WO2020159265A2 - Method for assessing angiogenesis in cancer tissue by using tissue clearing - Google Patents

Method for assessing angiogenesis in cancer tissue by using tissue clearing Download PDF

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WO2020159265A2
WO2020159265A2 PCT/KR2020/001443 KR2020001443W WO2020159265A2 WO 2020159265 A2 WO2020159265 A2 WO 2020159265A2 KR 2020001443 W KR2020001443 W KR 2020001443W WO 2020159265 A2 WO2020159265 A2 WO 2020159265A2
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cancer tissue
cancer
tissue
solution
blood vessels
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Korean (ko)
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WO2020159265A3 (en
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박영일
금상일
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주식회사 바이나리
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/48—Other medical applications
    • A61B5/4887—Locating particular structures in or on the body
    • A61B5/489—Blood vessels
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification
    • G01N2800/7014—(Neo)vascularisation - Angiogenesis

Definitions

  • the present invention relates to a method for evaluating angiogenesis in cancer tissue using tissue transparency.
  • Cancer is a disease that causes cells to multiply indefinitely and interfere with normal cell function. It is emerging as the biggest threat to public health, and due to various changes in the environment including aging, the threat of health and death due to cancer will increase. Seems to be.
  • One of the most important biological characteristics of cancer is that it can cause metastasis, which is considered to be the biggest obstacle to treating cancer, and the formation of new blood vessels plays an important role in the growth and metastasis of these cancer cells. Tumors are supplied with nutrients and oxygen necessary for growth and proliferation through new blood vessels, and new blood vessels that have penetrated to the tumors are metastased by giving metastatic cancer cells a chance to enter the blood circulation system.
  • angiogenesis inhibitors can be applied as therapeutic agents for these various angiogenesis-related diseases.
  • Research has been actively conducted to treat the diseases by suppressing it, and there is a need for research and development on a method for evaluating the degree of inhibition of angiogenesis in cancer tissues during the development of new drugs related to anticancer drugs.
  • tissue transparent technology can confirm the structure and protein expression without damaging the tissue
  • a technology capable of transparent tissue is developed in a variety of ways.
  • Existing tissue clearing techniques have been reported for the antigen preservation of tissues treated by Spatleholz, BABB, Scale S, iDISCO, which is a method for tissue clearing using organic solvents, and ACT (active CLARITY technology), which is a polymer injection method.
  • ACT active CLARITY technology
  • fluorescence and antigen retention are reduced.
  • ACT it has an antigen preservation of 90% or more, and it shows a higher preservation property compared to a method requiring binding to a hydrogel polymer in addition to a fixed protein such as CLARITY.
  • a strong tissue fixation process causes loss of antigenicity, so problems such as reduction of usable antibodies must be considered, and thus, various techniques need to be improved.
  • the recently developed CLARITY method uses a method to selectively remove lipids after making a kind of mesh support that holds a material such as DNA or protein by inserting a hydrogel in tissue to hold important materials for diagnosis.
  • Tissue transparency technologies that can create optically transparent and polymer-transmissive images, such as CLARITY and perfusion aided reagent release methods (PARS), have provided major advances in highly enhanced organ system imaging.
  • the clarity method has a disadvantage in that, as the hydrogel concentration increases, the degree of binding to the protein increases and the tissue becomes harder and thus the removal of lipids becomes difficult. This causes deposition of air and black particles on the tissue surface.
  • the existing Clarity method is a complicated process and requires a lot of additional equipment.
  • only one tissue can be transparent at a time, causing economic and time loss, and staining with antibodies has been unstable.
  • the present inventors do not require an expensive electrophoresis device and use a clearing solution that can increase the transparency of biological tissues without damaging various tissues, making cancer tissues transparent and observing the formation of new blood vessels in the cancer tissues. And invented a method to evaluate and use it for cancer-related research.
  • the present inventors prepare a clearing solution capable of increasing the transparency of biological tissues without damaging various tissues without the need for expensive electrophoresis devices, and using this to clear the cancerous tissues, thereby clarifying the fluorescence of blood vessels formed in the cancerous tissues.
  • An image was obtained, and a method of evaluating the formation of angiogenesis in cancer tissue was invented by analyzing the length and distribution of the angiogenesis.
  • an object of the present invention is to provide a method for evaluating angiogenesis in cancer tissue, comprising the following steps:
  • Another object of the present invention is to provide a method for screening a candidate for preventing or treating cancer, comprising the following steps:
  • the present invention provides a method for evaluating angiogenesis in cancer tissue, comprising the following steps:
  • the present invention provides a method for screening a candidate for cancer prevention or treatment, comprising the following steps:
  • the fixed solution may include sucrose.
  • the sucrose may have a concentration of 20% (w/v) to 100% (w/v).
  • the tissue clearing solution is N-Lauroylsarcosine sodium salt solution, CHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) , Urea (urea), and sodium chloride (NaCl).
  • the N-Lauroylsarcosine sodium salt solution has a concentration of 1% (v/v) to 30% (v/v), and CHAPS ( 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate) has a concentration of 10% (w/v) to 40% (w/v), and the urea has a concentration. 30% (w/v) to 70% (w/v), and the sodium chloride (NaCl) concentration may be 0.001% (w/v) to 1% (w/v).
  • the washing solution may include phosphate buffer saline (PBS) and sodium azide.
  • PBS phosphate buffer saline
  • the sodium azide may have a concentration of 0.001% (w/v) to 0.5% (w/v).
  • the mounting solution is composed of CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), urea, and sodium chloride (NaCl). It may include one or more selected from the group.
  • the concentration of CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) is 20% (w/v) to 60% (w/ v)
  • the concentration of urea (urea) is 10% (w/v) to 50% (w/v)
  • the concentration of sodium chloride (NaCl) may be 0.001% (w/v) to 3%. .
  • step (g) may be using an Imaris program.
  • the step of clearing the cancer tissue may include the following steps:
  • the method for evaluating the formation of new blood vessels in cancer tissue according to the present invention includes the step of making the cancer tissue transparent, and in the step, the blood vessel formed in the cancer tissue is made transparent by using the tissue clearing solution of the present invention. It can be clearly observed, and the length and distribution of blood vessels formed in the cancer tissue can be effectively analyzed and quantified through the Imaris program to evaluate the formation of new blood vessels in the cancer tissue.
  • the method for evaluating angiogenesis in cancer tissue of the present invention will be useful for evaluating the efficacy of inhibiting angiogenesis of drugs by analyzing the length and distribution of angiogenesis according to the treatment of drugs in a cancer animal model. It is expected.
  • FIG. 1 is a view showing neovascularization in cancer tissue formed by subcutaneously injecting a culture medium in which HT-29 colon cancer cells are cultured in a nude mouse according to one embodiment of the present invention.
  • Figure 2 is a result of analyzing the new blood vessels and the new blood vessels using the Imaris program in cancer tissue formed by subcutaneous injection of HT-29 colon cancer cell spheroid into nude mice according to an embodiment of the present invention. It is a figure showing.
  • the present invention provides a method for evaluating angiogenesis in cancer tissue, comprising the following steps:
  • the present invention provides a method for screening a candidate for cancer prevention or treatment, comprising the following steps:
  • angiogenesis used in the present invention means a process in which new blood vessels are generated from existing blood vessels.
  • angiogenesis is caused by a series of processes involving angiogenic factors, and in normal processes such as pear growth, reproduction, development and wound repair The regulation is performed smoothly and occurs, but if the regulation mechanism is abnormal, tumor growth and metastasis, diabetic retinopathy, neovascular glaucoma, rheumatoid arthritis, psoriasis (psoriasis), Kaposi's sarcoma, hemangiomas, chronic inflammation, and ulcers.
  • cancer is an aggressive property in which cells divide and grow, ignoring normal growth limits, an invasive property that penetrates surrounding tissue, and spreads to other parts of the body. It is a generic term for diseases caused by cells with metastatic characteristics.
  • the cancer is lung cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, anus near rectum cancer, breast cancer, fallopian tube Carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate Cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or urinary tract cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma and pituitary adenoma It may be any one or more selected from
  • spheroid used in the present invention is a small'spheroid' body made through 3D cell culture, and a spheroid is a small'tissue', which is divided into external and internal functions. It takes the form of replicating the functional units of an organization.
  • Spheroid culture is not only similar in nature and nature to animals or human tissues, but is also being developed as a platform that can be applied to research by applying it.
  • Spheroids made using cancer cells have a more similar shape to tumor tissues and show more similar results than cancer cells that have anti-cancer effects. It has been reported that when hepatocellular carcinoma cells are cultured as spheroids, hepatotoxicity test results closer to in vivo data can be obtained than results from the second plane.
  • the candidate substance refers to an unknown substance used for screening to confirm cancer prevention or treatment efficacy by analyzing the length and distribution of blood vessels formed in cancer tissues collected from a cancer animal model, for example, a chemical substance , Protein, (poly)nucleotide, antisense-RNA, siRNA (small interference RNA), or natural extracts, etc., but are not limited thereto.
  • the candidate substance may be an anticancer agent.
  • the step of clearing the cancer tissue may include the following steps:
  • the animal is not particularly limited in its kind, for example, a human, non-human primate, mouse, dog, cat, rabbit, horse, or cow, and according to a specific embodiment of the present invention, the animal is It can be a mouse.
  • the fixed solution may include sucrose (sucrose), the sucrose (sucrose) has a concentration of 20% (w/v) to 100% (w/v), 20% (w/v) To 70% (w/v), 20% (w/v) to 30% (w/v), 30% (w/v) to 40% (w/v), 40% (w/v) to 50 %(w/v), 50%(w/v) to 60%(w/v), 60%(w/v) to 70%(w/v), 70%(w/v) to 80%( w/v), or 80% (w/v) to 100% (w/v).
  • sucrose sucrose
  • sucrose sucrose
  • the sucrose (sucrose) has a concentration of 20% (w/v) to 100% (w/v), 20% (w/v) To 70% (w/v), 20% (w/v) to 30% (w/v), 30% (w/v) to 40% (w/v), 40% (w/v) to 50 %(w/v), 50%(w/v) to 60%(
  • the sample may be dehydrated by setting the concentration of sucrose to 20% (w/v) or more, and the sample covalently bound between organic substances with PFA (paraformaldehyde) may be more strongly fixed, but limited to the concentration. It does not work.
  • PFA paraformaldehyde
  • the tissue clearing solution is N-Lauroyl sarcosine sodium salt solution (N-Lauroylsarcosine sodium salt solution), CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane Sulfonate), urea (urea), and sodium chloride (NaCl).
  • the N-Lauroylsarcosine sodium salt solution has a concentration of 1% (v/v) to 30% (v/v) or 3% (v/v) to 20. % (v/v), and CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) has a concentration of 10% (w/v) to 40% (w/ v) or 10% (w/v) to 30% (w/v), wherein the urea has a concentration of 30% (w/v) to 70% (w/v) or 40% (w) /v) to 60% (w/v), and the concentration of sodium chloride (NaCl) is 0.001% (w/v) to 1% (w/v) or 0.01% (w/v) to 1% ( w/v), and according to a specific embodiment of the present invention, the N-Lauroylsarcosine sodium salt solution is 4% (v/v) or 15% (v/v) ),
  • the step (d) of the clearing using the tissue clearing solution uses a tissue clearing solution including 4% (v/v) of N-Lauroylsarcosine sodium salt solution.
  • the first clearing step or may include a second clearing step using a tissue clearing solution comprising 15% (v/v) of N-Lauroylsarcosine sodium salt solution.
  • the washing step, the second transparent step, and the washing step may be performed in order, but is not limited thereto.
  • the washing solution may include phosphate buffered saline (PBS) and sodium azide
  • the sodium azide has a concentration of 0.001% (w /v) to 0.5% (w/v) or 0.01% (w/v) to 0.5% (w/v)
  • the sodium azide is After clearing the concentration to 0.1% (w/v), after increasing the water content up to 30% in a dehydrated biotissue sample, dehydrating 15% to wash the organic matter that interferes with imaging attached to the tissue, but the concentration It is not limited to.
  • the mounting solution is one selected from the group consisting of CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), urea, and sodium chloride (NaCl). It may include the above.
  • the concentration of the CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) is 20% (w/v) to 60% (w/v) or 30% ( w/v) to 50% (w/v), and the urea has a concentration of 10% (w/v) to 50% (w/v) or 20% (w/v) to 50%.
  • the concentration of sodium chloride (NaCl) may be 0.001% (w/v) to 3% or 0.01% (w/v) to 2%, according to a specific embodiment of the present invention
  • the composition of the CHAPS and urea may include 40% (w/v) and 40% (w/v), respectively, and the concentration of sodium chloride (NaCl) is 0.1% (w /v) to 1% (w/v), but is not limited to the concentration.
  • step (g) may be to use an Imaris program.
  • a solution for clearing cancer tissue of a cancer animal model is prepared, and after the new blood vessels are stained in the cancer tissue of an animal transplanted with cancer cells, the cancer tissue is cleared with the solution (implementation) See example 1).
  • the length and distribution of new blood vessels were observed in the cleared cancer tissue using the Imaris program and quantitatively analyzed, thereby confirming the formation of new blood vessels in the cancer tissue. It was possible (see Example 2).
  • Example 1 Vascular staining formed in cancer tissue and clearing cancer tissue
  • a fixation solution a tissue clearing solution, a washing solution, and a mounting solution required to clear the cancer tissue of the cancer animal model were prepared, and the components of the solution are shown in Table 1 below.
  • a fixed solution containing sucrose having a concentration of 20% (w/v) or more as a component a fixed solution containing sucrose having a concentration of 20% (w/v) or more as a component
  • N-Lauroylsarcosine sodium salt at a concentration of 4% (v/v) or 15% (v/v) to minimize the degeneration of the fluorescent substance in the biological tissue sample by stabilizing the tissue deformation and ion strength by osmotic pressure.
  • solution 0.1% to 0.5% in 20% (w/v) CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) and 50% (w/v) urea
  • NaCl sodium chloride
  • PBS phosphate buffer saline
  • Configuration Composition One Fixing solution Sucrose (more than 20% (w/v)) 2 Tissue clearing solution N-Lauroylsarcosine sodium salt solution (4% (v/v) or 15% (v/v)), CHAPS (20% (w/v)), Urea (50% (w/v)), NaCl (0.1 to 0.5% (w/v)) 3 Washing solution PBS, sodium azied (0.1% (w/v)) 4 Mounting solution CHAPS (40% (w/v)), Urea (40% (w/v)), NaCl (0.1 to 1% (w/v))
  • the cancer tissue sample is placed in a fixing solution, shaking incubation until precipitation at 4°C/50 rpm, and the precipitated cancer tissue sample is 4% (v/v) N-Lauroylsarcosine sodium salt solution. It was put into a tissue clearing solution containing it, shaking incubation for 36 hours at 35°C/50 rpm, and repeated once under the same conditions.
  • the washed cancer tissue sample was placed in a mounting solution, shaking incubation at 35°C/50 rpm for 12 hours, and repeated once under the same conditions.
  • the cancer tissue sample treated with the mounting solution was centrifuge for 30 minutes at 1200 rpm to remove bubbles in the sample, and the cancer tissue sample was stored in an image chamber or stored at 4°C in 1X PBS.
  • the blood vessels formed in the entire cancer tissue that was made transparent by the method of Example 1-2 were observed with a lightsheet microscope, and the length and distribution of new blood vessels were observed using an Imaris 3D program.
  • FIG. 1 an image of blood vessels formed in cancer tissue of a mouse transplanted with a culture medium in which HT-29 colon cancer cells were cultured was shown in FIG. 1, and a mouse implanted with spheroid based on HT-29 colon cancer cells
  • FIG. 2 The image of blood vessels formed in the cancer tissues of FIG. 2 was shown, and a sharper and more precise result could be obtained from the cancer tissues of the mice implanted with spheroids.
  • angiogenesis could be analyzed to effectively evaluate the efficacy of anticancer drugs to inhibit angiogenesis.
  • the method for evaluating the formation of new blood vessels in cancer tissue can be used to evaluate the formation and development of new blood vessels in cancer tissue by effectively analyzing and quantifying the length and distribution of blood vessels formed in the cancer tissue. It is expected to be useful in evaluating the efficacy of inhibiting angiogenesis.

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Abstract

The present invention relates to a method for assessing angiogenesis in a cancer tissue by using tissue clearing. The method for assessing angiogenesis in a cancer tissue according to the present invention comprises the step of clearing the cancer tissue, wherein the cancer tissue is cleared with a tissue clearing solution of the present invention, whereby blood vessels formed in the cancer tissue can be clearly observed and the length and distribution of the blood vessels formed in the cancer tissue can be effectively analyzed and quantitated using the Imaris program to assess angiogenesis in the cancer tissue. In addition, the method for assessing angiogenesis in a cancer tissue according to the present invention analyzes the length and distribution of new vessels according to drug treatment in a cancer animal model and thus is expected to find advantageous applications in assessing the drug for inhibitory activity against angiogenesis.

Description

조직 투명화를 이용한 암 조직에서의 신생혈관 형성 평가 방법 Evaluation method of neovascularization in cancer tissue using tissue clarification
본 발명은 조직 투명화를 이용한 암 조직에서의 신생혈관 형성 평가 방법 등에 관한 것이다.The present invention relates to a method for evaluating angiogenesis in cancer tissue using tissue transparency.
본 출원은 2019년 02월 01일에 출원된 한국특허출원 제10-2019-0013802호에 기초한 우선권을 주장하며, 해당 출원의 명세서 및 도면에 개시된 모든 내용은 본 출원에 원용된다.This application claims priority based on Korean Patent Application No. 10-2019-0013802 filed on February 01, 2019, and all contents disclosed in the specification and drawings of the application are incorporated in this application.
암은 세포가 무한히 증식해 정상적인 세포의 기능을 방해하는 질병으로, 국민건강에 가장 큰 위협요인으로 대두되고 있으며, 고령화를 비롯하여 다양한 환경의 변화로 인해 암으로 인한 건강의 위협 및 사망은 더욱 증가될 것으로 보인다. 암의 가장 중요한 생물학적 특징 중 하나는 전이를 일으킬 수 있다는 것이며, 이는 암을 치료하는데 가장 큰 장애물로 여겨지고 있는데, 신생혈관 형성은 이러한 암세포의 성장과 전이에 중요한 역할을 한다. 종양은 신생혈관을 통하여 성장과 증식에 필요한 영양과 산소를 공급받으며, 또한 종양까지 침투한 신생혈관들은 전이하는 암세포가 혈액순환계로 들어가는 기회를 줌으로써 암세포가 전이(metastasis) 되도록 한다. Cancer is a disease that causes cells to multiply indefinitely and interfere with normal cell function. It is emerging as the biggest threat to public health, and due to various changes in the environment including aging, the threat of health and death due to cancer will increase. Seems to be. One of the most important biological characteristics of cancer is that it can cause metastasis, which is considered to be the biggest obstacle to treating cancer, and the formation of new blood vessels plays an important role in the growth and metastasis of these cancer cells. Tumors are supplied with nutrients and oxygen necessary for growth and proliferation through new blood vessels, and new blood vessels that have penetrated to the tumors are metastased by giving metastatic cancer cells a chance to enter the blood circulation system.
또한, 신생혈관 형성은 암 이외에 염증성 질환, 안과질환, 피부질환 등 여러 가지 질환과 관련되어 있어, 신생혈관 형성 억제제를 이러한 각종 혈관신생 관련 질환의 치료제로 적용할 수 있으므로, 최근에 신생혈관 형성을 억제시켜서 상기 질환들을 치료하려는 연구가 활발히 진행되고 있으며, 항암제 관련 신약 개발 과정에서 암 조직에서의 신생혈관 형성 억제 정도를 평가할 수 있는 방법 등에 관한 연구 개발이 필요한 실정이었다. In addition, since angiogenesis is associated with various diseases such as inflammatory diseases, ophthalmic diseases, and skin diseases in addition to cancer, angiogenesis inhibitors can be applied as therapeutic agents for these various angiogenesis-related diseases. Research has been actively conducted to treat the diseases by suppressing it, and there is a need for research and development on a method for evaluating the degree of inhibition of angiogenesis in cancer tissues during the development of new drugs related to anticancer drugs.
한편, 조직 투명화 기술은 조직의 손상 없이 구조 및 단백질 발현 등을 확인 할 수 있으므로 최근에 매우 다양한 방법으로 조직을 투명화 할 수 있는 기술이 개발되었다. 기존의 조직 투명화 기술은 유기용매를 이용한 조직 투명화 방법인 Spatleholz, BABB, Scale S, iDISCO법과, 폴리머 주입법인 ACT(active CLARITY technology)법에 의해 처리된 조직의 항원 보존성이 보고된 바 있다. ACT를 제외한 다른 방법의 경우 형광과 항원의 보존성이 감소하는 문제를 가지고 있다. ACT의 경우 90% 이상의 항원 보존성을 가지며, 이는 클라리티(CLARITY)와 같이 고정된 단백질에 추가로 하이드로젤 폴리머와의 결합을 필요로 하는 방법에 비하면 보다 높은 보존성을 보인다. 그러나 강한 조직 고정 과정은 항원성의 손실을 유발하여, 사용할 수 있는 항체가 감소하는 등의 문제점을 고려해야 하므로, 여러 가지 기술의 개선이 필요하다.On the other hand, since the tissue transparent technology can confirm the structure and protein expression without damaging the tissue, recently, a technology capable of transparent tissue is developed in a variety of ways. Existing tissue clearing techniques have been reported for the antigen preservation of tissues treated by Spatleholz, BABB, Scale S, iDISCO, which is a method for tissue clearing using organic solvents, and ACT (active CLARITY technology), which is a polymer injection method. For methods other than ACT, fluorescence and antigen retention are reduced. In the case of ACT, it has an antigen preservation of 90% or more, and it shows a higher preservation property compared to a method requiring binding to a hydrogel polymer in addition to a fixed protein such as CLARITY. However, a strong tissue fixation process causes loss of antigenicity, so problems such as reduction of usable antibodies must be considered, and thus, various techniques need to be improved.
최근에 개발된 클라리티(CLARITY)법은 조직 내 hydrogel을 넣어서 DNA나 단백질 등 진단에 중요한 material 등을 붙잡아 주는 일종의 그물망 지지체를 만든 뒤 지질만을 선택적으로 제거하는 방법을 이용한다. 클라리티(CLARITY) 및 관류 도움 시약 방출방법(PARS)과 같이 광학적으로 투명하고 고분자 투과가 가능한 이미지를 창출할 수 있는 조직투명성 기술은 매우 향상된 기관계 이미징에 있어서 주요한 진전을 제공하였다. The recently developed CLARITY method uses a method to selectively remove lipids after making a kind of mesh support that holds a material such as DNA or protein by inserting a hydrogel in tissue to hold important materials for diagnosis. Tissue transparency technologies that can create optically transparent and polymer-transmissive images, such as CLARITY and perfusion aided reagent release methods (PARS), have provided major advances in highly enhanced organ system imaging.
그러나, 상기 클라리티 방법은 hydrogel 농도가 높아지면 단백질과의 결합도가 많아 더 조직이 단단해져서 지질의 제거가 어려워 지기 때문에 투명화하는데 시간이 오래 걸리는 단점이 있다. 이는 조직 표면에 공기 및 검은 입자의 침착을 야기한다. 또한, 기존의 클라리티 법은 과정이 복잡하고 부가적인 장비가 많이 필요하다. 뿐만 아니라 한번에 한 조직만 투명화 할 수 있어 경제적, 시간적 손실을 유발하며 항체를 이용한 염색이 불안정한 문제가 있었다.However, the clarity method has a disadvantage in that, as the hydrogel concentration increases, the degree of binding to the protein increases and the tissue becomes harder and thus the removal of lipids becomes difficult. This causes deposition of air and black particles on the tissue surface. In addition, the existing Clarity method is a complicated process and requires a lot of additional equipment. In addition, only one tissue can be transparent at a time, causing economic and time loss, and staining with antibodies has been unstable.
이에, 본 발명자들은 고가의 전기영동 장치를 필요로 하지 않고 다양한 조직의 손상 없이 생체 조직의 투명성을 증가시킬 수 있는 투명화 용액을 이용하여 암 조직을 투명화하고, 상기 암 조직에서의 신생혈관 형성을 관찰하고 평가하는 방법을 발명하여 암 관련 연구 등에 활용하고자 하였다.Accordingly, the present inventors do not require an expensive electrophoresis device and use a clearing solution that can increase the transparency of biological tissues without damaging various tissues, making cancer tissues transparent and observing the formation of new blood vessels in the cancer tissues. And invented a method to evaluate and use it for cancer-related research.
본 발명자들은 고가의 전기영동 장치를 필요로 하지 않고 다양한 조직의 손상 없이 생체 조직의 투명성을 증가시킬 수 있는 투명화 용액을 제조하고, 이를 이용하여 암 조직을 투명화 함으로써 암 조직에서 형성되는 혈관의 선명한 형광 이미지를 획득하였으며, 상기 신생혈관의 길이 및 분포를 분석함으로써 암 조직에서의 신생혈관 형성을 평가하는 방법을 발명하였다.The present inventors prepare a clearing solution capable of increasing the transparency of biological tissues without damaging various tissues without the need for expensive electrophoresis devices, and using this to clear the cancerous tissues, thereby clarifying the fluorescence of blood vessels formed in the cancerous tissues. An image was obtained, and a method of evaluating the formation of angiogenesis in cancer tissue was invented by analyzing the length and distribution of the angiogenesis.
이에, 본 발명의 목적은 하기 단계를 포함하는, 암 조직에서의 신생혈관 형성 평가 방법을 제공하는 것이다:Accordingly, an object of the present invention is to provide a method for evaluating angiogenesis in cancer tissue, comprising the following steps:
(a) 암 세포를 배양한 배양액 또는 상기 암 세포를 기반으로 하는 스페로이드를 동물에 이식하여 암 조직을 형성하는 단계;(a) transplanting a culture medium in which cancer cells are cultured or a spheroid based on the cancer cells into an animal to form cancer tissue;
(b) 상기 암 조직이 형성된 동물에 렉틴(lectin)을 주입하여 혈관을 염색하고 암 조직을 채취하는 단계;(b) injecting lectin into the animal on which the cancer tissue is formed, staining blood vessels and collecting cancer tissue;
(c) 고정 용액으로 상기 동물의 암 조직 시료를 고정시키는 단계;(c) immobilizing the cancer tissue sample of the animal with a fixation solution;
(d) 조직 클리어링 용액으로 상기 고정된 시료에 있는 형광물질의 변성을 최소화하는 투명화 단계;(d) a clearing step to minimize denaturation of the fluorescent material in the fixed sample with a tissue clearing solution;
(e) 세척 용액으로 상기 형광물질의 변성을 최소화한 시료에 부착되어 있는 유기물을 씻어내는 세척 단계; (e) a washing step of washing the organic matter attached to the sample with minimal denaturation of the fluorescent material with a washing solution;
(f) 마운팅 용액으로 상기 세척된 시료를 고정시키는 단계; 및(f) fixing the washed sample with a mounting solution; And
(g) 상기 (a) 내지 (f) 단계를 거친 암 조직 시료에서 형성된 혈관의 길이 및 분포를 분석하는 단계.(G) analyzing the length and distribution of blood vessels formed in the cancer tissue samples subjected to the steps (a) to (f).
본 발명의 다른 목적은 하기 단계를 포함하는, 암 예방 또는 치료용 후보물질의 스크리닝 방법을 제공하는 것이다:Another object of the present invention is to provide a method for screening a candidate for preventing or treating cancer, comprising the following steps:
(a) 암 동물모델에 후보물질을 처리하는 단계;(a) treating the candidate material in a cancer animal model;
(b) 상기 암 동물모델에 렉틴(lectin)을 주입하여 혈관을 염색하고 암 조직을 채취하는 단계;(b) injecting lectin into the cancer animal model, dyeing blood vessels and collecting cancer tissue;
(c) 상기 암 조직을 투명화 시키는 단계;(c) making the cancer tissue transparent;
(d) 이마리스(Imaris) 프로그램을 통해 상기 투명화된 암 조직에서 형성된 혈관의 길이 및 분포를 분석하여 암 조직에서의 신생혈관 형성을 평가하는 단계; 및(d) analyzing the length and distribution of blood vessels formed in the cleared cancer tissue through the Imaris program to evaluate the formation of new blood vessels in the cancer tissue; And
(e) 상기 암 조직에서의 신생혈관 형성이 후보물질을 처리하기 전에 비해 억제된 경우 암 예방 또는 치료용 후보물질로 선별하는 단계.(e) When the formation of new blood vessels in the cancer tissue is suppressed compared to before treatment of the candidate substance, selecting as a candidate substance for preventing or treating cancer.
그러나, 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 본 발명이 속하는 기술 분야의 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned can be clearly understood by those having ordinary knowledge in the technical field to which the present invention belongs from the following description. There will be.
상기와 같은 목적을 달성하기 위해, 본 발명은 하기 단계를 포함하는, 암 조직에서의 신생혈관 형성 평가 방법을 제공한다:In order to achieve the above object, the present invention provides a method for evaluating angiogenesis in cancer tissue, comprising the following steps:
(a) 암 세포를 배양한 배양액 또는 상기 암 세포를 기반으로 하는 스페로이드를 동물에 이식하여 암 조직을 형성하는 단계;(a) transplanting a culture medium in which cancer cells are cultured or a spheroid based on the cancer cells into an animal to form cancer tissue;
(b) 상기 암 조직이 형성된 동물에 렉틴(lectin)을 주입하여 혈관을 염색하고 암 조직을 채취하는 단계;(b) injecting lectin into the animal on which the cancer tissue is formed, staining blood vessels and collecting cancer tissue;
(c) 고정 용액으로 상기 동물의 암 조직 시료를 고정시키는 단계;(c) immobilizing the cancer tissue sample of the animal with a fixation solution;
(d) 조직 클리어링 용액으로 상기 고정된 시료에 있는 형광물질의 변성을 최소화하는 투명화 단계;(d) a clearing step to minimize denaturation of the fluorescent material in the fixed sample with a tissue clearing solution;
(e) 세척 용액으로 상기 형광물질의 변성을 최소화한 시료에 부착되어 있는 유기물을 씻어내는 세척 단계; (e) a washing step of washing the organic matter attached to the sample with minimal denaturation of the fluorescent material with a washing solution;
(f) 마운팅 용액으로 상기 세척된 시료를 고정시키는 단계; 및(f) fixing the washed sample with a mounting solution; And
(g) 상기 (a) 내지 (f) 단계를 거친 암 조직 시료에서 형성된 혈관의 길이 및 분포를 분석하는 단계.(G) analyzing the length and distribution of blood vessels formed in the cancer tissue samples subjected to the steps (a) to (f).
또한, 본 발명은 하기 단계를 포함하는, 암 예방 또는 치료용 후보물질의 스크리닝 방법을 제공한다:In addition, the present invention provides a method for screening a candidate for cancer prevention or treatment, comprising the following steps:
(a) 암 동물모델에 후보물질을 처리하는 단계;(a) treating the candidate material in a cancer animal model;
(b) 상기 암 동물모델에 렉틴(lectin)을 주입하여 혈관을 염색하고 암 조직을 채취하는 단계;(b) injecting lectin into the cancer animal model, dyeing blood vessels and collecting cancer tissue;
(c) 상기 암 조직을 투명화 시키는 단계;(c) making the cancer tissue transparent;
(d) 이마리스(Imaris) 프로그램을 통해 상기 투명화된 암 조직에서 형성된 혈관의 길이 및 분포를 분석하여 암 조직에서의 신생혈관 형성을 평가하는 단계; 및(d) analyzing the length and distribution of blood vessels formed in the cleared cancer tissue through the Imaris program to evaluate the formation of new blood vessels in the cancer tissue; And
(e) 상기 암 조직에서의 신생혈관 형성이 후보물질을 처리하기 전에 비해 억제된 경우 암 예방 또는 치료용 후보물질로 선별하는 단계.(e) When the formation of new blood vessels in the cancer tissue is suppressed compared to before treatment of the candidate substance, selecting as a candidate substance for preventing or treating cancer.
본 발명의 일 구현예로서, 상기 고정 용액은 수크로오스(sucrose)를 포함할 수 있다.As an embodiment of the present invention, the fixed solution may include sucrose.
본 발명의 다른 구현예로서, 상기 수크로오스(sucrose)는 농도가 20%(w/v) 내지 100%(w/v)일 수 있다.As another embodiment of the present invention, the sucrose may have a concentration of 20% (w/v) to 100% (w/v).
본 발명의 또 다른 구현예로서, 상기 조직 클리어링 용액은 N-라우로일사르코신 나트륨염 용액(N-Lauroylsarcosine sodium salt solution), CHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), 우레아(urea), 및 염화나트륨(NaCl)으로 이루어진 군으로부터 선택된 하나 이상을 포함할 수 있다.As another embodiment of the present invention, the tissue clearing solution is N-Lauroylsarcosine sodium salt solution, CHAPS(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) , Urea (urea), and sodium chloride (NaCl).
본 발명의 또 다른 구현예로서, 상기 N-라우로일사르코신 나트륨염 용액(N-Lauroylsarcosine sodium salt solution)은 농도가 1%(v/v) 내지 30%(v/v)이고, CHAPS(3-[(3-콜아미도프로필)디메틸암모니오]-1-프로판설포네이트)는 농도가 10%(w/v) 내지 40%(w/v)이고, 상기 우레아(urea)는 농도가 30%(w/v) 내지 70%(w/v)이고, 상기 염화나트륨(NaCl)은 농도가 0.001%(w/v) 내지 1%(w/v)일 수 있다.In another embodiment of the present invention, the N-Lauroylsarcosine sodium salt solution has a concentration of 1% (v/v) to 30% (v/v), and CHAPS ( 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate) has a concentration of 10% (w/v) to 40% (w/v), and the urea has a concentration. 30% (w/v) to 70% (w/v), and the sodium chloride (NaCl) concentration may be 0.001% (w/v) to 1% (w/v).
본 발명의 또 다른 구현예로서, 상기 세척 용액은 인산완충식염수(phosphate buffer saline; PBS) 및 아지드화나트륨(sodium azide)을 포함할 수 있다.As another embodiment of the present invention, the washing solution may include phosphate buffer saline (PBS) and sodium azide.
본 발명의 또 다른 구현예로서, 상기 아지드화나트륨(sodium azide)은 농도가 0.001%(w/v) 내지 0.5%(w/v)일 수 있다.As another embodiment of the present invention, the sodium azide (sodium azide) may have a concentration of 0.001% (w/v) to 0.5% (w/v).
본 발명의 또 다른 구현예로서, 상기 마운팅 용액은 CHAPS(3-[(3-콜아미도프로필)디메틸암모니오]-1-프로판설포네이트), 우레아(urea), 및 염화나트륨(NaCl)으로 이루어진 군으로부터 선택된 하나 이상을 포함할 수 있다.As another embodiment of the present invention, the mounting solution is composed of CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), urea, and sodium chloride (NaCl). It may include one or more selected from the group.
본 발명의 또 다른 구현예로서, 상기 CHAPS(3-[(3-콜아미도프로필)디메틸암모니오]-1-프로판설포네이트)는 농도가 20%(w/v) 내지 60%(w/v)이고, 상기 우레아(urea)는 농도가 10%(w/v) 내지 50%(w/v)이고, 상기 염화나트륨(NaCl)은 농도가 0.001%(w/v) 내지 3%일 수 있다.As another embodiment of the present invention, the concentration of CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) is 20% (w/v) to 60% (w/ v), the concentration of urea (urea) is 10% (w/v) to 50% (w/v), and the concentration of sodium chloride (NaCl) may be 0.001% (w/v) to 3%. .
본 발명의 또 다른 구현예로서, 상기 (g) 단계는 이마리스(Imaris) 프로그램을 이용하는 것일 수 있다.As another embodiment of the present invention, step (g) may be using an Imaris program.
본 발명의 또 다른 구현예로서, 상기 암 조직을 투명화 시키는 단계는 하기 단계를 포함할 수 있다:As another embodiment of the present invention, the step of clearing the cancer tissue may include the following steps:
(가) 고정 용액으로 동물의 암 조직 시료를 고정시키는 단계;(A) fixing the cancer tissue sample of the animal with a fixed solution;
(나) 조직 클리어링 용액으로 상기 고정된 시료에 있는 형광물질의 변성을 최소화하는 투명화 단계;(B) a clearing step of minimizing denaturation of the fluorescent substance in the fixed sample with a tissue clearing solution;
(다) 세척 용액으로 상기 형광물질의 변성을 최소화한 시료에 부착되어 있는 유기물을 씻어내는 세척 단계; 및(C) a washing step of washing the organic matter attached to the sample with minimal degeneration of the fluorescent material with a washing solution; And
(라) 마운팅 용액으로 상기 세척된 시료를 고정시키는 단계.(D) fixing the washed sample with a mounting solution.
본 발명에 따른 암 조직에서의 신생혈관 형성을 평가하는 방법은 암 조직을 투명화 시키는 단계를 포함하며, 상기 단계에서 본 발명의 조직 투명화 용액을 이용하여 암 조직을 투명화 시킴으로써 암 조직에서 형성되는 혈관을 선명하게 관찰할 수 있고, 이마리스(Imaris) 프로그램을 통해 상기 암 조직에서 형성되는 혈관의 길이 및 분포를 효과적으로 분석하고 정량화하여 암 조직에서의 신생혈관 형성을 평가할 수 있다. 또한, 본 발명의 암 조직에서의 신생혈관 형성 평가 방법은 암 동물모델에서 약물의 처리에 따른 신생혈관의 길이 및 분포를 분석함으로써, 약물의 신생혈관 형성 억제 효능을 평가하는 데 유용하게 이용될 것으로 기대된다.The method for evaluating the formation of new blood vessels in cancer tissue according to the present invention includes the step of making the cancer tissue transparent, and in the step, the blood vessel formed in the cancer tissue is made transparent by using the tissue clearing solution of the present invention. It can be clearly observed, and the length and distribution of blood vessels formed in the cancer tissue can be effectively analyzed and quantified through the Imaris program to evaluate the formation of new blood vessels in the cancer tissue. In addition, the method for evaluating angiogenesis in cancer tissue of the present invention will be useful for evaluating the efficacy of inhibiting angiogenesis of drugs by analyzing the length and distribution of angiogenesis according to the treatment of drugs in a cancer animal model. It is expected.
도 1은 본 발명의 일구현예에 따라 HT-29 colon cancer cell을 배양한 배양액을 누드 마우스에 피하 주사하여 형성된 암 조직에서의 신생혈관을 나타낸 도면이다.1 is a view showing neovascularization in cancer tissue formed by subcutaneously injecting a culture medium in which HT-29 colon cancer cells are cultured in a nude mouse according to one embodiment of the present invention.
도 2는 본 발명의 일구현예에 따라 HT-29 colon cancer cell 스페로이드를 누드 마우스에 피하 주사하여 형성된 암 조직에서의 신생혈관 및 상기 신생혈관을 이마리스(Imaris) 프로그램을 사용하여 분석한 결과를 나타낸 도면이다. Figure 2 is a result of analyzing the new blood vessels and the new blood vessels using the Imaris program in cancer tissue formed by subcutaneous injection of HT-29 colon cancer cell spheroid into nude mice according to an embodiment of the present invention. It is a figure showing.
본 발명은 하기 단계를 포함하는, 암 조직에서의 신생혈관 형성 평가 방법을 제공한다:The present invention provides a method for evaluating angiogenesis in cancer tissue, comprising the following steps:
(a) 암 세포를 배양한 배양액 또는 상기 암 세포를 기반으로 하는 스페로이드를 동물에 이식하여 암 조직을 형성하는 단계;(a) transplanting a culture medium in which cancer cells are cultured or a spheroid based on the cancer cells into an animal to form cancer tissue;
(b) 상기 암 조직이 형성된 동물에 렉틴(lectin)을 주입하여 혈관을 염색하고 암 조직을 채취하는 단계;(b) injecting lectin into the animal on which the cancer tissue is formed, staining blood vessels and collecting cancer tissue;
(c) 고정 용액으로 상기 동물의 암 조직 시료를 고정시키는 단계;(c) immobilizing the cancer tissue sample of the animal with a fixation solution;
(d) 조직 클리어링 용액으로 상기 고정된 시료에 있는 형광물질의 변성을 최소화하는 투명화 단계;(d) a clearing step to minimize denaturation of the fluorescent material in the fixed sample with a tissue clearing solution;
(e) 세척 용액으로 상기 형광물질의 변성을 최소화한 시료에 부착되어 있는 유기물을 씻어내는 세척 단계; (e) a washing step of washing the organic matter attached to the sample with minimal denaturation of the fluorescent material with a washing solution;
(f) 마운팅 용액으로 상기 세척된 시료를 고정시키는 단계; 및(f) fixing the washed sample with a mounting solution; And
(g) 상기 (a) 내지 (f) 단계를 거친 암 조직 시료에서 형성된 혈관의 길이 및 분포를 분석하는 단계.(G) analyzing the length and distribution of blood vessels formed in the cancer tissue samples subjected to the steps (a) to (f).
또한, 본 발명은 하기 단계를 포함하는, 암 예방 또는 치료용 후보물질의 스크리닝 방법을 제공한다:In addition, the present invention provides a method for screening a candidate for cancer prevention or treatment, comprising the following steps:
(a) 암 동물모델에 후보물질을 처리하는 단계;(a) treating the candidate material in a cancer animal model;
(b) 상기 암 동물모델에 렉틴(lectin)을 주입하여 혈관을 염색하고 암 조직을 채취하는 단계;(b) injecting lectin into the cancer animal model, dyeing blood vessels and collecting cancer tissue;
(c) 상기 암 조직을 투명화 시키는 단계;(c) making the cancer tissue transparent;
(d) 이마리스(Imaris) 프로그램을 통해 상기 투명화된 암 조직에서 형성된 혈관의 길이 및 분포를 분석하여 암 조직에서의 신생혈관 형성을 평가하는 단계; 및(d) analyzing the length and distribution of blood vessels formed in the cleared cancer tissue through the Imaris program to evaluate the formation of new blood vessels in the cancer tissue; And
(e) 상기 암 조직에서의 신생혈관 형성이 후보물질을 처리하기 전에 비해 억제된 경우 암 예방 또는 치료용 후보물질로 선별하는 단계.(e) When the formation of new blood vessels in the cancer tissue is suppressed compared to before treatment of the candidate substance, selecting as a candidate substance for preventing or treating cancer.
본 발명에서 사용되는 용어 "신생혈관 형성(angiogenesis)"은 기존의 혈관에서부터 새로운 혈관이 생성되는 과정을 의미한다. 일반적으로 신생혈관 형성은 신생혈관 형성 유도인자 (angiogenic factor)가 관여하는 일련의 과정에 의해서 발생되며, 배 성장, 생식 (reproduction), 발달 (development) 및 상처 회복 (wound repair)과 같은 정상적인 과정에서는 그 조절이 원활하게 수행되며 발생되지만, 그 조절 메카니즘에 이상이 발생되는 경우에는 종양의 성장과 전이(metastasis), 당뇨병성 망막증(diabetic retinopathy), 신생혈관성 녹내장 (neovascular glaucoma), 류마티스성 관절염, 건선(psoriasis), 카포시 육종 (kaposi's sarcoma), 혈관종 (hemangiomas), 만성 염증(chronic inflammation), 궤양(ulcer)과 같은 다양한 질환들을 야기하게 된다.The term "angiogenesis" used in the present invention means a process in which new blood vessels are generated from existing blood vessels. In general, angiogenesis is caused by a series of processes involving angiogenic factors, and in normal processes such as pear growth, reproduction, development and wound repair The regulation is performed smoothly and occurs, but if the regulation mechanism is abnormal, tumor growth and metastasis, diabetic retinopathy, neovascular glaucoma, rheumatoid arthritis, psoriasis (psoriasis), Kaposi's sarcoma, hemangiomas, chronic inflammation, and ulcers.
본 발명에서 사용되는 용어 "암(cancer)"은 세포가 정상적인 성장 한계를 무시하고 분열 및 성장하는 공격적(aggressive) 특성, 주위 조직에 침투하는 침투적(invasive) 특성, 및 체내의 다른 부위로 퍼지는 전이적(metastatic) 특성을 갖는 세포에 의한 질병을 총칭하는 의미이다.The term “cancer,” as used herein, is an aggressive property in which cells divide and grow, ignoring normal growth limits, an invasive property that penetrates surrounding tissue, and spreads to other parts of the body. It is a generic term for diseases caused by cells with metastatic characteristics.
본 발명에 있어서, 상기 암은 폐암, 비소세포성폐암, 결장암, 골암, 췌장암, 피부암, 두부 또는 경부암, 피부 또는 안구내 흑색종, 자궁암, 난소암, 직장암, 위암, 항문부근암, 유방암, 나팔관암종, 자궁내막암종, 자궁경부암종, 질암종, 음문암종, 호지킨병(Hodgkin's disease), 식도암, 소장암, 내분비선암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 요도암, 음경암, 전립선암, 만성 또는 급성 백혈병, 림프구 림프종, 방광암, 신장 또는 수뇨관암, 신장세포 암종, 신장골반 암종, 중추신경계(CNS; central nervous system) 종양, 1차 CNS 림프종, 척수 종양, 뇌간 신경교종 및 뇌하수체 선종으로 이루어진 군에서 선택된 어느 하나 이상일 수 있으나, 이에 제한되지 않는다.In the present invention, the cancer is lung cancer, non-small cell lung cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, anus near rectum cancer, breast cancer, fallopian tube Carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate Cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or urinary tract cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma and pituitary adenoma It may be any one or more selected from the group consisting of, but is not limited thereto.
본 발명에서 사용되는 용어 "스페로이드(spheroid)"는 3차원 세포배양을 통하여 만들어지는 작은 '구'체로, 스페로이드는 일종의 작은 '조직'으로서, 외부와 내부가 구분되어 기능이 나뉘기도 하며, 조직의 기능적 단위를 모사하는 형태를 띤다. 스페로이드 배양은 동물이나 인체 조직이 가지고 있는 고유 형태나 성질이 비슷할 뿐만 아니라, 이를 적용하여 연구에 활용할 수 있는 플랫폼으로 개발되고 있다. 암세포를 이용하여 만들어진 스페로이드는 좀더 종양조직과 유사한 형태를 띠어 항암제에 대한 효능이 평면배양된 암세포에 비해 좀 더 비슷한 결과를 보여준다. 간암세포를 스페로이드로 배양할 경우, 2차 평면에서의 결과보다 더욱 in vivo 데이터와 가까운 간독성 실험결과를 얻을 수 있다고 보고되었다.The term "spheroid" used in the present invention is a small'spheroid' body made through 3D cell culture, and a spheroid is a small'tissue', which is divided into external and internal functions. It takes the form of replicating the functional units of an organization. Spheroid culture is not only similar in nature and nature to animals or human tissues, but is also being developed as a platform that can be applied to research by applying it. Spheroids made using cancer cells have a more similar shape to tumor tissues and show more similar results than cancer cells that have anti-cancer effects. It has been reported that when hepatocellular carcinoma cells are cultured as spheroids, hepatotoxicity test results closer to in vivo data can be obtained than results from the second plane.
본 발명에 있어서, 상기 후보물질은 암 동물모델로부터 채취한 암 조직에서 형성된 혈관의 길이 및 분포를 분석함으로써 암 예방 또는 치료 효능을 확인하기 위한 스크리닝에 이용되는 미지의 물질을 의미하며, 예컨대 화학물질, 단백질, (폴리)뉴클레오타이드, 안티센스-RNA, siRNA (small interference RNA), 또는 천연물 추출물 등이 포함될 수 있으나, 이에 제한되지 않는다. 본 발명에 있어서, 상기 후보물질은 항암제일 수 있다.In the present invention, the candidate substance refers to an unknown substance used for screening to confirm cancer prevention or treatment efficacy by analyzing the length and distribution of blood vessels formed in cancer tissues collected from a cancer animal model, for example, a chemical substance , Protein, (poly)nucleotide, antisense-RNA, siRNA (small interference RNA), or natural extracts, etc., but are not limited thereto. In the present invention, the candidate substance may be an anticancer agent.
본 발명에 있어서, 상기 암 조직을 투명화 시키는 단계는 하기 단계를 포함할 수 있다:In the present invention, the step of clearing the cancer tissue may include the following steps:
(가) 고정 용액으로 동물의 암 조직 시료를 고정시키는 단계;(A) fixing the cancer tissue sample of the animal with a fixed solution;
(나) 조직 클리어링 용액으로 상기 고정된 시료에 있는 형광물질의 변성을 최소화하는 투명화 단계;(B) a clearing step of minimizing denaturation of the fluorescent substance in the fixed sample with a tissue clearing solution;
(다) 세척 용액으로 상기 형광물질의 변성을 최소화한 시료에 부착되어 있는 유기물을 씻어내는 세척 단계; 및(C) a washing step of washing the organic matter attached to the sample with minimal degeneration of the fluorescent material with a washing solution; And
(라) 마운팅 용액으로 상기 세척된 시료를 고정시키는 단계.(D) fixing the washed sample with a mounting solution.
본 발명에 있어서, 상기 동물은 그 종류에 특별히 제한이 없으며, 예컨대 인간, 비-인간 영장류, 마우스, 개, 고양이, 토끼, 말, 또는 소일 수 있고, 본 발명의 구체적인 실시예에 따르면 상기 동물은 마우스일 수 있다.In the present invention, the animal is not particularly limited in its kind, for example, a human, non-human primate, mouse, dog, cat, rabbit, horse, or cow, and according to a specific embodiment of the present invention, the animal is It can be a mouse.
본 발명에 있어서, 상기 고정 용액은 수크로오스(sucrose)를 포함할 수 있으며, 상기 수크로오스(sucrose)는 농도가 20%(w/v) 내지 100%(w/v), 20%(w/v) 내지 70%(w/v), 20%(w/v) 내지 30%(w/v), 30%(w/v) 내지 40%(w/v), 40%(w/v) 내지 50%(w/v), 50%(w/v) 내지 60%(w/v), 60%(w/v) 내지 70%(w/v), 70%(w/v) 내지 80%(w/v), 또는 80%(w/v) 내지 100%(w/v)일 수 있다. 본 발명의 구체적인 실시예에 따르면 수크로오스의 농도를 20%(w/v) 이상으로 하여 시료를 탈수시키고 PFA(paraformaldehyde)로 유기물간 공유결합된 시료를 좀 더 강하게 고정할 수 있으나, 상기 농도에 한정되는 것은 아니다. In the present invention, the fixed solution may include sucrose (sucrose), the sucrose (sucrose) has a concentration of 20% (w/v) to 100% (w/v), 20% (w/v) To 70% (w/v), 20% (w/v) to 30% (w/v), 30% (w/v) to 40% (w/v), 40% (w/v) to 50 %(w/v), 50%(w/v) to 60%(w/v), 60%(w/v) to 70%(w/v), 70%(w/v) to 80%( w/v), or 80% (w/v) to 100% (w/v). According to a specific embodiment of the present invention, the sample may be dehydrated by setting the concentration of sucrose to 20% (w/v) or more, and the sample covalently bound between organic substances with PFA (paraformaldehyde) may be more strongly fixed, but limited to the concentration. It does not work.
본 발명에 있어서, 상기 조직 클리어링 용액은 N-라우로일사르코신 나트륨염 용액(N-Lauroylsarcosine sodium salt solution), CHAPS(3-[(3-콜아미도프로필)디메틸암모니오]-1-프로판설포네이트), 우레아(urea), 및 염화나트륨(NaCl)으로 이루어진 군으로부터 선택된 하나 이상을 포함할 수 있다.In the present invention, the tissue clearing solution is N-Lauroyl sarcosine sodium salt solution (N-Lauroylsarcosine sodium salt solution), CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane Sulfonate), urea (urea), and sodium chloride (NaCl).
이 때, 상기 N-라우로일사르코신 나트륨염 용액(N-Lauroylsarcosine sodium salt solution)은 농도가 1%(v/v) 내지 30%(v/v) 또는 3%(v/v) 내지 20%(v/v)일 수 있고, CHAPS(3-[(3-콜아미도프로필)디메틸암모니오]-1-프로판설포네이트)는 농도가 10%(w/v) 내지 40%(w/v) 또는 10%(w/v) 내지 30%(w/v)일 수 있고, 상기 우레아(urea)는 농도가 30%(w/v) 내지 70%(w/v) 또는 40%(w/v) 내지 60%(w/v)일 수 있고, 상기 염화나트륨(NaCl)은 농도가 0.001%(w/v) 내지 1%(w/v) 또는 0.01%(w/v) 내지 1%(w/v)일 수 있으며, 본 발명의 구체적인 실시예에 따르면 상기 N-라우로일사르코신 나트륨염 용액(N-Lauroylsarcosine sodium salt solution)은 4%(v/v) 또는 15%(v/v), CHAPS는 20%(w/v), 우레아는 50%(w/v)일 수 있고, 염화나트륨은 농도를 0.1%(w/v) 내지 0.5%(w/v)로 하여 삼투압에 의한 조직의 변형과 ion strength를 안정화시켜 시료에 있는 형광 물질의 변성을 최소화 할 수 있으나, 상기 농도에 제한되는 것은 아니다.In this case, the N-Lauroylsarcosine sodium salt solution has a concentration of 1% (v/v) to 30% (v/v) or 3% (v/v) to 20. % (v/v), and CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) has a concentration of 10% (w/v) to 40% (w/ v) or 10% (w/v) to 30% (w/v), wherein the urea has a concentration of 30% (w/v) to 70% (w/v) or 40% (w) /v) to 60% (w/v), and the concentration of sodium chloride (NaCl) is 0.001% (w/v) to 1% (w/v) or 0.01% (w/v) to 1% ( w/v), and according to a specific embodiment of the present invention, the N-Lauroylsarcosine sodium salt solution is 4% (v/v) or 15% (v/v) ), CHAPS may be 20% (w/v), urea may be 50% (w/v), and sodium chloride has a concentration of 0.1% (w/v) to 0.5% (w/v), resulting in osmotic pressure. Stabilization and ion strength can be stabilized to minimize denaturation of the fluorescent material in the sample, but is not limited to the concentration.
본 발명에 있어서, 상기 (d) 조직 클리어링 용액을 이용하는 투명화 단계는 4%(v/v)의 N-라우로일사르코신 나트륨염 용액(N-Lauroylsarcosine sodium salt solution)을 포함한 조직 클리어링 용액을 이용하는 제 1 투명화 단계, 또는 15%(v/v) 의 N-라우로일사르코신 나트륨염 용액(N-Lauroylsarcosine sodium salt solution)을 포함한 조직 클리어링 용액을 이용하는 제 2 투명화 단계를 포함할 수 있으며, 본 발명의 구체적인 실시예에 따르면, 상기 제 1 투명화 단계 이후 세척 단계, 제 2 투명화 단계, 및 세척 단계를 순서대로 실시할 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the step (d) of the clearing using the tissue clearing solution uses a tissue clearing solution including 4% (v/v) of N-Lauroylsarcosine sodium salt solution. The first clearing step, or may include a second clearing step using a tissue clearing solution comprising 15% (v/v) of N-Lauroylsarcosine sodium salt solution. According to a specific embodiment of the invention, after the first transparent step, the washing step, the second transparent step, and the washing step may be performed in order, but is not limited thereto.
본 발명에 있어서, 상기 세척 용액은 인산완충식염수(phosphate buffer saline; PBS) 및 아지드화나트륨(sodium azide)을 포함할 수 있으며, 상기 아지드화나트륨(sodium azide)은 농도가 0.001%(w/v) 내지 0.5%(w/v) 또는 0.01%(w/v) 내지 0.5%(w/v)일 수 있고, 본 발명의 구체적인 실시예에 따르면, 상기 아지드화나트륨(sodium azide)은 농도를 0.1%(w/v)로 하여 투명화 후 탈수된 생체조직 시료에 최대 30%까지 함수율을 증가시킨 후 15%를 탈수하여 조직에 붙어있는 이미징에 방해되는 유기물을 세척할 수 있으나, 상기 농도에 제한되지는 않는다.In the present invention, the washing solution may include phosphate buffered saline (PBS) and sodium azide, and the sodium azide has a concentration of 0.001% (w /v) to 0.5% (w/v) or 0.01% (w/v) to 0.5% (w/v), according to a specific embodiment of the present invention, the sodium azide (sodium azide) is After clearing the concentration to 0.1% (w/v), after increasing the water content up to 30% in a dehydrated biotissue sample, dehydrating 15% to wash the organic matter that interferes with imaging attached to the tissue, but the concentration It is not limited to.
본 발명에 있어서, 상기 마운팅 용액은 CHAPS(3-[(3-콜아미도프로필)디메틸암모니오]-1-프로판설포네이트), 우레아(urea), 및 염화나트륨(NaCl)으로 이루어진 군으로부터 선택된 하나 이상을 포함할 수 있다.In the present invention, the mounting solution is one selected from the group consisting of CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), urea, and sodium chloride (NaCl). It may include the above.
이 때, 상기 CHAPS(3-[(3-콜아미도프로필)디메틸암모니오]-1-프로판설포네이트)는 농도가 20%(w/v) 내지 60%(w/v) 또는 30%(w/v) 내지 50%(w/v)일 수 있고, 상기 우레아(urea)는 농도가 10%(w/v) 내지 50%(w/v) 또는 20%(w/v) 내지 50%(w/v)일 수 있고, 상기 염화나트륨(NaCl)은 농도가 0.001%(w/v) 내지 3% 또는 0.01%(w/v) 내지 2%일 수 있으며, 본 발명의 구체적인 실시예에 따르면 상기 마운팅 용액의 굴절률을 1.45로 맞추기 위해 상기 CHAPS 및 우레아의 조성은 각각 40%(w/v), 40%(w/v)로 포함할 수 있으며, 염화나트륨(NaCl)의 농도를 0.1%(w/v) 내지 1%(w/v)로 포함할 수 있으나, 상기 농도에 제한되는 것은 아니다.At this time, the concentration of the CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) is 20% (w/v) to 60% (w/v) or 30% ( w/v) to 50% (w/v), and the urea has a concentration of 10% (w/v) to 50% (w/v) or 20% (w/v) to 50%. (w/v), the concentration of sodium chloride (NaCl) may be 0.001% (w/v) to 3% or 0.01% (w/v) to 2%, according to a specific embodiment of the present invention In order to set the refractive index of the mounting solution to 1.45, the composition of the CHAPS and urea may include 40% (w/v) and 40% (w/v), respectively, and the concentration of sodium chloride (NaCl) is 0.1% (w /v) to 1% (w/v), but is not limited to the concentration.
본 발명의 또 다른 구현예로서, 상기 (g) 단계는 이마리스(Imaris) 프로그램을 이용하는 것일 수 있다.As another embodiment of the present invention, step (g) may be to use an Imaris program.
본 발명의 일 실시예에서는, 암 동물모델의 암 조직을 투명화하기 위한 용액을 제조하고, 암 세포를 이식한 동물의 암 조직에서의 신생혈관을 염색한 후 상기 용액으로 암 조직을 투명화하였다(실시예 1 참조).In one embodiment of the present invention, a solution for clearing cancer tissue of a cancer animal model is prepared, and after the new blood vessels are stained in the cancer tissue of an animal transplanted with cancer cells, the cancer tissue is cleared with the solution (implementation) See example 1).
본 발명의 다른 실시예에서는, 이마리스(Imaris) 프로그램을 이용하여 상기 투명화한 암 조직에서 신생혈관의 길이 및 분포를 관찰하고, 이를 정량적으로 분석하였으며, 이를 통해 암 조직에서의 신생혈관 형성을 확인할 수 있었다(실시예 2 참조).In another embodiment of the present invention, the length and distribution of new blood vessels were observed in the cleared cancer tissue using the Imaris program and quantitatively analyzed, thereby confirming the formation of new blood vessels in the cancer tissue. It was possible (see Example 2).
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred embodiments are provided to help understanding of the present invention. However, the following examples are only provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.
실시예Example 1. 암 조직에서 형성된 혈관 염색 및 암 조직 투명화 1. Vascular staining formed in cancer tissue and clearing cancer tissue
1-1. 고정 용액, 조직 1-1. Fixed solution, tissue 클리어링Clearing 용액, 세척 용액, 및 Solution, washing solution, and 마운팅Mounting 용액의 제조 Preparation of solution
우선, 암 동물모델의 암 조직을 투명화시키기 위해 필요한 고정 용액, 조직 클리어링 용액, 세척 용액, 및 마운팅 용액을 제조하였으며, 상기 용액의 구성 성분은 하기 표 1에 나타내었다.First, a fixation solution, a tissue clearing solution, a washing solution, and a mounting solution required to clear the cancer tissue of the cancer animal model were prepared, and the components of the solution are shown in Table 1 below.
구체적으로, 생체조직 시료를 탈수시키고 PFA(paraformaldehyde)로 유기물간 공유결합된 시료를 좀 더 강하게 고정시키기 위해, 농도가 20%(w/v) 이상인 수크로오스(sucrose)를 구성성분으로 하는 고정 용액(fixing solution)을 제조하였다. Specifically, in order to dehydrate the biological tissue sample and to more strongly fix the sample covalently bound between organic materials with PFA (paraformaldehyde), a fixed solution containing sucrose having a concentration of 20% (w/v) or more as a component ( fixing solution).
또한, 삼투압에 의한 조직의 변형과 ion strength를 안정화시켜 생체조직 시료에 있는 형광 물질의 변성을 최소화 하기 위해, 4%(v/v) 또는 15%(v/v) 농도의 N-Lauroylsarcosine sodium salt solution, 20%(w/v) CHAPS(3-[(3-콜아미도프로필)디메틸암모니오]-1-프로판설포네이트) 및 50%(w/v) 우레아(urea)에 0.1 내지 0.5%(w/v) 농도의 염화나트륨(NaCl)을 추가하여, 본 발명의 조직 클리어링 용액(tissue clearing solution)을 제조하였다. In addition, N-Lauroylsarcosine sodium salt at a concentration of 4% (v/v) or 15% (v/v) to minimize the degeneration of the fluorescent substance in the biological tissue sample by stabilizing the tissue deformation and ion strength by osmotic pressure. solution, 0.1% to 0.5% in 20% (w/v) CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) and 50% (w/v) urea By adding (w/v) concentration of sodium chloride (NaCl), a tissue clearing solution of the present invention was prepared.
이어서, 투명화 후 탈수된 생체조직 시료에 최대 30%까지 함수율을 증가시킨 후 15%를 탈수하여 조직에 붙어있는 이미징에 방해되는 유기물을 세척시킬 수 있도록 인산완충식염수(phosphate buffer saline; PBS)에 0.1%(w/v)의 아지드화나트륨(sodium azide)을 첨가하여 세척 용액(washing solution)을 제조하였다. Subsequently, 0.1% in phosphate buffer saline (PBS) to increase the water content to a maximum of 30% in dehydrated biotissue samples after clarification and then dehydrate 15% to wash organic matters that interfere with imaging attached to the tissues. A washing solution was prepared by adding% (w/v) sodium azide.
또한, 40%(w/v)의 CHAPS(3-[(3-콜아미도프로필)디메틸암모니오]-1-프로판설포네이트), 40%(w/v) 우레아(urea), 및 0.1 내지 1%(w/v) NaCl로 마운팅 용액(mounting solution)을 제조하여 굴절률을 1.45로 맞추었다.In addition, 40% (w/v) of CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), 40% (w/v) urea, and 0.1 to A mounting solution was prepared with 1% (w/v) NaCl to adjust the refractive index to 1.45.
구성Configuration 구성 성분Composition
1One Fixing solutionFixing solution Sucrose(20%(w/v)이상)Sucrose (more than 20% (w/v))
22 Tissue clearing solution Tissue clearing solution N-Lauroylsarcosine sodium salt solution(4%(v/v) 또는 15%(v/v)), CHAPS(20%(w/v)), Urea(50%(w/v)), NaCl(0.1 내지 0.5%(w/v))N-Lauroylsarcosine sodium salt solution (4% (v/v) or 15% (v/v)), CHAPS (20% (w/v)), Urea (50% (w/v)), NaCl (0.1 to 0.5% (w/v))
33 Washing solutionWashing solution PBS, sodium azied(0.1%(w/v))PBS, sodium azied (0.1% (w/v))
44 Mounting solutionMounting solution CHAPS(40%(w/v)), Urea(40%(w/v)), NaCl(0.1 내지 1%(w/v))CHAPS (40% (w/v)), Urea (40% (w/v)), NaCl (0.1 to 1% (w/v))
1-2. 암 조직에서 형성된 혈관 염색 및 암 조직 투명화1-2. Vascular staining formed in cancer tissue and clearing cancer tissue
HT-29 colon cancer cell을 배양한 배양액 및 상기 암 세포를 기반으로 하는 스페로이드를 각각 누드 마우스에 이식하고 4주간 키운 후 렉틴(lectin)을 cardiac injection 하고 perfusion하여 형성된 암 조직 샘플을 채취하였다. 상기로부터 채취한 암 조직 샘플은 상기 실시예 1-1에서 제조한 용액(표 1 참조)을 사용하여 투명화 하였다.A culture medium in which HT-29 colon cancer cells were cultured and a spheroid based on the cancer cells were transplanted into nude mice, grown for 4 weeks, and then lectin cardiac injection and perfusion were performed to collect cancer tissue samples. Cancer tissue samples collected from the above were clarified using the solution prepared in Example 1-1 (see Table 1).
구체적으로, 상기 암 조직 시료를 고정 용액(fixing solution)에 넣고 4℃/50 rpm에서 침전될 때까지 shaking incubation 하고, 침전된 암 조직 시료를 4%(v/v) N-Lauroylsarcosine sodium salt solution을 포함하는 조직 클리어링 용액(tissue clearing solution)에 넣고 35℃/50 rpm으로 36시간 동안 shaking incubation하고, 동일한 조건으로 1회 반복하였다.Specifically, the cancer tissue sample is placed in a fixing solution, shaking incubation until precipitation at 4°C/50 rpm, and the precipitated cancer tissue sample is 4% (v/v) N-Lauroylsarcosine sodium salt solution. It was put into a tissue clearing solution containing it, shaking incubation for 36 hours at 35°C/50 rpm, and repeated once under the same conditions.
상기 반응이 끝난 암 조직 시료에 50ml의 세척 용액(washing solution)을 넣고 4℃/50 rpm에서 4시간 동안 shaking incubation한 후(3회 반복), 15%(v/v)의 N-Lauroylsarcosine sodium salt solution을 포함하는 조직 클리어링 용액(tissue clearing solution)에 넣고 상기와 동일하게 shaking incubation 하고, 이를 세척 용액(washing solution)으로 다시 washing하였다. After 50 ml of washing solution was added to the cancer tissue sample after the reaction, shaking incubation was performed at 4°C/50 rpm for 4 hours (repeat 3 times), and 15% (v/v) of N-Lauroylsarcosine sodium salt It was put into a tissue clearing solution containing a solution, shaking incubation as described above, and then washed again with a washing solution.
상기 washing이 끝난 암 조직 시료는 마운팅 용액(mounting solution)에 넣고 35℃/50 rpm으로 12시간 shaking incubation 하고, 동일한 조건으로 1회 반복하였다.The washed cancer tissue sample was placed in a mounting solution, shaking incubation at 35°C/50 rpm for 12 hours, and repeated once under the same conditions.
그런 다음, 상기 마운팅 용액으로 처리한 암 조직 시료를 1200 rpm에서 30분 동안 centrifuge하여 시료 내 bubble을 제거하고, 암 조직 시료를 image chamber에서 보관하거나 1X PBS에 4℃에서 보관하였다.Then, the cancer tissue sample treated with the mounting solution was centrifuge for 30 minutes at 1200 rpm to remove bubbles in the sample, and the cancer tissue sample was stored in an image chamber or stored at 4°C in 1X PBS.
실시예Example 2. 2. 이마리스Imaris (( ImarisImaris ) 프로그램을 통한 신생혈관 길이 및 분포 분석) Analysis of new blood vessel length and distribution through the program
lightsheet 현미경으로 상기 실시예 1-2의 방법으로 투명화 시킨 전체 암 조직에서 형성된 혈관을 관찰하고, 이마리스(Imaris) 3D 프로그램을 이용하여 신생혈관의 길이 및 분포를 관찰하였다.The blood vessels formed in the entire cancer tissue that was made transparent by the method of Example 1-2 were observed with a lightsheet microscope, and the length and distribution of new blood vessels were observed using an Imaris 3D program.
구체적으로, lightsheet 현미경으로 관찰한 암 조직에서의 신생혈관 3차원 이미지에서 Imaris filaments를 이용하여 분석하고자 하는 영역을 설정한 후 혈관의 시작점(도 2에 나타낸 파란색 점), 분지점(도 2에 나타낸 붉은색 점), 끝점(도 2에 나타낸 녹색 점)을 구분하였다. 상기에서 선택한 영역과 3D 이미지의 매칭을 확인하고, 영상의 형광량을 분석하여 혈관의 지름 길이를 정확하게 관찰할 수 있었다.Specifically, after setting the region to be analyzed using Imaris filaments in a new blood vessel 3D image in cancer tissue observed with a lightsheet microscope, the starting point (blue dot shown in FIG. 2) and the branching point (shown in FIG. 2) Red dot) and end point (green dot shown in FIG. 2) were distinguished. The matching of the 3D image with the region selected above was confirmed, and the fluorescence amount of the image was analyzed to accurately observe the length of the diameter of the blood vessel.
그 결과, HT-29 colon cancer cell을 배양한 배양액을 이식한 마우스의 암 조직에서 형성된 혈관의 이미지는 도 1과 같이 나타났으며, HT-29 colon cancer cell을 기반으로 하는 스페로이드를 이식한 마우스의 암 조직에서 형성된 혈관의 이미지는 도 2와 같이 나타나, 스페로이드를 이식한 마우스의 암 조직에서 더 선명하고 정밀한 결과를 얻을 수 있었다.As a result, an image of blood vessels formed in cancer tissue of a mouse transplanted with a culture medium in which HT-29 colon cancer cells were cultured was shown in FIG. 1, and a mouse implanted with spheroid based on HT-29 colon cancer cells The image of blood vessels formed in the cancer tissues of FIG. 2 was shown, and a sharper and more precise result could be obtained from the cancer tissues of the mice implanted with spheroids.
또한, 스페로이드를 이식한 마우스의 암 조직에서 이마리스 프로그램을 사용하여 신생혈관의 분포를 정량적으로 분석하여 평균, 중앙값, 및 합계를 도 2에 나타내었다.In addition, the distribution of new blood vessels was quantitatively analyzed using the Imaris program in cancer tissues of mice transplanted with spheroids, and the mean, median, and total are shown in FIG. 2.
상기와 같이 암 조직에서 신생혈관의 길이 및 분포를 관찰함으로써, 신생혈관 형성을 분석하여 항암 약물의 신생혈관 형성 억제 효능을 효과적으로 평가할 수 있을 것으로 예상되었다.By observing the length and distribution of angiogenesis in cancer tissues as described above, it was expected that angiogenesis could be analyzed to effectively evaluate the efficacy of anticancer drugs to inhibit angiogenesis.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야 한다.The above description of the present invention is for illustration only, and those skilled in the art to which the present invention pertains can understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and not restrictive.
본 발명에 따른 암 조직에서의 신생혈관 형성을 평가하는 방법은 암 조직에서 형성되는 혈관의 길이 및 분포를 효과적으로 분석하고 정량화하여 암 조직에서의 신생혈관 형성을 평가하는데 이용할 수 있으며, 암 치료용 약물의 신생혈관 형성 억제 효능을 평가하는 데에도 유용하게 이용할 수 있을 것으로 기대된다.The method for evaluating the formation of new blood vessels in cancer tissue according to the present invention can be used to evaluate the formation and development of new blood vessels in cancer tissue by effectively analyzing and quantifying the length and distribution of blood vessels formed in the cancer tissue. It is expected to be useful in evaluating the efficacy of inhibiting angiogenesis.

Claims (12)

  1. 하기 단계를 포함하는, 암 조직에서의 신생혈관 형성 평가 방법:A method for evaluating angiogenesis in cancer tissue comprising the following steps:
    (a) 암 세포를 배양한 배양액 또는 상기 암 세포를 기반으로 하는 스페로이드를 동물에 이식하여 암 조직을 형성하는 단계;(a) transplanting a culture medium in which cancer cells are cultured or a spheroid based on the cancer cells into an animal to form cancer tissue;
    (b) 상기 암 조직이 형성된 동물에 렉틴(lectin)을 주입하여 혈관을 염색하고 암 조직을 채취하는 단계;(b) injecting lectin into the animal on which the cancer tissue is formed, staining blood vessels and collecting cancer tissue;
    (c) 고정 용액으로 상기 동물의 암 조직 시료를 고정시키는 단계;(c) immobilizing the cancer tissue sample of the animal with a fixation solution;
    (d) 조직 클리어링 용액으로 상기 고정된 시료에 있는 형광물질의 변성을 최소화하는 투명화 단계;(d) a clearing step to minimize denaturation of the fluorescent material in the fixed sample with a tissue clearing solution;
    (e) 세척 용액으로 상기 형광물질의 변성을 최소화한 시료에 부착되어 있는 유기물을 씻어내는 세척 단계; (e) a washing step of washing the organic matter attached to the sample with minimal denaturation of the fluorescent material with a washing solution;
    (f) 마운팅 용액으로 상기 세척된 시료를 고정시키는 단계; 및(f) fixing the washed sample with a mounting solution; And
    (g) 상기 (a) 내지 (f) 단계를 거친 암 조직 시료에서 형성된 혈관의 길이 및 분포를 분석하는 단계.(G) analyzing the length and distribution of blood vessels formed in the cancer tissue samples subjected to the steps (a) to (f).
  2. 제1항에 있어서,According to claim 1,
    상기 고정 용액은 수크로오스(sucrose)를 포함하는 것을 특징으로 하는, 암 조직에서의 신생혈관 형성 평가 방법.The fixed solution is characterized in that it contains sucrose (sucrose), a method for evaluating angiogenesis in cancer tissue.
  3. 제2항에 있어서,According to claim 2,
    상기 수크로오스(sucrose)는 농도가 20%(w/v) 내지 100%(w/v)인 것을 특징으로 하는, 암 조직에서의 신생혈관 형성 평가 방법.The sucrose (sucrose) is characterized in that the concentration is 20% (w / v) to 100% (w / v), the method of evaluating angiogenesis in cancer tissue.
  4. 제1항에 있어서,According to claim 1,
    상기 조직 클리어링 용액은 N-라우로일사르코신 나트륨염 용액(N-Lauroylsarcosine sodium salt solution), CHAPS(3-[(3-콜아미도프로필)디메틸암모니오]-1-프로판설포네이트), 우레아(urea), 및 염화나트륨(NaCl)으로 이루어진 군으로부터 선택된 하나 이상을 포함하는 것을 특징으로 하는, 암 조직에서의 신생혈관 형성 평가 방법.The tissue clearing solution is N-Lauroylsarcosine sodium salt solution, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), urea (urea), and sodium chloride (NaCl), characterized in that it comprises at least one selected from the group consisting of, neovascularization evaluation method in cancer tissue.
  5. 제4항에 있어서,According to claim 4,
    상기 N-라우로일사르코신 나트륨염 용액(N-Lauroylsarcosine sodium salt solution)은 농도가 1%(v/v) 내지 30%(v/v)이고, CHAPS(3-[(3-콜아미도프로필)디메틸암모니오]-1-프로판설포네이트)는 농도가 10%(w/v) 내지 40%(w/v)이고, 상기 우레아(urea)는 농도가 30%(w/v) 내지 70%(w/v)이고, 상기 염화나트륨(NaCl)은 농도가 0.001%(w/v) 내지 1%(w/v)인 것을 특징으로 하는, 암 조직에서의 신생혈관 형성 평가 방법.The N-Lauroylsarcosine sodium salt solution has a concentration of 1% (v/v) to 30% (v/v), and CHAPS(3-[(3-Colamido Propyl)dimethylammonio]-1-propanesulfonate) has a concentration of 10% (w/v) to 40% (w/v), and the urea has a concentration of 30% (w/v) to 70 % (w/v), and the sodium chloride (NaCl) concentration is 0.001% (w/v) to 1% (w/v), characterized in that the method of evaluating angiogenesis in cancer tissue.
  6. 제1항에 있어서,According to claim 1,
    상기 세척 용액은 인산완충식염수(phosphate buffer saline; PBS) 및 아지드화나트륨(sodium azide)을 포함하는 것을 특징으로 하는, 암 조직에서의 신생혈관 형성 평가 방법.The washing solution is characterized in that it comprises a phosphate buffer saline (PBS) and sodium azide (sodium azide), a method for evaluating the formation of new blood vessels in cancer tissue.
  7. 제6항에 있어서,The method of claim 6,
    상기 아지드화나트륨(sodium azide)은 농도가 0.001%(w/v) 내지 0.5%(w/v)인 것을 특징으로 하는, 암 조직에서의 신생혈관 형성 평가 방법.The sodium azide (sodium azide) has a concentration of 0.001% (w/v) to 0.5% (w/v), characterized in that the method of evaluating angiogenesis in cancer tissue.
  8. 제1항에 있어서,According to claim 1,
    상기 마운팅 용액은 CHAPS(3-[(3-콜아미도프로필)디메틸암모니오]-1-프로판설포네이트), 우레아(urea), 및 염화나트륨(NaCl)으로 이루어진 군으로부터 선택된 하나 이상을 포함하는 것을 특징으로 하는, 암 조직에서의 신생혈관 형성 평가 방법.The mounting solution comprises one or more selected from the group consisting of CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), urea, and sodium chloride (NaCl) A method for evaluating angiogenesis in cancer tissue.
  9. 제8항에 있어서,The method of claim 8,
    상기 CHAPS(3-[(3-콜아미도프로필)디메틸암모니오]-1-프로판설포네이트)는 농도가 20%(w/v) 내지 60%(w/v)이고, 상기 우레아(urea)는 농도가 10%(w/v) 내지 50%(w/v)이고, 상기 염화나트륨(NaCl)은 농도가 0.001%(w/v) 내지 3%인 것을 특징으로 하는, 암 조직에서의 신생혈관 형성 평가 방법.The CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) has a concentration of 20% (w/v) to 60% (w/v), and the urea Is a concentration of 10% (w / v) to 50% (w / v), the sodium chloride (NaCl) is characterized in that the concentration is 0.001% (w / v) to 3%, neovascularization in cancer tissue Formation evaluation method.
  10. 제1항에 있어서,According to claim 1,
    상기 (g) 단계는 이마리스(Imaris) 프로그램을 이용하는 것을 특징으로 하는, 암 조직에서의 신생혈관 형성 평가 방법.The (g) step, characterized in that using the Imaris (Imaris) program, a method for evaluating angiogenesis in cancer tissue.
  11. 하기 단계를 포함하는, 암 예방 또는 치료용 후보물질의 스크리닝 방법:A screening method for candidates for preventing or treating cancer, comprising the following steps:
    (a) 암 동물모델에 후보물질을 처리하는 단계;(a) treating the candidate material in a cancer animal model;
    (b) 상기 암 동물모델에 렉틴(lectin)을 주입하여 혈관을 염색하고 암 조직을 채취하는 단계;(b) injecting lectin into the cancer animal model, dyeing blood vessels and collecting cancer tissue;
    (c) 상기 암 조직을 투명화 시키는 단계;(c) making the cancer tissue transparent;
    (d) 이마리스(Imaris) 프로그램을 통해 상기 투명화된 암 조직에서 형성된 혈관의 길이 및 분포를 분석하여 암 조직에서의 신생혈관 형성을 평가하는 단계; 및(d) analyzing the length and distribution of blood vessels formed in the cleared cancer tissue through the Imaris program to evaluate the formation of new blood vessels in the cancer tissue; And
    (e) 상기 암 조직에서의 신생혈관 형성이 후보물질을 처리하기 전에 비해 억제된 경우 암 예방 또는 치료용 후보물질로 선별하는 단계.(e) When the formation of new blood vessels in the cancer tissue is suppressed compared to before treatment of the candidate substance, selecting as a candidate substance for preventing or treating cancer.
  12. 제11항에 있어서,The method of claim 11,
    상기 암 조직을 투명화 시키는 단계는 하기 단계를 포함하는 것을 특징으로 하는, 스크리닝 방법:Screening method, characterized in that the step of clearing the cancer tissue comprises the following steps:
    (가) 고정 용액으로 동물의 암 조직 시료를 고정시키는 단계;(A) fixing the cancer tissue sample of the animal with a fixed solution;
    (나) 조직 클리어링 용액으로 상기 고정된 시료에 있는 형광물질의 변성을 최소화하는 투명화 단계;(B) a clearing step of minimizing denaturation of the fluorescent substance in the fixed sample with a tissue clearing solution;
    (다) 세척 용액으로 상기 형광물질의 변성을 최소화한 시료에 부착되어 있는 유기물을 씻어내는 세척 단계; 및(C) a washing step of washing the organic matter attached to the sample with minimal degeneration of the fluorescent material with a washing solution; And
    (라) 마운팅 용액으로 상기 세척된 시료를 고정시키는 단계.(D) fixing the washed sample with a mounting solution.
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