CN118879636B - Cell strain AlmoR1 and application thereof - Google Patents
Cell strain AlmoR1 and application thereof Download PDFInfo
- Publication number
- CN118879636B CN118879636B CN202411358049.4A CN202411358049A CN118879636B CN 118879636 B CN118879636 B CN 118879636B CN 202411358049 A CN202411358049 A CN 202411358049A CN 118879636 B CN118879636 B CN 118879636B
- Authority
- CN
- China
- Prior art keywords
- almor1
- cell line
- cell
- mutation sites
- almor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 208000002154 non-small cell lung carcinoma Diseases 0.000 claims abstract description 27
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 claims abstract description 27
- 230000035772 mutation Effects 0.000 claims description 42
- 206010059866 Drug resistance Diseases 0.000 claims description 19
- 229940121647 egfr inhibitor Drugs 0.000 claims description 13
- 208000003174 Brain Neoplasms Diseases 0.000 claims description 6
- 206010059282 Metastases to central nervous system Diseases 0.000 claims description 6
- 102220014422 rs397517094 Human genes 0.000 claims description 5
- 229940121358 tyrosine kinase inhibitor Drugs 0.000 claims description 4
- 239000005483 tyrosine kinase inhibitor Substances 0.000 claims description 4
- 230000009456 molecular mechanism Effects 0.000 claims description 3
- 101150023956 ALK gene Proteins 0.000 claims description 2
- 101150039808 Egfr gene Proteins 0.000 claims description 2
- 101150105382 MET gene Proteins 0.000 claims description 2
- 238000010171 animal model Methods 0.000 claims description 2
- 108700021358 erbB-1 Genes Proteins 0.000 claims description 2
- 238000011056 performance test Methods 0.000 claims description 2
- 102220014476 rs1140475 Human genes 0.000 claims description 2
- 102200003067 rs1670283 Human genes 0.000 claims description 2
- 102220139466 rs1728828 Human genes 0.000 claims description 2
- 102200003068 rs1881420 Human genes 0.000 claims description 2
- 102200003069 rs1881421 Human genes 0.000 claims description 2
- 102220110099 rs2072454 Human genes 0.000 claims description 2
- 102220108577 rs2256740 Human genes 0.000 claims description 2
- 102220108578 rs2293564 Human genes 0.000 claims description 2
- 102220139478 rs3795850 Human genes 0.000 claims description 2
- 102220139140 rs397706189 Human genes 0.000 claims description 2
- 102220108581 rs4358080 Human genes 0.000 claims description 2
- 150000004917 tyrosine kinase inhibitor derivatives Chemical class 0.000 claims description 2
- 244000005700 microbiome Species 0.000 claims 1
- 229960003278 osimertinib Drugs 0.000 claims 1
- DUYJMQONPNNFPI-UHFFFAOYSA-N osimertinib Chemical compound COC1=CC(N(C)CCN(C)C)=C(NC(=O)C=C)C=C1NC1=NC=CC(C=2C3=CC=CC=C3N(C)C=2)=N1 DUYJMQONPNNFPI-UHFFFAOYSA-N 0.000 claims 1
- 210000004556 brain Anatomy 0.000 abstract description 58
- 206010027476 Metastases Diseases 0.000 abstract description 46
- 230000009401 metastasis Effects 0.000 abstract description 46
- 230000008685 targeting Effects 0.000 abstract description 12
- 238000004321 preservation Methods 0.000 abstract description 10
- 229940079593 drug Drugs 0.000 abstract description 9
- 239000003814 drug Substances 0.000 abstract description 9
- 238000009629 microbiological culture Methods 0.000 abstract description 6
- 210000004027 cell Anatomy 0.000 description 94
- 206010028980 Neoplasm Diseases 0.000 description 19
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 description 19
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 description 19
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 description 19
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 17
- 238000007917 intracranial administration Methods 0.000 description 17
- 201000005202 lung cancer Diseases 0.000 description 17
- 208000020816 lung neoplasm Diseases 0.000 description 17
- 238000011160 research Methods 0.000 description 14
- 238000012217 deletion Methods 0.000 description 10
- 230000037430 deletion Effects 0.000 description 10
- 238000000338 in vitro Methods 0.000 description 8
- 238000012360 testing method Methods 0.000 description 7
- 102100033793 ALK tyrosine kinase receptor Human genes 0.000 description 6
- 238000011065 in-situ storage Methods 0.000 description 6
- 230000003902 lesion Effects 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 238000011282 treatment Methods 0.000 description 6
- 108091092878 Microsatellite Proteins 0.000 description 5
- 238000011580 nude mouse model Methods 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 229940121646 third-generation egfr tyrosine kinase inhibitor Drugs 0.000 description 5
- 238000012795 verification Methods 0.000 description 5
- -1 MET Proteins 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 238000002513 implantation Methods 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 230000009826 neoplastic cell growth Effects 0.000 description 4
- 230000035755 proliferation Effects 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000002271 resection Methods 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 102100034540 Adenomatous polyposis coli protein Human genes 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 2
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 2
- 102100034157 DNA mismatch repair protein Msh2 Human genes 0.000 description 2
- 206010061818 Disease progression Diseases 0.000 description 2
- 102100030708 GTPase KRas Human genes 0.000 description 2
- 206010064571 Gene mutation Diseases 0.000 description 2
- 101001134036 Homo sapiens DNA mismatch repair protein Msh2 Proteins 0.000 description 2
- 101000605639 Homo sapiens Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform Proteins 0.000 description 2
- 101000984753 Homo sapiens Serine/threonine-protein kinase B-raf Proteins 0.000 description 2
- 229910015837 MSH2 Inorganic materials 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 102000007530 Neurofibromin 1 Human genes 0.000 description 2
- 108010085793 Neurofibromin 1 Proteins 0.000 description 2
- 108010011536 PTEN Phosphohydrolase Proteins 0.000 description 2
- 102000014160 PTEN Phosphohydrolase Human genes 0.000 description 2
- 102100038332 Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform Human genes 0.000 description 2
- 102100027103 Serine/threonine-protein kinase B-raf Human genes 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000005750 disease progression Effects 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 238000011503 in vivo imaging Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 238000002626 targeted therapy Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 206010071975 EGFR gene mutation Diseases 0.000 description 1
- 239000002146 L01XE16 - Crizotinib Substances 0.000 description 1
- 231100000002 MTT assay Toxicity 0.000 description 1
- 238000000134 MTT assay Methods 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 108020004485 Nonsense Codon Proteins 0.000 description 1
- 102000004278 Receptor Protein-Tyrosine Kinases Human genes 0.000 description 1
- 108090000873 Receptor Protein-Tyrosine Kinases Proteins 0.000 description 1
- 102100023361 SAP domain-containing ribonucleoprotein Human genes 0.000 description 1
- 101710139423 THO complex subunit 1 Proteins 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 229960000397 bevacizumab Drugs 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 230000008499 blood brain barrier function Effects 0.000 description 1
- 210000001218 blood-brain barrier Anatomy 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 210000001715 carotid artery Anatomy 0.000 description 1
- 229940121657 clinical drug Drugs 0.000 description 1
- KTEIFNKAUNYNJU-GFCCVEGCSA-N crizotinib Chemical compound O([C@H](C)C=1C(=C(F)C=CC=1Cl)Cl)C(C(=NC=1)N)=CC=1C(=C1)C=NN1C1CCNCC1 KTEIFNKAUNYNJU-GFCCVEGCSA-N 0.000 description 1
- 229960005061 crizotinib Drugs 0.000 description 1
- 238000012864 cross contamination Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000011066 ex-situ storage Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 238000011532 immunohistochemical staining Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 210000005240 left ventricle Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000037434 nonsense mutation Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 238000011338 personalized therapy Methods 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 230000008261 resistance mechanism Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
-
- 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
- G01N33/5044—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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Toxicology (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Oncology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The application discloses a cell strain AlmoR and application thereof, relates to the technical field of cell biology, and aims to solve the technical problem that a stable non-small cell lung cancer brain metastasis targeting drug-resistant cell strain is lacking in the prior art. The cell strain AlmoR is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) with a preservation address of North Star Xiyu No.1, 3 in the Korean region of Beijing, a preservation date of 2024, 05 month and 09 days, and a preservation number of CGMCC No. 45858.
Description
Technical Field
The application relates to the technical field of cell biology, in particular to a cell strain AlmoR1 and application thereof.
Background
Lung cancer is one of the most common tumors in clinic, and has a high incidence of brain metastasis, half of lung cancer patients can develop brain metastasis in the disease progression process, and the incidence of brain metastasis of driving gene positive (such as EGFR mutation, ALK fusion) lung cancer patients is as high as 70.3%. Once lung cancer brain metastasis is diagnosed, prognosis is extremely poor, and the average survival time is only 2-3 months.
In recent years, small molecule TKIs (tyrosine kinase receptor inhibitors) based on driving genes bring hope for treating brain metastasis due to good blood brain barrier penetrability and high local control rate. Wherein TKIs targeting EGFR and ALK can prolong the median survival time of patients to 10-19 months, and small molecule TKIs has become a first line drug for driving gene positive lung cancer brain metastasis patients according to guidelines. However, clinical studies have found that a large number of patients develop intracranial disease progression during long-term use of targeted therapies. At present, the treatment strategy after the TKIs treatment is progressed is mainly partial excision and radiotherapy, and has limited means and poor curative effect. Because of the specificity of the central system microenvironment, the therapeutic means of primary foci and the mechanism of action of drugs are different from intracranial resistance, so that there is a clinical lack of effective systemic therapeutic means to control the progression of intracranial lesions after resistance. However, due to the difficulty in acquiring intracranial drug resistant lesions, a mechanism understanding of the resistance of brain metastasis TKIs is still lacking.
The existing drug resistance models of the brain metastasis of the non-small cell lung cancer reported at home and abroad are mainly divided into an in-vivo mode and an in-vitro mode, wherein the in-vitro cell drug resistance induction mode is separated from a tumor microenvironment, and the in-vivo drug resistance induction models mostly lack intracranial transfer processes and cannot completely simulate the brain metastasis targeting drug resistance process of the non-small cell lung cancer of a human body. Therefore, a stable non-small cell lung cancer brain metastasis targeting drug-resistant cell strain is established, a good research tool can be provided for basic research of intracranial targeting therapy resistance, and a new thought is provided for treatment after non-small cell lung cancer brain metastasis targeting drug-resistant.
Disclosure of Invention
The application provides a cell strain AlmoR and application thereof, and aims to solve the technical problem that a stable non-small cell lung cancer brain metastasis targeting drug-resistant cell strain is lacking in the prior art.
In order to solve the technical problems, the embodiment of the application provides a cell strain AlmoR, wherein the cell strain AlmoR is preserved in the China general microbiological culture Collection center (China general microbiological culture Collection center) with a preservation address of No. 3 of the West-road No. 1 of North Star in the Korean area of Beijing, the preservation date is 2024, the month 05 and the day 09, and the preservation number is CGMCC No. 45858.
As some alternative embodiments of the application, the cell line AlmoR has EGFR E746_A750del (ex 19 del) mutation.
As some alternative embodiments of the application, the cell line AlmoR has 15 snp mutation sites and 2indel mutation sites on EGFR gene.
As some optional embodiments of the present application, the numbering of the snp mutation sites is rs712830、rs2072454、rs2270247、rs2075109、rs2075110、rs11506105、rs3735059、rs4947987、rs1558544、rs759162、rs17336919、rs6970262、rs1140475、rs139068680 and rs2692456, respectively;
the index mutation sites are numbered rs121913421 and rs34723095 respectively.
As some alternative embodiments of the application, the cell line AlmoR1 has 30 snp mutation sites and 5 indel mutation sites on the ALK gene.
As some optional embodiments of the present application, the numbering of the snp mutation sites is rs1728828、rs1881421、rs1881420、rs1670283、rs12619135、rs1728826、rs1670284、rs3738870、rs4666178、rs3820712、rs6748797、rs1625283、rs1534545、rs1569156、rs3795850、rs4666182、rs2276550、rs12714270、rs2256740、rs4666183、rs4589708、rs2272409、rs4666194、rs11127213、rs11127214、rs11127215、rs2293564、rs13428230、rs2256376 and rs4358080, respectively;
The index mutation sites are numbered rs1391843363, rs397706189, rs10623528, rs3832036 and rs540486879 respectively.
As some alternative embodiments of the application, the cell line AlmoR has 4 snp mutation sites and 1 indel mutation site on MET gene.
As some optional embodiments of the application, the numbering of the snp mutation sites is rs62469056, rs38860, rs13223756 and rs6947629 respectively, and the numbering of the indel mutation site is rs35212357.
As some alternative embodiments of the application, the cell line AlmoR1 is resistant to tyrosine kinase inhibitors, including 7.142. Mu.M IC50 for oxtinib and 7.289. Mu.M for amitinib.
On the other hand, the application also provides application of the cell strain AlmoR1, namely application in pharmacodynamic performance test of a tyrosine kinase inhibitor and research of molecular mechanism of EGFR inhibitor targeted drug resistance.
The application provides another application of the cell strain AlmoR as described above and in establishing a non-small cell lung cancer brain metastasis animal model.
Compared with the prior art, the cell strain AlmoR is a targeting drug-resistant cell strain which is extracted from the isolated craniocerebral tissue for the first time and has EGFR gene mutation, is of the same species source as the patient from which the isolated craniocerebral tissue is derived, has EGFR 19 exon deletion mutation in both primary cells and tumor tissue, and shows general drug resistance to the third generation EGFR-TKIs. Furthermore, almoR cell line showed good neoplasia in an in situ transplantation model in the brain. The primary cell strain can be continuously passaged in vitro, the current algebra is over 50 generations, and the in vitro continuous culture time is over 9 months. During passage, cell morphology, proliferation kinetics and genetic characteristics are stable. The shape of the recovered cells is kept unchanged after freezing and storing by liquid nitrogen, and the recovered cells are a stable cell strain. The cell strain AlmoR is preserved in China general microbiological culture Collection center (China general microbiological culture Collection center) with a preservation address of North Star Xiyu No.1, 3 in the Korean region of Beijing, a preservation date of 2024, 05 month and 09 days, and a preservation number of CGMCC No. 45858.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following description will make brief description of the drawings used in the description of the embodiments or the prior art. Like elements or portions are generally identified by like reference numerals throughout the several figures. In the drawings, elements or portions thereof are not necessarily drawn to scale.
Fig. 1 is a schematic diagram of obtaining a brain metastasis specimen of a human non-small cell lung cancer with the resistance of the EGFR-TKIs of the third generation, wherein part A represents a diagnosis and treatment time axis of a patient with the EGFR mutation and the non-small cell lung cancer, part B represents a craniocerebral magnetic resonance image of the patient at different treatment stages corresponding to the part A, the brain metastasis is a part pointed by a red arrow, and part C represents HE and immunohistochemical (Ki-67, CK) staining images of the brain metastasis specimen of the last operation (2023.7.23).
FIG. 2 is a schematic diagram showing the results of culturing and identifying AlmoR cell lines, wherein part A shows the morphology of the AlmoR cell line, the optical microscopes (magnification 10-fold and 20-fold, respectively) of the 1 st, 10 th, 20 th, 30 th, 40 th and 50 th generation AlmoR th cells of the cell line, the scale bar 200 μm, and part B shows the case of detecting the pan-CK expression of AlmoR th cells by immunofluorescence technique. The green fluorescence signal is pan-CK, the blue fluorescence signal is DAPI, and the C part and the D part represent Short Tandem Repeat (STR) analysis patterns of AlmoR cell lines, which show characteristic peaks of each locus.
FIG. 3 is a schematic representation of a AlmoR cell line with EGFR 19 exon deletion mutations, where parts A and B represent the number and mutation types of Single Nucleotide Variations (SNVs) and indels (indels) of the well-known lung cancer associated genes (TP 53, PTEN, PIK3CA, NF1, MSH2, MET, KRAS, EGFR, APC, ALK, MET, BRAF) detected in AlmoR cells.
FIG. 4 is a graph showing the results of drug resistance verification of AlmoR cell lines, wherein part A is a growth graph obtained by continuously monitoring proliferation of AlmoR cell lines by CCK8 test and drawing, part B is a graph showing sensitivity test of EGFR-mut cell PC9 to ametinib and oxatinib by MTT test, part C is a graph showing sensitivity test of AlmoR to ametinib and oxatinib by MTT test, and part D is a graph showing activation of EGFR and downstream survival signals AKT and ERK of AlmoR cells in control group, oxatinib-treated group and amotinib-treated group by Western blot verification.
FIG. 5 shows results of intracranial tumorigenicity verification of AlmoR cell lines, wherein part A is a 1 st, 4, 8, 12, 16, 20, 24, 28 and 32-day in vivo imaging image of a nude mouse AlmoR cell brain in situ tumor implantation for 3 weeks, part B is a AlmoR cell brain in situ implantation growth curve chart, part C is a AlmoR tumor-bearing nude mouse fresh cranium image, the nude mouse cranium is taken out at the end stage of the tumor-bearing nude mouse disease, the tumor is a blue arrow part, and part D is a mouse brain in situ tumor implantation HE and pan-CK staining image.
The achievement of the objects, functional features and advantages of the present application will be further described with reference to the accompanying drawings, in conjunction with the embodiments.
Detailed Description
The following description of the embodiments of the present application will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
The application relates to an EGFR inhibitor resistant human non-small cell lung cancer (NSCLC) brain metastasis cell line, named AlmoR1. The cell line is obtained by isolating and culturing from brain metastasis resistant tissue of a patient with EGFR mutant non-small cell lung cancer. AlmoR1 cell lines have EGFR 19 exon deletion mutations and exhibit broad resistance to EGFR-TKIs and have good intracranial neoplasia.
The establishment of the cell line is helpful for researching the molecular mechanism of EGFR inhibitor targeting drug resistance, and provides precious experimental materials for developing novel treatment methods. AlmoR1 cell line not only maintains the main molecular characteristics of original tumor EGFR 19 exon deletion mutation, but also shows the growth advantage in-vitro and in-vivo environment, so that the EGFR inhibitor is an ideal model for researching EGFR inhibitor drug resistance mechanism and drug screening. In addition, almoR cell line can be used for in vivo and in vitro drug sensitivity test, new drug research and targeted therapy research, and provides basis for the establishment of personalized therapy strategy.
The cell line AlmoR of the application is preserved in a cell bank, and the preservation number is CGMCC No.45858. The cell line is a first humanized lung cancer brain metastasis cell line, has driving gene mutation and acquires drug resistance to the EGFR-TKIs of the third generation, so that the cell line can be used as a tool cell for targeted drug resistance research, and has an pioneering significance in the field of targeted drug resistance research of lung cancer brain metastasis.
Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, and the use of NSCLC cell lines in research has been helpful in understanding its biological properties and in developing new therapies. The following are mutant cell lines ：ZX2021H、NCI-H2030、NCI-H1975、NCI-H2228、NCI-H2122、NCI-H23、Calu-3、PC9、NCI-H3255、NCI-H3122、NCI-H1792、NCI-H1435. used in existing non-small cell lung cancer studies, which are all sensitive to TKIs, which cannot truly mimic clinical drug resistance, and which currently do not have brain metastasis-derived cell lines. The cell line AlmoR is a NSCLC brain transfer cell line with mutation for resisting TKIs, and can provide a good research platform for intracranial targeting resistance of non-small cell lung cancer.
In addition, one of the problems that is currently critical to the study of brain metastases is the modeling problem of brain metastases. The modes of modeling brain metastases include in situ brain metastasis models (e.g., brain metastases from subcutaneous injection of melanoma, brain metastases from breast cancer cells from breast pad inoculation), ex situ brain metastasis models (tail vein injection, left ventricle injection, and carotid artery injection), and intracranial tumor implantation models. However, the currently available brain metastasis models cannot completely simulate the whole steps of brain metastasis, so that various defects exist in the modeling mode of brain metastasis. One of the important defects is that a lung cancer cell line is used in the brain metastasis modeling process, the intracranial tumor environment of the actual brain metastasis cannot be simulated, and research on brain metastasis targeting drug resistance is lacking currently. The AlmoR cell line of the invention is directly extracted from intracranial tumor tissues of NSCLC patients, so that the cell line is used for modeling, thereby being more consistent with the drug resistance reality condition of brain metastasis and better convincing.
In summary, the application aims to provide an EGFR mutant non-small cell lung cancer brain transfer cell strain with TKIs resistance, which provides a good research platform for basic research of intracranial targeting resistance of non-small cell lung cancer.
Example 1 obtaining a third generation EGFR-TKIs resistant human non-small cell lung cancer brain metastasis specimens to establish EGFR inhibitor resistant human non-small cell lung cancer brain metastasis cell lines, the present application first obtains an example of brain metastasis resistant tissue of EGFR mutant non-small cell lung cancer patients.
As shown in part a of fig. 1, the patient was diagnosed with lung cancer brain metastasis at month 6 of 2021 and underwent brain metastasis resection at month 7. The brain magnetic resonance was reviewed in 2022, 5 th month, to find new foci in the right brain hemisphere, and the puncture biopsy suggested that EGFR 19 exon deletion combined with MET amplification. Patients received both ametinib in combination with An Luoti < 2> (6 months), ametinib in combination with crizotinib (2 months), and ametinib (2 months) alone until lesion progression and a second brain metastasis resection (2023, 3, 10 days) and lesions also suggested 19 exon deletion combined MET amplification.
Subsequent to the administration of oxatinib in combination with bevacizumab treatment (3 months), lesions developed again on day 23 of 7 of 2023, the patient received a third brain transfer oncology resection (part B in fig. 1).
The last surgical specimens were subjected to HE and immunohistochemical staining to confirm that the lesions were non-small cell lung cancer brain metastasis tissues with proliferative activity (part C in fig. 1).
EXAMPLE 2 culture and identification of AlmR1 cell line A cell line was successfully constructed after 50 consecutive in vitro cultures by extracting the primary cell line from fresh brain metastasis tissue from the last surgery and purifying the primary cell line by differential adherence, designated AlmoR1.
As shown in part A of FIG. 2, the morphology of the 1 st, 10, 20, 30, 40 and 50 th generation cells was continuously collected by an optical microscope during the in vitro continuous culture of AlmoR1, and the observation result showed that the morphology of the cell line was stable.
The use of immunofluorescence technique to detect the broad expression of pan-CK by AlmoR cells, which was a preliminary demonstration of the fact that the cells were tumor cells of epithelial origin (FIG. 2, part B).
Next, STR identification was performed on AlmoR cell lines, and AlmoR cell line was found to have a unique STR profile by testing information from 8 STR loci (CSF 1PO, D13S317, D16S539, D5S818, D7S820, THO1, TPOX, vWA) and excluded the possibility of cross-contamination with other cells (fig. 2C-2D).
Taken together, the data demonstrate that the application establishes a new and stable lung cancer brain metastasis cell line.
Example 3 AlmoR1 cell line has EGFR 19 exon deletion mutation to determine the mutation site information of AlmoR cell line, it was subjected to whole exon sequencing.
Among the known mutation types of lung cancer-associated genes (TP 53, PTEN, PIK3CA, NF1, MSH2, MET, KRAS, EGFR, APC, ALK, MET, BRAF) are mainly single point mutations (SNVs) and indels (part A in FIG. 3 and part B in FIG. 3).
Further, the present application searches for EGFR, ALK, MET sites and finds that only EGFR E746_A750del site is mutated to a sense mutation, which is the most common type of 19 exon deletion (Table 1).
The mutation sites of ALK and MET are nonsense mutations, and are shown in tables 2-3. The above results suggest that the lung cancer brain metastasis cell line AlmoR1 constructed in the present application has EGFR 19 exon deletion mutation.
Table 1:
Table 2:
Table 3:
EXAMPLE 4 resistance verification of Almor1 cell line the proliferation of AlmoR cell lines was monitored continuously using the CCK8 experiment and a growth curve (part A in FIG. 4) was plotted which was S-shaped, suggesting that AlmoR cells increased exponentially and were in good condition. To confirm that AlmoR cells were resistant to the third generation EGFR-TKIs, the present application used MTT assays to examine the sensitivity of EGFR-mut cells PC9 and AlmoR1 to both ametinib and oxatinib, resulting in an IC50 of PC9 to the third generation EGFR-TKIs of 0.9. Mu.M and an IC50 of AlmoR1 to the third generation EGFR-TKIs of about 7. Mu.M (part B in FIG. 4 and part C in FIG. 4). Further, the present application demonstrates in Western blot experiments that ametinib and oxatinib can inhibit EGFR phosphorylation, but cannot inhibit activation of downstream survival signals AKT and ERK (part D in fig. 4). These results indicate that the primary lung cancer brain metastasis cell line AlmoR constructed by the present application is universally resistant to EGFR-TKIs.
EXAMPLE 5 intracranial tumorigenicity verification of AlmR1 cell line to assess whether AlmoR cells have intracranial tumorigenicity, the present application successfully constructed AlmoR/luc stably expressing green fluorescence using lentiviral transfection experiments, and constructed brain in situ-seeded tumor models using this cell line. After 3 weeks, the results of in vivo imaging of mice showed that (3/4,75%) nude mice had intracranially formed brain in-situ tumors, and that the tumor growth state was good (part a in fig. 5 and part B in fig. 5) as seen from the proliferation curve. Subsequently, the present application extracts the mouse brain in situ-tumor and performs HE, pan-CK staining, and the results show that the pathology type remains highly consistent with the characteristics of the brain metastasis tissue surgically excised by the patient (part C in fig. 5 and part D in fig. 5). Overall AlmoR1 cells showed good intracranial neoplasia.
In summary, the primary extracted non-small cell lung cancer brain metastasis cell line AlmoR of the present application has EGFR 19 exon deletion mutation, and exhibits broad resistance to EGFR-TKIs and good intracranial neoplasia. The cell line is a first humanized lung cancer brain metastasis cell line, has driving gene mutation and acquires drug resistance to the EGFR-TKIs of the third generation, so that the cell line can be used as a tool cell for targeted drug resistance research, and has an pioneering significance in the field of targeted drug resistance research of lung cancer brain metastasis.
The foregoing description is only of the preferred embodiments of the present application, and is not intended to limit the scope of the application, but rather is intended to cover any equivalents of the structures or equivalent processes disclosed herein or in the alternative, which may be employed directly or indirectly in other related arts.
Claims (9)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202411358049.4A CN118879636B (en) | 2024-09-27 | 2024-09-27 | Cell strain AlmoR1 and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202411358049.4A CN118879636B (en) | 2024-09-27 | 2024-09-27 | Cell strain AlmoR1 and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN118879636A CN118879636A (en) | 2024-11-01 |
| CN118879636B true CN118879636B (en) | 2024-12-03 |
Family
ID=93229038
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202411358049.4A Active CN118879636B (en) | 2024-09-27 | 2024-09-27 | Cell strain AlmoR1 and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118879636B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111218424A (en) * | 2019-12-12 | 2020-06-02 | 广州医科大学附属第一医院 | Osimertinib-resistant cell line NCI-H1975/AR and its application |
| CN115927187A (en) * | 2022-12-30 | 2023-04-07 | 河南省肿瘤医院 | Ametinib drug-resistant cell line NCI-H1975-AR and application thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2023524639A (en) * | 2020-04-22 | 2023-06-13 | アイオバンス バイオセラピューティクス，インコーポレイテッド | Manufacturing adjustment system and adjustment method of cells for patient-specific immunotherapy |
| AU2022212123A1 (en) * | 2021-01-29 | 2023-09-07 | Board Of Regents, The University Of Texas System | Methods of treating cancer with kinase inhibitors |
| CN116200340B (en) * | 2022-12-30 | 2023-08-15 | 河南省肿瘤医院 | Aftetinib-resistant human lung adenocarcinoma cell line PC9-AR and application thereof |
-
2024
- 2024-09-27 CN CN202411358049.4A patent/CN118879636B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111218424A (en) * | 2019-12-12 | 2020-06-02 | 广州医科大学附属第一医院 | Osimertinib-resistant cell line NCI-H1975/AR and its application |
| CN115927187A (en) * | 2022-12-30 | 2023-04-07 | 河南省肿瘤医院 | Ametinib drug-resistant cell line NCI-H1975-AR and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN118879636A (en) | 2024-11-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Hodgkinson et al. | Tumorigenicity and genetic profiling of circulating tumor cells in small-cell lung cancer | |
| US10844436B2 (en) | Use of double-stranded DNA in exosomes: a novel biomarker in cancer detection | |
| JP7519125B2 (en) | Probes and methods for detecting transcripts resulting from fusion genes and/or exon skipping - Patents.com | |
| WO2014061419A1 (en) | Novel cancer marker and utilization thereof | |
| Lohberger et al. | Establishment of a novel cellular model for myxofibrosarcoma heterogeneity | |
| CN110373471A (en) | Blood plasma excretion body tRFs marker and its application in breast cancer diagnosis | |
| CN109680064B (en) | Application of YTHDF2 gene in the diagnosis, prevention and treatment of urothelial carcinoma | |
| Wang et al. | Genome-wide CRISPR-Cas9 screening and identification of potential genes promoting prostate cancer growth and metastasis | |
| CN111187835A (en) | Pancreatic cancer target ERBB2 and its application in diagnosis and treatment | |
| Wang et al. | Dynamic viral integration patterns actively participate in the progression of BK polyomavirus-associated diseases after renal transplantation | |
| CN116463291A (en) | Human colorectal cancer primary focus and liver transfer focus pairing organoid cell line and culture method and application thereof | |
| Liu et al. | Establishment of a high-fidelity patient-derived xenograft model for cervical cancer enables the evaluation of patient’s response to conventional and novel therapies | |
| Yao et al. | Detecting EGFR mutations and ALK/ROS1 rearrangements in non‐small cell lung cancer using malignant pleural effusion samples | |
| Ida et al. | Clinicopathological and genetic analyses of small cell neuroendocrine carcinoma of the prostate: histological features for accurate diagnosis and toward future novel therapies | |
| Skakodub et al. | Genomic analysis and clinical correlations of non-small cell lung cancer (NSCLC) brain metastasis (BM) | |
| CN118879636B (en) | Cell strain AlmoR1 and application thereof | |
| CN109971832A (en) | It is a kind of to detect the kit of gene mutation, method and application thereof | |
| CN111793687A (en) | Inhibition of lung adenocarcinoma target CRTAC1 and its application | |
| CN111206102A (en) | A serum exosomal miRNA marker related to the diagnosis of liver cancer and its application | |
| CN110872624A (en) | A colorectal cancer marker and its application | |
| Cheng et al. | Establishment and characteristics of GWH04, a new primary human glioblastoma cell line | |
| Channir et al. | Genetic characterization of adenoid cystic carcinoma of the minor salivary glands: a potential familial occurrence in first-degree relatives | |
| CN114045286A (en) | Gene set for molecular typing of pancreatic cancer and its application | |
| Zhang et al. | Highly Sensitive Gold Nanoparticle Polymerase Chain Reaction in the Detection of Anaplastic Lymphoma Kinase-Positive Gastric Cancer from a Biopsy Specimen | |
| CN106635999A (en) | Establishment and application of MMHRL1 transgenic mouse liver tumor cell line |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |