KR20180130326A - Method for producing xenograft model derived from a patient of breast cancer - Google Patents

Abstract

It is an object of the present invention to provide a method of producing a breast cancer patient-derived tumor tissue xenograft model and a breast cancer patient-derived tumor tissue xenograft animal model transplanted with breast cancer tissue derived from a breast cancer (Leu1780Pro mutant) patient into an immunodeficient mouse. It is another object of the present invention to provide an information providing method for selecting a therapeutic breast cancer-tailored breast cancer patient using the animal model. The mammalian tumor xenograft model of the present invention can provide the same conditions for genetic, physiological, and environmental characteristics of cancer in Korean specific breast cancer patients. Therefore, screening for candidate anticancer drugs and breast cancer treatment in breast cancer patients Is expected to be greatly utilized.

Description

TECHNICAL FIELD The present invention relates to a method for producing a xenograft model derived from a breast cancer patient,

The present invention relates to a method for the preparation of a xenograft model derived from a breast cancer patient tumor using a breast cancer mutant (Leu1780Pro mutant).

Breast cancer refers to a mass consisting of cancerous cells in the breast, and generally refers to cancers arising from the breast ducts and lobes. Normal breast tissue consists of fat, connective tissue, and lymphatic ducts that support the mammary gland and mammary gland. The mammary gland consists of a lobster that produces milk, and a duct that connects the lobes to the nipple. Breast cancer can occur anywhere in the breast tissue and is more diverse than other cancers. Most of the breast cancers originate from the ductal and lobular cells, especially ductal epithelial cells. Breast cancer, like other cancers, can be transmitted systemically along the bloodstream and lymphatic system, without proper treatment, resulting in serious consequences.

According to the International Cancer Study in 2014, the incidence of cancer is higher in high-income countries, and Korea is classified as a high-income country along with North America and Western Europe. Breast cancer is a rapidly growing disease worldwide, with a 20% increase in its incidence in 2012 compared to 2008. Therefore, it is necessary to develop accurate diagnosis and treatment of breast cancer.

Breast cancer treatment will be applied with appropriate treatment methods such as surgery, radiotherapy, chemotherapy, endocrine therapy, and target treatment considering the age, stage, pathologic characteristics of the cancer, and psychological state of the patient. Recently, in order to treat with chemotherapy, PDX model (patient-derived xenograft model) using patient's cancer tissue has been actively developed in various pharmaceutical companies and laboratories (domestic patent application 2015-0008019). The model is suitable for assessing chemical sensitivity in vivo and is a good preclinical model to select the most appropriate treatment for each patient among various anticancer drugs. However, the development of the PDX model derived from Korean patients with breast cancer has not been developed yet.

Accordingly, the present inventors have completed the present invention by studying a method of producing a PDX animal model for cancer chemotherapy by transplanting a breast cancer tissue having a Korean specific breast cancer mutation isolated from breast cancer patients into a mouse.

It is an object of the present invention to provide a method for producing an animal model for chemotherapy by implanting a breast cancer tissue by a Korean specific breast cancer mutant obtained from breast cancer patients (Leu1780Pro mutant) into a mouse. Further, the present invention provides a method for screening candidate anticancer drug candidates using the same.

Hereinafter, various embodiments described herein will be described with reference to the drawings. In the following description, for purposes of complete understanding of the present invention, various specific details are set forth, such as specific forms, compositions and processes, and the like. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well-known processes and techniques of manufacture are not described in any detail, in order not to unnecessarily obscure the present invention. Reference throughout this specification to " one embodiment " or " embodiment " means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Accordingly, the appearances of the phrase " in one embodiment " or " an embodiment " in various places throughout this specification are not necessarily indicative of the same embodiment of the present invention. In addition, a particular feature, form, composition, or characteristic may be combined in any suitable manner in one or more embodiments.

Unless defined otherwise in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, " cancer " is characterized by uncontrolled cell growth, which causes abnormal cell growth to form a cell mass called a tumor that penetrates into surrounding tissues and, in severe cases, It says. Academicly, it is also called neoplasm. Cancer is an intractable chronic disease that, even if treated with surgery, radiation, and chemotherapy, does not result in fundamental healing, causes pain to the patient, and ultimately leads to death. There are various causes of cancer, And external factors. It is not known exactly how the normal cells transform into cancer cells through the mechanism, but it is known that a large number of cancers are affected by external factors such as environmental factors. Internal factors include genetic factors, immunological factors, and external factors include chemicals, radiation, and viruses. The genes involved in the development of cancer include oncogenes and tumor suppressor genes. Cancer occurs when the balance between them is broken down by internal or external embryos as described above.

As used herein, breast cancer is a human and other mammalian disease. Although the most common example in human beings is women, breast cancer may occasionally occur in men. In addition, breast cancer refers to a clinical diagnosis of breast cancer and includes all specific sub-phenotypes of breast cancer. For example, the breast cancer may be selected from the group consisting of Ductal Carcinoma In Situ (DCIS), microinvasive ductal carcinoma, invasive ductal carcinoma (IDC), aqueous carcinoma, invasive lobular carcinoma, ductal carcinoma, , Breast epithelial carcinoma, male breast cancer, follicular tumors of the breast, recurrent and metastatic breast cancer, or a combination thereof. Breast cancer is divided into stages 0, 1, 2, 3 and 4 according to the progression. The stage of breast cancer is determined by tumor size, axillary lymph node metastasis, and systemic metastasis including bone and lung involvement including neck lymph node metastasis. These three factors are important factors in determining prognosis. Breast cancer is a non-invasive breast cancer (cancer of the epithelium). Cancer cells are localized within the epithelium. The first stage of breast cancer is a tumor with a size of less than 2 cm and no lymph node metastasis. For example. Breast cancer stage 1 is an early stage of cancer progression, so there is less recurrence and metastasis, very high survival rate. Breast cancer stage 2 refers to the case when the tumor size is 2 ~ 5cm but not the lymph node metastasis or the lymph node metastasis but the tumor size is more than 5cm. The stage 3 breast tumor is less than 5cm in size but the lymph node metastasis is severe or the tumor size A case of large lymph node metastasis greater than 5 cm. The fourth stage of breast cancer, also called terminal stage, refers to cases of systemic metastasis such as lungs, bones, and liver. Breast cancer includes all aggressive breast cancer and cancer subtypes, including but not limited to triple negative breast cancer, grade 2 breast cancer, grade 3 breast cancer, lymph node positive (LN +) breast cancer, HER2 positive (HER2 +) and ER positive .

In the present specification, the term "cancer tissue section" refers to a section obtained from the cancer tissue to be discarded after the incision is performed when cancer incision is required for cancer patients for the purpose of benefiting the human body. By using the cancer tissue section, a customized therapeutic agent for a specific cancer tissue can be developed for the purpose of benefiting the human body of a cancer patient.

In the present specification, the term " metastasis " means that cancer cells leave the primary organs and go to other organs. The spread of cancer to other parts of the body is largely divided into the development of cancerous tissue in primary cancer, direct invasion of the surrounding organs, and distant metastasis along the blood vessels or lymph vessels to other organs far away. Metastasis can be regulated by inhibiting the expression of a gene associated with cancer development or inhibiting the protein activity of the gene.

As used herein, the term " neovascular " means a cell phenomenon in which vascular endothelial cells proliferate and reconstitute to form new blood vessels from an existing vascular network. In order for cancer cells to proliferate and grow, new blood vessels are required to supply oxygen and nutrients, so that inhibition of neovascularization can lead to cancer proliferation and inhibition of metastasis.

As used herein, the term "xenotransplantation" means a method of transplanting organs, organs, organs, tissues, cells, etc., of liver, heart, and kidney of another animal. For the purpose of the present invention, the xenotransplantation may be understood as a method of transplanting cancer cells isolated from a patient into an immunodeficient animal, but the present invention is not limited thereto.

As used herein, the term " immunodeficient mouse " refers to an animal model produced by manipulating some components constituting the immune system to artificially damage at the genetic level so that a normal immune system can not be realized. The immunodeficient animal including the immunodeficient mouse can be an animal in which a nervous system is formed. Preferably, an immunodeficient mammal can be used. More preferably, a mouse, a rat, a hamster, a guinea pig (NOD / shi-SCID / IL2R [gamma] null) mouse, NOD (non-obese diabetic) mouse, SCID Mouse, and the like, but are not particularly limited thereto.

As used herein, the term "patient-derived xenograft (PDX)" refers to a patient-customized animal model prepared by xenotransplantation of a patient-derived cancer cell or cancer tissue into an immunodeficient animal, The morphological environment is the same or similar, the genetic environment is the same or similar, and the expression characteristics of the marker proteins of cancer are the same, thereby providing conditions that directly reflect the genetic, physiological and environmental characteristics of cancer patients. Therefore, when an anticancer agent candidate determined to have anticancer effects in a patient-derived xenotransplantation animal model is administered to cancer patients who have received cancer cells or cancer tissues, the same results as those obtained by directly administering these candidate anticancer agents to patients can be obtained Therefore, the use of the patient-derived xenotransplantation animal model has an advantage that it is possible to confirm whether the anticancer drug can actually exhibit an appropriate effect on the patient.

As used herein, the term " transplantation " or " implantation culture " refers to a method of periodically transferring a cell or tissue part to a new culture container in order to continuously and continuously cultivate the cell or tissue in a healthy state, Of the culture medium is continuously cultured. As the number of cells in a cell or a tissue increases in a culture container having a limited space, it is used as a method for increasing the number of healthy cells because proliferation nutrients are consumed or pollutants accumulate after a certain period of time and cells naturally die. One passage (F1) is called a first-generation transplant (F1) in which the medium (culture container) is replaced once or the cell group is divided and cultured. For the purpose of the present invention, the culture container is a living system of immunodeficient mice, which is a nutrient for culturing cancer tissue derived from a patient who has been transplanted. Immunodeficient mice of the new individual are preferably used when transplanting the cultured cancer tissues (passages, for example F1 to F2), and are confused with the near system (such as crossbreeding or inbreeding) Is not desirable. The method of implantation can be carried out by any method known in the art without limitation, but preferably by mechanical separation or enzymatic separation.

As used herein, the term " mechanical method " or " mechanical decomposition method " means to physically or mechanically separate tissue as a cell mass, and any method known in the art may be used without limitation, ), A tissue chopper, a needle, a pipetting, an embryoid body divider (EBD), or a scrapper.

As used herein, the term " diagnosis " means identifying the presence or characteristic of a pathologic condition. For the purpose of the present invention, the diagnosis is to confirm the presence or absence of cancer and metastasis. The cancer can be diagnosed by the gross or cytological confirmation of the tissue from the cancerous or metastatic suspicious patient, and the cancerous or metastatic suspicious tissue specimen (clinically, cells, blood, sap, pleural fluid, ascites, A method for directly detecting a cancer-associated protein in the specimen, a method for detecting a cancer-associated protein in the specimen, a method for detecting a cancer-associated protein in the specimen, Cancer can be diagnosed by directly detecting nucleic acid. Diagnostic methods using antigen-antibody binding or directly detecting cancer-related proteins include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), Protein Chip (FACS), Immunoprecipitation Assay, Immunoprecipitation Assay, Ouchterlony Immunoprecipitation, Rocket Immunoelectrophoresis, (RT-PCR), competitive RT-PCR, and real-time RT-PCR are examples of methods for directly detecting a nucleic acid encoding a cancer-associated protein, (RT-PCR), RNase protection assay (RPA), northern blotting, or DNA chip. It is not.

In the present specification, the term " screening " is used to select a substance having a specific property aimed at from a candidate group made of various substances by a specific manipulation or evaluation method. For the purpose of the present invention, the screening of the present invention is a screening method in which when the cancerous growth is suppressed or the cancer is killed after administration of a candidate substance for breast cancer treatment to a cancer susceptible and cancer susceptible individual, cancer candidate It is a screening method of a therapeutic agent. Instrumental methods such as molecular biology assays, digital imaging, cytologic and histologic examinations can be used to confirm the therapeutic effect on breast cancer lesions or breast cancer cell metastasis suspect tissues.

As used herein, the term " anticancer agent candidate substance " means a substance used in screening in order to examine whether or not it has an inhibitory activity against growth or metastasis of tumor tissue. The substance may be an anticancer agent already known or may be an unknown substance. In addition, the candidate substance may include, but is not limited to, a chemical substance, DNA, siRNA, peptide, protein, antibody, natural extract and the like. A candidate substance to be analyzed by the screening method of the present invention is a single compound or a mixture (for example, a natural extract or a cell or a tissue culture) in which the compounds are combined. Candidate materials can be obtained from libraries of synthetic or natural compounds. Methods for obtaining libraries of such compounds are known in the art. Synthetic compound libraries are commercially available from Maybridge Chemical Co., Comgenex (USA), Brandon Associates (USA), Microsource (USA) and Sigma-Aldrich (USA) ) And MycoSearch (USA), but is not limited thereto. Candidate materials can be obtained by various combinatorial library methods known in the art and include, for example, biological libraries, spatially addressable parallel solid phase or solution phase libraries, Be obtained by the synthetic library method required, the 1-bead 1-compound library method, and the synthetic library method using affinity chromatography screening. Methods for synthesis of molecular libraries are described in DeWitt et al., Proc. Natl.Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994; Zuckermann et al., J. Med. Chem. 37, 2678, 1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew. Chem. Int. Ed. Engl. 33,2059,1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al., J. Med. Chem. 37, 1233, 1994, and the like. For example, anticancer candidate substances include bruceantin, pervilleine, betulinic acid,? -Lapachone, CDDO (2-cyano-3,12-dioxooleana -1,9-dien-28-oic acid, CB-Ph, obovatol, CB-Pic and Obo-Rd.

As used herein, " administration " means introducing the composition of the present invention to a patient by any appropriate method, and the administration route of the composition of the present invention may be administered through any conventional route so long as it can reach the target tissue have. But are not limited to, oral administration, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, intranasal administration, intrapulmonary administration, intrarectal administration, intraperitoneal administration, intraperitoneal administration, Do not. In the present invention, the effective amount may be determined depending on the kind of the disease, the severity of the disease, the kind and amount of the active ingredient and other ingredients contained in the composition, the type of the formulation and the age, body weight, general health condition, sex and diet, And the fraction of the composition, the duration of the treatment, the drug being co-administered, and the like. In the case of an adult, the therapeutic pharmaceutical composition may be administered once to the body in an amount of 50 ml to 500 ml, 0.1 ng / kg-10 mg / kg for a compound, 0.1 ng / kg for a monoclonal antibody to the protein, kg < / RTI > to 10 mg / kg. The administration interval may be 1 to 12 times a day, and once every 12 hours if administered 12 times a day. They may also be administered alone or in combination with other therapies known in the art, such as chemotherapeutic agents, radiation, and surgery. Other standard delivery methods such as biolistic delivery or ex vivo treatment may be used. In in vitro treatment, for example, antigen presenting cells (APCs), dendritic cells, peripheral blood mononuclear cells, or bone marrow cells may be obtained from a patient or a suitable donor and administered to the patient after being in vitro activated with the immunoconjugate have.

The present invention relates to a xenograft model derived from a breast cancer patient, which is obtained by transplanting a breast cancer tissue fragment isolated from a breast cancer patient subcutaneously in an immunodeficient mouse, wherein the breast cancer is breast cancer, breast cancer, breast cancer including Leu1780Pro mutant (rs80357474) Patient-derived xenograft animal model.

In one embodiment of the invention, the breast cancer patient is a TNBC condition or a Neo-TNBC condition. In another embodiment of the present invention, the breast cancer is not limited as long as it is preferably a breast cancer including a breast cancer mutation (Leu1780Pro mutant) or a breast cancer including a Korean specific mutation.

The present invention also relates to a method for the treatment of breast cancer, comprising the steps of: (a) dividing a breast cancer tissue isolated from a breast cancer patient into sections; And (b) transplanting the breast cancer tissue fragment of step (a) under subcutaneously in an F1 generation immunodeficient mouse, wherein the breast cancer is a breast cancer mutant Leu1780Pro mutant, (rs80357474). < / RTI > The present invention also provides a method for producing a xenotransplantation animal model derived from a breast cancer patient.

In one embodiment of the present invention, the immunodeficient mouse is NOG (NOD-SCID / IL-2Rγnull). In another embodiment of the present invention, the method further comprises the step of obtaining an implanted breast cancer tissue and re-implanting subcutaneously in a new immunodeficient mouse. In another embodiment of the invention, the breast cancer patient is a TNBC condition or a Neo-TNBC condition. In another embodiment of the present invention, the subcutaneously implanted breast cancer tissue section of F1 generation NOG (NOD-SCID / IL-2R? Null) mice further comprises growing to a size of 1500 mm ³ , wherein F2 generation NOG -SCID / IL-2R [gamma] null) further comprising the step of subcutaneously transplanted breast cancer tissue sections to a size of 1500 mm < ^{3 &} gt ;, wherein the subcutaneously injected breast cancer of F2 generation NOG (NOD-SCID / IL- And the tissue slice is grown to a size of 1500 mm < ^{3 &} gt ;. In another embodiment of the present invention, said step (a) further comprises treating with at least one compound selected from the group consisting of emptorisin B, penicillin, streptomycin and gentamicin. The compound may be a substance that is antimicrobial-treated with an antibiotic such as penicillin, streptomycin, and gentamicin, or may be a substance that treats an antifungal such as emptorisin B. Antibiotics or antifungal agents may be added in addition to the above materials. But are not limited to, those commonly used antibiotics or antifungal agents. In another embodiment of the present invention, the step (a) further comprises removing erythrocytes from the breast cancer tissue. In another embodiment of the present invention, the breast cancer is not limited as long as it is preferably a breast cancer including a breast cancer mutation (Leu1780Pro mutant) or a breast cancer including a Korean specific mutation.

Further, the present invention provides a method of treating cancer, comprising the steps of: (a) treating an animal cancer model with an anti-cancer candidate substance; And (b) selecting an anticancer candidate substance as a therapeutic agent for breast cancer of the breast cancer patient when the size of the breast cancer tissue is decreased or the breast cancer metastasis is inhibited by the therapeutic agent for breast cancer, Wherein the breast cancer is a breast cancer mutant, the breast cancer comprising the Leu1780Pro mutation (rs80357474).

In one embodiment of the invention, the breast cancer therapeutic agent is selected from the group consisting of DNA, siRNA, peptide, protein, antibody, natural extract, bruceantin, pervilleine, betulinic acid, beta-lapachone, CDDO (2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid), CB-Ph, obovatol, CB-Pic, Obo-Rd, trastuzumab and lapatinib ≪ / RTI > In another embodiment of the present invention, the therapeutic agent for breast cancer is preferably, but not limited to, any one or more selected from the group consisting of trastuzumab or lapatinib. In another embodiment of the present invention, the breast cancer is not limited as long as it is preferably a breast cancer including a breast cancer mutation (Leu1780Pro mutant) or a breast cancer including a Korean specific mutation.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for producing a xenograft animal model derived from a breast cancer patient tumor with high success rate using a breast cancer mutant (Leu1780Pro mutant). Also provided is a xenografted animal model of a breast cancer tumor tissue having the Korean specificity produced therewith. Thus, a xenograft animal model of patient-derived tumor tissue can provide conditions identical to genetic, physiological, and environmental properties of cancer in cancer patients. The patient-derived xenotransplantation animal model lacks immune function and can efficiently perform xenotransplantation from the human body. In addition, the xenotransplantation animal model derived from the breast cancer patient tumor of the present invention is an excellent animal model for studying the production, metastasis, proliferation, and the like of tumor in In vivo, and it is an effective animal model for screening of tumor therapy composition and in- And can be useful for establishing a personalized treatment strategy for each individual.

FIG. 1 is a graph showing the success rate and failure rate of F3 abnormality in TNBC patients among PDX models.
FIG. 2 is a graph showing the success rate and failure rate of F3 abnormality in Neo-TNBC patients among PDX models.
Figure 3 is a graph showing the tumor size over time for the control, carboplatin, Orafafer treatment in the breast cancer mutation (Leu1780Pro mutant).
Figure 4 is a graph showing the tumor size over time for the control, carboplatin, Orafafer treatment in a common breast cancer mutation (BRCA1 wild type).
Figure 5 is a photograph comparing tumor size for the control, carboplatin, and Orafafer treatment in the breast cancer mutation (Leu1780Pro mutant).
FIG. 6 is a photograph comparing tumor sizes for control, carboplatin, and oolafip treatment in a common breast cancer mutation (BRCA1 wild type).
Figure 7 is a graph showing the tumor size of the control and treatment groups over time in the breast cancer mutant (Leu1780Pro mutant).
Figure 8 is a graph showing the tumor size of the control and treatment groups over time in a common breast cancer mutation (BRCA1 wild type).

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example  1. Breast cancer mutation ( Leu1780Pro  mutant) confirmation

This study included at least one of the clinical features of HBOC (hereditary breast and ovarian cancer) that assessed the BRCA1 / 2 reproductive status in the Yonsei University Cancer Center from January 2008 to January 2016 Of the total. The clinical characteristics of the HBOC include: i) at least one of the first or second group associated with breast cancer and / or ovarian cancer, ii) breast cancer diagnosed before age 40, iii) bilateral breast cancer, iv) breast cancer and synchronous or retrograde ovarian cancer , And v) epithelial ovarian cancer. 745 included 596 breast tumors, 124 ovarian tumors, and 25 breast and ovarian tumors. The Helsinki Declaration on Medical Research Protocol and Ethics (World Medical Association Association of Helsinki) was maintained during the study. All participants in this study signed an informed consent form approved by the National Cancer Center Institutional Review Board (IRB No. NCCNCS 13717).

After extracting the patient genomic DNA from peripheral blood, the entire coding region of the BRCA1 and BRCA2 genes and the exon-intron boundary (+/- 20 basepairs) were analyzed by Sanger sequencing. The standard sequence used for the analysis is NM_007294.3 for BRCA1 and NM_000059.3 for BRCA2. The Mutation Technology Act complied with the naming system of the Human Genome Variation Society (http://www.hgvs.org/mutnomen). Variations were originally reported by the three-step system, and variants reported as disease-causing mutations or VUS were further evaluated and reclassified according to the ACMG guidelines as described below.

622 individuals from the Korean Standard Genome Database (KRGDB, http://152.99.75.168/KRGDB/) and 1,314 Koreans including 692 from the Therapeutic Etex Bio Institute (Suwon, Gyeonggi-do, Korea) laboratory database were searched for alleles. Missense mutation c.5339T> C (p.Leu1780Pro; rs80357474) was found to cause the amino acid substitution (Leu1780Pro) of proline to leucine in the BRCT region in the BRCA1 gene in breast cancer patients. A sufficient number of relevant population data were used to efficiently classify the mutations and identified the BRCA1 Leu1780Pro mutation as a possible pathogenic mutation in Korean patients (JNC et al., CANCER RESEARCH AND TREATMENT (CRT), https://doi.org/ 10.4143 / crt.2016.433).

The level of gene expression used herein may be an absolute or relative amount of an expressed gene or gene product comprising nucleic acids such as RNA, mRNA, cDNA and protein.

1-1. Patient Information

Patients enrolled from December 24, 2013 to October 26, 2016 were tested. This study was also conducted with the approval of the Institutional Review Committee. Based on the inclusion criteria, tumor size of 1 cm or more was identified as pathologic size based on preoperative FDG PET / CT, and full-width at half maximum (FWHM) for primary IDC and ALN metastases . The mean age of the patients was 50.5 ± 10.5 years (range: 30-76 years).

1-2. Patient-derived xenotransplantation (PDX) production

The cancer tissue obtained after surgery is placed in a medium containing three antibiotics (penicillin, streptomycin and gentamicin) and an antifungal agent (emptorisin B). The tumor tissue is washed in PBS and cut into small pieces of about ³ mm ³ . Five to ten small pieces are harvested and transplanted subcutaneously into NOD-SCID mouse or IL-2 Rγ null (F1 generation) mice. The size of the tumor is then measured twice a week using vernier calipers. When the size of the subcutaneously implanted tumor reached about 1500 mm ³ , the mice were sacrificed and the tumor was obtained and cut into 5-10 mm ³ sections, and then a new NOD-SCID mouse or IL-2 Rγ null (NOG) mouse ). The remaining sections are saved for further histological and genomic analysis. In the same way, the transplantation proceeds to the F3, F4, and F5 generations to increase the number of mice to which the tumor tissue has been transplanted.

Example 2. Characterization of patient-derived xenotransplantation animal (PDX)

2-1. First generation (F1) PDX model

NOD-SCID mouse or IL-2 Rγ null (NOG) mice are prepared. The patient - derived breast cancer tissues were engrafted from the wired fat pads. Mice without tumor growth were sacrificed for up to 6 months, and if the size of the tumor grew to 1500 mm ³ , the cancer tissues were collected again and transferred to the next generation.

2-2. Second generation (F2) PDX model

The F2 generation PDX model was prepared by re-transplanting cancer tissues collected from the first generation into NOD-SCID mouse or IL-2 Rγ null (NOG) mice. If the size of the transplanted tumor grew to 1500 mm ³ , Respectively.

2-3. Third generation (F3) PDX model

In the F3 generation of NOD-SCID mice or IL-2 Rγ null (NOG) mice, when the size of the tumor grew up to 1500 mm ³ after transplantation, cryo-preservation was performed at sacrifice. When the F3 generation was exceeded, it was regarded as a success model.

all subtype

Breast cancer included in the All subtype section includes all invasive breast cancer and breast cancer subtypes such as, but not limited to, the luminal subtype, the HER2 subtype, and the triple negative (TNBC) subtype.

Triple Negative Breast Cancer (TNBC)

As used herein, " triple negative breast cancer " (TNBC) is a breast cancer that is often taken from a patient who is an aggressive breast cancer subtype with no or significantly reduced expression of the estrogen receptor (ER) protein, progesterone receptor (PR) It refers to the gene state of the tissue section. TNBC and other aggressive breast cancers are typically insensitive to some of the most effective therapies and endocrine therapies available for treatment of breast cancer, including HER2-induced therapy, such as trastuzumab or lapatinib, such as tamoxifen and aromatase inhibitors.

Neo-TNBC

Refers to a TNBC breast cancer patient treated with preoperative neoadjuvant status before breast tissue sections were taken from patients with triple negative breast cancer. Examples of pre-operative adjuvant therapies used herein include various treatments such as curative resection, hamam chemotherapy, radiation therapy, immunocytochemistry, surveillance lymph node biopsy and / or axillary lymph node dissection or breast conserving surgery or modified radiotherapy . The chemotherapy may include taxane-based therapy consisting of doxorubicin and cyclophosphamide leading to docetaxel. Radiotherapy is a method of irradiating radiation to the breast and axilla or neck or internal mammary lymph nodes. Specifically, pre-operative adjuvant therapies may include providing hormone therapy or HER2 target therapy, such as trastuzumab, to hormone receptor positive or HER2-positive primary carcinoma patients. Patients will return to the hospital every 6 months for at least 3 years after initial treatment to perform routine physical examinations, mammography, breast ultrasound, and whole body bone scan, or FDG PET / CT and breast MRI 1 year and 3 years. Patients suspected of having recurrence were subjected to further examination in addition to conventional imaging. Survival was calculated from the date of first diagnosis or surgery to the recurrence or last follow-up date. Recurrence-free survival (RFS) is defined as ipsilateral breast or locally invasive recurrence; Remote recurrence; Or breast cancer, the cause of non-breast cancer, or death due to unknown causes mentioned in previous studies. Breast cancer was determined to be recurrent or metastatic based on the evaluation of the primary care physician as reported on the medical chart.

Patient and surgical sample

(Progesterone receptor) status, HER2 (Human Epithelial Growth Factor Receptor 2) status, Ki67, progress status, pre-operative adjuvant therapy, and the like, as well as patient age, tumor size, lymph node metastasis status, histologic grade, ER (estrogen receptor) Based on histopathology and clinical data, including information and tracking data including possible diseases and the like.

Stage classification according to breast cancer progression

The purpose of dividing cancer into cancers is to evaluate the progression and prognosis of the disease and to predict the outcome of treatment. The stage of breast cancer depends on the size of breast mass (T), axillary lymph node metastasis (N), and systemic metastasis (M) including bone, lung and liver including lymph node metastasis. (TNM) is an important factor in determining prognosis. The stage of breast cancer is classified into 0, 1, 2, 3, and 4 according to TNM.

Tumor size (T stage)

Tumor size (T stage) can be graded as follows.

- T0: No evidence of tumor

- Tis: Intranasal or lobular intraepithelial cancer

- T1: maximum diameter of tumor less than 2cm

- T2: maximum diameter of tumor is more than 2cm, less than 5cm

- T3: tumor has a maximum diameter greater than 5 cm

- T4: Tumor invades the chest wall or skin.

Lymph node metastasis

N stage according to degree of lymph node metastasis can be graded as follows.

- N0: No lymph node metastasis

- N1mi: fine transition of lymph nodes

- N1: 1 to 3 cases of metastatic lymph nodes

- N2: the number of lymph nodes that are transferred is 4 ~ 9

- N3: If the number of metastatic lymph nodes is 10 or more

M stage according to distant metastasis

- M0: no remote transition

- M1: distant metastases other than breast and adjacent organs

Histological grade

The histologic grade of breast cancer was determined by using a modified Bloom-Richardson grade. The grade was evaluated by microscopic examination of the tumor, and the score was determined according to the degree of tubular formation, the degree of nucleoli polymorphism, and the degree of mitosis It is classified as grade 3 according to the score after summing up. The higher the grade, the worse the patient's prognosis

c- erbB -2

c-erbB-2 is a receptor tyrosine-protein kinase, also referred to as HER2 (human epithelial cell growth factor receptor 2) or HER2 / neu. Amplification, also known as overexpression of the ERBB2 gene, occurs in about 15-30% of breast cancers. It is closely related to increased recurrence of disease and poor prognosis. Overexpression is also known to occur in aggressive forms of cervical cancer such as ovarian cancer, stomach cancer, lung adenocarcinoma, and endometrial cancer.

Ki67

The antigen Ki-67, also known as Ki67 or MKI67, is a protein encoded by the MKI67 gene (the antigen identified as the monoclonal antibody Ki-67) in man. The Ki-67 protein is a cell marker for proliferation and is involved in cell proliferation. Although Ki-67 antigen can only be detected in the nucleus during interphase of the cell cycle, most proteins in mitosis are rearranged to the chromosomal surface. The Ki-67 protein is present during all stages of the cell cycle (G1, S, G2 and mitosis) but not in the dormant cell (G0).

Example  3. PDX  How to Measure Success Rate

A total of 61 patients were used to construct the entire PDX model. When a breast cancer tissue section of a patient was transplanted into a nude mouse, a rejection of xenotransplantation occurred, or when the transplanted mouse died during the experiment, it was judged as a failure. The failure rate and the success rate were measured according to the characteristics of each breast cancer tissue section, expressed as a percentage of the corresponding population in the total population. (See Figures 1 and 2)

Statistical analysis

Allele frequencies and genotype frequencies were determined and the presence of Hardy-Weinberg equilibrium (HWE) was determined using single nucleotide polymorphisms. When p-value <0.05, Hardy-Weibull equilibrium was established. Linkage disequilibrium (LD) analysis (D ') was performed using the Haploview program version 3.2. The odds ratios (ORs), 95% confidence intervals (95% CI), and p-values of the genotypes and haplotypes were determined using the SAS program (SAS Institute, Cary, NC) Respectively. In addition, rigorous multiple analysis was performed with this pherenny analysis method (pc value). When the upper and lower values of the confidence interval do not include 1 and the p-value is less than 0.05, the correlation with the breast cancer, that is, the risk odds ratio, is judged to be significant. May be performed by applying various methods known in the art. For example, by one or more methods selected from the group consisting of Student's t-test, Chi-square test and multiple logistic regression analysis. , But is not limited thereto.

3-1. How to measure success rate in all subtype

As shown in Table 2, in all subtypes, the patient age was higher when formed from tissue when the age was 60 years or less than when it was older than 60 years. In this experiment, the success rate was high when the tumor size was T2 or T3, and the tumor size of T3 was preferable. Lymph node metastasis was found to be more successful when positive than negative. Histologic grade was higher in grade III than grade I or II. ER, PR, or c-erbB-2. Using Ki67 diagnostic markers, the success rate was higher for high Ki67 tumors (> 20) than low Ki67 tumors (≤ 20).

3-2. In the TNBC subtype  Check the success rate for

As shown in Table 3 above, in the case of the TNBC subtype, the patient age was higher when formed from tissue when the age was 60 years or less than when it was older than 60 years. In this experiment, the tumor size was the highest at T3 when the tumor size was T1 and T2. Lymph node metastasis was found to be more successful when positive than negative. Histologic grade was higher in grade III than grade I or II. Using Ki67 diagnostic markers, the success rate was higher for high Ki67 tumors (> 20) than low Ki67 tumors (≤ 20).

3-3. Neo- In the TNBC subtype  Check the success rate for

As shown in Table 4, in the case of the Neo-TNBC subtype, the patient age was higher in the case of formation from the tissue at the age of 60 years or less than in the case of over 60 years of age. In this experiment, the tumor size was the highest at T3 when the tumor size was T1 and T2. Lymph node metastasis was found to be more successful when positive than negative. Histologic grade was higher in grade III than grade I or II. Using Ki67 diagnostic markers, the success rate was higher for high Ki67 tumors (> 20) than low Ki67 tumors (≤ 20). In the course of the disease, the non-responder success rate was higher than the responders.

Example  4. Depending on processing settings PDX  Success rate

Add 1% penicillin streptomycin to RPMI 1640 media and transfer to the operating room. When the surgeon removes the tumor tissue from the operating room, it is transferred to the animal laboratory. In the PDX experiment, 6-12 week old IL-2 receptor gammar null mouse (NOG mouse) is used, and isoflurane anesthesia is performed and the tumor tissue is implanted in the mammary fat pad. This is called F1 mouse, and the size is measured using a caliper once a week (long axis * short axis ² * 0.5) for at least 6 months and at least one year. When the tumor size reaches 1500 ㎣, the transplant proceeds to the next generation. If not, the sacrifice is carried out and considered as fail. If successfully grown in F1, the tumor is harvested and then chopped to a size of approximately 5 * 5 * 5 (mm) and transplanted into a mouse mammary fat pad. This is called F2 mouse. For the remaining tumor tissue, a paraffin block is made, left fresh-frozen for sequencing, and cryo-preservation is carried out so that an F2 mouse can be transplanted again. In the case of cryo-preservation, DMSO and FBS (Fetal Bovine Serum) are mixed with media in a ratio of 1: 9 as in cell-preservation, then placed in a freezing container, and the temperature is gradually lowered to -70 ° C. ) For one day and then moved to the LN2 tank for storage. When the stock is released again, the tumor tissue should be sufficiently washed with PBS or free media (RPMI1640) and transplanted into the mouse. Then, when the tumor size of F2 mouse becomes 1500 ㎣ or more, it is transplanted into F3 mouse. If it is built up to F3, it is considered to be a successful PDX model. It can be used for various drug screening on behalf of the patient. When the tumor size of the F3 mouse is over 1500., The tumor does not pass on generation but is stored in fresh frozen for paraffin block preparation and sequencing, and the remaining tumor tissue is cryo-preserved and stored. In this successful PDX model, H & E staining and immunohistochemistry confirm the identity of histopathological characteristics with the patient. In the case of IHC, staining for ER (estrogen receptor), PR (progesterone receptor), HER2 (human epithelial receptor 2) and Ki67 proceeds.

Example  5. Anticancer drug treatment PDX  Reaction in the model

The PDX model with the breast cancer mutant Leu1780Pro was able to confirm a complete response in carboplatin (Paraplatin) as compared to Olaparib (Lynparza), an ovarian cancer treatment (Fig. 3 And Figure 4) is a graph showing percent increase or decrease in tumor size for each mouse in the group with the breast cancer mutant Leu1780Pro. It was confirmed that the growth of tumors in all the groups treated with carboplatin decreased from day 8 onwards and continuously decreased until sacrifice (see FIGS. 5 to 8).

In accordance with the above results in the above experiments, human xenotransplantation is effective for cancerous cancer which is difficult to obtain a sufficient cancer specimen. In particular, carcinomas with a low ablation rate are good candidates for PDX for obtaining specimens to produce high quality biodata because biopsy samples can be obtained only by biopsy. High-success rate PDX can be performed using NOD-SCID mouse or IL-2 Rγ null (NOG) mouse of the present invention such as breast cancer including breast cancer mutant Leu1780Pro, which is a mutation that is specific to Korean people in breast cancer, .

The xenografts derived from endoscopic ultrasound-guided fine-needle aspiration biopsy, tissues obtained through biopsy from hepatocellular lesions or tissues obtained through surgical resection can be transplanted into the model of the present invention step by step, and human xenotransplantation can be performed. Tumor studies in vivo can be done more efficiently.

Therefore, treating the anticancer agent candidates judged to have anticancer effect in the animal model to cancer cells or cancer-providing cancer patients can confirm the same effect as treating these anticancer agent candidates, so that the candidate anticancer drug candidate A great benefit is expected in substance screening and cancer treatment.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (11)

A xenograft model derived from a breast cancer patient, wherein the breast cancer tissue fragment isolated from a breast cancer patient is transplanted into an immunodeficient mouse subcutaneously,
Wherein said breast cancer is a breast cancer mutant. The method according to claim 1,
Wherein the breast cancer patient is a TNBC state or a Neo-TNBC state, the xenograft model derived from a breast cancer patient. (a) dividing the breast cancer tissue isolated from the breast cancer patient into sections; And
(b) transplanting the breast cancer tissue slice of step (a) under subcutaneously into an F1 generation immunodeficient mouse, comprising the steps of:
Wherein the breast cancer is a breast cancer mutant, the Leu1780Pro mutant (rs80357474). The method of claim 3,
Wherein said immunodeficient mouse is NOG (NOD-SCID / IL-2R [gamma] null). The method of claim 3,
The method of manufacture further comprises the step of obtaining an implanted breast cancer tissue and re-implanting subcutaneously in a new immunodeficient mouse. The method of claim 3,
Wherein the breast cancer patient is a TNBC state or a Neo-TNBC state. The method of claim 3,
The above method is the F1 generation NOG (NOD-SCID / IL- 2Rγnull) a breast tissue section transplanted under the skin of mice after the step (b) further comprises the step of growing to a size of 1500mm ^3, the 1500mm ³ (NOD-SCID / IL-2R [gamma] null) mouse subcutaneously transplanted breast cancer tissue slice to a size of 1500 mm &lt; ³ &gt; Further comprising the step of subcutaneously implanting a breast tumor tissue section of F2 generation NOG (NOD-SCID / IL-2Rγnull) mice to a size of 1500 mm ³ , the manufacture of a xenotransplantation animal model derived from a breast cancer patient Way. The method of claim 3,
Wherein said step (a) further comprises treating with at least one compound selected from the group consisting of amphotericin B, penicillin, streptomycin and gentamicin. The method of claim 3,
Wherein the method further comprises the step of removing erythrocytes from the breast cancer tissue in the step (a). (a) treating the animal model of claim 1 with a therapeutic agent for breast cancer; And
(b) selecting an anticancer candidate substance as the therapeutic agent for breast cancer in the breast cancer patient when the size of the breast cancer tissue is reduced or the breast cancer metastasis is inhibited by the therapeutic agent for breast cancer. 11. The method of claim 10,
The breast cancer therapeutic agent may be DNA, siRNA, peptide, protein, antibody, natural extract, bruceantin, pervilleine, betulinic acid,? -Lapachone, CDDO 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid, CB-Ph, obovatol, CB-Pic, Obo-Rd, trastuzumab and lapatinib , A method of providing information for selecting breast cancer patient-tailored breast cancer therapies.

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