WO2008093886A1 - Cellule dérivée de mcf7 - Google Patents

Cellule dérivée de mcf7 Download PDF

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Publication number
WO2008093886A1
WO2008093886A1 PCT/JP2008/051979 JP2008051979W WO2008093886A1 WO 2008093886 A1 WO2008093886 A1 WO 2008093886A1 JP 2008051979 W JP2008051979 W JP 2008051979W WO 2008093886 A1 WO2008093886 A1 WO 2008093886A1
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Prior art keywords
cells
mcf
gene
derived
mcf7
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PCT/JP2008/051979
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English (en)
Japanese (ja)
Inventor
Mika Imada
Hiroko Kojima
Masahiro Uchino
Takahiko Utsugi
Yasufumi Murakami
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Bio Matrix Research, Inc.
Tokyo University Of Science
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Priority to JP2008556219A priority Critical patent/JP5527573B2/ja
Publication of WO2008093886A1 publication Critical patent/WO2008093886A1/fr

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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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • 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
    • G01N33/5044—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 involving specific cell types

Definitions

  • the present invention relates to a highly invasive or highly metastatic M C F7-derived cell obtained by subculturing M C F7 cells, which are a low-metastatic human breast cancer-derived established cell line, under predetermined conditions.
  • metastasis and recurrence cannot be accurately predicted, it is necessary to carry out unnecessary anticancer drug treatment for a long time.
  • Many patients suffer from side effects.
  • prognostic markers such as the size of the tumor and the presence or absence of axillary lymph node metastasis, but the judgment accuracy is low and only about 30% of patients can predict the risk of recurrence of metastasis.
  • HER 2 discovered by the identification of drug-induced genes has been shown to be effective as a prognostic marker for breast cancer.
  • the monoclonal antibody tr a s t uz uma b against this receptor has the effect of reducing the risk of recurrence and prolonging life by administration to HER2-positive early breast cancer patients.
  • plasminogen activator and its inhibitory factor whose expression level increases when cancer invades, are also effective as prognostic markers, and have come to be clinically applied particularly as factors that regulate sensitivity to chemotherapy. .
  • metastasis occurs because distant metastasis or recurrence occurs in breast cancer patients that do not express these markers, and distant metastasis or recurrence occurs in breast cancer patients that are less sensitive to trastuzumab or chemotherapy.
  • a model for acquiring high metastatic potential of breast cancer-related cells was required.
  • Non-Patent Document 1 Lau a J. va n 't V e e r, Ho g y u e D a i, Mar k J. V a n e V i j v e r e a 1. (2002) Nat r e, Vo l 41 5, 530— 5 36 Disclosure of the Invention
  • cancer metastasis the steps of cancer cell adhesion, invasion, and migration are repeated. Cancer cells with high metastatic potential are highly adherent, invasive and mobile. In particular, since the first step of cancer metastasis is invasion from the basement membrane to surrounding tissues, cell invasiveness is deeply involved in the mechanism of metastasis generation and the early stage of acquiring metastatic potential. it is conceivable that.
  • an object of the present invention is to produce a model for acquiring high metastatic potential or high invasion ability of breast cancer-related cells.
  • the present inventors have seeded a low-metastatic human breast cancer-derived established cell line MCF 7 cell on Matrigel (trademark; the same shall apply hereinafter), and obtained cells having the ability to pass Matrigel.
  • the inventors succeeded in obtaining a novel cell line having high invasion and metastatic potential and different in morphology from MCF 7 cells, thereby completing the present invention.
  • MCF 7-derived cells which are cells derived from MCF 7 cells and have significantly higher invasion ability than MCF 7 cells;
  • the expression level of at least one gene selected from the group consisting of CD 44 gene, CXCL 12 gene, P LAU gene, and VEG is 1.5 times or more that of MCF 7 cells.
  • the MCF7-derived cell according to any one of [1] to [4] above;
  • At least one gene selected from the group consisting of a GFP gene, a YFP gene, a CFP gene, a ⁇ -galactosidase gene, a luciferase gene, and a pequolin gene has been introduced.
  • the MCF7-derived cell according to any one of
  • a screening system for cancer metastasis or recurrence-related factors or anticancer agents including the experimental animal according to [12] above;
  • a method for establishing a mutant cell line that has acquired high invasive ability or high metastatic ability from MCF 7 cells comprising seeding MCF 7 cells on a basement membrane preparation or the like, and then A method comprising collecting a cell having the ability to pass through a membrane preparation or an analogue thereof and then seeding the cells again on a basement membrane preparation or an analogue thereof at least 7 times.
  • Fig. 1 shows an outline of a method for establishing a highly invasive cell line using a Matrigel invension chamber.
  • Figure 2 shows an image of cells that have passed through the Matrigel invasion chamber fixed and stained with crystal violet.
  • Figure 3 shows an image obtained by microscopically observing the cells that have passed through Matrigel and moved downwards at a magnification of 100 times.
  • Figure 4 shows the average number of cells that have passed through Matrigel and migrated downward.
  • FIG. 5 shows the phase contrast images of MCF 7 cells (A) and MCF 7-14 cells (B).
  • FIG. 6 shows the growth curves of MCF 7 cells and MCF 7-14 cells.
  • FIG. 7 shows the results of Wo n d he a 1 in g assembly for MCF 7 cells and MCF 7-14 cells.
  • FIG. 8 shows the results of detecting the MMP activity by the zymogram method when cultured in a cell culture dish containing no matrigel.
  • FIG. 9 shows the results of detection of MMP activity by the zymogram method when cultured in a Matrigel invasion chamber.
  • FIG. 10 shows the results of examining the expression levels of the HER 2 gene and the ESTR gene by Western plot analysis.
  • FIG. 11 shows an overview of the cell transplantation method.
  • Figure 12 shows the anatomical results of mice 4 weeks after transplantation of MCF 7—GFP cells (A, C) and MCF 7-14—GFP cells (B, D).
  • a and B are anatomical images
  • C and D are the results of fluorescence observation of the behavior of GFP-expressing cells.
  • Figure 13 shows the results of observation of lungs removed 4 weeks after transplantation of MCF 7-GFP cells (A, C, E) and MCF 7-14-GFP P cells (B, D, F). Show. A and B are anatomical images of the lungs, C and D are fluorescence observation images, and E and F are the results of preparing frozen sections from the extracted lungs and immunostaining them with anti-GFP antibodies.
  • Figure 14 shows the results of observation of the transplantation site (A, C) and proximal lymph nodes (B, D) of the mammary gland removed 4 weeks after the mice transplanted with MCF 7-GFP cells.
  • Fig. 15 shows the results of observation of the transplantation site (A, C) and proximal lymph nodes (B, D) of the mammary gland excised 4 weeks after the mice transplanted with MCF 7-14-GFP cells.
  • Fig. 16 shows the results of excision of the peripheral part of the pancreas 4 weeks after transplantation from MCF7_14-GFP cells transplanted mice.
  • Figure 17 shows the results of bright field and fluorescence observations of a tumor of the pancreas excised from a mouse transplanted with MCF 7-14-GFP cells 4 weeks after transplantation, and frozen tissue sections prepared.
  • Figure 18 shows a frozen section of the mass of the MCF 7-GF P cell transplanted part and MCF 7-14-GF P cell transplanted part of the pancreas from the mouse 4 weeks after transplantation. The result of having performed immunostaining of a marker is shown.
  • Figure 19 shows the mouse abdomen (A, B), excised pancreatic region (C, D), and brain (E, F) 1 to 2 weeks after transplantation of MCF 7-14 GFP cells into mice. The result of having observed is shown.
  • a and B the arrow indicates the transplant site, and the circled area indicates the periphery of the pancreas.
  • the reference numerals indicate 1 ... Matrigel invasion chamber, 1O ... cell force-receptor, 1 2 ... constitutene, 1 3 ... matrigenore, 14 ... cell, 2 ... dish, respectively.
  • the MCF 7-derived cell according to the present invention is a mutant strain of MCF 7 cell that has acquired high invasiveness.
  • MCF 7 cells derived from human breast cancer are inherently poorly invasive and therefore low metastatic, and even when transplanted into nude mice, primary lesions form but do not metastasize.
  • the MCF7-derived cells of the present invention are mutant strains that have acquired significantly higher invasiveness than the original MCF7 cells.
  • “having significantly higher invasive ability” means that the invasive ability measured by any one of various methods for measuring invasiveness of cells is more statistically than the invasive ability of MC F 7 cells. Means significantly higher.
  • the invasiveness of cells can be evaluated by, for example, the ability to pass through Matrigel that provides an environment similar to the basement membrane in vivo. MCF 7-derived cells are seeded on Matrigel, and the ratio of the number of cells that passed Matrigel to the number of seeded cells is 50 times or more, preferably 1 compared to the same test with MCF 7 cells. It can be said that invasiveness is high when it is 00 times or more, more preferably 500 times or more.
  • the MCF 7-derived cells of the present invention have a significantly higher metastatic potential than the original MCF 7 cells.
  • MC F 7 cells have low metastasis, and even when transplanted to nude mice, primary foci are formed but metastasis is not caused.
  • the MCF 7-derived cells of the present invention Has a mutation that acquires high metastatic potential.
  • “having a significantly high metastatic ability” means that the metastatic ability obtained by any one of various methods for evaluating the metastatic ability of cells is statistically more significant than the metastatic ability of MCF 7 cells. Means high.
  • metastasis to adjacent lymph nodes is observed in at least 50% or more, preferably 60% or more, more preferably 70% or more. It can be said that the performance is high.
  • the MCF7-derived cells of the present invention are characterized by being HER2-negative.
  • HER 2 negative means that the HER 2 protein is below the detection limit of the standard Western plotting method.
  • the HER 2 gene and protein are often expressed in breast cancer-derived malignant tumors with high metastatic potential, and are often used as diagnostic markers for diagnosis and treatment.
  • the MCF7-derived cells of the present invention have high invasion ability and high metastasis ability, they are HER 2 negative related to metastasis ability. This suggests that it is likely to be used for this research.
  • the MCF7-derived cells of the present invention are E STR positive despite having high invasive ability or high metastatic ability.
  • ESTR is an estrogen receptor
  • ESTR positive means that a clear band can be detected by standard Western blotting.
  • the MCF7-derived cell of the present invention is metastasized as a breast cancer model with low sensitivity to hormonal treatment. This suggests that it is likely to be used in studies of relapse and recurrence.
  • the expression level of at least one gene selected from the group consisting of CD44 gene, CXCL12 gene, PLAU gene, and VEGF gene is 1 ⁇ !
  • ⁇ 7 in the MCF7-derived cell of the present invention 1.5 times more than cells. gene Although there are various methods for measuring the expression level of MCF7, it may be 1.5 times or more that of MCF7 cells when measured by any method.
  • the CD44 gene is preferably 2 times or more
  • the CXCL12 gene is preferably 5 times or more
  • the PLAAU gene is preferably 10 times or more
  • the VEGF gene is preferably 1.8 times or more. All of these genes are known to be expressed in large amounts in cells having metastatic potential.
  • the MCF 7-derived cell of the present invention forms a spheroid.
  • MCF 7 cells When MCF 7 cells are cultured, they usually become confluent in 4-5 days, and when they are detached from the container and die, the MCF 7-derived cells of the present invention formed spheroids and could be cultured for more than 10 days. This suggests that long-term culture and high-density culture are possible. Such a property is useful when a specific substance such as protein or nucleic acid is extracted from the MCF7-derived cells of the present invention for research.
  • the MCF 7-derived cells of the present invention have the ability to form a primary tumor nest at the transplanted site when transplanted into an experimental animal.
  • cancer cells such as MCF 7 cells are known to often detach in several days after transplantation due to increased expression of cancer-specific antigens. Is stably established in transplanted individuals.
  • stable establishment means, for example, a case where at least 50%, preferably 6.0%, more preferably 70%, more preferably 80% of the transplanted individuals do not peel for 28 days or more.
  • the MCF7-derived cells of the present invention have the ability to form metastatic foci in the proximal lymph nodes, distant lymph nodes, or brain when transplanted into experimental animals.
  • Such MCF 7-derived cells can be used, for example, by seeding MCF 7 cells on a basement membrane preparation having the same properties as the basement membrane or the like and passing through the basement membrane preparation or the like. It can be obtained by recovering cells in which a spontaneous mutation has occurred spontaneously.
  • the ability to pass through a basement membrane preparation or the like Cells having higher invasive ability or metastatic ability by repeating the process of seeding cells having the same again on the basement membrane preparation or the like and recovering cells having the ability to pass the basement membrane preparation or the like MC F 7-derived cell lines can be generated.
  • An example of a basement membrane preparation or the like can include, for example, Matrigel, which is a solubilized extract from EHS mouse tumor cells rich in extracellular matrix protein (EC M).
  • the step of seeding MCF 7 cells on Matrigel, then collecting the cells that pass Matrigel and seeding them again on Matrigel is at least 7 times, preferably 8 times, more preferably By repeating 9 times, most preferably 10 times, it is possible to obtain MCF 7-derived cells that have acquired a sufficiently high invasive ability or metastatic ability.
  • Examples of analogs of basement membrane preparations include agarose or collagen gel.
  • MCF 7-derived cell line is limited in its ability to migrate during culturing due to fiber components in the medium, and is considered to have low invasive and metastatic potential. It is also possible to obtain F7-derived cells.
  • MCF7-derived cells of the present invention examples include the MCF 7-14 cell line deposited with the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology.
  • F ERM B P-1 0 9 4 4 These cells are likely to provide useful information for cancer metastasis research, prognostic markers, drug discovery target searches, and the like.
  • analysis at the gene level such as DNA microarrays by subtraction
  • detailed analysis at the protein level such as two-dimensional electrophoresis and mass spectrometry can be used to acquire high metastatic potential and high invasion ability. Factors that are considered specific are likely to be identified.
  • by capturing factors that mutate in the early stages of metastasis it becomes possible to predict the prognosis of whether the cancer is metastatic at the same time as the discovery of the initial cancer, which is a useful diagnostic criterion for formulating treatment strategies such as the administration method of anticancer drugs.
  • Probability is high. In addition to its potential as a new prognostic marker, the identified factors themselves may become target molecules for cancer treatment. If a factor that is expressed specifically in metastatic cells can be discovered, it will also contribute to drug discovery, such as the development of a drug delivery system that targets disseminated cancer cells floating in the blood before the formation of metastases and induces cell death Big. In addition, a lot of information obtained by comprehensive analysis of metastasis from various angles such as gene level and protein level provides many clues to elucidate the mechanism of metastasis. A deeper understanding of the molecular mechanism of metastasis formation will lead to the development of new cancer treatment strategies.
  • the present invention also provides an MCF7-derived cell into which at least one gene selected from the group consisting of a GFP gene, a YFP gene, a CFP gene, a ⁇ -galactosidase gene, a luciferase gene, and a equorin gene has been introduced.
  • Such cells can be prepared by a known gene transfer method or a method similar thereto, and the phenotype of each gene can be used for various studies and is useful.
  • the present invention also provides an experimental animal in which the aforementioned MCF 7-derived cells are transplanted. Such a laboratory animal can be produced by a known method or a method similar thereto.
  • the experimental animal is useful for studies of metastasis and recurrence as a breast cancer model. is there.
  • experimental animals that can be used include mice, rats, hamsters, gerbils, moss / lemots, usagis, dogs, cats, peta, monkeys, birds, fishes, amphibians, and the like.
  • the present invention also provides a screening system for cancer metastasis or recurrence-related factors or anticancer agents, comprising the above-described MCF7-derived cells.
  • a screening system containing cells can be produced by a known method or a method according to it.
  • This screening system includes MCF 7-derived cells that are less sensitive to conventional breast cancer treatment methods, so it is possible to screen for invasion inhibitors, discover new breast cancer metastasis or recurrence-related factors, and new breast cancer It is useful to contribute to the discovery of therapeutic / preventive drugs.
  • the present invention also provides a screening system for cancer metastasis or recurrence-related factors or anticancer agents, including experimental animals transplanted with the above-mentioned M C F7 -derived cells.
  • a screening system containing experimental animals can be prepared by a known method or a method equivalent thereto. This screening system is useful for screening metastasis inhibitors.
  • the present invention also provides a screening method for establishing a mutant cell line that has acquired high invasiveness or high metastatic ability from MCF 7 cells.
  • this method after seeding MCF 7 cells on a basement membrane preparation or an analogue thereof, the cells having the ability to pass through the basement membrane preparation or an analogue thereof are recovered, and then again the basement membrane preparation or an analogue thereof. It is characterized by repeating the seeding process on the material at least 7 times.
  • Invasion of cells derived from MCF 7 'As a method of improving metastasis, there are methods using artificial mutagens such as inducing point mutations, but there are mutation patterns that can be introduced. Therefore, the invasiveness cannot be made sufficiently high.
  • MC F 7 Itadatsuki 3 (Br ea sta d eno rc a c i n oma; Hu m a n) was cultured in a 10% FB S (Eq i tech h BIO company) -containing R PM I 1640 (S I GMA company) medium. Cells were generated every 3 days according to known methods.
  • MC F 7 cells 14 were treated with Matrigel invasion channel 1 (Becton Dickinson) The seeds were sown on the Norreyansat 10 according to a known method. Matrigel 13 is coated on the membrane 12 and the cell 14 having the ability to pass through the matrix migrates to the lower side of the membrane 12 at the bottom of the cell culture kinase 10. After 60 hours, cells (infiltrating cells) that migrated to the lower side of the membrane 12 were collected by trypsin-EDTA treatment. The recovered cells 14 were cultured in dish 2 until they became subconfluent, and were seeded again in the matrigel inversion chamber 1-1. The above operation was repeated 14 times. Also membrane 1 2 The cells that migrated to the lower side were stained with crystal violet and the whole image was observed. At the same time, 5 fields were selected under the microscope to count the number of migrated cells.
  • Figs. Fig. 2 shows a crystal violet stained image
  • Fig. 3 shows a microscopic image observed at a magnification of 100x
  • Fig. 4 shows the number of migrated cells.
  • c o n t r o 1 indicates MCF 7 cells, and the numbers indicate the number of passes through Matrigel.
  • MCF 7-14 cells a highly invasive cell group
  • Figure 5 shows the phase contrast images of MCF 7 and MCF 7-14 cells. Comparing MCF 7 cells ( Figure 5A) and MCF 7-14 cells ( Figure 5 B), MCF 7-14 cells were more rounded. In addition, MCF 7 cells detached when confluent, but MCF 7-14 cells formed spheroids and continued to proliferate.
  • MC F 7 cells and MC F 7-14 cells were seeded on 96-well plates with 5x10 3 cells Z-wells, and the number of cells after 1, 2, 3 and 4 days was counted by MTT method. The result is shown in FIG.
  • the vertical axis represents the number of cells, and the horizontal axis represents the number of culture days. There was no significant difference in cell growth rate between MCF 7 cells and MCF 7-14 cells.
  • FIG. 7A Comparing MCF 7 cells (Fig. 7A) and MCF 7-14 cells (Fig. 7 B) at 0, 1, 2, 3, and 4 days after grooving, MCF 7-14 cells were grooved faster. Is buried.
  • Figure 7C shows the average (%) of the three fields of distance traveled by the cells.
  • FIG. 7D and FIG. 7E are magnified images of MCF 7 cells and MCF 7-14 cells after 3 days, respectively. In MCF 7-14 cells (Fig. 7E), the actin stress fibers were actively extended.
  • MMP matrix metaprotease
  • MC F 7 cells and MC F 7-14 cells were seeded at a concentration of 2.5 ⁇ 10 5 cells Z-well in a normal medium without Matrigel and a medium with Matrigel. The next day, the medium was replaced with a 500 ⁇ 1 serum-free medium, and the medium was collected after 1, 2, and 3 hours. The collected medium was centrifuged and concentrated using Microcon (registered trademark) YM30 (Millipore). Concentrated medium and sample buffer were mixed and electrophoresed at 25 mA for 2 hours at 4 ⁇ using a 10% polyacrylamide gel. 2.
  • Microcon registered trademark
  • the gel was placed in a vat filled with 5% triton X—100 containing buffer, and the SDS was removed twice while slowly permeating at room temperature for 30 minutes. Furthermore, the cells were incubated with gelatinase activating solution in 37 mouths for 24 hours. Thereafter, the cells were stained with CBB staining solution for 30 minutes, and further decolorized with a decolorizing solution for 30 minutes.
  • Fig. 8 shows the results when cultured in a cell culture dish containing no Matrigel
  • Fig. 9 shows the results when cultured in a Matrigel incubation chamber.
  • Each figure A is a stained image of the gel used in the zymogram method
  • each figure B and C are analyzed by image analysis software to show the activity of MMP 9 and MMP 2, respectively. It is the result of measurement.
  • MCF 7-14 cells expressed active MMP even when the medium was cultured without containing Matrigel or when the medium was cultured while infiltrating with Matrigel. The amount was' high.
  • RNA was extracted from MC F 7 cells and MC F 7-14 cells according to a known method, and Gene C hip (registered trademark) Human Genome U 1 3 3 P lus 2.0 Ar ray ( A ff yme tr
  • Tables 1-4 The results are shown in Tables 1-4. Among the 1495 genes that fluctuated more than twice, 13 genes (Table 1) were involved in can c cer, and 13 genes (Table 2) were involved in metastasis. Among 136 genes that fluctuated 0.5 times or less, 7 genes (Table 3) were involved in can ccer, and 0 genes were involved in metastasis. Further suggested to be involved in breast cancer
  • epidermal growth factor receptor erythroblastic leukemia viral
  • fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinoc te growth factor receptor, craniofacial dysostosis 1,
  • Crouzon syndrome Pfeiffer syndrome, Jackson-Weiss syndrome
  • fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1,
  • CXCL12 chemokine C-X-C moti ⁇ ligand 12 (stromal cell-derived fector 1) integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen
  • CD29 includes MDF2 ( MSK12)
  • tissue inhibitor of metalloproteinaae 1 erythroid potentiating
  • MCF 7 cells and established MCF 7-14 cells were transfected with a GFP expression plasmid vector according to a known method, and the GFP expression cells were screened for about 1 week. Cloning 30 clones using the cloning ring, and selection medium (4 00 g / m 1 G4 1 8 (manufactured by SI GMA) supplemented medium) and maintenance medium (50 / i gZml) for about 3 weeks. A GGFP-expressing stable strain was established by repeated cultivation in a G4 18 supplemented medium. Hereinafter, they are referred to as MCF 7-GF P cells and MC F 7-14 GFP cells, respectively.
  • MCF 7 cells MCF 7-14 cells, MCF 7—GFP cells, MCF 7
  • the protein was extracted from 14 GFP cells, and SDS polyacrylamide gel electrophoresis was performed according to a known method using a polyacrylamide gel having a concentration gradient of 2 to 15%.
  • the separated protein was transferred to PVDF membrane, blocked with 5% skim milk, and reacted with anti-HER 2 antibody or anti-estrogen receptor antibody. Further, it was reacted with an HRP-crosslinked secondary antibody and detected by ECL.
  • MC F 7— GFP cells (control) and established MC F 7— 1 4— GFP cells are grown to a final concentration of 8.6 ⁇ 10 7 cells Zml.
  • the transplanted cell solution was prepared by mixing 1: 1 with Tarreduce Tomato Trigenole (Becton Dickinson). As shown in FIG. 11, this transplanted cell solution 100 ⁇ 1 was transplanted into the fourth mammary gland of a female BALBZcAL c 1 -nu / nu strain mouse. After transplantation, the behavior of GFP-expressing cells was analyzed during dissection. The results of observation of mice 4 weeks after transplantation are shown in FIG.
  • Figure 1 2A and C are MCF7-GFP cells
  • B and D are mice transplanted with MCF7-14 GFP cells
  • a and B are the whole images
  • C and D are A and B, respectively.
  • This is a fluorescent observation image.
  • MC F7-GFP cells no metastasis was observed although tumors formed at the transplant site.
  • MCF7_14-GFP cells are localized to the GFP-expressing cells that have been disseminated in the peritoneum, and relatively large GFP-expressing cells in the abdomen. Cell clumps were observed. 10. Confirmation of metastasis by staining
  • the mammary gland was removed and frozen sections were prepared according to a known method. This section was subjected to HE staining and immunostaining with anti-GFP polyclonal antibody and anti-BRC A2 monoclonal antibody according to a known method.
  • Figure 13 shows the results of observing the whole lung region and tissue sections immunostained with anti-G F P antibody in mice transplanted with MC F 7 -GF P cells and MC F 7-14 G F P cells.
  • A, C and E are related to MC F 7—GFP cells
  • B, D and F are related to MC F 7—14—GFP cells.
  • Anatomical images C and D are fluorescence observation images
  • E and F are the results of immunostaining the frozen sections with anti-GFP antibody. As shown in the figure, tumors and GFP expressing cells were not observed in the mouse lung region.
  • FIG. 14 shows the results of observation of the transplantation site (A, C) and proximal lymph node (B, D) of the mammary gland extracted from a mouse 4 weeks after MCF 7-GFP cell transplantation.
  • a and B show the results of HE staining
  • C and D show the results of immunostaining with anti-GFP antibody.
  • M large number of GFP-expressing cells were observed at the transplantation site in mice transplanted with CF7-GFP cells, but no metastasis was observed in the mammary lymph nodes.
  • Fig. 15 shows the results of observation of transplanted sites (A, C) and proximal lymph nodes (B, D) of 3 ⁇ 4 gland excised from mice 4 weeks after MCF 7-14 transplantation of GFP cells .
  • a and B show HE staining
  • C and D show immunostaining results with anti-GFP antibody.
  • MCF 7-14 One GFP cell-transplanted mice showed GFP-expressing cells at both the transplant site and mammary lymph node, and it was observed that proximal lymph node metastasis, which is also a prognostic marker of metastasis, occurred. It was.
  • Figure 16 shows the results of excision and observation of the area around the pancreas for more detailed investigation of the metastatic foci of the MCF 7-14 GFP cell transplanted mice shown in Figure 12.
  • A is the whole mouse image
  • B is the anatomical image around the excised pancreas
  • C and D are the results of fluorescence observation of the sites corresponding to A and B, respectively. It was observed that the lymph nodes of the pancreas were enlarged and many GFP-expressing cells were localized.
  • Fig. 17A, B pancreatic tumor
  • Fig. 17D prepared frozen tissue sections (thickness, and HE-stained
  • Fig. 17 E fluorescence observation of GFP
  • transplanted part (AC) from MCF 7—GF P cell transplanted mice 4 weeks after transplantation transplanted from MCF 7-14 GF P cell transplanted mice 4 weeks after transplantation (D—F) and pancreatic mass (G—I) were removed, frozen sections were prepared, and cytokeratin 7 (RT 7; A, D, G), KRT 19 (B, E, H) and KRT 20 (C, F, I) were immunostained.
  • mouse anti-KRT 7 monoclonal antibody ZYMED Laboratories, 18 — 0234; dilution factor 50 ⁇ , mouse anti-KRT 19 monoclonal antibody (SANTA CRUZ, sc-6 278; 50 ⁇ ) and mouse anti-KRT 20 monoclonal antibody (SANTA CR UZ, sc—523 20; 50 times)
  • Histfine Mouse Stain Kit Naichirei
  • DAKO 3'-daminobenzidine tetrahydrochloride
  • MCF 7-GFP GFP-transfected strain of MCF 7 cells
  • FIG. 19 shows the result of observing mice 2 weeks after MCF 7-14 GFP cell transplantation 1.
  • the peripheral part of the pancreas (C, D) was removed from the abdomen (A, B) of the mouse, and the brain (E, F) was further removed.
  • A, C, and E are anatomical images
  • B ', D, and F are fluorescence observation images. From these results, metastatic tumors with a diameter of 1 cm or more were observed in the lymph nodes near the pancreas, and the localization of GFP-expressing cells was confirmed in the brain.
  • the MCF 7-derived cells according to the present invention have a high invasion ability or a high metastasis ability despite being derived from MCF 7 cells. Since such mutations are induced in a natural environment without using drugs or the like, the mechanism of metastasis generation and the MCF 7-derived cells according to the present invention are the first to acquire metastatic potential. It is suitable for research to detect prognostic markers such as metastasis and recurrence by capturing changes at the stage of life. It is a diagnostic kit for invasion / metastasis-specific factors and cancer metastasis and prognosis prediction. It can also be used for development.

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  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Toxicology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention a pour but de produire un modèle qui acquiert une haute capacité métastatique et une haute capacité invasive d'une cellule liée au cancer du sein. Plus spécifiquement, l'invention concerne une cellule dérivée de MCF7, qui est dérivée d'une cellule MCF7 et présente une capacité invasive ou une capacité métastatique significativement plus élevée, comparativement à une cellule MCF7. La cellule dérivée de MCF7 peut être produite par collecte d'une cellule MCF7 qui acquiert une capacité de traverser une préparation de membrane basale, telle que Matrigel ou analogue.
PCT/JP2008/051979 2007-01-30 2008-01-30 Cellule dérivée de mcf7 WO2008093886A1 (fr)

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Cited By (4)

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Publication number Priority date Publication date Assignee Title
WO2010067722A1 (fr) * 2008-12-08 2010-06-17 株式会社バイオマトリックス研究所 Procédé pour évaluer le degré de malignité d’un cancer du sein, et kit pour l’évaluation
WO2010098471A1 (fr) 2009-02-27 2010-09-02 株式会社バイオマトリックス研究所 Procédé d'immunisation utilisant une cellule cancéreuse
WO2012026615A1 (fr) 2010-08-25 2012-03-01 株式会社バイオマトリックス研究所 Procédé de production d'anticorps à l'aide de cellules cancéreuses
WO2012028958A2 (fr) 2010-09-01 2012-03-08 株式会社バイオマトリックス研究所 Anticorps pour un marqueur du cancer colorectal

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010067722A1 (fr) * 2008-12-08 2010-06-17 株式会社バイオマトリックス研究所 Procédé pour évaluer le degré de malignité d’un cancer du sein, et kit pour l’évaluation
JP2010130993A (ja) * 2008-12-08 2010-06-17 Bio Matrix Research Inc 乳癌の悪性度の評価方法ならびに該評価方法に用いるアレイおよびキット
WO2010098471A1 (fr) 2009-02-27 2010-09-02 株式会社バイオマトリックス研究所 Procédé d'immunisation utilisant une cellule cancéreuse
WO2012026615A1 (fr) 2010-08-25 2012-03-01 株式会社バイオマトリックス研究所 Procédé de production d'anticorps à l'aide de cellules cancéreuses
WO2012028958A2 (fr) 2010-09-01 2012-03-08 株式会社バイオマトリックス研究所 Anticorps pour un marqueur du cancer colorectal

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JPWO2008093886A1 (ja) 2010-05-20

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