell adhesion to lymphatic endothelial cells. We also examined the possibility that cancer cells, particularly malignant breast cancer cells, release chemicals that create an environment in which cancer cells are prone to micrometastasis before metastasis in sentinel lymph nodes and the closest imported lymphatic vessels.
- the present invention has been made to solve the above-mentioned problems, and is a simple kit capable of detecting cancer cells that have lymphatic metastasis from the primary cancer focus to lymph nodes, particularly sentinel lymph nodes, in a simple and accurate manner in a short time.
- lymph nodes particularly sentinel lymph nodes
- an environment that facilitates the attachment of minute cancer cells is constructed, and drug transport that can selectively reach the drug used for testing and treatment into the sentinel lymph node to which the minute cancer cells are attached It aims at providing an agent.
- the sentinel lymph node metastasis cancer cell detection kit according to claim 1, which is made to achieve the above object, is characterized in that a human lymphatic vessel-derived endothelial cell line is attached to a substrate. To do.
- the sentinel lymph node metastasis cancer cell detection kit according to claim 2 is the kit according to claim 1, wherein the human lymphatic endothelial cell line perfuses the extracted human lymphatic lumen with a protease solution. It is characterized by being endothelial cells collected by peeling off.
- the sentinel lymph node metastasis cancer cell detection kit according to claim 3 is the kit according to claim 1, wherein the adhesion is mediated by adhesion between the human lymphatic endothelial cell line and cancer cells metastasized from the primary tumor.
- An immunoassay detection agent for detecting a molecule by an antigen-antibody reaction is added.
- the sentinel lymph node metastasis cancer cell detection kit according to claim 4 is the kit according to claim 1, characterized in that the adhesion molecule is activated and expressed.
- the sentinel lymph node metastasis cancer cell detection kit according to claim 5 is the kit according to claim 3, wherein the adhesion molecule is ICAM-1 or E-selectin.
- the sentinel lymph node metastasis cancer cell detection kit according to claim 6 is the kit according to claim 3, characterized in that the adhesion molecule is bound via its ligand.
- the sentinel lymph node metastasis cancer cell detection kit according to claim 7 is the kit according to claim 6, wherein the ligand is CD11a, CD11b and / or CD11c.
- the drug transporter into a sentinel lymph node wherein an antibody and / or a ligand for an adhesion molecule that mediates the attachment of metastatic cancer cells from a primary cancer site to lymphatic endothelial cells in the sentinel lymph node. It is exposed from the surface of the colloidal particles or is suspended.
- the drug transport agent according to claim 9 is the drug transport agent according to claim 8, wherein the antibody is an anti-ICAM-1 antibody and / or an anti-E-selectin antibody.
- the drug transport agent according to claim 10 is the drug transport agent according to claim 8, characterized in that the ligand is an ICAM-1 ligand.
- the drug transport agent according to claim 11 is the drug transport agent according to claim 10, wherein the ligand is CD11a, CD11b and / or CD11c.
- the drug transport agent according to claim 12 is the drug transport agent according to claim 8, wherein the colloidal particles contain a fluorescent agent, a contrast agent, a therapeutic agent, and / or a metastatic cancer cell adhesion enhancer. Or it is exposed.
- the drug transport agent according to claim 13 is the drug transport agent according to claim 8, wherein the colloidal particles are biodegradable resin micelle particles, synthetic resin micelle particles, or liposomes.
- the sentinel lymph node metastasis cancer cell detection kit of the present invention can detect cancer cells that have undergone lymphatic metastasis from the primary cancer focus to the lymph node, particularly the sentinel lymph node, easily and accurately. In addition, because it can be detected in a short time, during resection of the primary cancer, only the lymph nodes to which malignant cancer cells have already metastasized or to which minute cancer cells have been attached are reliably dissected, and cancer recurrence occurs. It can contribute to prevention.
- the drug transporter of the present invention is a drug that is used for the examination and treatment of cancer because the cancer transporter selectively collects in the sentinel lymph node to which the cancer cells have metastasized or the minute cancer cells adhere, and easily binds to the cancer cells. Can selectively reach cancer cells in the sentinel lymph node.
- the sentinel lymph node metastasis cancer cell detection kit of the present invention is derived from human lymphatic vessels collected by detaching them by perfusing a protease solution such as trypsin solution or collagenase solution into the lumen of lymphatic vessels, in particular, isolated human lymphatic vessels.
- a protease solution such as trypsin solution or collagenase solution
- An endothelial cell line is applied to a substrate by seeding or the like.
- the human lymphatic vessel is preferably a human collecting lymphatic vessel, particularly a human axillary lymph node importing lymphatic vessel.
- the human lymphatic vessel-derived endothelial cell line is preferably one that has been cultured in a hypoxic environment after collecting the endothelial cells. Under this low oxygen environment, the oxygen concentration is preferably 1 to 10%, more preferably 3 to 7%, and particularly preferably 5%. Since the oxygen concentration in the lymph fluid in the human body is much lower than the oxygen concentration in the blood, the culture conditions in such a hypoxic environment are optimal for culturing human lymphatic endothelial cell lines. it is conceivable that.
- collagenase solution used for isolating human collecting lymphatic-derived endothelial cell lines examples include collagenase II solution (manufactured by Worthington, USA; product number S2B5456). The concentration thereof is preferably from 0.01 to 0.1%, particularly preferably 0.05%.
- the perfusion rate of the human collecting lymphatic lumen may be any rate as long as at least the enzyme action of collagenase II can be expressed, and the perfusion can be temporarily stopped to detach the endothelial cells. It is also possible to peel the endothelial cells while perfusing.
- the composition of the collagenase II solution is not particularly limited as long as collagenase II is contained at the aforementioned concentration.
- Human lymphatic vessel-derived endothelial cell lines include HUVEC (human umbilical vein endothelial cells: manufactured by Takara Bio Inc .; product number # CC-2517) and HMVEC (human microvascular endothelial cells) collected from human subcutaneous tissue. Similar to Product No. # CC-2505), it is not derived from a single cell and has the property that the number of possible passages is about 10 passages.
- the human lymphatic vessel-derived endothelial cell line is removed from mycoplasma.
- the endothelial cells are preferably purified by adding a mycoplasma removing agent to invert the mycoplasma and then purifying.
- the mycoplasma removing agent is not particularly limited, and any method or substance may be used as long as at least mycoplasma removal is possible, and the concentration of the mycoplasma removing agent is not particularly limited.
- the mycoplasma removal may be performed at any passage, but it is preferably performed at the second to fifth passages, preferably the second passage.
- mycoplasma remover for cultured cells.
- Mynox manufactured by Minerva Biolabs; trade name
- MC-210 manufactured by Dainippon Pharmaceutical Co., Ltd .; trade name
- the presence or absence of mycoplasma infection may be measured, and it may be measured at any passage before the removal of mycoplasma, but preferably at the second to fifth passages, particularly preferably at the second passage. It is.
- Mycoplasma infection measurement may be performed using Mycoplasma infection measurement kit Mycoplasma Plus PCR PCR Primer Set (trade name, manufactured by Stratagene, USA), but any method or substance can be used as long as at least the presence of mycoplasma infection can be measured. It may be used, and a plurality of methods may be used in combination.
- This human lymphatic endothelial cell line was received by the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1-1-1 East Tsukuba, Ibaraki Prefecture) on January 18, 2006, and received the domestic accession number FERM P-20768. The application was transferred to the International Depository on January 16, 2009, and the receipt number FERM ABP-11089 was assigned to the same center.
- this kit is seeded on a substrate on which an endothelial cell line that is a human lymphatic cell line can grow.
- a substrate any culture plate, culture slide glass, or semipermeable membrane may be used, and any shape or modified state of the culture surface may be used.
- the culture medium used for the base material of this kit may have any composition as long as it is at least a culture medium capable of growing cell lines.
- EGM-2 manufactured by Sanko Junyaku Co., Ltd.
- EGM-2 is a medium for vascular endothelial cells. Is mentioned.
- cytokines such as hFGF, VEGF, R 3 -IGF-1, hEGF, VFGF-C, VEGF-D and PDGF-BB
- various vitamins such as ascorbic acid, steroids such as hydrocortisone, and serum
- the number of endothelial cells derived from human lymphatic vessels on the substrate is preferably 1 ⁇ 10 4 to 1 ⁇ 10 5 cells / cm 2 depending on the area of the seeding substrate.
- the sentinel lymph node metastasis cancer cell detection kit is an adhesive intercellular adhesion molecule that mediates the adhesion of metastasis cancer cells to the human lymphatic vessel-derived endothelial cells and the substrate to which the human lymphatic vessel-derived endothelial cell line is attached. It is preferable that an adhesion molecule such as (ICAM-1) or E-selectin is activated and expressed. In particular, the adhesion molecule is more preferably ICAM-1.
- an adhesion molecule that mediates adhesion to metastasized cancer cells to human lymphatic endothelial cells may be bound via its ligand.
- a ligand include CD11a, CD11b, and CD11c, which are ligands of ICAM-1.
- the sentinel lymph node metastasis cancer cell detection kit preferably further comprises an immunoassay detection agent that immunostains adhesion molecules.
- Such immunoassay detection agents are used for indirect immunohistochemical observation using antigen-antibody reaction.
- the sentinel lymph node metastasis cancer cell detection kit is used as follows. To the base material of the kit, lymph fluid, for example, sentinel lymph node fluid collected from a breast cancer patient is added. If there are metastasized cancer cells, for example, micrometastasized cells, from the primary tumor in the lymph, the metastasized cancer cells adhere to the human lymphatic vessel-derived endothelial cells via adhesion molecules on the human lymphatic vessel-derived endothelial cells. When the adhesion molecule is detected with the antibody by immunoassay, since there are micrometastasis cells in the lymph, it can be confirmed that there are metastasis cancer cells in the sentinel lymph nodes.
- lymph fluid for example, sentinel lymph node fluid collected from a breast cancer patient is added. If there are metastasized cancer cells, for example, micrometastasized cells, from the primary tumor in the lymph, the metastasized cancer cells adhere to the human lymphatic vessel-derived endothelial cells via adhesion molecules on the human lymphatic vessel-
- the drug transporter into the sentinel lymph node of the present invention is an antibody against an adhesion molecule that mediates the attachment of metastatic cancer cells from the primary cancer site to lymphatic endothelial cells in the sentinel lymph node, such as anti-ICAM-1 antibody,
- the anti-E-selectin antibody is exposed from the surface of the colloidal particles.
- a ligand for the adhesion molecule for example, CD11a, CD11b and / or CD11c may be exposed from the surface of the colloidal particle.
- anti-ICAM-1 antibodies anti-E-selectin antibodies
- ICAM-1 ligands such as CD11a, CD11b, and CD11c may be commercially available.
- ICAM-1 agonists are expressed on the surface of cancer cells and are found to be bound to adhesion molecules expressed on lymph node endothelial cells. Thus, drug delivery can be performed in a site-specific manner. Since ICAM-1 is expressed on endothelial cells in lymph nodes where cancer cells have metastasized, it intervenes to bind to it.
- the colloidal particles include biodegradable resin micelle particles, synthetic resin micelle particles, and liposome particles, and are preferably so-called nanoparticles having an average particle size of 5 to 500 nm. If the particle size is less than 5 nm, it is rapidly excreted in the living body, whereas if it exceeds 500 nm, it is excluded as a foreign substance in the living body. When it is about 200 nm, it is easy to be taken in by a smooth muscle cell exposed especially in a gap formed between cells at a vascular injury site or in a blood vessel.
- the drug delivery agent preferably contains 0.5 to 2.0% of the colloidal particles.
- biodegradable resin particles include suspended polylactic acid particles.
- the synthetic resin particles include polystyrene beads having an average particle diameter of about 200 nm.
- the liposome include those made of fat or phospholipid and having a diameter of 50 to 800 nm, preferably 200 to 400 nm.
- the drug transporting agent may contain a medicinal substance such as a fluorescent agent, a contrast agent, a therapeutic agent, and / or a metastatic cancer cell adhesion enhancer, or may be contained in or bound to the colloidal particle. It is preferable.
- the drug transporter may be suspended while these medicinal substances and the above-mentioned antibodies and ligands bind or interact with each other.
- the drug transporting agent include those containing colloidal particles including those obtained by binding a medicinal substance as a fluorescent agent to ICAM-1 ligands such as CD11a, CD11b or CD11c, and adhere to cells in vitro. It can be used for observation with a fluorescence microscope or used for transport to a desired living body site in vivo.
- the fluorescent agent include fluorescein isothiocyanate (FITC), and a dye for live cell staining, Calcein-AM (manufactured by Doujin Chemical Laboratory, Inc .; trade name).
- the drug transporting agent may contain colloidal particles including, for example, a chemokine combined with a near-infrared coloring pigment such as indocyanine green.
- contrast agent examples include gadolinium compounds for nuclear magnetic resonance imaging diagnosis and iodine compounds for X-ray tomography.
- therapeutic agents include vascular endothelial cell proliferation promoters, vascular smooth muscle cell proliferation inhibitors, anti-inflammatory agents, and anticancer agents. It may be a metastatic cancer cell adhesion enhancer.
- This drug transport agent is used as follows. When this drug transporter is administered to the primary lesion of cancer or a lymph vessel in the vicinity thereof, it reaches the sentinel lymph node by lymphatic circulation. If cancer cells such as micro cancer cells have metastasized to lymphatic endothelial cells or sentinel lymph node endothelial cells downstream of the primary tumor, adhesion molecules such as ICAM-1 that mediate adhesion to cancer cells It is expressed on endothelial cells. The adhesion molecule antibody of the drug transport agent induces an antigen-antibody reaction and binds to the adhesion molecule.
- the adhesion molecule ligand of the drug transport agent selectively causes an enzymatic reaction as if it were a key and a keyhole, and binds to the adhesion molecule.
- the fluorescent agent, contrast agent, therapeutic agent, or metastatic cancer cell adhesion enhancer exudes from the colloidal particle surface of the drug transport agent and is released or absorbed by the metastatic cancer cells to cause fluorescence or contrast. It also exhibits medicinal effects.
- the location of sentinel lymphatic vessels with metastasis to which drug transporters are selectively concentrated can be confirmed on the image by, for example, ultrasonic diagnosis, nuclear magnetic resonance imaging (MRI), or X-ray tomography (CT).
- MRI nuclear magnetic resonance imaging
- CT X-ray tomography
- Only one or two micrometastatic cancer cells attached to the sentinel lymph node can develop a microenvironment inside the lymph node that facilitates microcarcinoma metastasis and express the adhesion molecule, It can be detected and the lymph nodes to be dissected can be identified accurately and quickly.
- an adhesive molecule antibody or an adhesive molecule ligand exposed on the surface of an MRI contrast medium containing a metal such as gadolinium can be mentioned.
- this drug transporter can enhance the adhesion of micrometastasis cells trapped in the sentinel lymph node so that they do not metastasize to the downstream lymph vessels. Furthermore, since this drug transporter has sustained release properties, the drug efficacy can be maintained for a long time.
- EGM-2 endothelial growth medium
- FBS fetal bovine serum
- the human breast cancer sentinel lymph node imported lymphatic vessel-derived lymphatic endothelial cells thus obtained were stored in 10% FBS-containing EGM-2 and used after being subcultured for the fifth to seventh passages.
- Lymphatic endothelial cells were cultured at 37 ° C. under atmospheric conditions of 5% oxygen, 5% carbon dioxide and 90% nitrogen gas.
- a human collecting lymphatic vessel-derived endothelial cell line was isolated and cultured as follows.
- a breast cancer patient who also obtained consent received donation of human collecting lymphatic vessels, which are imported lymphatic vessels of human axillary lymph nodes, with the surrounding tissues removed at the time of surgery attached.
- tissues such as fat and capillaries around the lymphatic vessels were exfoliated under a stereomicroscope.
- a thin polyethylene tube was inserted into the lumen of the human collecting lymph vessel and left in place for perfusion, washing and cell collection. The lumen was washed by perfusion with phosphate buffered saline (PBS solution).
- PBS solution phosphate buffered saline
- This lumen was filled with 0.05% collagenase II solution (manufactured by Worthington, USA; product number S2B5456). It was allowed to stand in a 37 ° C. incubator for a period in which endothelial cell detachment was observed, mainly 10 minutes.
- As a medium and medium supplement 500 ml of growth medium bullet kit EGM-2 for vascular endothelial cells (Sanko Junyaku Co., Ltd .; product number CC-3162) and fetal bovine serum (FBS) (manufactured by Japan Bioserum; product number) S1560) 40 ml was used.
- the human collecting lymphatic lumen was perfused with 1 ml of EGM-2 supplemented with 10% FBS, and the detached endothelial cells were collected in this lumen.
- the endothelial cell collection solution was put into a tube and centrifuged at 2000 rpm for 5 minutes to collect endothelial cell components. Thereby, a human collecting lymphatic vessel-derived endothelial cell line could be isolated.
- the storage conditions of the human lymphatic endothelial cell line were as follows: the endothelial cell line was recovered with 0.25% trypsin solution, and a cell suspension was prepared using a cryopreservation solution consisting of 10% dimethyl sulfoxide (DMSO) and 90% FBS. After that, it is put in a freezing tube, cooled in stages using a bicell, and finally stored in liquid nitrogen.
- the melting conditions and the culturing conditions at the time of thawing were as follows: a cryotube was thawed in a 37 ° C.
- a cell suspension is prepared using 2 and cultured in the same manner as in the above-mentioned endothelial cell line culture conditions.
- VEGF-R3 vascular endothelial growth factor receptor 3
- the human collecting lymphatic vessel-derived endothelial cell line transferred to the culture system was seeded on a slide glass. When the culture was continued and became confluent, it was fixed with 10% formalin. After blocking with a PBS solution containing 0.1% bovine serum albumin (BSA), VEGF-R3 (manufactured by Santa Cruz, USA; product number sc-637), which is a specific marker of lymphatic endothelial cells, is used as a primary antibody. : 100 diluted with 0.1% BSA-added PBS solution, and allowed to stand at 4 ° C. overnight.
- BSA bovine serum albumin
- LYVE-1 lymphatic vessel endothelial hyaluronan receptor-1
- LYVE-1 lymphatic vessel endothelial hyaluronan receptor-1
- TNF- ⁇ tumor necrosis factor- ⁇
- IL-1 ⁇ interleukin-1 ⁇
- the negative control without a stimulating factor showed an unclear green fluorescent color and expressed less F-actin, whereas it was stimulated with TNF- ⁇ or IL-1 ⁇ .
- the product showed a clear green fluorescent color, confirming an increase in the expression of F-actin.
- Morphological features, culture properties, and physiological properties of other human lymphatic endothelial cell lines (1.5.1. Morphological properties)
- Shape and size Shows a polygon that is characteristic of cultured endothelial cells, and exhibits a paving stone-like arrangement in a single layer. The size is around 50 ⁇ m in diameter.
- Polymorphism Endothelial cells show almost the same morphology, and no polymorphism is observed.
- FBS fetal bovine serum
- cytokines and growth factors The concentrations of specific cytokines and growth factors in the respective culture supernatants of MDA-MB-231 and MCF-7 were measured using an enzyme-linked immunosorbent assay (ELISA). First, those supernatants were collected, and cancer cells were seeded in DMEM / F12 containing 10% FBS. On the next day, the medium was replaced with DMEM / F12 containing no FBS and cultured overnight. The culture was centrifuged at 2,000 rpm for 5 minutes at 4 ° C. and then stored frozen at ⁇ 20 ° C. to measure cytokines and growth factors.
- ELISA enzyme-linked immunosorbent assay
- TNF- ⁇ , TGF- ⁇ 1, INF- ⁇ , IL-1 ⁇ , IL-6, IL-12, basic FGF, PDGF-BB, VEGF-A and VEGF-C was measured by a private laboratory (SRL, Inc.) Measured by outsourcing.
- the detection limits were 5 pg / mL for tumor necrosis factor- ⁇ (TNF- ⁇ ), 5 ng / mL for tumor growth factor- ⁇ 1 (TGF- ⁇ 1), 0.1 U / mL for interferon- ⁇ (INF- ⁇ ), Interleukin-1 ⁇ (IL-1 ⁇ ) is 10 pg / mL, IL-6 is 0.2 pg / mL, IL-12 is 7.8 pg / mL, basic fibroblast growth factor (bFGF) is 10 pg / mL, platelets Derived growth factor-BB (PDGF-BB) is 31.2 pg / mL, vascular endothelial growth factor-A (VEGF-A) is 20 pg / mL, and VEGF-C is 109 pg / mL.
- TNF- ⁇ tumor necrosis factor- ⁇
- TGF- ⁇ 1 tumor growth factor- ⁇ 1
- INF- ⁇ interferon- ⁇
- luciferin luciferase reaction was measured by Luminescent Cell Viability Assay using CellTiter-Glo (manufactured by Promega, USA; registered trademark).
- E-selectin / CD63E (dilution 10 ⁇ g / mL, manufactured by R & D® Systems, USA), P-selectin / CD63P (diluted 10 ⁇ g / mL, manufactured by R & D® Systems, USA), VCAM-1 / Cultured overnight with primary polyclonal human antisera of CD106 (diluted 10 ⁇ g / mL, R & D® Systems, USA) or ICAM-1 / CD54 (diluted 10 ⁇ g / mL, manufactured by R & D® Systems, USA) at 4 ° C. overnight.
- Cancer cells were infiltrated with 0.1% Triton X before primary staining. After washing 3 times with PBS, the cells were cultured for 1 hour at room temperature using Alexa Fluor 488 anti-mouse immunoglobulin G (manufactured by Invitrogen, USA) diluted 1: 100. The cell nuclei of the cultured cells are stained with a counterstain, and ProLong Gold non-bleaching reagent added with 4 ', 6-diamidine-2-phenylindole (DAPI) (Molecular Probes, USA) is added, followed by fluorescence microscopy (Leica, Switzerland). Observed) and photographed.
- Alexa Fluor 488 anti-mouse immunoglobulin G manufactured by Invitrogen, USA
- DAPI 6-diamidine-2-phenylindole
- Block-Ace manufactured by Sumitomo Dainippon Pharma Co., Ltd.
- Block-Ace manufactured by Sumitomo Dainippon Pharma Co., Ltd.
- PECAM Primary polyclonal human antiserum platelet endothelial cell adhesion molecule
- LYVE lymphatic endothelial hyaluronan receptor
- VEGF R3 endothelial growth factor receptor 3
- E-selectin / CD62E (1:50 diluted, R & D systems, USA
- P-selectin / CD62P (1:50 diluted, USA, R & D systems
- VCAM vascular cell adhesion Molecule
- the cultured cells Prior to staining, the cultured cells were permeabilized with 0.1% Triton-X. Next, the cells were stained with AlexaFluor 488 chicken anti-rabbit immunoglobulin G or Alexa Fluor 488 donkey anti-mouse immunoglobulin G (manufactured by Invitrogen, USA). The cell nuclei of the cultured cells were counterstained and sampled with 4 ′, 6-diamidine-2-phenylindole (DAPI) -containing anti-fading agent ProLong Gold (manufactured by Molecular Probe, USA). The cultured cells were observed and photographed with a fluorescence microscope (Leica, Germany).
- DAPI 6-diamidine-2-phenylindole
- sentinel lymph node tissue that had been frozen fresh was fixed with 100% acetone at 4 ° C. Endogenous peroxidase activity was inhibited with 0.3% H 2 O 2 for 30 minutes at room temperature.
- the tissue was cultured for 1 hour at room temperature in the presence of primary polyclonal antiserum E-selectin (R & D systems, USA) and ICAM-1 (R & D systems, USA), then horseradish peroxidase-labeled anti-rabbit immunoglobulin G and The cells were cultured at room temperature for 30 minutes in the presence of anti-mouse immunoglobulin G (manufactured by Nichirei Co., Ltd.).
- the reaction product was stained with a DAB kit (manufactured by Nichirei Co., Ltd.). Cell nuclei of sentinel lymph node tissue were also counterstained with hematoxylin staining. The sentinel lymph node tissue was observed with an optical microscope (manufactured by Leica, Germany) and photographed.
- chemokines To examine the effects of chemokines on the immunohistochemical expression of adhesion molecules on human lymphatic endothelial cells, 1 mL of 3% FBS-containing EBM-2 containing various concentrations of chemokines was added to the starvation medium, followed by human lymphatic endothelial cells Were stimulated for 4, 18 and 48 hours. Various chemokine concentrations were prepared by diluting each chemokine with an appropriate amount of 3% FBS-containing EBM-2.
- TNF tumor necrosis factor
- Block-ace manufactured by Dainippon Sumitomo Pharma Co., Ltd. was replaced with the primary antiserum to serve as a negative control.
- Human lymphatic endothelial cells were formed in a monolayer on a 35 mm culture dish coated with type I collagen, and cultured at 37 ° C. in oxygen 5%, carbon dioxide 5% and nitrogen 90% until confluence. Lymphatic endothelial cells were preserved in serum starvation medium with EBM-2 containing 3% FBS. A given culture dish was treated with 10 ng / mL CCL2 for 18 hours. In some experiments, culture dishes were stimulated with serum starvation medium pre-neutralized with 10 ng / m CCL2 with 1.0 ⁇ g / mL specific CCL2 antibody.
- the culture dish was treated with 10 ng / mL CCL2 for 18 hours, and then treated with anti-human ICAM-1 antibody (manufactured by R & D systems, USA) for 30 minutes.
- MDA-MB-231 and MCF-7 stained with PKH26 fluorescent dye (manufactured by SIGMA, USA) were seeded at 1 ⁇ 10 5 cells per culture dish at 37 ° C. Incubated for 30 minutes. Cells that did not adhere were removed by aspiration and the culture dish was washed 3 times with EBM-2. The adhesion of cancer cells was quantified by enlarging 100 times using a microscope (manufactured by Leica, Germany) and measuring the number of cells.
- PKH26 fluorescent dye manufactured by SIGMA, USA
- CD11a LFA-1
- CD11b Mac-1 on MDA-MB231 and MCF-7 breast cancer cell lines
- Most of the immunohistochemical methods are the same as those used for the experiment of lymphatic marker (FIG. 9).
- polyclonal antisera against CD11a (1:50 diluted, AnaSpace, USA) and CD11b (1:50 diluted, R & D Systems, USA) were used.
- RNA was extracted from cultured human lymphatic endothelial cells using Isogen reagent (Nippon Gene Co., Ltd.) according to the manufacturer's instructions. The concentration of each RNA was calculated from the 260 nm absorption by a spectrophotometer. The extracted RNA was reverse transcribed with M-MLV reverse transcriptase (Ambion, USA).
- Quantitative RT-PCR was performed using a light cycler rapid thermal cycler system (Roche Diagnostics, UK). A 20 ⁇ L volume containing 0.5 ⁇ M primer, Taq DNA polymerase, and buffer was added to SYBR Premix Ex Taq (manufactured by Takara Bio Inc .; SYBR is a registered trademark), and reacted.
- the PCR operation includes a 10-second alteration, followed by a 45-step process of alteration at 95 ° C. for 5 seconds and annealing at 60 ° C. for 20 seconds 45 times. After further standing at 72 ° C., the fluorescent product was detected.
- the negative control is a PCR reaction without using cDNA.
- the PCR product obtained from each primer pair was subjected to melting temperature curve analysis, and the quantitative data was analyzed using Light Cycler analysis software. The results are shown as a normalized index of ICAM-1 mRNA expression relative to cyclophilin A.
- the membrane was reacted with an anti-ICAM-1 antiserum (diluted 1: 1000, manufactured by Cell Signaling Technology, USA) and then incubated with an anti-rabbit immunoglobulin G horseradish peroxidase-conjugated antibody.
- the membrane was re-reacted with a monoclonal anti-actin antibody (Santa Cruz, USA) and then visualized with an ECL-Western blot detection system (Amersham BioScience, UK).
- the supernatants were dialyzed with two types of tubular dialysis membranes (manufactured by Spectrum Medical Industries, USA) for molecular dialysis having a molecular weight of 1,000 or 500. Dialysis was performed overnight at 4 ° C. by placing the tubular dialysis membrane in a buffer (DMEM-F12 (1: 1) medium). The supernatant obtained in the tubular dialysis membrane was used for bioassay. This supernatant does not contain a substance having a molecular weight of less than 1,000 or less than 500.
- the culture dish was stimulated with 10 ⁇ 7 M ATP or MDA-MB-231 cell culture supernatant for 48 hours and then treated with anti-human ICAM-1 antibody (manufactured by R & D Systems, USA) for 30 minutes.
- Breast cancer cells stained with PKH26 fluorescent dye (manufactured by SIGMA, USA) were added at 5 ⁇ 10 4 cells per culture dish and cultured at 37 ° C. for 30 minutes. Cells that did not adhere were removed by aspiration and the culture dish was washed 3 times with DMEM / F12. Cell adhesion was quantified by enlarging 100 times using a microscope (Leica, Switzerland) and counting the number of cells.
- the salts used are all manufactured by Wako Pure Chemical Industries, Ltd. ATP and suramin are from SIGMA, USA, and DPCPX and DMPX are from Research Biologicals.
- DCPPX was diluted with ethanol
- DMPX was diluted with dimethyl sulfoxide (DMSO).
- the concentrations of ethanol and DMSO are set so as not to affect the survival of cultured cells.
- Cellular chemokines were obtained from R & D systems (USA). The reagent concentration is indicated by the final concentration in the culture dish.
- FIG. 1 is a representative of the expression results of adhesion molecules E-selectin, P-selectin, VCAM-1 and ICAM-1 on lymphatic endothelial cells when stimulated with MDA-MB-231 cell culture supernatant. It is a typical immunohistochemical micrograph. When cultured overnight in a starvation medium containing 3% FBS, as shown in FIGS.
- FIGS. 1E and 1F when stimulated with MDA-MB-231 cell culture supernatant for 4 hours, almost all cultured lymphatic endothelial cells were found to have E-selectin antiserum. Since the immunological activity staining was clearly observed with P-selectin antiserum, the expression of E-selectin and P-selectin on human lymphatic endothelial cells was remarkably induced. As shown in FIGS. 1G and 1H, even when stimulated with MDA-MB-231 cell culture supernatant for 4 hours, the immunoreactivity staining with VCAM-1 antiserum and ICAM-1 antiserum was observed in human lymphatic endothelial cells. The expression of VCAM-1 and ICAM-1 was hardly triggered because it was only slightly observed.
- FIG. 2 shows E-selectin, P-selectin, VCAM-1 and ICAM-1 which are adhesion molecules on human lymphatic endothelial cells when stimulated with MDA-MB-231 supernatant or MCF-7 supernatant for 48 hours. It is the typical immunohistochemical micrograph about the expression result of.
- E-selectin, P-selectin, VCAM-1 and ICAM-1 were cultured on cultured human lymphatic endothelial cells.
- the expression of these adhesion molecules was not induced at all or almost.
- FIG. 2H when stimulated with MDA-MB-231 supernatant for 48 hours, almost all cultured lymphatic endothelial cells were clearly marked by ICAM-1 antiserum. Since it was immunostained, significant expression of ICAM-1 was induced on human lymphatic endothelial cells, and as shown in P of FIG. 2, the supernatant diluted 1 / 10,000 times The immune response was somewhat diminished, but nevertheless strong immunoactivity staining was observed, leading to overexpression of ICAM-1 produced by the diluted serum.
- Table 1 shows the concentrations of TNF- ⁇ , TGF- ⁇ 1, INF- ⁇ , IL-1 ⁇ , IL-6, and IL-12 cytokines in the MDA-MB-231 culture supernatant or MCF-7 culture supernatant.
- BFGF, PDGF-BB, VEGF-A, and VEGF-C growth factor concentrations and measurement data are collectively shown.
- Table 2 summarizes each ATP concentration in the MDA-MB-231 supernatant, or in the medium in the presence or absence of ATP (10 ⁇ 8 , 10 ⁇ 7 and 10 ⁇ 6 M).
- FIG. 9 shows lymphatic markers such as VEGF-R3 (FIG. C), LYVE-1 (FIG. D), Prox-1 (FIG. E), and podoplanin (FIG. F) on cultured cells. It is a typical photomicrograph. The cultured cells were strongly stained with the antisera of VEGF-R3, Prox-1, podoplanin, and PECAM-1 (Fig. B), whereas the antibody against LYVE-1 slightly subtracted the cultured cells. It was only stained (Fig. D).
- FIG. 9A is a representative photomicrograph of a phase difference image of cultured cells. These results indicate that the cultured cells are imported lymphatic human lymphatic endothelial cells that are closest to the sentinel lymph node of breast cancer patients.
- FIG. 10 shows the influence of chemokines on the expression of adhesion molecules on human lymphatic endothelial cells.
- Fig. A shows 10 ng / mL CCL1 (1 to 4 in Fig. A), 10 ng / mL CCL2 (5 to 8 in Fig. A), and 10 ng / mL CCL12 (9 to 12 in Fig. A).
- 10 ng / mL CCL21 (13 to 16 in FIG. A)
- FIG. 8A As shown in FIG. 8A, almost all cultured lymphatic endothelial cells were clearly stained with ICAM-1 antiserum only when stimulated with 10 ng / mL CCL2 for 18 hours. Significant expression of ICAM-1 was observed on the cells. As shown in FIGS. 1, 5, 9, 13, and 3, 7, 11, and 15, no or little expression of E-selectin or VCAM-1 was observed on cultured lymphatic endothelial cells. . In contrast, as shown in FIGS. 4, 12, and 16 on the lymphatic endothelial cells stimulated with 10 ng / mL CCL1, 10 ng / mL CCL12, and 10 ng / mL CCL21, respectively, ICAM ⁇ Slight staining was observed with 1 antiserum.
- FIG. 11A shows the effect of stimulation time on the immunohistological expression of adhesion molecules mediated by CCL2 on cultured human lymphatic endothelial cells.
- FIGS. 1 to 16 show that when cultured human lymphatic endothelial cells were stimulated with 10 ng / mL of CCL2 for 0 hour, 4 hours, 18 hours, or 48 hours, respectively, on the cells.
- E-selectin Fig. 1, 5, 9, 13
- P-selectin Fig. 2, 6, 10, 14
- VCAM-1 Fig. 3, 7, 11, 15
- ICAM-1 Fig.
- FIG. 4 is a representative photomicrograph showing the influence of stimulation time on the immunohistochemical expression of each adhesion factor in FIGS.
- FIG. 11B is a graph summarizing the effect of stimulation time on stimulation with 10 ng / mL CCL2 on ICAM-1 mRNA levels in cultured human lymphatic endothelial cells.
- FIG. 5B shows that when human lymphatic endothelial cells were stimulated with 10 ng / mL CCL2 for 0 hour, 1 hour, 4 hours, and 18 hours, the ICAM-1 mRNA level of the cells was reverse transcription polymerase chain reaction. Evaluation by (RT-PCR) shows the effect of stimulation time. ** indicates a significant difference of p ⁇ 0.01, * indicates a significant difference of p ⁇ 0.05, and NS indicates no significant difference.
- CCL2-mediated expression of ICAM-1 mRNA was markedly increased 1 hour after stimulation. The increase in CCL2-mediated expression of the ICAM-1 mRNA reached almost the maximum expression level after about 4 hours from the stimulation and continued to increase slightly until 18 hours after the stimulation.
- FIG. 12 shows the effect of 10 pg / mL to 10 ng / mL of CCL2 on the immunohistochemical expression of ICAM-1 on human lymphatic endothelial cells.
- Fig. A shows 10 pg / mL (1 in Fig. A), 100 pg / mL (2 in Fig. A), 1 ng / mL (3 in Fig. A), and 10 ng / mL (4 in Fig. A).
- FIG. 12B shows a gray scale image of a specific area of human lymphatic endothelial cells of 5 specimens for each of the 5 specimens.
- the density measurement data of each micrograph obtained by Scion-Image analysis is summarized and expressed in relative units (average density / pixel). This is shown in the graph.
- FIG. B there was no significant difference between 10 pg / mL (1 in FIG. B) and 100 pg / mL (2 in FIG. B), but 10 pg / mL (1 in FIG. B).
- 1 ng / mL (3 in Fig. B) and 10 ng / mL (4 in Fig. B) were significantly different at p ⁇ 0.01, respectively.
- FIG. 13 shows the effect of CCL2 neutralization on the expression of ICAM-1 mediated by CCL2 on cultured human lymphatic endothelial cells.
- Figure A shows the effect of 10 ng / mL CCL2 in the presence and absence of 1.0 ⁇ g / mL specific CCL2 antibody (3 in Figure A) and in the absence (2 in Figure A), respectively. It is the typical microscope picture shown about.
- a in FIG. 1A is a photomicrograph showing a negative control obtained with a serum starvation culture medium (EBM-2 containing 3% FBS).
- EBM-2 serum starvation culture medium
- FIG. 13B is a gray scale image of a specific area of human lymphatic endothelial cells, 5 samples each, and measured density, and the density measurement data of each micrograph by Scion Image analysis is summarized and expressed in relative units (average density / pixel). This is shown in the graph. The vertical axis is the same as in FIG. 12B. ** indicates a significant difference at p ⁇ 0.01, and * indicates a significant difference at p ⁇ 0.05. As is clear from FIG. 13B, p ⁇ 0.01 significantly increased in the case of CCL210 ng / mL treatment than in the negative control (B1), and neutralized after CCL210 ng / mL treatment than in the case of CCL210 ng / mL treatment. In some cases, there was a significant decrease at p ⁇ 0.05.
- FIG. 13C is a photograph showing a part of the result of a typical electrophoresis subjected to Western blot analysis.
- FIG. 2C when 18 ng / mL CCL2 stimulated for 18 hours, significant expression of ICAM-1 was observed on cultured human lymphatic endothelial cells, but as shown in FIG. 3C.
- the expression of ICAM-1 mediated by CCL2 was remarkably suppressed by CCL2 neutralization treatment.
- 1 of the same figure C shows a negative control.
- FIG. 14 shows breast cancer cell lines MCF-7 (I) and MDA-MB-231 (II), when specific CCL2 antibody is not present (I-2 and II-2) and when it is present (CCL2 Neutralization: I-3 and II-3), and in the presence of anti-ICAM-1 antibodies (I-4 and II-4), respectively, an adhesion test was conducted on the effect of 10 ng / mL CCL2 It is the graph which summarized data.
- I is shown as a white bar graph for cancer cell MCF-7
- II is shown as a hatched bar graph for cancer cell MDA-MD-231.
- the vertical axis represents the normalized number of adherent cancer cells in one field of view (magnification is 100 times). ** indicates a significant difference of p ⁇ 0.01.
- I-2 and II-2 in the figure when human lymphatic endothelial cells were stimulated in vitro with 10 ng / mL CCL2 for 18 hours, MCF-7 (I-2) and MDA-MD- The adhesion of 231 (II-2) was significantly increased at each p ⁇ 0.01 compared to the respective DMEM / F12 negative control.
- FIG. 15 shows representative immunohistochemical expression of CD11a (FIG. A, B) and MCD11b (FIG. D, E) on human breast cancer cell lines MCF-7 and MDA-MB-231. It is a micrograph.
- FIGS. C and F are representative photomicrographs in the case of a negative control not containing the primary antibody of CD11a and CD11b, respectively. Significant immunohistochemical expression of both CD11a and CD11b was observed on MCF-7 and MDA-MB-231 cells.
- FIG. 16 shows E-selectin in a cancer cell metastatic sentinel lymph node tissue frozen immediately after removal from a breast cancer patient and a cancer cell untransferred sentinel lymph node tissue frozen immediately after removal from the patient (FIG. C, C, D) and representative micrographs showing immunohistochemical expression of ICAM-1 (FIG. E, F, G, H).
- FIG. 5A is a photomicrograph showing representative hematoxylin-eosin staining of sentinel lymph node tissue with cancer cell metastasis, and FIG.
- FIGS. B is a sentinel lymph node tissue with no cancer cell metastasis.
- FIGS. F and H marked immunohistochemical expression of ICAM-1 was observed on the sentinel lymph node tissue to which cancer cells had metastasized.
- FIGS. E and G almost no expression of ICAM-1 was observed on sentinel lymph node tissue that had not been metastasized from cancer cells and was excised from the same breast cancer patient.
- FIGS. C and D no or little expression of E-selectin was observed on the sentinel lymph node tissue regardless of the presence or absence of cancer cell metastasis.
- "*" in the same figure B, D, F, and H shows the range of cancer cell metastasis in the sentinel lymph node.
- lymph nodes are the site where malignant tumors usually metastasize first.
- the lymph node acts as a barrier that physically inhibits the passage of cancer cells therein and also as a barrier that biochemically inhibits the growth of cancer therein.
- the dramatic success of sentinel lymph node navigation surgery in clinical settings means that local lymph nodes have an effective filtering mechanism that provides a physical barrier to migrating cancer cells. Suggests.
- the primary cancer site affects the microenvironment of cancer tissue before metastasis.
- the concentrations of IL-6, VEGF-A and VEGF-C in the MDA-MB-231 supernatant are much higher than those obtained from the supernatant of MCF-7.
- cytokines and growth factors elicit only a slight expression of ICAM-1 on human lymphatic endothelial cells and differ from MDA-MB-231 supernatant-mediated ICAM-1 expression on lymphatic endothelial cells .
- MDA-MB-231 When chemical treatment was performed by dialysis of a substance having a molecular weight of less than 1,000, MDA-MB-231 supernatant-mediated ICAM-1 expression on human lymphatic endothelial cells was completely reduced. In contrast, MDA-MB-231 on human lymphatic endothelial cells is not affected by heat treatment, enzymatic degradation of the MDA-MB-231 supernatant with protease, or pretreatment of dialyzing a substance having a molecular weight of less than 500. Supernatant-mediated ICAM-1 expression had little effect.
- the ATP concentration in the MDA-MB-231 supernatant was significantly higher than that obtained from the medium alone.
- 10 ⁇ 8 and 10 ⁇ 7 M ATP was found to be present in human lymphatic endothelial cells, as was the case with MDA-MB-231 supernatant. Induced ICAM-1 expression.
- Pretreatment with 10 ⁇ 7 and 10 ⁇ 6 M suramin markedly demonstrated ATP and MDA-MB-231 supernatant mediated ICAM-1 expression in lymphatic endothelial cells Declined. Depending on the suramin concentration, it is known to selectively block P2X and P2Y receptors.
- ATP which induces selective ICAM-1 expression on human lymphatic endothelial cells by activation of purinergic P2X and P2Y receptors on lymphatic endothelial cells, is converted to MDA-MB-231, a malignant human breast cancer cell line. Seems to be released or leaking.
- cytokines and growth factors such as IL-6, VEGF-A and VEGF-C have no or little effect on ICAM-1 expression in human lymphatic endothelial cells.
- the molecular weight is ATP (molecular weight 551.1) in the range of 500 to 1,000.
- the malignant melanoma cell line B16-BL6 releases non-peptidic substances with a molecular weight of less than 1,000, thereby producing or releasing endogenous nitric oxide from lymphatic endothelial cells.
- the mitochondrial ATP-selective K + channel in muscle cells appears to significantly stop lymphatic pumping activity.
- ATP controls the cessation of lymphatic pumping activity. It is endothelium-dependent or endothelium-independent reactions that induce and control and inhibit pump activity of isolated rat lymphatic vessels with ATP. Thus, the ATP-mediated inhibitory reaction is accompanied by the production and release of some endogenous nitric oxide in lymphatic endothelial cells and the activation of ATP-selective K + channels in lymph smooth muscle. It seems. High concentrations of ATP released or leaked from malignant cancer cells such as MDA-MB-231 and B16-BL6 diffuse to the interstitial site, invade lymph capillaries, disrupt the active lymph transport mechanism, It is presumed that a pre-metastatic environment suitable for micrometastasis of cancer cells to lymph nodes is constructed.
- ATP seems to cause lymphatic control and decrease in lymph pump activity, reduce lymph flow, and form edema of cancer tissue.
- Microenvironmental edema in cancer tissues appears to negatively affect cancer cell re-diffusion and contribute to the development of micrometastasis in sentinel lymph nodes.
- Another important aspect of the present invention is that treatment with MDA-MB-231 supernatant for 48 hours causes enhanced adhesion between cancer cells and human lymphatic endothelial cells in vitro. Stimulation with 10 ⁇ 7 M ATP markedly increased the adhesion of cancer cells to lymphatic endothelial cells, and the reaction was almost the same as that caused by MDA-MB-231 supernatant.
- MDA-MB-231 a human malignant breast cancer cell line, releases or leaks a large amount of ATP and selectively adheres to local lymph nodes and / or on lymphatic endothelial cells in the vicinity. It is thought that it forms molecules and promotes adhesion of cancer cells to lymphatic endothelial cells. This is also due to the finding that the promotion of cancer cell adhesion to human lymphatic endothelial cells mediated by ATP or MDA-MB-231 supernatant is significantly reduced by treatment with anti-ICAM-1 antibody. It is strongly suggested.
- ATP-induced ICAM-1 overexpression in human lymphatic endothelial cells appears to contribute to the establishment of a pre-metastatic environment and the induction of micrometastasis of cancer cells in local lymph nodes.
- the adhesion of leukocytes to vascular endothelial cells is an important process, and is accompanied by gradual increase and infiltration of leukocytes to the site of tissue damage, infection or lesion formation.
- ICAM-1 expressed in endothelial cells is one of the cell surface glycoproteins that is a major contributor to the cell adhesion process. ICAM-1 is essentially expressed in endothelial cells but can significantly elicit responses to pro-inflammatory mediators such as tumor necrosis factor (TNF) - ⁇ and interleukin (IL) -1 ⁇ Is.
- TNF tumor necrosis factor
- IL interleukin
- ICAM-1 is characteristically expressed in lymphatic endothelial cells compared to vascular endothelial cells. It was suggested that it is not. Furthermore, when the concentration of proinflammatory mediators such as TNF- ⁇ and IL-1 ⁇ in the culture supernatant of MDA-MB-231 was measured, it was confirmed that ICAM-1 was overexpressed in human lymphatic endothelial cells There was no significant increase.
- P2X 7 receptor expression in chronic lymphocytic leukemia evaluation systems have been identified, ATP inhibits the growth of white blood cells in the leukemia evaluation system.
- P2X 7 receptor mRNA expression occurs frequently in most types of leukemia, but a decrease in P2X 7 receptor action is observed.
- P2X receptor subtype that contributes to ATP suppression in malignant melanoma in early squamous cell carcinoma and prostate bladder cancer.
- P2Y 1 and P2Y z receptors interposed growth or anti-proliferative, P2X 5 receptors mediated cell differentiation, actually antiproliferative P2X 7 is interposed apoptotic cell death.
- ATP released or leaked from malignant cancer cells having high metastasis ability is used to construct a pre-metastasis environment in the local lymph node and malignant. It is the first clinically used, taking advantage of its important role in the development of highly metastatic micrometastasis of cancer cells.
- Chemokines are soluble low molecular weight proteins that bind to cognate G-protein coupled receptors and usually cause cellular responses such as directional migration or chemotaxis. Tumor cells secrete chemokines and react to chemokines. Chemokines promote tumor growth by affecting endothelial cell recruitment, disruption of immunological surveillance, and neoplastic leukocyte chemokine properties, thereby distorting immunoediting. Metastasize or grow tumors.
- the CXCL12-CXCR4 axis promotes metastasis to distant organs, and the CCL21-CCR7 pair rather promotes metastasis to lymph nodes. These two chemokine ligand-receptor systems are important transmitters for metastasis of cancer cells of various malignant tumors and are important indicators in chemotherapy.
- Local lymph node is the site where a malignant tumor usually metastasizes first.
- the dramatic success of sentinel lymph node navigation surgery in clinical settings suggests that local lymph nodes have an effective filter function that provides a physical barrier to metastatic cancer cells. is doing.
- the primary tumor affects the microenvironment of the tumor tissue before metastasis.
- chemokine CCL2 is ICAM-1 on cultured human lymphatic endothelial cells isolated from imported lymphatic vessels closest to the sentinel lymph node of breast cancer patients.
- ICAM-1AM mRNA levels also increased markedly up to 18 hours.
- Immunohistochemical expression of ICAM-1 mediated by CCL2 on lymphatic endothelial cells increases up to 1 ng / mL in a concentration-dependent manner, and its expression is markedly reduced by neutralization of CCL2 by specific CCL2 antibodies did.
- CCL2 in vitro for 18 hours, the attachment of cancer cells, MDA-MB-231 and MCF-7, to human lymphatic endothelial cells was markedly promoted.
- the CCL2-mediated response in the adhesion test was significantly reduced by CCL2 neutralization treatment or addition treatment with anti-ICAM-1 antibody.
- CCL2 cascade kinase cascade kinase cascade kinase cascade kinase cascade kinase cascade kinase cascade kinase cascade kinase cascade kinase cascade kinase cascade kinase cascade kinase cascade kinase cascade kinase cascade kinase cascade kinase mRNA and ICAM-1 protein in cultured human lymphatic endothelial cells
- cancer cells in vitro are MDA-MD- It is considered that the adhesion of 231 and MCF-7 to cultured lymphatic endothelial cells is promoted by overexpression of ICAM-1 in an in vitro micrometastasis experimental model. Therefore, overexpression of ICAM-1 mediated by CCL2 on human lymphatic endothelial cells is thought to contribute to the establishment of an environment suitable for cancer cell micrometastasis to local lymph nodes and the cause of micrometastasis.
- monocytes should migrate to the tumor site by microcirculation and lymphatic circulation, at least prior to progression in the host immune response. And administering specific chemokines to recruit monocytes gradually induces an anti-cancer host response.
- CCL2 induces overexpression of ICAM-1 on human lymphatic endothelial cells of sentinel lymph nodes and / or the closest imported lymphatic vessels and promotes the interaction between lymphatic endothelial cells and malignant tumor cells That is very noteworthy.
- the CCL2 concentration in the MDA-MB-231 cell culture supernatant and the MCF-7 cell culture supernatant was measured by enzyme-linked immunosorbent assay (ELISA analysis) and found to be less than 62.5 pg / mL. It has also been shown that lipopolysaccharide induces ICAM-1 and CCL2 expression on cultured human lymphatic endothelial cells isolated from skin microlymphatic vessels. However, the source of CCL2 that causes overexpression of ICAM-1 in sentinel lymph nodes is not always clear.
- ICAM-1 expression by tumor cells is a major factor that promotes metastatic progression.
- studies on tumor microvessels in which leukocytes and endothelial cells are adhered have revealed that adhesion interaction is reduced under basic conditions and cytokine-stimulated conditions. This is consistent with immunohistochemical findings and cytofluorescence findings that endothelial ICAM-1 expression is reduced in tumor microvessels. Inhibiting ICAM-1 in the endothelium will allow tumor cells to escape immune monitoring by circulating lymphocytes and promote tumor progression.
- CCL2 induces overexpression of ICAM-1 on human lymphatic endothelial cells residing in imported lymphatic vessels closest to the sentinel lymph node of breast cancer patients, and then promoted production of ICAM.
- ICAM-1 was found to mediate cancer cell adhesion to human lymphatic endothelial cells and completed the present invention.
- Counter-receptors and ligands for ICAM-1 such as CD11a and CD11b could be clearly observed on the breast cancer cells MDA-MB-231 and MCF-7 used in the in vitro adhesion test.
- significant immunohistochemical expression only in ICAM-1 but not E-selectin was observed on malignant tumor cell metastatic sentinel lymph node tissues that had been frozen immediately after harvesting from breast cancer patients. Therefore, it is first shown that CCL2 plays an important role in constructing a microenvironment suitable for micrometastasis of cancer cells in local lymph nodes.
- the sentinel lymph node metastasis cancer cell detection kit of the present invention is useful for identifying a lymph node to be dissected before or during the operation of removing the primary tumor.
- the drug transporter of the present invention can selectively reach a lymph node where cancer cells have metastasized, it is useful as a marker or a quantitative reagent for treatment or diagnosis. Moreover, since it can reach the lymph node to which the metastasized cancer cells are attached, it can be used for the progression and prevention of malignant cancer.
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Abstract
Description
ヒトリンパ管内皮細胞(LEC)の単離及び培養は、乳癌患者のセンチネルリンパ節に最も近接している輸入リンパ管を用い、Kawai Yらの手法(非特許文献1、及びLymphatic Research and Biology(リンパティック リサーチ アンド バイオロジー)、2008年、第6巻、p.15~27)に準じて行った。その実験プロトコルは、信州大学医学部内の医倫理委員会で承認されたものである。その乳癌患者に研究の目的やリスクに関し全て告知したうえで、同意書を提出してもらってある。
信州大学医学部乳腺外科内での乳癌患者の乳腺内分泌外科手術によるセンチネルリンパ節生検症例のうち、その手術前に同意が得られた患者から摘出したセンチネルリンパ節輸入リンパ管の内腔に、無菌ポリエチレン管を挿入し、予め37℃に温めたトリプシン・エチレンジアミン四酢酸溶液500U/mlで10分間灌流した。その内腔を酵素分解した後、その内腔に存在している内皮細胞を含んだ液を得た。それに10%ウシ胎仔血清(FBS)を含有する内皮増殖培地(EGM)-2(米国Clonetics社製)入りの遠心分離管に、静かに流し込んだ。それを、4℃で5分間、2,000r.p.m.で遠心分離した。上澄みを取り除き、得られた沈殿している細胞成分をEGM-2培地に再懸濁させ、それを、I型コラーゲン(新田ゼラチン株式会社製)でコーティングした直径35mmの培養皿(米国Corning社製)に付した。このようにして得られたヒト乳癌センチネルリンパ節輸入リンパ管由来リンパ管内皮細胞を、10%FBS含有EGM-2に保存し、第5~7継代培養して使用した。リンパ管内皮細胞を、酸素5%、二酸化炭素5%及び窒素ガス90%の大気条件下、37℃で培養した。
酸素5%という低酸素環境下と酸素20%という通常の酸素環境下とでそれぞれ96時間かけてこのヒトリンパ管由来内皮細胞株を培養した場合に、顕微鏡でそれぞれの1視野の細胞数を数えることにより、酸素濃度毎の内皮細胞株の増殖能の相違を検討した。その結果、通常の酸素環境下での内皮細胞株の培養よりも低酸素環境下での内皮細胞株の培養の方が、一層優れた増殖能を示していた。
ヒトリンパ管由来内皮細胞株の保存条件は、0.25%トリプシン液で内皮細胞株を回収し、10%ジメチルスルホキシド(DMSO)および90%FBSからなる凍結保存液を用いて細胞懸濁液を調製した後、凍結チューブに入れ、バイセルを用いて段階的に冷却し、最終的に液体窒素内で保存するというものである。その融解条件および融解時培養条件は、凍結チューブを37℃の恒温槽で融解させ、2000r.p.m.で5分間遠心して内皮細胞株を回収し、細胞培養液として10%FBS添加EGM-2を用いて細胞懸濁液を調製し、前記の内皮細胞株の培養の条件と同様にして培養するというものである。
(1.4 A. CD31(platelet endothelial cell adhesion molecule:PECAM)による免疫染色観察)
培養系に移行したヒト集合リンパ管由来内皮細胞株をスライドガラス上に播種した。培養を継続し、コンフルエントになったところで、これを10%ホルマリンにて固定した。0.1%ウシ血清アルブミン(BSA)添加PBS溶液にてブロッキングした後、一次抗体として内皮細胞のマーカーであるCD31(米国Santa Cruz社製;品番sc-8306)を1:100に0.1%BSA添加PBS溶液で希釈して加え、4℃で一晩静置した。その一次抗体をPBS溶液で洗浄した後に、この一次抗体に見合う二次抗体として色素で蛍光標識してあるヤギ抗ウサギ免疫グロブリンG-FITC(米国Santa Cruz社製;品番sc-2012)を0.1%BSA添加PBS溶液で1:100に希釈して加え、室温で1時間静置した。その二次抗体をPBS溶液で洗浄した後、Mobi GLOW Mounting Medium(独国MoBiTec社;品番MGM01)に封入して蛍光顕微鏡で観察したところ、この内皮細胞株は、緑の蛍光色を示し、CD31に対して陽性であることが確認された。
培養系に移行したヒト集合リンパ管由来内皮細胞株をスライドガラス上に播種した。培養を継続し、コンフルエントになったところで、これを10%ホルマリンにて固定した。0.1%ウシ血清アルブミン(BSA)添加PBS溶液にてブロッキングした後、一次抗体としてリンパ管内皮細胞の特異的なマーカーであるVEGF-R3(米国Santa Cruz社製;品番sc-637)を1:100に0.1%BSA添加PBS溶液で希釈して加え、4℃で一晩静置した。その一次抗体をPBS溶液で洗浄した後に、この一次抗体に見合う二次抗体として蛍光標識してあるヤギ抗ウサギ免疫グロブリンG-FITC(米国Santa Cruz社製;品番sc-2012)を0.1%BSA添加PBS溶液で1:100に希釈して加え、室温で1時間静置した。その二次抗体をPBS溶液で洗浄した後、Mobi GLOW Mounting Medium(独国MoBiTec社;品番MGM01)に封入し、蛍光顕微鏡で観察したところ、この内皮細胞株は、緑の蛍光色を示し、VEGF-R3に対して陽性であることが確認された。
一次抗体としてリンパ管内皮細胞の特異的な別のマーカーであるLYVE-1(米国Santa Cruz社製;品番sc-19316)を用い、二次抗体として蛍光標識であるロバ抗ヤギ免疫グロブリンG-FITC(米国Santa Cruz社製;品番sc-2024)を用いたこと以外は、VEGF-R3による免疫染色と同様に観察したところ、この内皮細胞株は、緑の蛍光色を示し、LYVE-1に対して陽性であることが確認された。これらのCD31、VEGF-R3およびLYVE-1の免疫染色の観察の結果から明らかなように、リンパ管由来内皮細胞株は培養系に移行させても、CD31、VEGF-R3およびLYVE-1の発現が認められ、内皮細胞の生物学的特性が維持されていた。したがって、リンパ管由来内皮細胞株の分離・培養が樹立できたと確認された。
培養系に移行したヒト集合リンパ管由来内皮細胞株をスライドガラス上に播種した。培養を継続し、コンフルエントになったところで、刺激因子として腫瘍壊死因子-α(TNF-α)(米国SIGMA社製;品番T-0157)の10ng/ml、インターロイキン-1β(IL-1β)(米国PeproTech社製;品番200-01B)の1ng/mlおよび10ng/mlのそれぞれを3%FBS添加EBM-2(三光純薬株式会社;品番CC-3156)にて溶解して細胞上に付加し、2時間37℃で培養した。これを、3.7%ホルマリンにて固定した。これをPBS溶液にて洗浄し、0.1% Triton X-100(米国SIGMA社製;品番X-100)添加PBS溶液内で5分間静置した。PBS溶液にて洗浄し、phalloidin-FITC抗体(米国SIGMA社製;品番P-5282)を0.1%BSA添加PBS溶液で1:500に希釈して加え、室温で2時間静置した。PBS溶液にて洗浄した後、Mobi GLOW Mounting Mediumに封入した。また、刺激因子を用いないこと以外は同様にしてネガティブコントロールを調製した。これらを蛍光顕微鏡で観察し比較したところ、刺激因子がないネガティブコントロールは不明瞭な緑の蛍光色を示しF-アクチンの発現が少なかったのに対し、TNF-αやIL-1βで刺激されたものは明瞭な緑の蛍光色を示しF-アクチンの発現の増加が確認された。
(1.5.1. 形態的性質)
(1)形、大きさ
培養内皮細胞の特徴である多角形を示し、単層で敷石状配列を呈する。大きさは直径50μm前後である。
(2)多形性
内皮細胞はほぼ同様の形態を示しており、多形性は認められない。
(1.5.2. 培養的性質)
(1)培地
血管内皮細胞用増殖培養液であるEGM-2に、最終濃度が10%になるよう牛胎児血清(Fetal Bovine Serum:FBS)を添加したものを用いている。
(2)培養条件
37℃の細胞培養器を用い、5%酸素、5%二酸化炭素、90%窒素環境下で培養を行う。
(3)生理学的性質
(3.1)リンパ管内皮細胞マーカーの発現
本細胞は、内皮細胞マーカーであるCD31、リンパ管内皮細胞マーカーであるProx-1・LYVE-1・podoplaninの発現が確認できた。
(3.2)生物学的特長
細胞の倍加時間は約48時間であり、リンパ管新生因子であるbasic-FGF、VEGF、VEGF-Cで増殖能の亢進が確認できた。
ヒトの乳癌細胞株である高転移株のMDA-MB-231及び低転移株のMCF-7は、米国American Type Culture Collection社から購入したものを用いた。この癌細胞を、10%FBS含有ダルベッコ変法イーグル培地(DMEM)/養分混合F12ハム培地で保存した。リンパ管内皮細胞を、酸素5%、炭酸ガス5%、及び窒素ガス90%の大気条件下、37℃で培養した。癌細胞を、酸素21%、炭酸ガス5%、及び窒素ガス74%の標準酸素条件下、37℃で培養した。
(2.1 サイトカイン及び増殖因子の測定)
MDA-MB-231とMCF-7との夫々の培養上清中の特定のサイトカイン及び増殖因子の濃度を、酵素結合免疫吸着法(Enzyme-Linked Immuno Sorbent Assay:ELISA)を用いて測定した。先ずそれらの上清を回収し、癌細胞を10%FBS含有DMEM/F12に播種した。翌日その培地を、FBS未含有のDMEM/F12に置換し、一晩培養した。その培養液を、4℃で5分間、2,000r.p.m.で遠心分離した後、サイトカイン及び増殖因子を測定するために-20℃で冷凍保存した。TNF-α、TGF-β1、INF-γ、IL-1β、IL-6、IL-12、basic FGF、PDGF-BB、VEGF-A及びVEGF-Cの濃度を、民間検査機関(株式会社エスアールエル)に委託して測定した。検出限界は夫々、腫瘍壊死因子-α(TNF-α)が5pg/mL、腫瘍成長因子-β1(TGF-β1)が5ng/mL、インターフェロン-γ(INF-γ)が0.1U/mL、インターロイキン-1β(IL-1β)が10pg/mL、IL-6が0.2pg/mL、IL-12が7.8pg/mL、塩基性線維芽細胞増殖因子(bFGF)が10pg/mL、血小板由来成長因子-BB(PDGF-BB)が31.2pg/mL、血管内皮成長因子-A(VEGF-A)が20pg/mL、及びVEGF-Cが109pg/mLである。
MDA-MB-231培養液と、ATP(10-8M、10-7M、及び10-6M)含有又はATP非含有培養液(何れもDMEM/F12)との夫々の上清中のATP濃度について、CellTiter-Glo(米国Promega社製;登録商標)による発光細胞生存試験(Luminescent Cell Viability Assay)で、ルシフェリン・ルシフェラーゼ反応測定をした。具体的には、100μLのMDA-MB-231培養上清、又は100μLのATP含有又は非含有の培養上清を、96穴培養皿に添加し、100μLのルシフェリン・ルシフェラーゼ溶液を加え、その発光を照度計(大日本住友製薬株式会社製)により検出した。
間接免疫組織化学的方法を用いて、E-セレクチン、P-セレクチン、血管細胞接着分子(VCAM)-1、及び細胞間接着分子(ICAM)-1のようなヒトリンパ管内皮細胞の接着分子に対して、癌細胞株である2種類のMDA-MB-231とMCF-7との培養上清の作用について検討した。10%FBS含有DMEM/F12に癌細胞を播種した。翌日その培地を、3%FBS含有DMEM/F12に置換し、一晩培養した後、回収した。回収した溶液を、4℃で5分間、2,000r.p.m.で遠心分離した。ヒトリンパ管内皮細胞での接着分子発現に対するこれら上清の作用について解析するために、回収した各溶液1mLを、ヒトリンパ管内皮細胞の3%FBS含有EBM-2飢餓培地に加えた後、48時間培養した。また、異なる濃度(1/100に希釈、又は1/10,000に希釈)の上清溶液を、3%FBS含有EBM-2の適量に加えたものについても、同様にして培養した。
乳癌患者から摘出された培養ヒトリンパ管内皮細胞や乳癌患者から摘出した直後に凍結しておいたセンチネルリンパ節について、免疫組織化学的観察を行った。具体的には、培養リンパ管内皮細胞を、10%ホルマリン含有リン酸緩衝生理食塩水(PBS)により常温で固定した。一次ポリクローナルヒト抗血清血小板内皮細胞接着分子(PECAM)-1(1:100に希釈、米国BD Biosciences社製)、リンパ管内皮ヒアルロン酸受容体(LYVE)-1(1:50に希釈、独国RELIATech社製)、Prox-1(1:50に希釈、米国AngioBio社製)、ポドプラニン(1:50に希釈、米国AngioBio社製)、内皮増殖因子受容体3(VEGF R3)(1:50に希釈、米国Santa Cruz社製)、E-セレクチン/CD62E(1:50に希釈、米国R&D systems社製)、P-セレクチン/CD62P(1:50に希釈、米国R&D systems社製)、血管細胞接着分子(VCAM)-1/CD106(1:50に希釈、米国R&D systems社製)、又は細胞間接着分子(ICAM)-1/CD54(1:50に希釈、米国R&D systems社製)の存在下、細胞を室温で4時間培養した。染色前に、0.1%トリトン-Xで、培養細胞に浸透化処理を施した。次に、AlexaFluor488ニワトリ抗ウサギ免疫グロブリンG又はAlexaFluor488ロバ抗マウス免疫グロブリンG(米国Invitrogen社製)で、その細胞を染色した。培養細胞の細胞核を、対比染色し、4',6-ジアミジン-2-フェニルインドール(DAPI)含有退色防止薬ProLong Gold(米国Molecular Probe社製)で標本にした。培養細胞を、蛍光顕微鏡(Leica社、独国)で観察し、撮影した。
I型コラーゲンでコーティングした35mmの培養皿上で、ヒトリンパ管内皮細胞を単層に形成し、37℃で、酸素5%、二酸化炭素5%及び窒素90%にて、コンフルエントになるまで培養した。3%FBS含有EBM-2を有する血清飢餓培地で、リンパ管内皮細胞を保存した。所定の培養皿を、10ng/mLのCCL2で18時間、処理した。幾つかの実験では、培養皿を、特異的CCL2抗体1.0μg/mLで10ng/mのCCL2を予め中和した血清飢餓培地で刺激した。
ICAM-1 cDNAに対する定量的逆転写酵素ポリメラーゼ連鎖反応法(RT-PCR)により、0時間、1時間、4時間、18時間におけるICAM-1 mRNAの発現を評価した。Isogen試薬(株式会社日本ジーン)を用いそのメーカーの使用説明書に従って、培養ヒトリンパ管内皮細胞から全RNAを抽出した。分光光度計による260nm吸収から、各RNAの濃度を計算した。抽出したRNAをM-MLV逆転写酵素(米国Ambion社製)で逆転写した。RT-PCR分析を行うsuperscript first-strand synthesis kit(米国Invitrogen社製)には、1.0μg/mLの全RNAを用いた。ICAM-1のフォワードプライマー及びリバースプライマーとサイクロフィリンAとを夫々特異的プローブとして用いた。夫々、ICAM-1(タカラバイオ株式会社製)、サイクロフィリンA;5'-TTCGTGCTCTGAGCACTGGAG-3'(forward:配列表の配列番号1)、5'-GGACCCGTATGCTTTAGGATGAAG-3'(reverse:配列表の配列番号2)である。cDNAを5倍に希釈してから、PCR増幅を行った。Light Cycler rapid thermal cycler system(英国Roche Diagnostics社製)を用いて、定量的RT-PCRを行った。0.5μMのプライマー、Taq DNA ポリメラーゼ、緩衝液が含有された20μL容量を、SYBR Premix Ex Taq(タカラバイオ株式会社製;SYBRは登録商標)に添加して、反応させた。PCRの操作には、10秒間の変質、次いで95℃で5秒間の変質と60℃で20秒間のアニール化とを45回繰返す工程とが、含まれているものである。さらに72℃で放置した後に、蛍光生成物を検出した。ネガティブコントロールは、cDNAを用いずにPCR反応させたものである。増幅特異性を確認するため、各プライマー対から得られるPCR生成物を、融解温度曲線分析にかけ、Light Cycler分析ソフトウエアを用いて、定量データを解析した。その結果を、サイクロフィリンAに対するICAM-1 mRNAの発現の標準化指数として示した。
培養ヒトリンパ管内皮細胞上でCCL2が介在したICAM-1発現を定量分析するために、ウェスタンブロット法を行った。細胞を、M-PER哺乳類タンパク質抽出試薬(米国Thermo Scientific社製)中に、溶解させ、14,000r.p.m.で10分間遠心分離した。全細胞溶解物の内の15μgの試料を、ドデシル硫酸ナトリウム-ポリアクリルアミド電気泳動(SDS-PAGE)用SDS試料緩衝液で分析し、ポリフッ化ビニリデン(PVDF)膜(アトー株式会社製)に転写して、45分間インキュベートした。抗ICAM-1抗血清(1:1000に希釈、米国Cell Signaling Technology社製)でその膜に反応させた後、抗ウサギ免疫グロブリンG ホースラディッシュペルオキシダーゼ共役抗体とインキュベートした。モノクローナル抗アクチン抗体(米国Santa Cruz社)でその膜に再反応させた後、ECL-ウェスタンブロッド検出システム(英国Amersham Bio Science社製)で、可視化した。
(3.1 調製)
MDA-MB-231細胞から放出された刺激物質の化学的特性、ヒトリンパ管内皮細胞でのICAM-1の免疫組織化学発現のおける化学的処理又は物理的処理が施された上清の作用について評価するために、48時間刺激した。上清を30分間80℃で加熱処理、又はプロテアーゼであるプロナーゼE(1μg/mL、米国SIGMA社製)で、37℃で一晩、処理した。また、分子量1,000又は500の物質透析用の2種類の管状透析膜(米国Spectum Medical Industries社製)により、それら上清を透析した。透析は、管状透析膜を緩衝剤(DMEM-F12(1:1)培地)に入れて、4℃で一晩かけて行った。管状透析膜内に得られた上清を、バイオアッセイに用いた。この上清は、分子量1,000未満、又は500未満の物質を含まないものである。
I型コラーゲンでコーティングした直径35mmの培養皿上で、ヒトリンパ管内皮細胞を単層に形成してコンフルエントになるまで、37℃で、酸素5%、炭酸ガス5%、及び窒素ガス90%の条件下、培養した。それらリンパ管内皮細胞を、3%FBS含有EBM-2血清飢餓培地で保存した。培養皿を、10-7MのATP又はMDA-MB-231細胞培養上清で48時間刺激した。さらに、10-6Mのスラミンを、10-7MのATP又はMDA-MB-231細胞培養上清で48時間刺激する際に、同時に添加した。
なお、用いた塩類は、全て和光純薬工業株式会社製のものである。ATP及びスラミンは米国SIGMA社製、DPCPX及びDMPXはResearch Biologicalsのものである。DPCPXをエタノールで希釈し、DMPXをジメチルスルホキシド(DMSO)で希釈して用いた。エタノール及びDMSOの濃度は、培養細胞の生存に影響を及ぼさない程度にしてある。細胞ケモカインは、R&D systems社(米国)から入手したものである。試薬濃度は、培養皿での最終濃度で、表記してある。
全ての結果を、平均±平均の標準誤差で示した。統計学的有意性に関し、不対スチューデントt検定により解析し、p<0.05のとき有意差ありと、判断した。p<0.01のとき極めて有意差がある。
(5.1 ヒトリンパ管内皮細胞上での接着分子の発現に関するMDA-MB-231上清又はMCF-7上清の影響)
図1は、MDA-MB-231細胞培養上清で刺激したときのリンパ管内皮細胞上での接着分子であるE-セレクチン、P-セレクチン、VCAM-1及びICAM-1の発現結果についての代表的な免疫組織化学的顕微鏡写真である。3%FBS含有飢餓培地で一晩培養したところ、無刺激、即ち0時間では、図1のA、B、C、Dに示すように、培養ヒトリンパ管内皮細胞上でのE-セレクチン、P-セレクチン、VCAM-1及びICAM-1の各接着分子につき、その免疫活性染色が全く又は殆んど観察されなかったことから、ヒトリンパ管内皮細胞上でそれら接着分子の発現は、全く又は殆んど惹起されていなかった。なお3%FBS含有DMEM/F12培地で48時間培養しても、同様に、ヒトリンパ管内皮細胞上で、接着分子は、全く発現していなかった。
表1に、MDA-MB-231培養上清又はMCF-7培養上清中、TNF-α、TGF-β1、INF-γ、IL-1β、IL-6、IL-12の各サイトカインの濃度と、bFGF、PDGF-BB、VEGF-A、VEGF-Cの各成長因子の濃度との測定データを、纏めて示す。
MDA-MB-231上清中、又はATP(10-8、10-7及び10-6M)存在下若しくは非存在下での培地中の各ATP濃度を、纏めて表2に示す。
図4のA-1~A-4に示すように、MDA-MB-231上清をプロテアーゼによる酵素分解処理や加熱処理を行っても、また分子量500未満のものを除去する透析膜で前処理をおこなっても、ヒトリンパ管内皮細胞上での上清媒介ICAM-1発現の有意な効果は認められなかった。また、同図のB-1~B-4に示すように、これらの場合とMDA-MB-231上清のみの場合との間に有意差がなかった(何れもn=5)。
ATPの分子量が551.1であるという事実に基づき、ヒトリンパ管内皮細胞上で付着分子の発現におけるATPの影響を検討した。その結果を、図5のA-1及びA-2に示す。10-8又は10-7MのATPで48時間処理したところ、ヒトリンパ管内皮細胞上のICAM-1の顕著な発現が惹き起こされ、MDA-MB-231上清で48時間処理することによって産生されたリンパ管内皮細胞上のICAM-1の発現と極めて似ていた。
図6のA-1~A-3に示すように、MDA-MB-231上清で刺激するのと同時に10-7又は10-6Mのスラミンで48時間処理したところ、ヒトリンパ管内皮細胞上でMDA-MB-231上清媒介ICAM-1発現の顕著な減退が惹き起こされた。また、同図B-1~B-3に示すように、MDA-MB-231上清のみの場合と、同時にスラミンで処理した場合との間に、p<0.01で有意差があった(何れもn=5)。
図7のA-1~A-3で示すように、インビトロで10-7MのATPで48時間刺激した場合、ヒトリンパ管内皮細胞への癌細胞の付着が、ネガティブコントロールであるDMEM/ハムF12培養液のみの場合に比べ顕著に増大した。一方、ヒトリンパ管内皮細胞への癌細胞の付着増加は、10-6Mのスラミンで同時に刺激することによって減退した。同図のB-2に示すATPで刺激した場合には、同図のB-1に示すDMEM/ハムF12培養液のみの場合及び同図のB-3に示すATPの刺激と同時にスラミンで処理した場合との間に、p<0.01で両者に有意差があった(何れもn=5)。
次いで、MDA-MB-231上清又はATPにより促進されるインビトロでの癌細胞とヒトリンパ管内皮細胞との付着を、ICAM-1抗体処理による抑制の可能性を調べた。図8のA-1~A-3に示すように、10-7MのATPで48時間刺激した場合、培養液DMEM/F12のみの場合よりも、ヒトリンパ管内皮細胞への癌細胞の付着が顕著に増大した。付着性試験での10-7MのATPによる増加は、抗ICAM-1抗体による処理で顕著に減少した。同図B-2で示すATP刺激の場合には、B-1の培養液DMEM/F12のみの場合やB-3の抗ICAM-1抗体処理の場合との間に、p<0.01で両者に有意差があった(何れもn=5)。
図9は、培養細胞上における、VEGF-R3(同図C)、LYVE-1(同図D)、Prox-1(同図E)、及びポドプラニン(同図F)のようなリンパ管マーカーの代表的な顕微鏡写真である。VEGF-R3、Prox-1、ポドプラニン、及びPECAM-1(同図B)の抗血清により、培養細胞が強く染色されたのに対し、LYVE-1に対する抗体では、極僅かに培養細胞を微かに染色しただけであった(同図D)。なお、図9Aは、培養細胞の位相差画像についての代表的な顕微鏡写真である。これらの結果は、培養細胞が、乳癌患者のセンチネルリンパ節に最も近接している輸入リンパ管のヒトリンパ管内皮細胞であることを示している。
図10に、ヒトリンパ管内皮細胞上での接着分子の発現におけるケモカイン類の影響を、示す。同図Aは夫々、10ng/mLのCCL1(同図Aの1~4)、10ng/mLのCCL2(同図Aの5~8)、10ng/mLのCCL12(同図Aの9~12)、及び10ng/mLのCCL21(同図Aの13~16)の各ケモカイン類で培養ヒトリンパ管内皮細胞を18時間刺激した場合、その細胞上での接着分子の免疫組織化学的発現を示す代表的な顕微鏡写真である。同図Aの8に示すように、10ng/mLのCCL2で18時間刺激した場合にのみ、ほぼ全ての培養リンパ管内皮細胞がICAM-1抗血清により、はっきりと染色されており、リンパ管内皮細胞上でICAM-1の顕著な発現が認められた。同図Aの1、5、9、13、及び3、7、11、15に示すように、培養リンパ管内皮細胞上にE-セレクチン又はVCAM-1の発現は、全く又は殆ど認められなかった。それとは対照的に、10ng/mLのCCL1、10ng/mLのCCL12、10ng/mLのCCL21で夫々刺激したリンパ管内皮細胞上に、同図Aの4、12、16に示すように、ICAM-1抗血清により、僅かに染色されているのが認められた。
mRNAの発現における刺激時間の影響)
図11Aは、培養ヒトリンパ管内皮細胞上でのCCL2介在による接着分子の免疫組織学的発現において、刺激時間が与える影響を示している。同図Aの1~16は、10ng/mLのCCL2を介在させて培養ヒトリンパ管内皮細胞を、0時間、4時間、18時間、又は48時間の各時間、刺激した場合、その細胞上でのE-セレクチン(同図1、5、9、13)、P-セレクチン(同図2、6、10、14)、VCAM-1(同図3、7、11、15)、及びICAM-1(同図4、8、12,16)の各接着因子の免疫組織化学的発現について刺激時間による影響を示す代表的な顕微鏡写真である。
図12は、ヒトリンパ管内皮細胞上でのICAM-1の免疫組織化学的発現に関して、10pg/mL~10ng/mLのCCL2の濃度が与える影響を示すものである。同図Aは、10pg/mL(同図Aの1)、100pg/mL(同図Aの2)、1ng/mL(同図Aの3)、及び10ng/mL(同図Aの4)の各濃度のCCL2で夫々ヒトリンパ管内皮細胞を18時間刺激した場合、その細胞上でのICAM-1の発現について濃度による影響を示す代表的な顕微鏡写真である。同図Aの1に示すように、10pg/mLのCCL2による刺激では、僅かにではあるが培養ヒトリンパ管内皮細胞上に明らかなICAM-1の発現を、惹き起こしていた。リンパ管内皮細胞上でのCCL2介在によるICAM-1の発現は、濃度依存的に最大1ng/mLまで増加し、1ng/mL及び10ng/mLのCCL2による刺激は、ほぼ全ての培養リンパ管内皮細胞上にICAM-1の顕著な発現を、惹き起こした。
図13は、培養ヒトリンパ管内皮細胞上でのCCL2介在によるICAM-1の発現において、CCL2の中和が与える影響を示すものである。同図Aは、1.0μg/mLの特異的なCCL2抗体が存在する場合(同図Aの3)と存在しない場合(同図Aの2)と夫々において、10ng/mLのCCL2が及ぼす影響について示した代表的な顕微鏡写真である。同図Aの1は、血清飢餓培養培地(3%FBS含有EBM-2)により得られたネガティブコントロールを示す顕微鏡写真である。同図Aの3に示すように、特異的CCL2抗体によりCCL2を中和すると、培養ヒトリンパ管内皮細胞上でのCCL2介在によるICAM-1の免疫組織化学的発現が、顕著に減退した。
10ng/mLのCCL2含有培地をCCL2で中和するか否かで、夫々癌細胞の付着数を計測した。図14は、乳癌細胞株であるMCF-7(I)及びMDA-MB-231(II)を用い、特異的CCL2抗体が存在しない場合(I-2及びII-2)と存在する場合(CCL2中和:I-3及びII-3)、及び抗ICAM-1抗体(I-4及びII-4)が存在する場合の夫々において、10ng/mLのCCL2が及ぼす影響について付着性試験を行ったデータを纏めたグラフである。同図のIに癌細胞MCF-7について白抜き棒グラフで示し、同図のIIに癌細胞MDA-MD-231について斜線付き棒グラフで示す。縦軸は、一視野(倍率は100倍)の接着癌細胞の正規化数を示す。**はp<0.01の有意差ありを示している。同図のI-2及びII-2に示すように、ヒトリンパ管内皮細胞に対し、インビトロで10ng/mLのCCL2で18時間刺激した場合、MCF-7(I-2)、及びMDA-MD-231(II-2)の付着が、夫々のDMEM/F12であるネガティブコントロールに比べ、夫々p<0.01で有意に増大した。また、同図のI-3及びII-3に示すように、10ng/mLのCCL2で刺激後、CCL2の中和した場合は、10ng/mLのCCL2で18時間刺激した場合よりも、ヒトリンパ管内皮細胞への癌細胞の付着の増加が、p<0.01で有意に減退した。このように、10ng/mLのCCL2介在反応又は各乳癌細胞の場合と顕著に異なる。
次に、10ng/mLのCCL2介在によるヒトリンパ管内皮細胞への癌細胞MCF-7及びMDA-MD-231の付着の促進を、ICAM-1抗血清で阻害するかについて検討した。その結果を示す図14のI-4及びII-4のように、ヒトリンパ管内皮細胞に対し、インビトロで10ng/mLのCCL2で18時間刺激した後、抗ICAM-1抗体で処理した場合は、10ng/mLのCCL2で18時間刺激した場合よりも、ヒトリンパ管内皮細胞への癌細胞の付着の増加が、p<0.01で有意に減退した。
ICAM-1のカウンター受容体/リガンドを評価するため、ヒト乳癌細胞株であるMCF-7及びMDA-MB-231に対するCD11a(LFA-1)及びCD11b(Mac-1)の免疫組織化学的発現について、検討した。図15は、ヒト乳癌細胞株であるMCF-7及びMDA-MB-231上でのCD11a(同図A、B)及びMCD11b(同図D、E)の免疫組織化学的発現を示す代表的な顕微鏡写真である。同図C及びFは、夫々、CD11a及びCD11bの一次抗体を含まないネガティブコントロールの場合の代表的な顕微鏡写真である。CD11a及びCD11bの両方の顕著な免疫組織化学的発現が、MCF-7及びMDA-MB-231細胞上に認められた。
図16は、乳癌患者から摘出した直後に凍結した癌細胞転移センチネルリンパ節組織、及び同患者から摘出した直後に凍結した癌細胞未移転センチネルリンパ節組織の夫々におけるE-セレクチン(同図C、D)及びICAM-1(同図E、F、G、H)の免疫組織化学的発現を示す代表的な顕微鏡写真である。同図Aは癌細胞転移のあるセンチネルリンパ節組織、同図Bは癌細胞未転移のセンチネルリンパ節組織の代表的なヘマトキシリン-エオシン染色を示す顕微鏡写真である。同図F及びHに示すように、癌細胞が転移したセンチネルリンパ節組織上では、ICAM-1の顕著な免疫組織化学的発現が、認められた。これとは対照的に、同図E及びGに示すように、同じ乳癌患者から摘出した癌細胞未転移のセンチネルリンパ節組織上では、ICAM-1の発現が、殆ど認められなかった。一方、同図C及びDに示すように、癌細胞の転移の有無に関わらず、センチネルリンパ節組織上でE-セレクチンの発現は、全く又は殆どみられなかった。なお、同図B,D,F,H中の「*」はセンチネルリンパ節での癌細胞転移範囲を示す。
局所リンパ節は、悪性腫瘍が通常最初に転移する部位である。リンパ節は、そこでの癌細胞の通過を物理的に阻害するバリアとして働き、またそこでの癌の成長を生化学的に阻害するバリアとしても働く。センチネルリンパ節ナビゲーション手術が臨床現場で劇的な成功を収めているということは、移行している癌細胞に対する物理的なバリアとなる有効なフィルター機構を局所リンパ節が有していることを、示唆している。一方、癌原発巣が、転移前に癌組織の微小環境に影響を及ぼしていることが知られている。しかし、転移前の局所リンパ節中の分子がリンパ節への微小転移に適した環境を構築できるか否かについては、今まで明らかにされていなかった。しかし、局所リンパ節へ癌細胞の微小転移に適した前転移の環境を構築するのに重要な化学物質を悪性癌細胞が放出することが分かったので、そのことを利用した本発明のセンチネルリンパ節内転移癌細胞検出キット及び薬物輸送剤が、臨床的に有用であることが、明らかとなった。
極めて高い転移能を有するヒト悪性乳癌細胞系であるMDA-MB-231の上清をヒトリンパ管内皮細胞上で48時間処理すると、選択的にICAM-1の発現が、惹き起こされる。上清を1/10,000倍に希釈してさえ、ICAM-1の免疫反応の強度は極めて強い。一方、低い転移能を有するヒト乳癌細胞系であるMCF-7の上清は、ヒトリンパ管内皮細胞上で、全く又は殆んどICAM-1の発現を惹き起こさない。
本発明のもう一つの重要な点は、MDA-MB-231上清で48時間処理すると、インビトロで癌細胞とヒトリンパ管内皮細胞との付着の促進が惹き起こされるということである。10-7MのATPで刺激しても、リンパ管内皮細胞への癌細胞の付着が顕著に増加し、その反応はMDA-MB-231上清で惹き起こされるのと略同様であった。
最近、癌細胞によるICAM-1発現は転移進行を促進する主要因子であると報告された。一方、白血球-癌毛細管内皮細胞接着の研究により、一次的サイトカイン刺激条件下、接着の影響が低減することが明らかになっている。癌毛細管内での内皮のICAM-1発現が減退するとされた免疫組織化学的な細胞蛍光の結果とも矛盾がない。
アデニンヌクレオチドの抗腫瘍活性は、既に知られている。担腫瘍マウスへの腹腔内ATP投与により、様々な急性悪性癌腫に対して顕著な抗腫瘍活性が認められた。ATPは、マウスでのネズミ結腸腺腫やヒトすい臓癌腫の成長を阻害する。インビトロでの前立腺癌細胞の成長は、P2受容体を介するATPにより90%も阻害されるが、サブタイプがその効果に介在していることやそれが直接的な抗増殖性効果や前アポトーシス効果であることは、未だにはっきりしていない。細胞外のATPは、アデノシンやATPが一部介在しているヒトHL-60白血病細胞の分化の誘発及び増殖を、抑制する。慢性リンパ性白血病評価系でのP2X7受容体発現が、確認されており、ATPは、この白血病評価系での白血球の増殖を抑制する。P2X7受容体mRNAの発現は殆んどの種類の白血病において頻発するが、P2X7受容体作用の低下が認められる。
ケモカイン類は、可溶性低分子量タンパク質であり、同族のG-タンパク共役受容体と結合して、通常、方向性移動又は走化性のような細胞反応を引き起こす。腫瘍細胞は、ケモカイン類を分泌したり、ケモカイン類に反応したりする。ケモカイン類は、内皮細胞漸増や免疫学的監視機能の破壊や腫瘍性白血球ケモカイン特性に影響によって腫瘍の増殖を促進して、免疫編集を歪めるものであり、遊離されたケモカイン類は、遠隔部位へ腫瘍を転移させたり増殖させたりする。
腫瘍細胞によるICAM-1発現は、転移進行を促進する主要因子である。一方、白血球と内皮細胞とが接着した腫瘍微小血管の研究から、基礎条件及びサイトカイン刺激条件下で接着相互作用が低減することが明らかになっている。このことは、腫瘍微小血管内での内皮のICAM-1発現が減退するという免疫組織化学的な知見や細胞蛍光の知見とも矛盾していない。内皮でのICAM-1を抑制することは、リンパ球の循環による免疫学的監視から腫瘍細胞が免れさせることになり、腫瘍の進行を促進させることになる。乳癌での炎症性所見に似た内皮でのICAM-1発現促進が腫瘍血管系で惹き起こされているという報告があるが、ヒトリンパ管内皮細胞上での接着分子の発現については、明らかではなかった。
Claims (13)
- ヒトリンパ管由来内皮細胞株が基材に付されていることを特徴とするセンチネルリンパ節内転移癌細胞検出キット。
- 前記ヒトリンパ管由来内皮細胞株が、摘出されたヒトリンパ管の内腔にプロテアーゼ液を灌流することにより剥離させて採取した内皮細胞であることを特徴とする請求項1に記載のセンチネルリンパ節内転移癌細胞検出キット。
- 前記ヒトリンパ管由来内皮細胞株と癌原発巣から転移した癌細胞との付着を介在する接着分子を、抗原抗体反応により検出するイムノアッセイ検出剤が、添加されることを特徴とする請求項1に記載のセンチネルリンパ節内転移癌細胞検出キット。
- 前記接着分子が、活性化されて発現していることを特徴とする請求項3に記載のセンチネルリンパ節内転移癌細胞検出キット。
- 前記接着分子が、ICAM-1、又はE-セレクチンであることを特徴とする請求項3に記載のセンチネルリンパ節内転移癌細胞検出キット。
- 前記接着分子が、それのリガンドを介して結合していることを特徴とする請求項3に記載のセンチネルリンパ節内転移癌細胞検出キット。
- 前記リガンドが、CD11a、CD11b及び/又はCD11cであることを特徴とする請求項6に記載のセンチネルリンパ節内転移癌細胞検出キット。
- センチネルリンパ節で癌原発巣からの転移癌細胞がリンパ管内皮細胞へ付着するのを介在する接着分子に対する抗体及び/又はリガンドが、コロイド粒子の表面から露出し、又は懸濁されていることを特徴とするセンチネルリンパ節内への薬物輸送剤。
- 前記抗体が、抗ICAM-1抗体、及び/又は抗E-セレクチン抗体であることを特徴とする請求項8に記載の薬物輸送剤。
- 前記リガンドが、ICAM-1リガンドであることを特徴とする請求項8に記載の薬物輸送剤。
- 前記リガンドが、CD11a、CD11b及び/又はCD11cであることを特徴とする請求項10に記載の薬物輸送剤。
- 前記コロイド粒子に、蛍光剤、造影剤、治療剤、及び/又は転移癌細胞接着増強剤が、含有され及び/又は露出されていることを特徴とする請求項8に記載の薬物輸送剤。
- 前記コロイド粒子が、生体分解性樹脂ミセル粒子、合成樹脂ミセル粒子、又はリポソームであることを特徴とする請求項8に記載の薬物輸送剤。
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EP09722615A EP2251694A4 (en) | 2008-02-29 | 2009-01-28 | KIT FOR DETECTING CANCER CELLS METASTASING IN SENTINEL LYMPHATIC GANGLION |
US12/919,185 US20110045060A1 (en) | 2008-02-29 | 2009-01-28 | Kit for detecting cancer cells metastasizing into sentinel lymph node |
JP2010503794A JP4719930B2 (ja) | 2008-02-29 | 2009-01-28 | センチネルリンパ節内転移癌細胞検出用薬物輸送剤 |
CA2717489A CA2717489A1 (en) | 2008-02-29 | 2009-01-28 | Kit for detecting cancer cells metastasizing into sentinel lymph node |
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Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101975860A (zh) * | 2010-10-20 | 2011-02-16 | 江阴诺格生物科技有限公司 | 前哨淋巴结快速诊断试剂盒 |
JP2011169873A (ja) * | 2010-02-22 | 2011-09-01 | Nagasaki Univ | 癌転移の検出方法および検出用キット |
WO2012002388A1 (ja) * | 2010-07-01 | 2012-01-05 | 国立大学法人信州大学 | センチネルリンパ節への薬物輸送剤 |
JP2013539454A (ja) * | 2010-07-13 | 2013-10-24 | バイオインヴェント インターナショナル アーベー | 再発した癌患者の治療におけるicam−1に対する抗体の使用 |
JP2015130836A (ja) * | 2014-01-15 | 2015-07-23 | 地方独立行政法人 大阪府立病院機構 | リンパ節郭清により得られた組織片中のリンパ節判別方法 |
US9803013B2 (en) | 2005-12-12 | 2017-10-31 | Bioinvent International Ab | Biological materials and uses thereof |
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RU2015102026A (ru) * | 2012-06-26 | 2016-08-10 | Ф.Хоффманн-Ля Рош Аг | Биомаркеры в плазме крови для комбинированных терапий с использованием бевацизумаба при лечении рака молочной железы |
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Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2007222155A (ja) | 2006-01-27 | 2007-09-06 | Shinshu Univ | ヒトリンパ管由来細胞株およびそれを用いた診断キット |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5284931A (en) * | 1987-05-04 | 1994-02-08 | Dana Farber Cancer Institute | Intercellular adhesion molecules, and their binding ligands |
WO1999013918A2 (en) * | 1997-09-18 | 1999-03-25 | University Of Pittsburgh | Icam-1 selective echogenic microbubbles |
AU2002211649A1 (en) * | 2000-10-11 | 2002-04-22 | Targesome, Inc. | Targeted therapeutic agents |
AU2002245629A1 (en) * | 2001-03-08 | 2002-09-24 | Targesome, Inc. | Stabilized therapeutic and imaging agents |
JP4934802B2 (ja) * | 2005-05-30 | 2012-05-23 | 国立大学法人信州大学 | リンパ系の細胞接着物質、リンパ系薬物輸送材およびそれを含有する薬剤 |
-
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Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2007222155A (ja) | 2006-01-27 | 2007-09-06 | Shinshu Univ | ヒトリンパ管由来細胞株およびそれを用いた診断キット |
Non-Patent Citations (7)
Title |
---|
ATSUSHI TOGAWA: "Tahatsusei Kotsuzuishu to Saibo Secchaku Bunshi", RINSHO KETSUEKI, vol. 45, no. 7, 30 July 2004 (2004-07-30), pages 509 - 517 * |
KAWAI ET AL., LYMPHATIC RESEARCH AND BIOLOGY, vol. 5, 2007, pages 115 - 126 |
KAWAI Y. ET AL., LYMPHATIC RESEARCH AND BIOLOGY, vol. 5, 2007, pages 115 - 126 |
KEIKO KAWAI ET AL.: "Lymph-kan to Lymph-kan Naihi Saibo no Keitai, Kino, Seibutsugakuteki Tokusei", EXPERIMENTAL MEDICINE, vol. 26, no. 6, 1 April 2008 (2008-04-01), pages 849 - 854 * |
SCOTT R. C. ET AL.: "Targeted delivery of antibody conjugated liposomal drug carriers to rat myocardial infarction", BIOTECH.BIOENG., vol. 96, no. 4, 1 March 2007 (2007-03-01), pages 795 - 802 * |
See also references of EP2251694A4 * |
SUMIO MATSUMOTO: "E-selectin, Sialyl Lewis-X, Oyobi Sialyl Lewis-A", G.I. RES., vol. 14, no. 2, 1 April 2006 (2006-04-01), pages 198 - 201 * |
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US9803013B2 (en) | 2005-12-12 | 2017-10-31 | Bioinvent International Ab | Biological materials and uses thereof |
JP2011169873A (ja) * | 2010-02-22 | 2011-09-01 | Nagasaki Univ | 癌転移の検出方法および検出用キット |
WO2012002388A1 (ja) * | 2010-07-01 | 2012-01-05 | 国立大学法人信州大学 | センチネルリンパ節への薬物輸送剤 |
JPWO2012002388A1 (ja) * | 2010-07-01 | 2013-08-29 | 国立大学法人信州大学 | センチネルリンパ節への薬物輸送剤 |
JP2013539454A (ja) * | 2010-07-13 | 2013-10-24 | バイオインヴェント インターナショナル アーベー | 再発した癌患者の治療におけるicam−1に対する抗体の使用 |
JP2016175906A (ja) * | 2010-07-13 | 2016-10-06 | バイオインヴェント インターナショナル アーベー | 再発した癌患者の治療におけるicam−1に対する抗体の使用 |
CN101975860A (zh) * | 2010-10-20 | 2011-02-16 | 江阴诺格生物科技有限公司 | 前哨淋巴结快速诊断试剂盒 |
JP2015130836A (ja) * | 2014-01-15 | 2015-07-23 | 地方独立行政法人 大阪府立病院機構 | リンパ節郭清により得られた組織片中のリンパ節判別方法 |
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CA2717489A1 (en) | 2009-09-24 |
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JP4719930B2 (ja) | 2011-07-06 |
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