WO2009157627A1 - Procédé de criblage d’inhibiteurs d’invasion de cellules cancéreuses et système de criblage de ceux-ci - Google Patents

Procédé de criblage d’inhibiteurs d’invasion de cellules cancéreuses et système de criblage de ceux-ci Download PDF

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WO2009157627A1
WO2009157627A1 PCT/KR2008/007636 KR2008007636W WO2009157627A1 WO 2009157627 A1 WO2009157627 A1 WO 2009157627A1 KR 2008007636 W KR2008007636 W KR 2008007636W WO 2009157627 A1 WO2009157627 A1 WO 2009157627A1
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cancer
chamber
cancer cell
cell
tissue
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PCT/KR2008/007636
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English (en)
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Da-Woon Jung
Jin Mi Kim
Zhong Min Che
Young-Tae Chang
Jin Kim
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Industry-Academic Cooperation Foundation, Yonsei University
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Priority claimed from KR1020080127159A external-priority patent/KR101008602B1/ko
Application filed by Industry-Academic Cooperation Foundation, Yonsei University filed Critical Industry-Academic Cooperation Foundation, Yonsei University
Priority to US13/000,483 priority Critical patent/US20110104741A1/en
Publication of WO2009157627A1 publication Critical patent/WO2009157627A1/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
    • G01N33/5005—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
    • G01N33/5011—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 for testing antineoplastic activity
    • 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
    • G01N33/502—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 for testing non-proliferative effects
    • G01N33/5029—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 for testing non-proliferative effects on cell motility
    • 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

Definitions

  • the present invention relates to a screening method for inhibitors of cancer cell invasion and a screening system thereof.
  • BACKGROUND OF TECHNIQUE Mature cancer tissues have a characteristics to invade into adjacent tissues from a lesion developed and proliferate, called as invasion. Invasion and metastasis of cancer cells is an indispensible relationship and the invasion of cancer cells could be considered as an early stage of metastasis.
  • the metastasis of cancer cells is mainly composed of two processes: (a) degradation of extracellular matrix by overproduction of a protease; and (b) oriented migration of cancer cells.
  • a theory that the metastasis of cancer cells is occurred by induction of cells in an adjacent micro-environment, not cancer cell alone, has been commonly accepted.
  • a fibroblast is a representative stromal cell.
  • the experimental studies how fibroblasts influence on epithelia or epithelial carcinomas and on a novel cancer therapy targeting fibroblasts have been actively carried out.
  • the epithelial cells rapidly acquired the characteristics of cancer cells such as excessive cell proliferation, loss of cell suicide program or tissue invasion capability where fibroblasts are adjacent to epithelial cells initiating metastasis to cancer cells in breast cancer (Sadlonova et a/. 2005).
  • fibroblasts derived from prostate cancer promotes proliferation of prostate cancer cells and affects differentiation of cancer cells (Orimo et al. 2005).
  • tumor cells were invaded into connective tissues only in the presence of fibroblast when oral squamous cell carcinoma (OSCC) cell line was cultured in a three-dimensional manner (Che et al, 2006).
  • OSCC oral squamous cell carcinoma
  • the present inventors have intensive studies to develop a high-throughput screening method (HTS) for isolating a novel inhibitor to cancer cell invasion.
  • HTS high-throughput screening method
  • a highly specific inhibitor to cancer cell invasion could be screened in a high-throughput manner where a substance having not only an inhibitory activity to cancer cell invasion but also no cytotoxicity to fibroblasts is selected under conditions in which carcinoma-associated fibroblasts (CAFs) and cancer cells are co-cultured in a multi-chamber (e.g, Boyden chamber) and then a candidate is added to the culture, based on the fact that CAFs induce cancer cell invasion.
  • CAFs carcinoma-associated fibroblasts
  • a multi-chamber e.g, Boyden chamber
  • a screening method for an inhibitor to cancer cell invasion comprising the steps of: (a) co-culturing cancer cells and a carcinoma-associated fibroblasts (CAFs) in a multi-chamber containing a upper-chamber, a lower-chamber and a porous filter separating the upper-chamber from the lower-chamber; in which each cancer cells and CAFs is inoculated into the upper-chamber and the lower-chamber of the multi-chamber, and then a candidate is added to the upper-chamber; and (b) measuring the number of cancer cells passing the porous filter.
  • CAFs carcinoma-associated fibroblasts
  • the present inventors have intensive studies to develop a high-throughput screening method (HTS) for isolating a novel inhibitor to cancer cell invasion.
  • HTS high-throughput screening method
  • a highly specific inhibitor to cancer cell invasion could be screened in a high-throughput manner where a substance having not only an inhibitory activity to cancer cell invasion but also no cytotoxicity to fibroblasts is selected under the condition in which carcinoma-associated fibroblasts (CAFs) and cancer cells are co-cultured in a multi-chamber (e.g, Boyden chamber) and then a candidate is added to the culture, based on the fact that CAF induce cancer cell invasion.
  • CAFs carcinoma-associated fibroblasts
  • a multi-chamber e.g, Boyden chamber
  • CAFs cancer cells in a multi-chamber containing a upper-chamber, a lower-chamber and a porous filter separating the upper-chamber from the lower-chamber; in which each cancer cells and CAFs is inoculated into the upper-chamber and the lower-chamber of the multi-chamber, and then a candidate is added to the upper-chamber.
  • the screening method of the inhibitor to cancer cell invasion in the present invention is based on the fact that CAFs induce cancer cell invasion where CAFs and cancer cells are co-cultured.
  • the screening method of the present invention is carried out according to suitable modifications of a Boyden chamber assay or a transwell assay analyzing a cell migration or invasion by a conventional chemotaxis using a multi-chamber.
  • carcinoma- associated fibroblasts are incubated in the lower chamber of transwell, and cancer cells are cultured in the upper chamber of transwell, so as to induce cancer cell invasion. It is analyzed whether a candidate compound added inhibits cancer cell invasion.
  • the cancer cell inoculated to the upper-chamber includes stomach cancer cell, liver cancer cell, lung cancer cell, breast cancer cell, ovarian cancer cell, bronchogenic cancer, nasopharyngeal cancer cell, laryngeal cancer cell, pancreatic cancer cell, bladder cancer cell, colon cancer cell, uterine cervical cancer cell, prostate cancer cell, renal cancer cell or oral squamous cell carcinoma (OSCC) cell, but not limited to.
  • the term "carcinoma-associated fibroblasts (CAFs)” refers to a fibroblast located in a tissue surrounding a cancer or a malignant tumor.
  • the CAF may be feasibly separated from specimens obtained by cutting a particular malignant tumor tissue.
  • the malignant tumor tissue as an isolation source of CAF used in the present invention includes lung cancer tissue, skin cancer tissue, stomach cancer tissue, intestinal cancer tissue, colorectal cancer tissue, pancreatic cancer tissue, liver cancer tissue, thyroid cancer tissue, uterine cancer tissue, cervical cancer tissue, ovarian cancer tissue, testicular cancer tissue, prostate cancer tissue, breast cancer tissue and oral cancer tissue, but not limited to.
  • the carcinoma-associated fibroblasts are cultured after inoculation into the lower-chamber of the multi-chamber, inducing cancer cell invasion of the upper-chamber.
  • CAF not a chemo-attractant is used to induce cancer cell invasion different to a conventional transwell-based invasion assay.
  • the porous filter is coated with collagen and more preferably, collagen type I.
  • the porous filter is coated with collagen, not MatrigelTM (a gelatin-containing protein mixture secreted from mouse tumor cells) used in a conventional transwell assay.
  • collagen not MatrigelTM (a gelatin-containing protein mixture secreted from mouse tumor cells) used in a conventional transwell assay.
  • the coating by collagen not MatrigelTM may exclude an influence of a cytokine or chemo-attractant capable of being contained in MatrigelTM on cancer cell invasion induced by CAF.
  • the method of this invention may permit to screen a highly specific inhibitor to cancer cell invasion
  • the term “candidate” refers to a substance which is expected to have cytotoxicity to cancer cells perse, not CAFs, or to inhibit secretion of, or to impede function of a cytokine or a chemokine inducing cancer cell invasion secreted from CAFs.
  • the candidate of the present invention includes, but not limited to, a synthetic compound, a natural compound, a low-molecular-weight compound, a nucleic acid (e.g., DNA, RNA, PNA and aptamer), a protein, a sugar and a lipid.
  • the candidate inhibiting cancer cell invasion may be selected by measuring the number of cancer cells passing the porous filter.
  • the measurement of the cell number passing the porous filter is carried out according to a method that after cells passing the porous filter are immobilized and stained, the cell number immobilized is counted.
  • a method for staining a cell may be carried out.
  • the method includes a utilization of a Hoechst 33258 or crystal violet dye, but not limited to.
  • the present invention further includes a step determining the candidate as the inhibitor to cancer cell invasion when the number of cancer cells passing the porous filter in a candidate-treated group is smaller than in that in a candidate-untreated group. Where the candidate inhibits cancer cell invasion, the number of the cancer cells passing the porous filter will be smaller in a candidate-treated group than in a candidate-untreated group.
  • the cytotoxicity of the candidate to CAFs inoculated into the lower-chamber is measured, and thus the candidate representing cytotoxicity to CAFs is not selected.
  • the candidates representing cytotoxicity to CAFs are not a specific toxic-substance to cancer cells.
  • a candidate having a specific inhibitory activity to cancer cells may be screened.
  • the cytotoxicity of candidates to CAFs may be carried out according to a MTT assay measuring cell viability of carcinoma-associated fibroblasts.
  • a screening system for an inhibitor to cancer cell invasion wherein each cancer cells and CAFs is inoculated into an upper-chamber and a lower-chamber in a multi-chamber containing the upper-chamber, the lower-chamber and a porous filter separating the upper- chamber from the lower-chamber.
  • the screening system in which the porous filter is coated with collagen.
  • the present invention relates to a screening method for inhibitors of cancer cell invasion and a screening system thereof. According to the screening system and screening method using the same, the inhibitor to cancer cell invasion is able to be screened in a high-throughput manner.
  • Fig. 1 shows compounds having an inhibitory activity on cancer cell invasion selected from a tagged triazine compound library.
  • Fig. 2 schematically represents a system to screen a molecule with a low molecular weight regulating tumor-stroma interactions.
  • a filter with a pore size of 8 ⁇ m was coated with collagen type I (45 g/30 L).
  • 2 x IQ 4 OSCC cells were inoculated into an upper well of transwell having the porous filter, and each CAF of 2 x 10 4 , CAF-conditioned media (CM), or CCL7 was added to a lower well of transwell having the porous filter.
  • the cells passing the filter were immobilized and stained with 0.25% crystal violet. The cells were counted under a light microscope.
  • Fig. 1 schematically represents a system to screen a molecule with a low molecular weight regulating tumor-stroma interactions.
  • FIG. 3 is a schematic diagram representing that a compound (S06) screened according to the method of the present invention prevents invasion and proliferation of a CAF-induced oral cancer cell line (YD-IOB) and reduces secretion of CCL7 by CAF.
  • Cancer cell line (YD-IOB) was inoculated into an upper well of transwell having the porous filter (pore size; 8 ⁇ m), and CAF was added to a lower well of transwell. To measure cell invasion, the cells passing a collagen-coated transwell membrane were immobilized and stained. The cells were counted under a light microscope. Cell invasion was visualized using a light microscope (Fig. 3A (i)), and cells invaded were quantitated by a cell counting (Fig. 3A (ii)).
  • a cell viability of the oral cancer cell line (YD-IOB) was measured for 48 hrs after adding the compound (S06) (Fig. 2B).
  • S06 the compound
  • Fig. 2B a cell viability of the oral cancer cell line
  • Figs. 4-6 show graphs to test the sensitivity to cytotoxicity of the triazine compound (S06) in oral cancer cell lines (YDlOB, HSC-2, HSC-3 and Ca9.22) using the MTT assay.
  • Fig. 7 represents a result to test the sensitivity to cytotoxicity of the triazine compound (S06) in colon cancer cell lines (HCT-116, DLD-I and HT-29) using the MTT assay.
  • Fig. 8 represents a result to test the sensitivity to cytotoxicity of the triazine compound (S06) in lung cancer cell lines (A549, NCI-H596 and NCI-H460) using the MTT assay.
  • Fig. 9 represents a result to test the sensitivity to cytotoxicity of the triazine compound (S06) in breast cancer cell lines (MDA-MB-231, MDA-MB-435 and MCF-7) using the MTT assay.
  • Fig. 10 shows graphs to test the sensitivity to cytotoxicity of the triazine compound (S06) in prostate cancer cell line (PC-3) using the MTT assay.
  • Fig. 11 is a histogram representing that the triazine compound (S06) has no influence on cell viability of CAF.
  • S06 triazine compound
  • Fig. 14 represents a histogram adding conditioned media recovered from YD-
  • Triazine Library Compounds 1040 compounds from a tagged triazine library were dissolved in DMSO (dimethyl sulfoxide) at a concentration of 5 mM. Tagged triazine library compounds and preparation method thereof are described in a conventional reference (Facilitated forward chemical genetics using tagged triazine library and zebrafish embryo screening, Khersonsky, S. M; Jung, D. W.; Kang, T. W.; Walsh, D. P.; Moon, H. S.; Jo, H.; Jacobson,E. M.; Shett ⁇ , V.; Neubert, T. A.; Chang, Y. T; J. Am. Chem. Soc. 2003, 125, 11804-11805). For a transwell invasion assay, an anti- invasive effect was measured by treating a candidate triazine compound with a final concentration of 5 ⁇ M.
  • OSCC Oral Squamous Cell Carcinoma
  • HSC-2, HSC-3 and Ca9.22 cell lines registered in Japanese Cell Line Bank were used as oral squamous cell carcinoma (OSCC) cells in the experiments.
  • HSC-2, HSC-3 or Ca9.22 cell line was incubated at 37°C incubator maintaining 5% CO 2 in DMEM:Hams-F12 (3:1) supplemented with 1% penicillin/streptomycin and 10% FBS (fetal bovine serum).
  • YD-IOB cell line was cultured in media with the above composition supplemented with cholera toxin (0.1 mg/ml, Sigma), hydrocortisone (0.4 mg/ml, Sigma), insulin (5 mg/ml, Sigma), apo- transferrin (5 mg/ml, Sigma) and 3,3',5-triiodo-l-thyronine (2 mg/ml, Sigma) additionally.
  • HT-29 and DLD-I cell lines were incubated in RPMI 1640 supplemented with 1% penicillin/streptomycin and 10% FBS.
  • HCT-116 cell line was incubated in DMEM supplemented with 1% penicillin/streptomycin and 10% FBS.
  • NCI-H596, NCI-H460 and A549 cell lines were kindly provided from Yonsei Cancer Research Institute. NCI-H596 and NCI-H460 cell lines were incubated in
  • RPMI 1640 supplemented with 1% penicillin/streptomycin and 10% FBS.
  • A549 cell line was incubated in DMEM supplemented with 1% penicillin/streptomycin and
  • MCF-7, MDA-MB-231 and MDA-MB-435 cell lines were purchased from ATCC (American Type Culture Collection) and incubated in DMEM supplemented with 1% penicillin/streptomycin and 10% FBS.
  • PC-3 cell line kindly provided from Yonsei Cancer Research Institute was incubated in Hams-F12 supplemented with 1% penicillin/streptomycin and 10% FBS.
  • a method to screen an inhibitor to cancer cell invasion was performed according to suitable modifications of a transwell assay.
  • the invasion of cancer cells was measured in a 24-transwell plate (Corning Inc.)-
  • a filter with a pore diameter of 8 ⁇ m was coated with collagen type I, and CAF (carcinoma-associated fibroblasts) were seeded into a lower well beneath the porous filter at a density of 2.1 x 10 4 cells/well.
  • media were replaced with serum- free media and 2 x IO 4 cancer cells diluted in serum-free media were loaded on an upper well above the filter coated with collagen.
  • a triazine compound to be screened was added to the upper side of the filter.
  • cells passing the porous filter were immobilized with 10% formalin and stained with 0.25% crystal violet. The number of cells was counted under a microscope.
  • MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay was carried out.
  • the cells were seeded in 96-well plate at a density of 8 x 10 3 cells/well. After culturing overnight, a triazine compound as a candidate to be screened was added to the plate and further incubated for 24 hrs or 48 hrs. Media were replaced with serum-free media containing MTT reagents and further incubated for 3 hrs. The culture solution was removed and DMSO was added. Formazan crystal formed was dissolved and the absorbance was measured at 540 nm.
  • CCL7 ELISA The amount of CCL7 secreted into cell culture media was measured using a
  • CAF human CCL7 ELISA kit
  • CAF human CCL7 ELISA kit
  • the amount of CXCL7 secreted into cell culture media was measured using a Human CCL7 ELISA kit (R&D Systems).
  • the method to harvest cell culture media was carried out according to the method as same as the above-described CCL7 ELISA.
  • the amount of IL-8 secreted into cell culture media was measured using a Human CCL7 ELISA kit (R&D Systems).
  • the method to harvest cell culture media was carried out according to the method as same as the above-described CCL7 ELISA.
  • the screening method used for selection was as follows: CAF were cultured in a lower well of a transwell and OSCC cells as cancer cell were incubated in an upper well of a transwell. OSCC cell invasion was induced to investigate whether a candidate compound added inhibit cell invasion (Fig. 2). After cancer cells passing a collagen-coated filter located between the upper and lower compartment of transwell were fixed and stained, the extent to invasion of cancer cells was judged by measuring the number of stained cells. In addition, MTT assay of candidate compounds for CAF as well as measurement of the invasion-inhibitory activity described above were carried out to exclude selection of candidate compounds having an inhibitory activity caused from non-specific cytotoxicity. The compounds representing cytotoxicity to carcinoma-associated fibroblasts by a MTT assay was kept out.
  • Ten compounds selected as an invasion inhibitor finally were thought to have a specific cytotoxicity to cancer cells, or to prevent secretion of a cytokine or a chemokine, or to impede function of a cytokine or a chemokine inducing cancer cell invasion secreted from fibroblasts.
  • the cytotoxicity to each cancer cells, fibroblasts and normal cells, and the effect on secretion of MCP-3/CCL7 (Monocyte Chemotactic Protein-3), CXCL8/IL-8, CXCLl/GRO- ⁇ among cytokines secreted from fibroblasts were re-examined in ten compounds selected.
  • MCP-3/CCL7 Monocyte Chemotactic Protein-3
  • CXCL8/IL-8 CXCLl/GRO- ⁇ among cytokines secreted from fibroblasts
  • the ability of the compound S06 inhibiting a cell viability was investigated using a MTT assay, demonstrating that the cell viability of each oral cancer (Figs. 4- 6), colon cancer (Fig. 7), lung cancer (Fig. 8), breast cancer (Fig. 9) or prostate cancer (Fig. 10) cell line was decreased. However, it was demonstrated that the cell viability is sustained up to 90% by 50 ⁇ M (24 hrs) treatment in carcinoma-associated fibroblasts (Fig. 11), above 90% and above 60% by 20 ⁇ M (24 hrs) and 80 ⁇ M (24 hrs) treatment in normal epithelial cells, respectively (Fig. 12).
  • the present inventors investigated how triazine compound (S06) represented by the formula 6 has an effect on cytokine secretion in carcinoma-associated fibroblasts. Consequently, secretion of MCP-3/CCL7 and CXCLl/GRO- ⁇ increased by adding conditioned media to fibroblast culture system was decreased in a concentration-dependent manner by treating the compound S06 of formula 6 (Fig. 13 and Fig. 14) while secretion of CXCL8/IL-8 was not changed (Fig. 15).
  • CCL7 (Chemokine C-C motif ligand 7) is a cytokine known as a CC chemokine that was previously called monocyte-specific chemokine 3 (MCP3).
  • CCL7 specifically attracts chemotaxis of monocytes, and regulates macrophage function. It is produced by certain tumor cell lines and by macrophages.
  • the secretion of CCL7 was enhanced by tumor stimulation in carcinoma-associated fibroblast used in the present invention. It is thought that CCL7 secreted improves migration of cancer cells, accelerating cancer cell invasion.
  • CXCLl (Chemokine C-X-C motif ligand 1) is a small cytokine belonging to the CXC chemokine family that was previously called GROl oncogene, Neutrophil- activating protein 3 (NAP-3) and melanoma growth stimulating activity, alpha (MSGA- ⁇ ).
  • CXCLl is secreted by human melanoma cells, has mitogenic properties and is implicated in melanoma pathogenesis.
  • CXCLl is a cytokine having various activities such as angiogenes ⁇ s, inflammation, wound healing, and tumorigenesis. In addtion, CXCLl is secreted by fibroblasts used in the present invention and is accelerated by cancer cell stimulation. It is thought that CXCLl plays a function in enhancing invasion and proliferation of cancer cells.
  • the compounds screened in the present invention have not only a selective cytotoxicity to cancer cells but also inhibit secretion of cytokines from fibroblasts, which function to promote cancer cell invasion, providing much more remarkable treatment efficacy on cancer.

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Abstract

La présente invention concerne un procédé de criblage d’un inhibiteur d’invasion de cellules cancéreuses, comprenant les étapes consistant à : a) cocultiver des cellules cancéreuses et des fibroblastes associés à un carcinome (CAF) dans une multi-chambre contenant une chambre supérieure, une chambre inférieure et un filtre poreux séparant la chambre supérieure de la chambre inférieure; dans lequel chaque cellule cancéreuse et CAF est inoculé dans la chambre supérieure et la chambre inférieure de la multi-chambre, et ensuite un candidat est ajouté à la chambre supérieure; et (b) mesurer le nombre de cellules cancéreuses traversant le filtre poreux. Selon le système de criblage et le procédé de criblage utilisant celui-ci, l’inhibiteur d’invasion de cellules cancéreuses peut être criblé avec un rendement élevé.
PCT/KR2008/007636 2008-06-23 2008-12-24 Procédé de criblage d’inhibiteurs d’invasion de cellules cancéreuses et système de criblage de ceux-ci WO2009157627A1 (fr)

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EP2443225A2 (fr) * 2009-06-18 2012-04-25 Kiyatec, Llc Système bioréacteur
EP2443225A4 (fr) * 2009-06-18 2013-01-02 Kiyatec Llc Système bioréacteur
US9575055B2 (en) 2009-06-18 2017-02-21 Kiyatec Inc. Co-culture bioreactor system
EP3330368A1 (fr) * 2009-06-18 2018-06-06 Kiyatec Inc. Système de bioréacteur
US10466232B2 (en) 2009-06-18 2019-11-05 Kiyatec Inc. Co-culture bioreactor system
EP3936606A1 (fr) * 2009-06-18 2022-01-12 Kiyatec Inc. Système de bioréacteur
US9423234B2 (en) 2012-11-05 2016-08-23 The Regents Of The University Of California Mechanical phenotyping of single cells: high throughput quantitative detection and sorting
US10302408B2 (en) 2012-11-05 2019-05-28 The Regents Of The University Of California Mechanical phenotyping of single cells: high throughput quantitative detection and sorting
CN118389632A (zh) * 2024-04-12 2024-07-26 首都医科大学附属北京中医医院 莲蛇药对对mda-mb-231细胞抑制机理的研究方法及其应用

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