JP6700048B2 - Anti-cancer drug evaluation method - Google Patents
Anti-cancer drug evaluation method Download PDFInfo
- Publication number
- JP6700048B2 JP6700048B2 JP2016008264A JP2016008264A JP6700048B2 JP 6700048 B2 JP6700048 B2 JP 6700048B2 JP 2016008264 A JP2016008264 A JP 2016008264A JP 2016008264 A JP2016008264 A JP 2016008264A JP 6700048 B2 JP6700048 B2 JP 6700048B2
- Authority
- JP
- Japan
- Prior art keywords
- anticancer agent
- cells
- cancer
- tumor cells
- cell
- 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
- 239000002246 antineoplastic agent Substances 0.000 title claims description 124
- 229940041181 antineoplastic drug Drugs 0.000 title claims description 20
- 238000011156 evaluation Methods 0.000 title claims description 14
- 210000004027 cell Anatomy 0.000 claims description 157
- 210000004881 tumor cell Anatomy 0.000 claims description 75
- 238000000034 method Methods 0.000 claims description 50
- 238000001514 detection method Methods 0.000 claims description 41
- 230000009471 action Effects 0.000 claims description 35
- 239000012472 biological sample Substances 0.000 claims description 35
- 238000002372 labelling Methods 0.000 claims description 34
- 239000000758 substrate Substances 0.000 claims description 33
- 239000003814 drug Substances 0.000 claims description 29
- 229940079593 drug Drugs 0.000 claims description 25
- 238000000926 separation method Methods 0.000 claims description 22
- 230000000694 effects Effects 0.000 claims description 15
- 210000004369 blood Anatomy 0.000 claims description 14
- 239000008280 blood Substances 0.000 claims description 14
- 239000007788 liquid Substances 0.000 claims description 12
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 claims description 11
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 claims description 11
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 claims description 11
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 claims description 11
- 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 claims description 11
- 238000011161 development Methods 0.000 claims description 8
- 230000005484 gravity Effects 0.000 claims description 8
- 229960000575 trastuzumab Drugs 0.000 claims description 8
- 229960001972 panitumumab Drugs 0.000 claims description 7
- 239000002136 L01XE07 - Lapatinib Substances 0.000 claims description 5
- 229960004891 lapatinib Drugs 0.000 claims description 5
- BCFGMOOMADDAQU-UHFFFAOYSA-N lapatinib Chemical compound O1C(CNCCS(=O)(=O)C)=CC=C1C1=CC=C(N=CN=C2NC=3C=C(Cl)C(OCC=4C=C(F)C=CC=4)=CC=3)C2=C1 BCFGMOOMADDAQU-UHFFFAOYSA-N 0.000 claims description 5
- 239000002118 L01XE12 - Vandetanib Substances 0.000 claims description 4
- 229960005395 cetuximab Drugs 0.000 claims description 4
- 229960000241 vandetanib Drugs 0.000 claims description 4
- UHTHHESEBZOYNR-UHFFFAOYSA-N vandetanib Chemical compound COC1=CC(C(/N=CN2)=N/C=3C(=CC(Br)=CC=3)F)=C2C=C1OCC1CCN(C)CC1 UHTHHESEBZOYNR-UHFFFAOYSA-N 0.000 claims description 4
- 239000005411 L01XE02 - Gefitinib Substances 0.000 claims description 3
- 239000005551 L01XE03 - Erlotinib Substances 0.000 claims description 3
- 229960001686 afatinib Drugs 0.000 claims description 3
- ULXXDDBFHOBEHA-CWDCEQMOSA-N afatinib Chemical compound N1=CN=C2C=C(O[C@@H]3COCC3)C(NC(=O)/C=C/CN(C)C)=CC2=C1NC1=CC=C(F)C(Cl)=C1 ULXXDDBFHOBEHA-CWDCEQMOSA-N 0.000 claims description 3
- 229960001433 erlotinib Drugs 0.000 claims description 3
- AAKJLRGGTJKAMG-UHFFFAOYSA-N erlotinib Chemical compound C=12C=C(OCCOC)C(OCCOC)=CC2=NC=NC=1NC1=CC=CC(C#C)=C1 AAKJLRGGTJKAMG-UHFFFAOYSA-N 0.000 claims description 3
- XGALLCVXEZPNRQ-UHFFFAOYSA-N gefitinib Chemical compound C=12C=C(OCCCN3CCOCC3)C(OC)=CC2=NC=NC=1NC1=CC=C(F)C(Cl)=C1 XGALLCVXEZPNRQ-UHFFFAOYSA-N 0.000 claims description 3
- 229960002584 gefitinib Drugs 0.000 claims description 3
- 229960002087 pertuzumab Drugs 0.000 claims description 3
- 208000005443 Circulating Neoplastic Cells Diseases 0.000 claims description 2
- 238000007789 sealing Methods 0.000 claims 1
- 239000000126 substance Substances 0.000 description 84
- 239000000243 solution Substances 0.000 description 43
- 206010028980 Neoplasm Diseases 0.000 description 35
- 239000000523 sample Substances 0.000 description 27
- 201000011510 cancer Diseases 0.000 description 25
- 102000011782 Keratins Human genes 0.000 description 20
- 108010076876 Keratins Proteins 0.000 description 20
- 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 17
- 230000008859 change Effects 0.000 description 14
- 206010009944 Colon cancer Diseases 0.000 description 13
- 208000029742 colonic neoplasm Diseases 0.000 description 13
- 210000000265 leukocyte Anatomy 0.000 description 13
- 206010006187 Breast cancer Diseases 0.000 description 12
- 208000026310 Breast neoplasm Diseases 0.000 description 12
- 238000005119 centrifugation Methods 0.000 description 12
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 11
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 11
- -1 urine Substances 0.000 description 10
- 230000007423 decrease Effects 0.000 description 9
- 210000003743 erythrocyte Anatomy 0.000 description 9
- 239000004698 Polyethylene Substances 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- 239000000725 suspension Substances 0.000 description 7
- 229940125644 antibody drug Drugs 0.000 description 6
- 210000000170 cell membrane Anatomy 0.000 description 6
- 238000004891 communication Methods 0.000 description 6
- 230000003287 optical effect Effects 0.000 description 6
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical group N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 5
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 5
- 229930195725 Mannitol Natural products 0.000 description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 5
- 239000006285 cell suspension Substances 0.000 description 5
- 230000000857 drug effect Effects 0.000 description 5
- 239000000594 mannitol Substances 0.000 description 5
- 235000010355 mannitol Nutrition 0.000 description 5
- 238000012545 processing Methods 0.000 description 5
- 239000004743 Polypropylene Substances 0.000 description 4
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 4
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 4
- 239000011230 binding agent Substances 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 230000005684 electric field Effects 0.000 description 4
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 4
- 239000007850 fluorescent dye Substances 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 229920001155 polypropylene Polymers 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 229940124597 therapeutic agent Drugs 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 108090001008 Avidin Proteins 0.000 description 3
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 description 3
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 3
- 206010025323 Lymphomas Diseases 0.000 description 3
- 108010090804 Streptavidin Proteins 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- 229960000397 bevacizumab Drugs 0.000 description 3
- 229960002685 biotin Drugs 0.000 description 3
- 235000020958 biotin Nutrition 0.000 description 3
- 239000011616 biotin Substances 0.000 description 3
- 210000005266 circulating tumour cell Anatomy 0.000 description 3
- 239000012141 concentrate Substances 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 201000005202 lung cancer Diseases 0.000 description 3
- 208000020816 lung neoplasm Diseases 0.000 description 3
- 239000002829 mitogen activated protein kinase inhibitor Substances 0.000 description 3
- 229960004641 rituximab Drugs 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 102100033793 ALK tyrosine kinase receptor Human genes 0.000 description 2
- 101710168331 ALK tyrosine kinase receptor Proteins 0.000 description 2
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- MLDQJTXFUGDVEO-UHFFFAOYSA-N BAY-43-9006 Chemical compound C1=NC(C(=O)NC)=CC(OC=2C=CC(NC(=O)NC=3C=C(C(Cl)=CC=3)C(F)(F)F)=CC=2)=C1 MLDQJTXFUGDVEO-UHFFFAOYSA-N 0.000 description 2
- 206010005003 Bladder cancer Diseases 0.000 description 2
- 102100024217 CAMPATH-1 antigen Human genes 0.000 description 2
- 108010065524 CD52 Antigen Proteins 0.000 description 2
- 229920002307 Dextran Polymers 0.000 description 2
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 2
- 206010018910 Haemolysis Diseases 0.000 description 2
- 101000934338 Homo sapiens Myeloid cell surface antigen CD33 Proteins 0.000 description 2
- 101000851376 Homo sapiens Tumor necrosis factor receptor superfamily member 8 Proteins 0.000 description 2
- 208000008839 Kidney Neoplasms Diseases 0.000 description 2
- 239000002147 L01XE04 - Sunitinib Substances 0.000 description 2
- 239000005511 L01XE05 - Sorafenib Substances 0.000 description 2
- 239000005536 L01XE08 - Nilotinib Substances 0.000 description 2
- 239000003798 L01XE11 - Pazopanib Substances 0.000 description 2
- 108090001090 Lectins Proteins 0.000 description 2
- 102000004856 Lectins Human genes 0.000 description 2
- 102100025243 Myeloid cell surface antigen CD33 Human genes 0.000 description 2
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 2
- 206010033128 Ovarian cancer Diseases 0.000 description 2
- 206010061535 Ovarian neoplasm Diseases 0.000 description 2
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 2
- 229940079156 Proteasome inhibitor Drugs 0.000 description 2
- 206010038389 Renal cancer Diseases 0.000 description 2
- 208000005718 Stomach Neoplasms Diseases 0.000 description 2
- 102100036857 Tumor necrosis factor receptor superfamily member 8 Human genes 0.000 description 2
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 2
- 108091008605 VEGF receptors Proteins 0.000 description 2
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 2
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 2
- 102100033177 Vascular endothelial growth factor receptor 2 Human genes 0.000 description 2
- MCMNRKCIXSYSNV-UHFFFAOYSA-N Zirconium dioxide Chemical compound O=[Zr]=O MCMNRKCIXSYSNV-UHFFFAOYSA-N 0.000 description 2
- 230000004308 accommodation Effects 0.000 description 2
- 229960000548 alemtuzumab Drugs 0.000 description 2
- 230000001093 anti-cancer Effects 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 229960003005 axitinib Drugs 0.000 description 2
- RITAVMQDGBJQJZ-FMIVXFBMSA-N axitinib Chemical compound CNC(=O)C1=CC=CC=C1SC1=CC=C(C(\C=C\C=2N=CC=CC=2)=NN2)C2=C1 RITAVMQDGBJQJZ-FMIVXFBMSA-N 0.000 description 2
- 229920001222 biopolymer Polymers 0.000 description 2
- 229960000455 brentuximab vedotin Drugs 0.000 description 2
- 238000011088 calibration curve Methods 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 229920001577 copolymer Polymers 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 238000001647 drug administration Methods 0.000 description 2
- 201000004101 esophageal cancer Diseases 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 238000001215 fluorescent labelling Methods 0.000 description 2
- 206010017758 gastric cancer Diseases 0.000 description 2
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 2
- 230000008588 hemolysis Effects 0.000 description 2
- 239000012212 insulator Substances 0.000 description 2
- NBQNWMBBSKPBAY-UHFFFAOYSA-N iodixanol Chemical compound IC=1C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C(I)C=1N(C(=O)C)CC(O)CN(C(C)=O)C1=C(I)C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C1I NBQNWMBBSKPBAY-UHFFFAOYSA-N 0.000 description 2
- 201000010982 kidney cancer Diseases 0.000 description 2
- 239000002523 lectin Substances 0.000 description 2
- 208000032839 leukemia Diseases 0.000 description 2
- 201000007270 liver cancer Diseases 0.000 description 2
- 208000014018 liver neoplasm Diseases 0.000 description 2
- 230000033001 locomotion Effects 0.000 description 2
- 238000004020 luminiscence type Methods 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 229960001346 nilotinib Drugs 0.000 description 2
- HHZIURLSWUIHRB-UHFFFAOYSA-N nilotinib Chemical compound C1=NC(C)=CN1C1=CC(NC(=O)C=2C=C(NC=3N=C(C=CN=3)C=3C=NC=CC=3)C(C)=CC=2)=CC(C(F)(F)F)=C1 HHZIURLSWUIHRB-UHFFFAOYSA-N 0.000 description 2
- 229960004378 nintedanib Drugs 0.000 description 2
- XZXHXSATPCNXJR-ZIADKAODSA-N nintedanib Chemical compound O=C1NC2=CC(C(=O)OC)=CC=C2\C1=C(C=1C=CC=CC=1)\NC(C=C1)=CC=C1N(C)C(=O)CN1CCN(C)CC1 XZXHXSATPCNXJR-ZIADKAODSA-N 0.000 description 2
- 201000002528 pancreatic cancer Diseases 0.000 description 2
- 208000008443 pancreatic carcinoma Diseases 0.000 description 2
- 229960000639 pazopanib Drugs 0.000 description 2
- CUIHSIWYWATEQL-UHFFFAOYSA-N pazopanib Chemical compound C1=CC2=C(C)N(C)N=C2C=C1N(C)C(N=1)=CC=NC=1NC1=CC=C(C)C(S(N)(=O)=O)=C1 CUIHSIWYWATEQL-UHFFFAOYSA-N 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 229920000729 poly(L-lysine) polymer Polymers 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 239000003207 proteasome inhibitor Substances 0.000 description 2
- 230000005855 radiation Effects 0.000 description 2
- 229960002633 ramucirumab Drugs 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 229960003787 sorafenib Drugs 0.000 description 2
- 201000011549 stomach cancer Diseases 0.000 description 2
- 229960001796 sunitinib Drugs 0.000 description 2
- WINHZLLDWRZWRT-ATVHPVEESA-N sunitinib Chemical compound CCN(CC)CCNC(=O)C1=C(C)NC(\C=C/2C3=CC(F)=CC=C3NC\2=O)=C1C WINHZLLDWRZWRT-ATVHPVEESA-N 0.000 description 2
- 238000001356 surgical procedure Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 229940121358 tyrosine kinase inhibitor Drugs 0.000 description 2
- 239000005483 tyrosine kinase inhibitor Substances 0.000 description 2
- 201000005112 urinary bladder cancer Diseases 0.000 description 2
- KKVYYGGCHJGEFJ-UHFFFAOYSA-N 1-n-(4-chlorophenyl)-6-methyl-5-n-[3-(7h-purin-6-yl)pyridin-2-yl]isoquinoline-1,5-diamine Chemical compound N=1C=CC2=C(NC=3C(=CC=CN=3)C=3C=4N=CNC=4N=CN=3)C(C)=CC=C2C=1NC1=CC=C(Cl)C=C1 KKVYYGGCHJGEFJ-UHFFFAOYSA-N 0.000 description 1
- 102000001714 Agammaglobulinaemia Tyrosine Kinase Human genes 0.000 description 1
- 108010029445 Agammaglobulinaemia Tyrosine Kinase Proteins 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 206010061424 Anal cancer Diseases 0.000 description 1
- 208000007860 Anus Neoplasms Diseases 0.000 description 1
- 206010073360 Appendix cancer Diseases 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- 108010074708 B7-H1 Antigen Proteins 0.000 description 1
- 102000008096 B7-H1 Antigen Human genes 0.000 description 1
- 206010005949 Bone cancer Diseases 0.000 description 1
- 208000018084 Bone neoplasm Diseases 0.000 description 1
- 208000003174 Brain Neoplasms Diseases 0.000 description 1
- 108091007914 CDKs Proteins 0.000 description 1
- 206010008342 Cervix carcinoma Diseases 0.000 description 1
- 102000003903 Cyclin-dependent kinases Human genes 0.000 description 1
- 108090000266 Cyclin-dependent kinases Proteins 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- ZBNZXTGUTAYRHI-UHFFFAOYSA-N Dasatinib Chemical compound C=1C(N2CCN(CCO)CC2)=NC(C)=NC=1NC(S1)=NC=C1C(=O)NC1=C(C)C=CC=C1Cl ZBNZXTGUTAYRHI-UHFFFAOYSA-N 0.000 description 1
- 101150029707 ERBB2 gene Proteins 0.000 description 1
- 206010014733 Endometrial cancer Diseases 0.000 description 1
- 206010014759 Endometrial neoplasm Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- BRLQWZUYTZBJKN-UHFFFAOYSA-N Epichlorohydrin Chemical compound ClCC1CO1 BRLQWZUYTZBJKN-UHFFFAOYSA-N 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- 208000022072 Gallbladder Neoplasms Diseases 0.000 description 1
- 206010051066 Gastrointestinal stromal tumour Diseases 0.000 description 1
- 238000002738 Giemsa staining Methods 0.000 description 1
- 206010067807 Gingival cancer Diseases 0.000 description 1
- 102100041003 Glutamate carboxypeptidase 2 Human genes 0.000 description 1
- 208000017604 Hodgkin disease Diseases 0.000 description 1
- 208000010747 Hodgkins lymphoma Diseases 0.000 description 1
- 101000914324 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 5 Proteins 0.000 description 1
- 101000914321 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 7 Proteins 0.000 description 1
- 101000892862 Homo sapiens Glutamate carboxypeptidase 2 Proteins 0.000 description 1
- 101000692455 Homo sapiens Platelet-derived growth factor receptor beta Proteins 0.000 description 1
- 101000617725 Homo sapiens Pregnancy-specific beta-1-glycoprotein 2 Proteins 0.000 description 1
- 101000997832 Homo sapiens Tyrosine-protein kinase JAK2 Proteins 0.000 description 1
- 239000002067 L01XE06 - Dasatinib Substances 0.000 description 1
- 239000002145 L01XE14 - Bosutinib Substances 0.000 description 1
- 239000002146 L01XE16 - Crizotinib Substances 0.000 description 1
- 239000002144 L01XE18 - Ruxolitinib Substances 0.000 description 1
- 239000002177 L01XE27 - Ibrutinib Substances 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 206010027406 Mesothelioma Diseases 0.000 description 1
- BAQCROVBDNBEEB-UBYUBLNFSA-N Metrizamide Chemical compound CC(=O)N(C)C1=C(I)C(NC(C)=O)=C(I)C(C(=O)N[C@@H]2[C@H]([C@H](O)[C@@H](CO)OC2O)O)=C1I BAQCROVBDNBEEB-UBYUBLNFSA-N 0.000 description 1
- 208000003445 Mouth Neoplasms Diseases 0.000 description 1
- 102000015728 Mucins Human genes 0.000 description 1
- 108010063954 Mucins Proteins 0.000 description 1
- 208000034578 Multiple myelomas Diseases 0.000 description 1
- 101100381978 Mus musculus Braf gene Proteins 0.000 description 1
- 208000015914 Non-Hodgkin lymphomas Diseases 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 108010004729 Phycoerythrin Proteins 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 102100026547 Platelet-derived growth factor receptor beta Human genes 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 102100022019 Pregnancy-specific beta-1-glycoprotein 2 Human genes 0.000 description 1
- 206010036790 Productive cough Diseases 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 229940123690 Raf kinase inhibitor Drugs 0.000 description 1
- 208000037323 Rare tumor Diseases 0.000 description 1
- 102100029986 Receptor tyrosine-protein kinase erbB-3 Human genes 0.000 description 1
- 101710100969 Receptor tyrosine-protein kinase erbB-3 Proteins 0.000 description 1
- 208000004337 Salivary Gland Neoplasms Diseases 0.000 description 1
- 206010061934 Salivary gland cancer Diseases 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 208000000453 Skin Neoplasms Diseases 0.000 description 1
- 208000024313 Testicular Neoplasms Diseases 0.000 description 1
- 206010057644 Testis cancer Diseases 0.000 description 1
- 208000024770 Thyroid neoplasm Diseases 0.000 description 1
- 239000004012 Tofacitinib Substances 0.000 description 1
- 206010062129 Tongue neoplasm Diseases 0.000 description 1
- 102100033444 Tyrosine-protein kinase JAK2 Human genes 0.000 description 1
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 1
- 208000002495 Uterine Neoplasms Diseases 0.000 description 1
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 1
- 201000005188 adrenal gland cancer Diseases 0.000 description 1
- 208000024447 adrenal gland neoplasm Diseases 0.000 description 1
- 229960001611 alectinib Drugs 0.000 description 1
- KDGFLJKFZUIJMX-UHFFFAOYSA-N alectinib Chemical compound CCC1=CC=2C(=O)C(C3=CC=C(C=C3N3)C#N)=C3C(C)(C)C=2C=C1N(CC1)CCC1N1CCOCC1 KDGFLJKFZUIJMX-UHFFFAOYSA-N 0.000 description 1
- YVPYQUNUQOZFHG-UHFFFAOYSA-N amidotrizoic acid Chemical compound CC(=O)NC1=C(I)C(NC(C)=O)=C(I)C(C(O)=O)=C1I YVPYQUNUQOZFHG-UHFFFAOYSA-N 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 210000004381 amniotic fluid Anatomy 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000009175 antibody therapy Methods 0.000 description 1
- 239000012635 anticancer drug combination Substances 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 201000011165 anus cancer Diseases 0.000 description 1
- 208000021780 appendiceal neoplasm Diseases 0.000 description 1
- 210000001367 artery Anatomy 0.000 description 1
- 102000004441 bcr-abl Fusion Proteins Human genes 0.000 description 1
- 108010056708 bcr-abl Fusion Proteins Proteins 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 102000005936 beta-Galactosidase Human genes 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 229960001467 bortezomib Drugs 0.000 description 1
- GXJABQQUPOEUTA-RDJZCZTQSA-N bortezomib Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)B(O)O)NC(=O)C=1N=CC=NC=1)C1=CC=CC=C1 GXJABQQUPOEUTA-RDJZCZTQSA-N 0.000 description 1
- 229960003736 bosutinib Drugs 0.000 description 1
- UBPYILGKFZZVDX-UHFFFAOYSA-N bosutinib Chemical compound C1=C(Cl)C(OC)=CC(NC=2C3=CC(OC)=C(OCCCN4CCN(C)CC4)C=C3N=CC=2C#N)=C1Cl UBPYILGKFZZVDX-UHFFFAOYSA-N 0.000 description 1
- 239000003990 capacitor Substances 0.000 description 1
- 229960002438 carfilzomib Drugs 0.000 description 1
- 108010021331 carfilzomib Proteins 0.000 description 1
- BLMPQMFVWMYDKT-NZTKNTHTSA-N carfilzomib Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC(C)C)C(=O)[C@]1(C)OC1)NC(=O)CN1CCOCC1)CC1=CC=CC=C1 BLMPQMFVWMYDKT-NZTKNTHTSA-N 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 229960001602 ceritinib Drugs 0.000 description 1
- VERWOWGGCGHDQE-UHFFFAOYSA-N ceritinib Chemical compound CC=1C=C(NC=2N=C(NC=3C(=CC=CC=3)S(=O)(=O)C(C)C)C(Cl)=CN=2)C(OC(C)C)=CC=1C1CCNCC1 VERWOWGGCGHDQE-UHFFFAOYSA-N 0.000 description 1
- 201000010881 cervical cancer Diseases 0.000 description 1
- 201000004948 cheek mucosa cancer Diseases 0.000 description 1
- 208000006990 cholangiocarcinoma Diseases 0.000 description 1
- 239000008119 colloidal silica Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 229960005061 crizotinib 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
- 229940043378 cyclin-dependent kinase inhibitor Drugs 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 229960002465 dabrafenib Drugs 0.000 description 1
- BFSMGDJOXZAERB-UHFFFAOYSA-N dabrafenib Chemical compound S1C(C(C)(C)C)=NC(C=2C(=C(NS(=O)(=O)C=3C(=CC=CC=3F)F)C=CC=2)F)=C1C1=CC=NC(N)=N1 BFSMGDJOXZAERB-UHFFFAOYSA-N 0.000 description 1
- 229960002448 dasatinib Drugs 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 229960002086 dextran Drugs 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229960005223 diatrizoic acid Drugs 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000000428 dust Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 239000012997 ficoll-paque Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000012041 food component Nutrition 0.000 description 1
- 201000010175 gallbladder cancer Diseases 0.000 description 1
- 201000011243 gastrointestinal stromal tumor Diseases 0.000 description 1
- 229960003297 gemtuzumab ozogamicin Drugs 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229960005150 glycerol Drugs 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 229910052736 halogen Inorganic materials 0.000 description 1
- 150000002367 halogens Chemical class 0.000 description 1
- 201000010536 head and neck cancer Diseases 0.000 description 1
- 208000014829 head and neck neoplasm Diseases 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 229940022353 herceptin Drugs 0.000 description 1
- 229960001507 ibrutinib Drugs 0.000 description 1
- XYFPWWZEPKGCCK-GOSISDBHSA-N ibrutinib Chemical compound C1=2C(N)=NC=NC=2N([C@H]2CN(CCC2)C(=O)C=C)N=C1C(C=C1)=CC=C1OC1=CC=CC=C1 XYFPWWZEPKGCCK-GOSISDBHSA-N 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 229910010272 inorganic material Inorganic materials 0.000 description 1
- 239000011147 inorganic material Substances 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 229960004359 iodixanol Drugs 0.000 description 1
- 229960001025 iohexol Drugs 0.000 description 1
- NTHXOOBQLCIOLC-UHFFFAOYSA-N iohexol Chemical compound OCC(O)CN(C(=O)C)C1=C(I)C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C1I NTHXOOBQLCIOLC-UHFFFAOYSA-N 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 229960005386 ipilimumab Drugs 0.000 description 1
- 239000012948 isocyanate Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- FZWBNHMXJMCXLU-BLAUPYHCSA-N isomaltotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)O1 FZWBNHMXJMCXLU-BLAUPYHCSA-N 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 229960003784 lenvatinib Drugs 0.000 description 1
- WOSKHXYHFSIKNG-UHFFFAOYSA-N lenvatinib Chemical compound C=12C=C(C(N)=O)C(OC)=CC2=NC=CC=1OC(C=C1Cl)=CC=C1NC(=O)NC1CC1 WOSKHXYHFSIKNG-UHFFFAOYSA-N 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 208000012987 lip and oral cavity carcinoma Diseases 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 239000006249 magnetic particle Substances 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229960005108 mepolizumab Drugs 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 208000037819 metastatic cancer Diseases 0.000 description 1
- 208000011575 metastatic malignant neoplasm Diseases 0.000 description 1
- 229960000554 metrizamide Drugs 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 229950007699 mogamulizumab Drugs 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 229940051875 mucins Drugs 0.000 description 1
- 229960003301 nivolumab Drugs 0.000 description 1
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 1
- 210000004882 non-tumor cell Anatomy 0.000 description 1
- 238000012758 nuclear staining Methods 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 229950005751 ocrelizumab Drugs 0.000 description 1
- 229960002450 ofatumumab Drugs 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 229920000620 organic polymer Polymers 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 201000008968 osteosarcoma Diseases 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 102000051624 phosphatidylethanolamine binding protein Human genes 0.000 description 1
- 108700021017 phosphatidylethanolamine binding protein Proteins 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 229920001481 poly(stearyl methacrylate) Polymers 0.000 description 1
- 229920000058 polyacrylate Polymers 0.000 description 1
- 229920002239 polyacrylonitrile Polymers 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 239000004417 polycarbonate Substances 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920000193 polymethacrylate Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 229920000915 polyvinyl chloride Polymers 0.000 description 1
- 239000004800 polyvinyl chloride Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 102000009929 raf Kinases Human genes 0.000 description 1
- 108010077182 raf Kinases Proteins 0.000 description 1
- 229960003876 ranibizumab Drugs 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 229960000215 ruxolitinib Drugs 0.000 description 1
- HFNKQEVNSGCOJV-OAHLLOKOSA-N ruxolitinib Chemical compound C1([C@@H](CC#N)N2N=CC(=C2)C=2C=3C=CNC=3N=CN=2)CCCC1 HFNKQEVNSGCOJV-OAHLLOKOSA-N 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 210000000582 semen Anatomy 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 208000037968 sinus cancer Diseases 0.000 description 1
- 201000000849 skin cancer Diseases 0.000 description 1
- YEENEYXBHNNNGV-XEHWZWQGSA-M sodium;3-acetamido-5-[acetyl(methyl)amino]-2,4,6-triiodobenzoate;(2r,3r,4s,5s,6r)-2-[(2r,3s,4s,5r)-3,4-dihydroxy-2,5-bis(hydroxymethyl)oxolan-2-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound [Na+].CC(=O)N(C)C1=C(I)C(NC(C)=O)=C(I)C(C([O-])=O)=C1I.O[C@H]1[C@H](O)[C@@H](CO)O[C@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 YEENEYXBHNNNGV-XEHWZWQGSA-M 0.000 description 1
- 210000003802 sputum Anatomy 0.000 description 1
- 208000024794 sputum Diseases 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 229960004793 sucrose Drugs 0.000 description 1
- 201000003120 testicular cancer Diseases 0.000 description 1
- 201000002510 thyroid cancer Diseases 0.000 description 1
- 229960001350 tofacitinib Drugs 0.000 description 1
- UJLAWZDWDVHWOW-YPMHNXCESA-N tofacitinib Chemical compound C[C@@H]1CCN(C(=O)CC#N)C[C@@H]1N(C)C1=NC=NC2=C1C=CN2 UJLAWZDWDVHWOW-YPMHNXCESA-N 0.000 description 1
- 201000006134 tongue cancer Diseases 0.000 description 1
- 229960004066 trametinib Drugs 0.000 description 1
- LIRYPHYGHXZJBZ-UHFFFAOYSA-N trametinib Chemical compound CC(=O)NC1=CC=CC(N2C(N(C3CC3)C(=O)C3=C(NC=4C(=CC(I)=CC=4)F)N(C)C(=O)C(C)=C32)=O)=C1 LIRYPHYGHXZJBZ-UHFFFAOYSA-N 0.000 description 1
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 1
- 150000004917 tyrosine kinase inhibitor derivatives Chemical class 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 206010046766 uterine cancer Diseases 0.000 description 1
- 229960003862 vemurafenib Drugs 0.000 description 1
- GPXBXXGIAQBQNI-UHFFFAOYSA-N vemurafenib Chemical compound CCCS(=O)(=O)NC1=CC=C(F)C(C(=O)C=2C3=CC(=CN=C3NC=2)C=2C=CC(Cl)=CC=2)=C1F GPXBXXGIAQBQNI-UHFFFAOYSA-N 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
本発明は、抗癌剤の薬効を評価する方法であり、特に好適には腫瘍細胞の抗癌剤作用部を標識物質により標識し、抗癌剤適用による光強度の変化から、抗癌剤が腫瘍細胞に対して効果的に作用しているかを判断する方法に関する。 The present invention is a method for evaluating the drug efficacy of an anticancer agent, particularly preferably an anticancer agent acting part of a tumor cell is labeled with a labeling substance, and the anticancer agent effectively acts on the tumor cell from a change in light intensity due to the application of the anticancer agent. It is about how to determine if it is working.
腫瘍細胞が保有するレセプターなどのタンパク質を標的とし、当該標的タンパク質を有する腫瘍細胞に対し特異的に作用する抗癌剤が、分子標的治療薬として開発されている。例えば、乳癌細胞等のＨＥＲ２／ｎｅｕに対するトラスツズマブ、大腸癌細胞等のＥＧＦＲに対するセツキシマブ、パニツムマブ、リンパ腫のＣＤ２０に対するリツキシマブ、非小細胞肺癌、卵巣癌細胞等の血管内皮細胞増殖因子ＶＥＧＦに対するベバシズマブなどが挙げられる（非特許文献１）。これらの抗癌剤の腫瘍細胞に対する効果は、ＣＴ、ＭＲＩ検査などに代表される画像診断での腫瘍組織の縮小や腫瘍マーカーの低下等で判断されている。 An anticancer agent that targets a protein such as a receptor possessed by a tumor cell and specifically acts on a tumor cell having the target protein has been developed as a molecular target therapeutic agent. Examples include trastuzumab for HER2/neu of breast cancer cells, cetuximab for pancreatum mab, EGFR of colon cancer cells, rituximab for CD20 of lymphoma, bevacizumab for vascular endothelial growth factor VEGF of non-small cell lung cancer, ovarian cancer cells, etc. (Non-patent document 1). The effect of these anti-cancer agents on tumor cells is judged by the reduction of tumor tissue and the reduction of tumor markers in image diagnosis represented by CT and MRI examinations.
抗癌剤の効果をＣＴやＭＲＩ等による画像診断で評価する場合、１カ月程度の期間をおいて腫瘍サイズの増大もしくは縮小が明確になるのを待つ必要があるため、短期間もしくはリアルタイムでの薬効判断には不適である。また腫瘍マーカーは、腫瘍細胞そのものを直接検出していないため、腫瘍以外の因子による偽陽性が発生したり、病態悪化に相関しない偽陰性が発生することから、薬効を正確に評価し得るには至っていない。 When evaluating the effects of anti-cancer agents by CT or MRI imaging, it is necessary to wait for a period of about one month until the tumor size increases or decreases, so it is possible to judge the drug efficacy in a short period or in real time. Is not suitable for. In addition, since tumor cells do not directly detect tumor cells themselves, false positives due to factors other than tumors and false negatives that do not correlate with the deterioration of the disease state occur, so that the drug efficacy can be accurately evaluated. I haven't arrived.
一方で、近年、血中循環腫瘍細胞（Ｃｉｒｃｕｌａｔｉｎｇ Ｔｕｍｏｒ Ｃｅｌｌ，ＣＴＣ）を使用する癌の診断方法や予後決定方法の臨床応用が提案されている（特許文献１）。 On the other hand, in recent years, clinical application of a cancer diagnosis method and a prognosis determination method using circulating blood tumor cells (Circulating Tumor Cell, CTC) has been proposed (Patent Document 1).
抗癌剤の投薬治療期間に亘り、一定量の血液中に含まれるＣＴＣ数を測定することで、その増減から投薬された抗癌剤の薬効を推測することが可能になってきている。しかしながら、ＣＴＣ数に基づいて薬効を推測するには、一定期間に亘り投薬することが必要であり、投薬前に薬効を推測することはできなかった。またＣＴＣ数の増減のみに基づいて実際に投薬された抗癌剤が腫瘍細胞に対して作用しているかについては評価することが困難であった。そこで、本発明は、ある抗癌剤の投薬前に、その抗癌剤の薬効を評価する方法を提供すること、さらにはある抗癌剤が実際に投与された場合にその薬効を正確に評価することを目的とする。 By measuring the number of CTCs contained in a certain amount of blood over the period of anti-cancer drug administration and treatment, it has become possible to estimate the drug effect of the anti-cancer drug administered from the increase and decrease. However, in order to estimate the drug effect based on the CTC number, it was necessary to administer the drug for a certain period, and it was not possible to estimate the drug effect before the drug administration. Further, it was difficult to evaluate whether or not the actually administered anticancer drug acts on tumor cells based on only the increase/decrease in the number of CTCs. Therefore, the present invention aims to provide a method for evaluating the drug efficacy of an anticancer drug before the administration of the anticancer drug, and further to accurately evaluate the drug efficacy of the anticancer drug when actually administered. ..
前記目的に鑑みて完成された本発明は、抗癌剤の薬効を評価する方法であり、特に好適には腫瘍細胞の抗癌剤作用部を標識物質により標識し、抗癌剤適用による光強度の変化から、抗癌剤の薬効を評価する方法に関している。 The present invention completed in view of the above-mentioned object is a method for evaluating the drug efficacy of an anticancer agent, particularly preferably an anticancer agent acting part of a tumor cell is labeled with a labeling substance, from a change in light intensity due to the application of the anticancer agent, It relates to a method for evaluating drug efficacy.
本発明の方法は、希少な腫瘍細胞を検出可能であり、かつ腫瘍細胞への抗癌剤の薬効を直接検出できるため、画像診断と比較して高感度であり、腫瘍マーカーに対してより精度の高い薬効評価が可能となる。本発明の抗癌剤の薬効を評価する方法は、抗癌剤が、投与対象である各患者にとって有効な治療効果を発揮するか否かを、投与前に又は投与期間中若しくは期間終了後に評価することを可能にする。これにより、適切な抗癌剤の選択が可能になると共に、副作用を最小にする処方で治療を行うことが可能になる。 The method of the present invention can detect rare tumor cells, and can directly detect the drug effect of an anticancer agent on tumor cells, and thus has high sensitivity as compared with image diagnosis and is more accurate for tumor markers. The drug efficacy can be evaluated. The method for evaluating the drug efficacy of the anticancer agent of the present invention can evaluate whether or not the anticancer agent exerts an effective therapeutic effect on each patient to be administered, before administration, or during or after the administration period. To This allows the selection of an appropriate anti-cancer agent and allows treatment with a regimen that minimizes side effects.
本発明は、患者に対する抗癌剤の薬効を評価する方法であって、
抗癌剤を患者から得た生物試料に適用する抗癌剤適用工程と、
生物試料から腫瘍細胞を濃縮する濃縮工程と、
前記濃縮工程で得られた前記濃縮液に含まれる生体試料を基板上に展開する展開工程と、
前記基板上に展開した腫瘍細胞に存在する抗癌剤の作用部を標識して、光強度を検出する検出工程と、
検出された光強度に基づき、抗癌剤の効果を決定する評価工程、
を備えることを特徴とする抗癌剤の評価方法に関する。
The present invention is a method for evaluating the efficacy of an anticancer drug on a patient,
An anticancer agent applying step of applying an anticancer agent to a biological sample obtained from a patient,
An enrichment step of enriching tumor cells from a biological sample,
A development step of developing a biological sample contained in the concentrated liquid obtained in the concentration step on a substrate,
A detection step of detecting the light intensity by labeling the action part of the anticancer agent present in the tumor cells spread on the substrate,
An evaluation step of determining the effect of the anticancer agent based on the detected light intensity,
And a method for evaluating an anticancer agent.
本発明の別の態様は、患者に対する抗癌剤の薬効を評価する方法であって、
抗癌剤を投与された患者から得られた生物試料から腫瘍細胞を濃縮する濃縮工程と、
前記濃縮工程で得られた前記濃縮液に含まれる生体試料を基板上に展開する展開工程と、
前記基板上に展開した腫瘍細胞に存在する抗癌剤の作用部を標識して、光強度を検出する検出工程と、
検出された光強度に基づき、抗癌剤の効果を決定する評価工程、
を備えることを特徴とする抗癌剤の評価方法に関する。
Another aspect of the present invention is a method for evaluating the efficacy of an anticancer drug on a patient,
An enrichment step of enriching tumor cells from a biological sample obtained from a patient administered an anti-cancer agent,
A development step of developing a biological sample contained in the concentrated liquid obtained in the concentration step on a substrate,
A detection step of detecting the light intensity by labeling the action part of the anticancer agent present in the tumor cells spread on the substrate,
An evaluation step of determining the effect of the anticancer agent based on the detected light intensity,
And a method for evaluating an anticancer agent.
本発明は、個別の癌を患う又は患う恐れのある患者に対して、抗癌剤の薬効を評価することができる。抗癌剤の中でも特に分子標的薬は、癌細胞に含まれる１又は数種のタンパク質を標的とする薬であることから、癌細胞の種類又は変異などにより、分子標的薬に対する効果が大きく異なることがある。例えばモノクローナル抗体を含む抗体医薬である場合、癌細胞中のタンパク質の抗原部位に変異が生じてしまうと、抗体医薬としての作用が弱くなってしまう。発明の抗癌剤の薬効を評価する方法は、抗癌剤が、投与対象である各患者にとって有効な治療効果を発揮するか否かを、投与前に又は投与期間中若しくは期間終了後に評価することを可能にする。これにより、適切な抗癌剤又は抗癌剤の組合せの選択が可能になると共に、副作用を最小にする処方で治療の選択が可能になる。 INDUSTRIAL APPLICABILITY The present invention can evaluate the drug efficacy of an anticancer agent for patients suffering from or at risk of suffering from individual cancer. Among the anti-cancer agents, the molecular-targeted drug is a drug that targets one or several kinds of proteins contained in cancer cells, and therefore, the effect on the molecular-targeted drug may vary greatly depending on the type or mutation of the cancer cells. .. For example, in the case of an antibody drug containing a monoclonal antibody, if a mutation occurs in the antigenic site of a protein in cancer cells, the action as an antibody drug will be weakened. The method for evaluating the drug efficacy of the anticancer agent of the invention makes it possible to evaluate whether the anticancer agent exerts an effective therapeutic effect for each patient to be administered, before administration, or during or after the administration period. To do. This allows the selection of an appropriate anti-cancer agent or combination of anti-cancer agents as well as the choice of treatment with a regimen that minimizes side effects.
本発明の別の態様では、本発明の評価方法により、患者に対する抗癌剤の薬効を評価し、その評価に従い優れた薬効を有する抗癌剤又はその組合せを選択して、患者に投与することを含む、患者の治療方法にも関する。また、本発明の更なる態様では、上述の各工程を含む、患者が患う癌がどの抗癌剤に対し感受性であるかについて患者を診断する方法にも関する。 In another aspect of the present invention, the evaluation method of the present invention evaluates the drug efficacy of an anticancer agent for a patient, selects an anticancer agent or a combination thereof having an excellent drug efficacy according to the evaluation, and comprises administering to the patient. It also relates to the treatment method of. Further, a further aspect of the present invention also relates to a method of diagnosing a patient as to which anticancer agent the cancer afflicted by the patient is susceptible to, which comprises the steps described above.
本発明において患者とは、癌を患っている患者又は癌の手術、投薬、放射線治療などの癌治療を受けた経験のある患者であってもよいし、癌治療後に、転移癌を患う恐れのある患者であってもよい。本発明を診断方法として用いることもできることから、癌を患う恐れのある患者も含まれる。本発明の患者が患っている癌は、任意の癌であってよく、例えば白血病、リンパ腫、ホジキン病、非ホジキンリンパ腫、多発性骨髄腫などの造血細胞悪性腫瘍、脳腫瘍、乳がん、子宮体がん-子宮、子宮頚がん、卵巣がん、食道癌、胃癌、虫垂癌、大腸癌、肝臓癌、胆嚢癌、胆管癌、膵臓癌、副腎癌、消化管間質腫瘍、中皮腫-口腔底癌、歯肉癌、舌癌、頬粘膜癌などの喉頭癌口腔癌、頭頚部癌、唾液腺癌、副鼻腔癌、甲状腺癌、腎臓がん、肺癌、骨肉腫、骨癌、前立腺癌、精巣腫瘍、腎臓癌、膀胱癌、皮膚癌、肛門癌が挙げられるがこれらに限定されることを意図しない。 In the present invention, the patient may be a patient suffering from cancer or a patient who has undergone cancer treatment such as surgery, medication, or radiation treatment for cancer, or may have metastatic cancer after cancer treatment. It may be a patient. Since the present invention can be used as a diagnostic method, it also includes patients at risk of suffering from cancer. The cancer afflicted by the patient of the present invention may be any cancer, for example, leukemia, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, hematopoietic cell malignancies such as multiple myeloma, brain tumor, breast cancer, endometrial cancer. -Uterus, cervical cancer, ovarian cancer, esophageal cancer, gastric cancer, appendix cancer, colon cancer, liver cancer, gallbladder cancer, cholangiocarcinoma, pancreatic cancer, adrenal cancer, gastrointestinal stromal tumor, mesothelioma-oral floor Lung cancer such as cancer, gingival cancer, tongue cancer, buccal mucosa cancer, oral cancer, head and neck cancer, salivary gland cancer, sinus cancer, thyroid cancer, kidney cancer, lung cancer, osteosarcoma, bone cancer, prostate cancer, testicular cancer, It includes but is not limited to kidney cancer, bladder cancer, skin cancer, anal cancer.
本発明において、生物試料とは、細胞を含む試料であれば任意の試料のことをいい、生体から取得された生体試料並びに細胞培養や組織培養などで得られる培養試料を含む。生体試料としては、尿、血液、血漿、血清、唾液、精液、糞便、痰、髄液、腹水、羊水等の生体試料、細胞の凝集物、腫瘍、リンパ節又は動脈といった器官や組織に由来する試料が挙げられる。培養試料としては、細胞培養物、組織培養物、又はそれらの培養液が挙げられる。本発明は、生物試料中に含まれる腫瘍細胞に対する抗癌剤の作用を評価するために使用することができる。腫瘍細胞としては、任意の癌由来の細胞であってよいが、より詳細には、血液やリンパ液を通じて遠隔転移する癌細胞である血中循環腫瘍細胞（CTC）であり、例えば胃癌、大腸癌、食道癌、肝臓癌、肺癌、すい臓癌、膀胱癌、子宮癌（上皮性腫瘍）由来のＣＴＣ細胞、及び、血液に含まれるリンパ球や白血球の腫瘍細胞（リンパ腫、白血病）が例示できる。別の態様では、生検により取得した腫瘍から得た細胞の懸濁物を試料として用いることもできる。 In the present invention, the biological sample refers to any sample as long as it is a sample containing cells, and includes a biological sample obtained from a living body and a culture sample obtained by cell culture, tissue culture and the like. The biological sample is derived from biological samples such as urine, blood, plasma, serum, saliva, semen, feces, sputum, cerebrospinal fluid, ascites fluid, amniotic fluid, cell aggregates, tumors, organs or tissues such as lymph nodes or arteries. Examples include samples. Examples of the culture sample include cell cultures, tissue cultures, and culture solutions thereof. The present invention can be used to evaluate the effect of anticancer agents on tumor cells contained in biological samples. Tumor cells may be cells derived from any cancer, but more specifically, circulating blood tumor cells (CTC), which are cancer cells that metastasize through blood or lymph, such as gastric cancer, colon cancer, Examples thereof include CTC cells derived from esophageal cancer, liver cancer, lung cancer, pancreatic cancer, bladder cancer, uterine cancer (epithelial tumor), and lymphocyte and leukocyte tumor cells (lymphoma, leukemia) contained in blood. In another embodiment, a suspension of cells obtained from a tumor obtained by biopsy can be used as a sample.
本発明で評価される抗癌剤は、癌の治療に用いられる任意の抗癌剤であってよい。腫瘍細胞に存在する抗癌剤の作用部を標識する観点から、分子標的治療薬であることが好ましく、抗体医薬がより好ましい。 The anticancer agent evaluated in the present invention may be any anticancer agent used for the treatment of cancer. From the viewpoint of labeling the action site of the anticancer agent present in tumor cells, it is preferably a molecularly targeted therapeutic agent, and more preferably an antibody drug.
癌治療用の分子標的治療薬としては、市場されている又はこれから市場される任意の分子標的治療薬が挙げられ、例えば、イマニチブ、ゲフィチニブ、エルロチニブ、アファチニブ、ダサチニブ、ボスチニブ、ニロチニブ、バンデタニブ、スニチニブ、アキシチニブ、パゾパニブ、レンバチニブ、ラパチニブ、ニンテダニブ、ニロチニブ、クリゾチニブ、セリチニブ、アレクチニブ、ルキソリチニブ、トファシチニブ、イブルチニブなどのチロシンキナーゼ阻害剤や、ソラフェニブ、ベムラフェニブ、ダブラフェニブなどのＲａｆキナーゼ阻害薬や、パルボシクリブなどのＣＤＫ阻害薬や、トラメチニブなどのＭＥＫ阻害薬、ボルテゾミブ、カルフィルゾミブなどのプロテアソーム阻害剤が挙げられる。 Examples of molecular targeted therapeutic agents for treating cancer include any molecular targeted therapeutic agents that are on the market or will be marketed from the market, for example, imanitinib, gefitinib, erlotinib, afatinib, dasatinib, bosutinib, nilotinib, vandetanib, sunitinib, Tyrosine kinase inhibitors such as axitinib, pazopanib, lenvatinib, lapatinib, nintedanib, nilotinib, crizotinib, ceritinib, alectinib, ruxolitinib, tofacitinib, ibrutinib, and K, and sorafenib, vemurafenib, abrafenib, dabrafenib And MEK inhibitors such as trametinib and proteasome inhibitors such as bortezomib and carfilzomib.
癌治療用の抗体医薬としては、市場されている又はこれから市場される任意の抗体医薬が挙げられ、例えばリツキシマブ、セツキシマブ、ブレンツキシマブ ベドチン、トラスツズマブ、ベバシズマブ、メポリズマブ、ゲムツズマブオゾガマイシン、モガムリズマブ、ペルツズマブ、アレムツズマブ、イノツズマブ、パニツムマブ、オファツムマブ、イピリムマブ、ラムシルマブ、ニボルマブなどが挙げられる。 Examples of the antibody drug for treating cancer include any antibody drug that is on the market or is to be marketed, and examples thereof include rituximab, cetuximab, brentuximab vedotin, trastuzumab, bevacizumab, mepolizumab, gemtuzumab ozogamicin, Examples include mogamulizumab, pertuzumab, alemtuzumab, inotuzumab, panitumumab, ofatumumab, ipilimumab, ramucirumab, nivolumab and the like.
腫瘍細胞に存在する抗癌剤の作用部は、抗癌剤の種類に応じて適宜選択することができる。例えば、チロシンキナーゼ阻害剤の場合、チロシンキナーゼ、例えばＢｃｒ−Ａｂｌチロシンキナーゼ、ＫＩＴチロシンキナーゼ、ＥＧＦＲチロシンキナーゼ、ＲＥＴチロシンキナーゼ、ＶＥＧＦＲチロシンキナーゼ、ＰＤＧＦＲキナーゼ、退形成性リンパ腫キナーゼ、未分化リンパ腫キナーゼ、ＪＡＫ２チロシンキナーゼ、ブルトン型チロシンキナーゼなどが作用部となりうる。Ｒａｆキナーゼ阻害剤の場合、Ｒａｆキナーゼ、Ｂ−Ｒａｆ酵素が作用部となり、ＣＤＫ阻害剤は、サイクリン依存性キナーゼ、ＭＥＫ阻害剤はＭＥＫが、プロテアソーム阻害剤は、プロテアソームが作用部となる。抗体医薬の場合、その抗体が結合する抗原が作用部となる。検出を容易にする観点から、作用部は、細胞表面に現れている膜タンパク質であることが好ましい。 The action site of the anticancer agent present in the tumor cells can be appropriately selected according to the type of the anticancer agent. For example, in the case of a tyrosine kinase inhibitor, a tyrosine kinase such as Bcr-Abl tyrosine kinase, KIT tyrosine kinase, EGFR tyrosine kinase, RET tyrosine kinase, VEGFR tyrosine kinase, PDGFR kinase, anaplastic lymphoma kinase, anaplastic lymphoma kinase, JAK2. Tyrosine kinase, Bruton's tyrosine kinase, etc. can be the action site. In the case of Raf kinase inhibitor, Raf kinase and B-Raf enzyme serve as action sites, CDK inhibitors serve as cyclin-dependent kinases, MEK inhibitors serve as MEK, and proteasome inhibitors serve as proteasomes as action sites. In the case of an antibody drug, the antigen bound by the antibody serves as the action site. From the viewpoint of facilitating detection, the action portion is preferably a membrane protein appearing on the cell surface.
抗癌剤適用工程
抗癌剤適用工程は、患者から得た生物試料に対し、抗癌剤を適用するin vitroの工程である。抗癌剤の適用の前に生物試料を、希釈、濃縮、培養などの前処理にかけていてもよい。本発明では、抗癌剤の適用工程は、抗癌剤の評価が可能となる範囲で任意の段階で行われてもよい。一例として、濃縮工程の前に抗癌剤適用工程が行われてもよいし、濃縮工程の後に抗癌剤適用工程が行われてもよく、また展開工程の後に抗癌剤適用工程が行われてもよい。適用される抗癌剤の濃度は、適用する抗癌剤の種類に応じて任意に設定することができる。例えば、通常の処方に従いその抗癌剤が患者に投与された場合に達成できる血中濃度と同程度の濃度、またはそれ以上若しくはそれ以下の濃度を設定することができるが、これらの濃度に限定されるものではない。
Step of Applying Anticancer Agent The step of applying an anticancer agent is an in vitro step of applying an anticancer agent to a biological sample obtained from a patient. Prior to application of the anti-cancer agent, the biological sample may be subjected to pretreatment such as dilution, concentration and culture. In the present invention, the step of applying the anticancer agent may be carried out at any stage as long as the anticancer agent can be evaluated. As an example, the anticancer agent application step may be performed before the concentration step, the anticancer agent application step may be performed after the concentration step, and the anticancer agent application step may be performed after the development step. The concentration of the applied anti-cancer agent can be arbitrarily set according to the type of the applied anti-cancer agent. For example, it is possible to set a concentration similar to the blood concentration that can be achieved when the anticancer drug is administered to a patient according to a usual prescription, or a concentration higher or higher, but not limited to these concentrations. Not a thing.
本発明の別の態様では、抗癌剤適用工程の代わりに、抗癌剤を患者に投与する工程を含んでもよいし、抗癌剤適用工程を含まない代わりに、抗癌剤を投与された患者から取得された生物試料を評価方法に供することもできる。この場合、投与された抗癌剤の薬効を評価することができる。生物試料の取得タイミングは、抗癌剤の投与後、任意にタイミングで行うことができるが、腫瘍サイズの減少や、ＣＴＣ数の減少が生じる前に取得することが好ましい。また、抗癌剤治療期間にわたり、経時的に生物試料を取得することで、抗癌剤に対する薬効の変化、すなわち癌の耐性獲得を評価することができる。抗癌剤に対する耐性を獲得したことが確認できた場合、新たな治療方針、例えば手術、放射線治療、別の種類の抗癌剤の投薬などを検討して、癌治療に役立てることができる。 In another aspect of the present invention, in place of the anti-cancer agent application step, may include the step of administering an anti-cancer agent to the patient, instead of not including the anti-cancer agent application step, a biological sample obtained from the patient to whom the anti-cancer agent was administered. It can also be used as an evaluation method. In this case, the efficacy of the administered anticancer drug can be evaluated. The biological sample can be obtained at any timing after the administration of the anticancer agent, but it is preferable to obtain the biological sample before the decrease in tumor size or the decrease in CTC number occurs. Further, by obtaining biological samples over time during the anticancer agent treatment period, it is possible to evaluate the change in drug efficacy against the anticancer agent, that is, the acquisition of cancer resistance. When it is confirmed that the resistance to the anticancer drug is acquired, a new treatment policy such as surgery, radiation treatment, administration of another kind of anticancer drug, etc. can be considered to be useful for the cancer treatment.
濃縮工程
濃縮工程は、生物試料中から生きた腫瘍細胞の検出を容易にするために分離及び/又は濃縮をする工程である。濃縮方法は、生物試料中から生きた腫瘍細胞以外の成分を低減することで生きた腫瘍細胞をより選択的に回収できれば特に制限はない。例えば、腫瘍細胞と腫瘍細胞以外の成分とのサイズの違いを利用して分離濃縮するフィルター法、細胞表面の抗体発現プロファイルの違いを利用し、抗体磁性粒子で濃縮する磁気ビーズ法、細胞間の比重差を利用した比重法が例示できる。なかでも比重法は、短時間で選択的に生きた腫瘍細胞を濃縮できることから特に好ましい。
Concentration step The concentration step is a step of separating and/or concentrating in order to facilitate detection of live tumor cells in the biological sample. The concentration method is not particularly limited as long as live tumor cells can be more selectively recovered by reducing components other than live tumor cells in the biological sample. For example, a filter method that separates and concentrates by utilizing the size difference between tumor cells and components other than tumor cells, a magnetic bead method that concentrates by antibody magnetic particles by utilizing the difference in antibody expression profile on the cell surface, an inter-cell A specific gravity method utilizing a difference in specific gravity can be exemplified. Among them, the specific gravity method is particularly preferable because it can selectively concentrate live tumor cells in a short time.
以下、比重法を用いた濃縮工程について詳細を示す。
本発明おける比重分離では、腫瘍細胞と腫瘍細胞以外の成分が比重差によって分離できれば特に制限はない。密度勾配溶液は、それ自身で又は遠心分離によって密度勾配を形成する液体状の物質であり、目的とする細胞の密度（比重）を特定し、その分離に適当なものを選択して使用すれば良い。選択の指標としては、例えば栄養成分、ｐＨ、等張性等を例示できる。具体的にはショ糖、グリセロール、デキストラン、メトリザミド、イオディキサノール、ショ糖とエピクロロヒドリンの共重合体、ポリビニルピロリドンの被膜をもつコロイド状シリカ粒子、スクロースポリマー、ジアトリゾ酸、イオヘキソール、ニコデンツ等のイオン性又は非イオン性のものが例示できる。市販されている密度勾配溶液として、ＧＥヘルスケア バイオサイエンス社製の商品名Ｆｉｃｏｌｌ、Ｆｉｃｏｌｌ−Ｐａｑｕｅ又はＰｅｒｃｏｌｌ、Ａｘｉｓ−Ｓｈｉｅｌｄ ＰｏＣ ＡＳ社製の商品名Ｌｙｍｐｈｏｐｒｅｐ、Ｐｏｌｙｍｏｒｐｈｐｒｅｐ又はＯｐｔｉＰｒｅｐが例示できる。
Details of the concentration step using the specific gravity method will be described below.
The specific gravity separation in the present invention is not particularly limited as long as tumor cells and components other than tumor cells can be separated by the difference in specific gravity. The density gradient solution is a liquid substance that forms a density gradient by itself or by centrifugation, and if the density (specific gravity) of the target cells is specified and an appropriate one is selected and used for the separation, good. Examples of the selection index include nutritional components, pH, isotonicity, and the like. Specifically, sucrose, glycerol, dextran, metrizamide, iodixanol, a copolymer of sucrose and epichlorohydrin, colloidal silica particles with a polyvinylpyrrolidone coating, sucrose polymer, diatrizoic acid, iohexol, nicodentz. Examples thereof include ionic and nonionic ones. Examples of commercially available density gradient solutions include GE Healthcare Bioscience's trade name Ficoll, Ficoll-Paque or Percoll, and Axis-Shield PoC AS trade name Lymphoprep, Polymorphprep or OptiPrep.
濃縮工程を実施するための器具として、例えば図１の細胞分離濃縮構造体を用いることができる。分離濃縮構造体１は、２及び３の２つの筒状部材からなる。分離濃縮構造体の上部を構成する筒状部材２は開口を有し、筒状部材３は、一端が閉塞して底部５を形成している。筒状部材２及び３は、それぞれ開口又は底部の反対の端に連通開口６が設けられ、該両部材が連結された場合に両筒状部材の内部空間が連通し、全体として一つの分離濃縮構造体を形成する。 As an instrument for carrying out the concentration step, for example, the cell separation concentration structure shown in FIG. 1 can be used. The separation/concentration structure 1 is composed of two tubular members 2 and 3. The tubular member 2 forming the upper portion of the separation/concentration structure has an opening, and the tubular member 3 has one end closed to form a bottom portion 5. The tubular members 2 and 3 each have a communication opening 6 provided at the opposite end of the opening or bottom, and when the two members are connected, the internal spaces of the two tubular members communicate with each other, so that one separation/concentration is achieved as a whole. Form a structure.
密度勾配溶液は、分離濃縮構造体１において、その底部（筒状部材３の閉塞端５）から分離部近傍まで注入する。より具体的には、分離濃縮構造体を静置した場合に、密度勾配溶液の液面高さが上側の筒状部材２の連通口端より高くなる（筒状部材２側になる）、すなわち、下側の筒状部材（筒状部材３）を分離した際に、遠心分離操作により密度勾配溶液を通過して筒状部材３の閉塞端５側に移動した成分を密度勾配溶液の大半とともに筒状部材３に、密度勾配溶液上に維持された目的成分（細胞）を筒状部材２に維持された状態で分離できる程度、好ましくは１ｍｍ程度、高くなるよう注入する。その後、生体試料溶液を密度勾配溶液の上に重層し、開口部をキャップ４で密閉し、遠心分離操作を行う。遠心分離操作は、一般には１０００から２０００×ｇ程度の低速で実施すれば良いが、目的とする細胞の密度や使用する密度勾配溶液の密度を勘案し、密度勾配溶液の上に維持される条件を選択する。例えば目的とする細胞が腫瘍細胞であり、上記のような遠心を行うのであれば、腫瘍細胞の種類に応じて密度勾配溶液の密度を１．０６０〜１．０９５ｇ／ｍＬの範囲に設定することができる。密度勾配溶液の密度は、腫瘍細胞の濃縮率を高める観点から、１．０７５ｇ/ｍｌ以上が好ましく、１．０８０ｇ/ｍｌ以上がさら好ましい。腫瘍細胞の濃縮率を高める観点から、１．１００ｇ／ｍｌ以下が好ましく、１．０９６以下がより好ましく、１．０９３以下がさらに好ましい。より具体的には密度勾配溶液の密度は１．０８２〜１．０９１ｇ／ｍＬの範囲で設定することができる。密度勾配溶液の浸透圧は、２００〜４５０ｍＯｓｍ/ｋｇの範囲で設定することができ、３００〜４００ｍＯｓｍ/ｋｇがより好ましい。溶液のｐＨは細胞が損傷を受けない範囲で任意に選択することができ、例えば６．８〜７．８の範囲に調整することが例示できる。 The density gradient solution is injected from the bottom portion (closed end 5 of the tubular member 3) of the separation and concentration structure 1 to the vicinity of the separation portion. More specifically, when the separation/concentration structure is left stationary, the liquid level height of the density gradient solution becomes higher than the communication port end of the upper tubular member 2 (toward the tubular member 2 side), that is, When the lower tubular member (the tubular member 3) is separated, the components that have passed through the density gradient solution and moved to the closed end 5 side of the tubular member 3 by the centrifugal separation operation together with most of the density gradient solution. The target component (cells) maintained on the density gradient solution is injected into the tubular member 3 so that the target component (cells) can be separated while being maintained in the tubular member 2, preferably about 1 mm higher. Then, the biological sample solution is overlaid on the density gradient solution, the opening is sealed with the cap 4, and the centrifugation operation is performed. The centrifugation operation may be generally performed at a low speed of about 1000 to 2000×g, but the conditions to be maintained on the density gradient solution in consideration of the density of target cells and the density of the density gradient solution to be used. Select. For example, if the target cells are tumor cells and the centrifugation as described above is performed, the density of the density gradient solution should be set in the range of 1.060 to 1.095 g/mL according to the type of tumor cells. You can From the viewpoint of increasing the concentration rate of tumor cells, the density of the density gradient solution is preferably 1.075 g/ml or more, more preferably 1.080 g/ml or more. From the viewpoint of increasing the concentration rate of tumor cells, 1.100 g/ml or less is preferable, 1.096 or less is more preferable, and 1.093 or less is further preferable. More specifically, the density of the density gradient solution can be set in the range of 1.082 to 1.091 g/mL. The osmotic pressure of the density gradient solution can be set in the range of 200 to 450 mOsm/kg, and more preferably 300 to 400 mOsm/kg. The pH of the solution can be arbitrarily selected within a range where cells are not damaged, and it can be exemplified that the pH is adjusted to a range of 6.8 to 7.8.
遠心分離操作により、密度勾配溶液の密度より大きな密度を有する成分は密度勾配溶液の勾配層を通過して下側の筒状部材（筒状部材３）中に移動する。一方、密度勾配溶液より小さな密度の目的とする細胞は、上側の筒状部材（筒状部材２）内の密度勾配溶液の上に維持される。そこで開口部の密閉を維持したまま連結された筒状部材を図１で示した状態となるように分離すれば、上側の筒状部材（筒状部材２）中に目的とする細胞を含む分画を回収することができる。この分画は、例えばキャップ４を取り外すことによって密閉状態を開放することで下方へ滴下させる等すれば、特別の熟練を要することなく容易に回収できる。一方、下側の筒状部材（筒状部材３）中に移動した分画については、例えば当該筒状部材とともに廃棄等することができる。 By the centrifugation operation, a component having a density higher than that of the density gradient solution passes through the gradient layer of the density gradient solution and moves into the lower tubular member (tubular member 3). On the other hand, target cells having a smaller density than the density gradient solution are maintained on the density gradient solution in the upper tubular member (cylindrical member 2). Therefore, if the connected tubular members are separated into the state shown in FIG. 1 while maintaining the airtightness of the opening, the upper tubular member (cylindrical member 2) will contain the target cells. Images can be collected. This fraction can be easily collected without requiring special skill by, for example, dropping the cap 4 to open the closed state and dropping the fraction downward. On the other hand, the fraction moved into the lower tubular member (the tubular member 3) can be discarded, for example, together with the tubular member.
本発明においては、標的細胞に特異的に結合する物質を添加し、又は、標的細胞以外の成分に特異的に結合する物質を添加することにより、標的細胞を更に効率的に分離することができる。なお、特異的に結合する物質と多孔質シリカ粒子等の比較的密度が小さい物質を結合させれば、見かけ上の密度を小さくすることができる。このように、密度を調整する目的で使用する物質としては、前記多孔質シリカ粒子に加え、例えば、ポリエチレン、ポリプロピレン、ポリビニルクロリド、ポリアクリロニトリル、ポリアクリレート、ポリメタクリレート、ポリカルボネート等のポリビニル化合物に代表される有機ポリマー、ポリスチレンラテックス、ナイロン、ポリテレフタレート等の共重合体、ガラス、シリカ、ジルコニア等の無機材料、セルロース、デキストラン、アガロース、セルロース、セファロース等の生体ポリマー、赤血球などの生体試料が例示でき、標的とする細胞等と特異的に結合する物質として抗体、抗原、ペプチド、ポリペプチド、成長因子、サイトカイン、レクチン等の生体高分子を例示できる（以下、「特異的に結合する物質」と「密度を調整する目的で使用する物質」が結合し、標的細胞もしくは標的細胞以外の成分の密度を調整する物質を「結合剤」と記載する）。 In the present invention, a target cell can be further efficiently separated by adding a substance that specifically binds to a target cell or by adding a substance that specifically binds to a component other than the target cell. .. The apparent density can be reduced by binding a substance that specifically binds to a substance having a relatively low density such as porous silica particles. Thus, as the substance used for the purpose of adjusting the density, in addition to the porous silica particles, for example, polyvinyl compounds such as polyethylene, polypropylene, polyvinyl chloride, polyacrylonitrile, polyacrylate, polymethacrylate, polycarbonate. Representative examples include organic polymers, polystyrene latex, nylon, copolymers such as polyterephthalate, inorganic materials such as glass, silica, zirconia, biopolymers such as cellulose, dextran, agarose, cellulose and sepharose, and biological samples such as red blood cells. As a substance that specifically binds to a target cell or the like, an antibody, an antigen, a peptide, a polypeptide, a growth factor, a cytokine, a biopolymer such as a lectin can be exemplified (hereinafter, referred to as a “specifically binding substance”). A substance that binds to a “substance used for adjusting the density” and adjusts the density of target cells or components other than the target cells is referred to as a “binding agent”).
濃縮工程には、標的細胞を濃縮することを目的として、さらに選択工程を含んでもよい。このような選択工程としては、例えば血液試料を用いた場合に、溶血処理が行われる。溶血工程が行われることで、赤血球の細胞数を減じることができ、それにより標的細胞が選択的に濃縮される。かかる選択工程は、濃縮のための遠心分離の後に行われてもよいし、遠心分離前に行われてもよい。選択工程後に、さらに遠心分離が行われてもよい。 The concentration step may further include a selection step for the purpose of concentrating the target cells. As such a selection step, for example, when a blood sample is used, hemolysis treatment is performed. By performing the hemolysis step, the number of red blood cells can be reduced, thereby selectively enriching the target cells. Such a selection step may be performed after centrifugation for concentration or may be performed before centrifugation. Further centrifugation may be performed after the selection step.
回収した細胞は、希釈、懸濁などの処理をした上で、スライドへの塗布もしくはウェルへ捕捉した細胞を顕微鏡観察する手法やフローサイトメトリー法などを用いて測定することができる。特に本発明で使用する誘電泳動力によって基板上に備えた複数の保持孔に１つずつ細胞を捕捉して測定する手法は、高感度かつ高精度に１細胞ごとを観察、解析できる点でより好ましい。 The collected cells can be measured by a method such as diluting or suspending, and then applying to a slide or microscopically observing the cells captured in a well or a flow cytometry method. In particular, the method of capturing and measuring cells one by one in a plurality of holding holes provided on the substrate by the dielectrophoretic force used in the present invention is more sensitive and highly accurate in observing and analyzing each cell. preferable.
展開工程
展開工程は、濃縮工程を経て得られた濃縮液を基板上に展開することにより行われる。濃縮液を展開することで、濃縮液に含まれる細胞を検出に適した間隔で基板上に分布させることができる。非凝集状態で展開させることが好ましく、展開前に濃縮液を十分に懸濁しておくことが好ましい。展開工程は、細胞を検出に適した間隔で基板上に分布させることができれば任意の手法を用いることができ、単に濃縮液を基板上に適用するのみであってもよいが、必要に応じてさらなる処理を行ってもよい。一例として、濃縮液を基板上に適用後に、振動や誘導泳動力をあたえることにより、細胞を展開することもできる。細胞を均一に展開するために、基板上に保持孔があけられていることが好ましく、各保持孔につき、概ね一個の細胞を配置することで、その後の検出工程にて標的細胞の検出が容易になる。所望される細胞の展開密度に応じて、濃縮液中の細胞数を計数し、適切な細胞数が展開されるように希釈されてもよく、また展開に供する濃縮液を計量して展開することもできる。
Development Step The development step is performed by developing the concentrated liquid obtained through the concentration step on the substrate. By developing the concentrated solution, cells contained in the concentrated solution can be distributed on the substrate at intervals suitable for detection. It is preferable to develop in a non-aggregated state, and it is preferable to sufficiently suspend the concentrated liquid before development. In the developing step, any technique can be used as long as cells can be distributed on the substrate at an interval suitable for detection, and the concentrated solution may simply be applied on the substrate, but if necessary, Further processing may be performed. As an example, cells can be expanded by applying vibration or inductive migration force after applying the concentrated solution onto the substrate. In order to spread cells evenly, it is preferable to have holding holes on the substrate. By placing approximately one cell in each holding hole, it is easy to detect target cells in the subsequent detection step. become. Depending on the desired cell expansion density, the number of cells in the concentrated solution may be counted and diluted so that an appropriate number of cells may be expanded, and the concentrated solution to be expanded may be measured and expanded. You can also
本発明の展開工程に用いる器具として、例えば、図２の生物試料検出構造体を使用することができる。本構造体は、後の検出工程において細胞の存在を示す物質により発せられる光を検出するために細胞をそれぞれ保持する複数の保持孔（貫通孔）７を有する構造体８であって、平板状の基板９上に配置されている。また、前記基板および上蓋基板１０は透光性材料からなり、前記基板の前記保持孔側および上蓋基板の表面に設けられた電極はＩＴＯなどの透明電極であることが好ましい。これにより、保持孔から発せられる光を基板の上側もしくは下側から観察することが可能となる。保持孔は絶縁体膜１１から構成されているが、遮光膜１２を備えていてもよい。遮光膜を設けることにより、例えば絶縁体膜自体の自家蛍光に起因するバックグラウンドノイズや隣接する保持孔からの漏れ光に起因するクロストークノイズなどの光ノイズを低減することができ、各保持孔内の観察対象物質により発せられる光のみを高感度かつ高精度に検出することができる。 As the instrument used in the developing step of the present invention, for example, the biological sample detection structure shown in FIG. 2 can be used. This structure is a structure 8 having a plurality of holding holes (through holes) 7 for holding cells in order to detect light emitted by a substance indicating the presence of cells in a subsequent detection step, and is a flat plate shape. Are arranged on the substrate 9. Further, it is preferable that the substrate and the upper lid substrate 10 are made of a translucent material, and that the electrodes provided on the holding hole side of the substrate and on the surface of the upper lid substrate are transparent electrodes such as ITO. This makes it possible to observe the light emitted from the holding hole from above or below the substrate. The holding hole is composed of the insulator film 11, but may be provided with the light shielding film 12. By providing the light-shielding film, it is possible to reduce optical noise such as background noise caused by autofluorescence of the insulator film itself and crosstalk noise caused by leaked light from an adjacent holding hole. Only the light emitted by the substance to be observed inside can be detected with high sensitivity and high accuracy.
また、前記構造体は、前記保持孔の上に前記細胞を含む懸濁液を収容する収容部１３を備えており、前記保持孔が前記収容部と連通するように設けられている。また収容部には細胞懸濁液を導入する導入口１４、細胞懸濁液を排出する排出口１５を備える。
保持孔内へ細胞を捕捉する方法としては、誘電泳動力を利用する。この誘電泳動力により、生きた細胞を数秒程度の極めて短い時間で多数の保持孔に捕捉することができる。誘電泳動力を細胞に作用させるには、収容部及び保持孔を懸濁液で満たした状態で、保持孔の部分に電気力線が集中するような交流電界をかければよい。かかる交流電界を印加するための構成として、図２の構造体の他に図３に示すように、前記基板の前記保持孔側の表面に、互いに異なる保持孔に対応する位置にそれぞれ配置される一対の電極（櫛状電極１８）を構成する電極１６、１７が設けられ、前記保持孔は、前記保持孔の上表面から前記基板上の櫛状電極まで延在する、という構成を採用することができる。いずれの構成の場合も、保持孔の底部に電極を露出させ、２つの電極の間に所定の波形を有する交流電圧を印加することで、誘電泳動力により懸濁液中の細胞を保持孔内へ捕捉することが可能である。また保持孔をアレイ状に配置することで、電極間に印加した電圧によって生じる電界がすべての保持孔にほぼ均等に生じることになり、すべての保持孔に対して同じように細胞を誘導し捕捉することができる。
In addition, the structure includes an accommodating portion 13 that accommodates the suspension containing the cells above the retaining hole, and the retaining hole is provided so as to communicate with the accommodating portion. Further, the accommodating portion is provided with an inlet 14 for introducing the cell suspension and an outlet 15 for discharging the cell suspension.
A dielectrophoretic force is used as a method for capturing cells in the holding holes. Due to this dielectrophoretic force, living cells can be captured in a large number of holding holes in an extremely short time of about several seconds. In order to apply the dielectrophoretic force to the cells, it is sufficient to apply an AC electric field such that the lines of electric force are concentrated on the holding holes while the containing portion and the holding holes are filled with the suspension. As a structure for applying such an AC electric field, as shown in FIG. 3 in addition to the structure shown in FIG. 2, they are arranged on the surface of the substrate on the side of the holding holes at positions corresponding to different holding holes. The electrodes 16 and 17 forming a pair of electrodes (comb-shaped electrodes 18) are provided, and the holding hole extends from the upper surface of the holding hole to the comb-shaped electrode on the substrate. You can In any of the configurations, by exposing the electrode at the bottom of the holding hole and applying an AC voltage having a predetermined waveform between the two electrodes, the cells in the suspension are held in the holding hole by the dielectrophoretic force. Can be captured. Also, by arranging the holding holes in an array, the electric field generated by the voltage applied between the electrodes is generated almost uniformly in all the holding holes, and the cells are similarly guided and trapped in all the holding holes. can do.
図４は本発明に用いる検出装置を示した図である。本発明に用いる検出装置の一例として、図２に示した基板９、上蓋基板１０と、前記電極に誘電泳動力２４を発生させるための交流電圧を印加する交流電源１９と、前記交流電源からの電圧印加後に、前記構造体の保持孔に捕捉された細胞２３の存在を示す物質により発せられる光２０を検出する検出部２１とを備える。検出部の一例としては蛍光顕微鏡を例示できる。 FIG. 4 is a diagram showing a detection device used in the present invention. As an example of the detection device used in the present invention, the substrate 9 and the upper lid substrate 10 shown in FIG. 2, an AC power supply 19 for applying an AC voltage for generating the dielectrophoretic force 24 to the electrodes, and an AC power supply from the AC power supply. And a detector 21 for detecting the light 20 emitted by the substance showing the presence of the cells 23 trapped in the holding holes of the structure after the voltage is applied. A fluorescence microscope can be illustrated as an example of the detection unit.
前記構造体一対の電極には、導電線２２を介して交流電源が接続される。交流電源は、保持孔に細胞を移動させ、捕捉する誘電泳動力に必要な電界を発生させるのに十分な電圧を電極間に印加できればよい。具体的には、ピーク電圧が１Ｖから２０Ｖ程度で、周波数１０ｋＨｚから１０ＭＨｚ程度の正弦波、矩形波、三角波、台形波等の波形の交流電圧を印加できる電源が例示できる。特に細胞を移動させ、１つの保持孔に１個の細胞のみを捕捉し得る周波数および波形として周波数１００ｋＨｚから３ＭＨｚの矩形波を使用すること特に好ましい。かかる波形の交流電圧としては、矩形波は、波形が正弦波、三角波、台形波である場合に比べて、瞬時に設定したピーク電圧に到達するため、細胞を保持孔に向けて速やかに移動させることが可能となり、２個以上の細胞が重なるように保持孔に入る確率を低くできる（１つの保持孔に１個の細胞のみを捕捉し得る確率が高くなる）。細胞は電気的にコンデンサーと見なすことができるが、矩形波のピーク電圧が変化しない間は、保持孔に捕捉された細胞には電流が流れ難くなって電気力線が生じ難くなり、この結果、細胞を捕捉した保持孔には誘電泳動力が発生し難くなる。従って、一度保持孔に細胞が捕捉されると、別の細胞が同一の保持孔に捕捉される確率は低くなり、代わりに電気力線が生じ誘電泳動力が発生している他の保持孔（細胞が捕捉されていない、空の保持孔）に、順次、細胞が捕捉される。 An AC power source is connected to the pair of electrodes of the structure through a conductive wire 22. The AC power supply only needs to be able to apply a voltage sufficient between the electrodes to move the cells to the holding holes and generate an electric field necessary for the dielectrophoretic force to be captured. Specifically, a power supply that can apply an AC voltage having a peak voltage of about 1 V to 20 V and a waveform of a frequency of about 10 kHz to 10 MHz, such as a sine wave, a rectangular wave, a triangular wave, or a trapezoidal wave can be exemplified. In particular, it is particularly preferable to use a rectangular wave having a frequency of 100 kHz to 3 MHz as a frequency and a waveform capable of moving cells and capturing only one cell in one holding hole. As the AC voltage having such a waveform, the rectangular wave instantaneously reaches the set peak voltage as compared with the case where the waveform is a sine wave, a triangular wave, or a trapezoidal wave, so that the cells are moved quickly toward the holding hole. This makes it possible to reduce the probability that two or more cells enter the retention hole so that they overlap (there is a higher probability that only one cell can be captured in one retention hole). Although cells can be electrically regarded as capacitors, as long as the peak voltage of the rectangular wave does not change, it becomes difficult for electric current to flow through the cells trapped in the holding holes, and electric lines of force are less likely to occur. The dielectrophoretic force is less likely to be generated in the holding holes that have captured the cells. Therefore, once a cell is trapped in the holding hole, the probability that another cell is trapped in the same holding hole is low, and instead, electric lines of force are generated and dielectrophoretic force is generated in other holding holes ( The cells are sequentially captured in empty holding holes (where the cells are not captured).
なお、本発明に用いる検出装置では、直流成分を有しない交流電圧を発生する電源を採用することが好ましい。直流成分を有する交流電圧を印加すると、直流成分により発生した静電気力（電気泳動力）により細胞が特定の方向に偏った力を受けて移動し、誘電泳動力による細胞捕捉が困難になるからである。また直流成分を有する交流電圧を印加すると、細胞を含有する懸濁液に含まれるイオンが電極表面で電気反応を生じて発熱し、細胞が熱運動を起こすため誘電泳動力による動きを制御できなくなり、保持孔に移動させて捕捉することが困難になる。なお、直流成分を有する交流電圧とは、周波数デューティ比が５０％でない電圧、オフセットを有する電圧、周期が極端に長い（例えば１秒以上）電圧などをいう。 In addition, in the detection device used in the present invention, it is preferable to employ a power source that generates an AC voltage having no DC component. When an AC voltage with a DC component is applied, the electrostatic force (electrophoretic force) generated by the DC component causes cells to move with a biased force in a specific direction, making it difficult to capture cells by dielectrophoretic force. is there. Also, when an AC voltage with a DC component is applied, the ions contained in the cell-containing suspension generate an electric reaction on the electrode surface to generate heat, causing the cells to undergo thermal motion, making it impossible to control the movement due to dielectrophoretic force. , It becomes difficult to move to the holding hole and capture. The AC voltage having a DC component means a voltage having a frequency duty ratio of not 50%, a voltage having an offset, a voltage having an extremely long cycle (for example, 1 second or more), and the like.
検出工程
検出工程は、基板上に展開された腫瘍細胞に存在する抗癌剤の作用部を標識して、光強度を検出する工程である。この検出は、腫瘍細胞に存在する作用部を、標識物質で標識し、標識に応じた光源を導入して、光強度を検出することができる。検出工程では、腫瘍細胞以外の細胞から、腫瘍細胞を識別するための検出を同時に行ってもよい。腫瘍細胞を識別するために、腫瘍細胞に特異的に発現する物質や非腫瘍細胞に特異的に発現する物質などの標識物質により標識して検出してもよいし、腫瘍細胞のサイズ、形状などの外観により識別を行うこともできる。
Detecting Step The detecting step is a step of labeling the action portion of the anticancer agent existing in the tumor cells spread on the substrate and detecting the light intensity. In this detection, the action part existing in the tumor cells can be labeled with a labeling substance, and a light source corresponding to the label can be introduced to detect the light intensity. In the detection step, detection for distinguishing tumor cells from cells other than tumor cells may be performed simultaneously. In order to identify tumor cells, they may be labeled with a labeling substance such as a substance that is specifically expressed in tumor cells or a substance that is specifically expressed in non-tumor cells, or the size and shape of tumor cells, etc. It is also possible to identify by the appearance of.
本発明において標識物質は、細胞の表面又は内部の特定物質と結合可能であり、かつ検出工程で検出可能な標識物質を使用する。特定物質とは、特定の性質を有する細胞（以下、「標的細胞」と記載する）の表面又は内部に特異的に存在する/又は存在しない物質であって、標的細胞の識別を可能にする物質のことをいう。標的細胞に特異的に存在するとは、標的細胞には存在し、かつ、標的細胞以外には存在しないか、あるいは、標的細胞以外の細胞よりも標的細胞に多く存在することをいう目的の細胞である腫瘍細胞を赤血球から識別するために利用する、核染色物質例えばＤＡＰＩ等や、腫瘍細胞を白血球やリンパ球から識別するための白血球マーカー、例えばＣＤ４５等も特定物質に含まれる。また、腫瘍細胞に存在する抗癌剤の作用部も、特定物質に含まれる。腫瘍細胞の特定物質としては、例えば、ＣＤ２０、ＣＤ３０、ＣＤ３３、ＣＤ５２、ＨＥＲ２、ＨＥＲ３、ＥＧＦＲ、ＶＥＧＦＲ、ＰＤ−Ｌ１、ＣＥＡ、Ｍｕｃｉｎｓ、ＰＳＭＡ、サイトケラチン（ＣＫ）等が挙げられる。 In the present invention, as the labeling substance, a labeling substance that can bind to a specific substance on the surface or inside of cells and that can be detected in the detection step is used. The specific substance is a substance that specifically exists/is not present on the surface or inside of a cell having a specific property (hereinafter, referred to as “target cell”), and is a substance that enables the identification of the target cell. I mean. To be specifically present in a target cell means that the target cell is present in the target cell and is not present in other than the target cell, or is present in the target cell more than in the cells other than the target cell. Specific substances include nuclear stains such as DAPI, which are used to distinguish certain tumor cells from red blood cells, and leukocyte markers such as CD45, which distinguish tumor cells from white blood cells and lymphocytes. In addition, the action part of the anticancer agent existing in the tumor cells is also included in the specific substance. Specific examples of tumor cells include CD20, CD30, CD33, CD52, HER2, HER3, EGFR, VEGFR, PD-L1, CEA, Mucins, PSMA, cytokeratin (CK) and the like.
上で挙げられた特定物質のうち、ＣＤ２０は、イブリツモマブチウキセタン、リツキシマブ、オクレリズマブ等の作用部であり、ＣＤ３０は、ブレンツキシマブ ベドチン等の作用部であり、ＣＤ３３は、ゲムツズマブオソガマイシン等の作用部であり、ＣＤ５２は、アレムツズマブ等の作用部であり、ＨＥＲ２は、ラパチニブ、トラスツズマブ、ペルツズマブ等の作用部であり、ＥＧＦＲは、ゲフィチニブ、エルロチニブ、アファチニブ、バンデタニブ、ラパチニブ、セツキシマブ、パニツムマブ等の作用部であり、ＶＥＧＦはバンデタニブ、アキシチニブ、スニチニブ、パゾパニブ、ニンテダニブ、ソラフェニブ、ベバシズマブ、ラニビズマブ、ラムシルマブ等の作用部である。 Among the specific substances listed above, CD20 is the action part of ibritumomabuchiuxetane, rituximab, ocrelizumab, etc., CD30 is the action part of brentuximab vedotin, etc., and CD33 is gemtuzumaboo. CD52 is an action part of alemtuzumab, HER2 is an action part of lapatinib, trastuzumab, pertuzumab, etc., EGFR is gefitinib, erlotinib, afatinib, vandetanib, lapatinib, cetuximab, VEGF is an action part of vandetanib, axitinib, sunitinib, pazopanib, nintedanib, sorafenib, bevacizumab, ranibizumab, ramucirumab and the like.
前記特定物質は、例えば特定物質に特異的に結合する標識物質を用いて標識することにより観察（検出）することができる。つまり、特定物質に特異的に結合する認識物質と光学的に検出可能なシグナルを発する標識とを結合して標識物質とする。本発明の特定物質を検出する標識物質は、標的細胞が検出できれば特に制限はない。標識物質は、細胞を構成する成分の存在を示すことのできるあらゆる物質をさす。代表的な標識物質として、蛍光、リン光、もしくは発光などの性質に基づいて検出可能な標識（例えば蛍光色素）を前記した抗体、リガンド、レクチン等の特定物質と特異的に結合可能な物質とを結合したものが例示できる。また、標識物質は、光を発する反応、例えば蛍光、もしくはリン光を発する物質を生成する反応、又は発光反応を触媒する物質を特定物質と特異的に結合可能なものに結合したものであってもよい。例えば蛍光色素としては、ＦＩＴＣ（フルオレセインイソシアネート）、ＰＥ（フィコエリスリン）、ローダミン等が挙げられる。また、前記反応を触媒する物質としては、パーオキシダーゼ、β−ガラクトシダーゼ、アルカリフォスファターゼ、ルシフェラーゼ等が挙げられる。 The specific substance can be observed (detected) by labeling with a labeling substance that specifically binds to the specific substance. That is, a recognition substance that specifically binds to a specific substance and a label that emits an optically detectable signal are combined to form a labeling substance. The labeling substance for detecting the specific substance of the present invention is not particularly limited as long as the target cell can be detected. The labeling substance refers to any substance capable of showing the presence of components constituting cells. As a typical labeling substance, a substance capable of specifically binding to a specific substance such as an antibody, a ligand, a lectin, etc., which can be detected based on properties such as fluorescence, phosphorescence, or luminescence (for example, a fluorescent dye) An example is one in which The labeling substance is a substance that binds a substance capable of specifically binding to a specific substance, which is a substance that catalyzes a reaction that emits light, for example, a reaction that produces a substance that emits fluorescence or phosphorescence, or a luminescence reaction. Good. Examples of the fluorescent dye include FITC (fluorescein isocyanate), PE (phycoerythrin), and rhodamine. Moreover, examples of the substance that catalyzes the reaction include peroxidase, β-galactosidase, alkaline phosphatase, and luciferase.
標識物質は、前記した通り、それ自体が特定物質と結合又は反応するものであっても、特定物質に特異的に結合する物質と光学的に検出可能なシグナルを発する標識とを結合したものであってもよい。光学的に検出可能なシグナルを発する標識を特定物質と特異的に結合する物質と結合する場合は、両者を公知の化学的な方法等によって直接結合しても良いし、特定物質と特異的に結合する物質に対して結合する物質を介して間接的に結合しても良い。例えば、特定物質に特異的に結合する物質とビオチンを結合しておき、シグナルを発生する物質をアビジン又はストレプトアビジンと結合しておくことも例示できる。この場合、シグナルを発生する物質と結合したアビジン又はストレプトアビジンは、特定物質に特異的に結合した物質と結合されたビオチンと結合し、結果的に（特定物質）−（特定物質に特異的に結合する物質）−（ビオチン）−（アビジン又はストレプトアビジン）−（シグナルを発生する標識）という複合体を形成することにより、特定物質は、標識物質により間接的に標識される。このような間接的な場合もまた、本発明における標識物質に包含される。 As described above, the labeling substance is a substance in which a substance that specifically binds to a specific substance and a label that emits an optically detectable signal are bound, even if it itself binds or reacts with the specific substance. It may be. When binding a label that emits an optically detectable signal to a substance that specifically binds to a specific substance, both may be directly bound by a known chemical method or the like, or specifically to the specific substance. It may be indirectly bound to the substance to be bound via a substance to be bound. For example, a substance that specifically binds to a specific substance may be bound to biotin, and a substance that generates a signal may be bound to avidin or streptavidin. In this case, avidin or streptavidin bound to a substance that generates a signal binds to biotin bound to a substance specifically bound to a specific substance, and as a result, (specific substance)-(specific substance specific The specific substance is indirectly labeled with the labeling substance by forming a complex of (binding substance)-(biotin)-(avidin or streptavidin)-(label generating a signal). Such indirect cases are also included in the labeling substance of the present invention.
本発明で用いる検出工程は、腫瘍細胞に存在する作用部を標識物質により標識し、光強度の増減が検出できれば特に制限はない。好ましくは、前記基板と、基板の観察領域に光を照射し、発生する光学シグナルを検出する光学検出手段を備えることにより、生体試料を観察可能な装置を使用すればよい。光源には、ハロゲンランプ、水銀ランプ、メタルはライドランプ、レーザー、ＬＥＤ等を用いることができ、光源からの光は、必要に応じて光学フィルターやミラー、レンズ等によって構成される光学手段により観察領域に導光されればよい。このような検出工程を行うことができるシステムとして、蛍光顕微鏡システムが挙げられる。 The detection step used in the present invention is not particularly limited as long as the action site existing in tumor cells is labeled with a labeling substance and an increase or decrease in light intensity can be detected. Preferably, a device capable of observing a biological sample may be used by including the substrate and an optical detection unit that irradiates an observation region of the substrate with light and detects an optical signal generated. As the light source, a halogen lamp, a mercury lamp, a metal, a ride lamp, a laser, an LED, or the like can be used, and the light from the light source is observed by an optical means including an optical filter, a mirror, a lens, or the like as necessary. The light may be guided to the area. A fluorescence microscope system is an example of a system capable of performing such a detection step.
光学シグナル情報として、広い波長域での透過光、反射光による光強度分布から構成される像（明視野）や、蛍光物質が発する蛍光から検出した蛍光強度、蛍光強度のピーク値、蛍光強度の最小値、蛍光強度の平均値、蛍光強度の積分値などの蛍光強度から算出された数値などが例示できる。明視野情報は、細胞形態に関する情報が取得可能であれば、特に制限されない。例えば、細胞の直径、細胞の面積、細胞の体積、細胞の周囲長、真円度などが例示できる。 As optical signal information, an image (bright field) composed of light intensity distribution by transmitted light and reflected light in a wide wavelength range, fluorescence intensity detected from fluorescence emitted by a fluorescent substance, peak value of fluorescence intensity, and fluorescence intensity Examples include a minimum value, an average value of fluorescence intensity, a numerical value calculated from fluorescence intensity such as an integrated value of fluorescence intensity, and the like. The bright field information is not particularly limited as long as the information on the cell morphology can be acquired. For example, the diameter of a cell, the area of a cell, the volume of a cell, the perimeter of a cell, the circularity, etc. can be illustrated.
一般的な検出工程では、腫瘍細胞に存在する作用部を標識物質により標識し、検出すると同時に、別の標識物質を用いるか又は明視野観察により、腫瘍細胞を特定してもよい。一例として、ＦＩＴＣやＰＥで標識された抗体、例えば抗ＣＫ抗体を用いて腫瘍細胞を蛍光標識してもよいし、腫瘍細胞以外の細胞を識別する蛍光標識物質、例えば抗ＣＤ４５抗体や核染色物質（ＤＡＰＩ等）で蛍光標識した後、蛍光顕微鏡等により観察することが挙げられる。より具体的に、本発明では、ＣＴＣを検出するために、ＤＡＰＩ波長（ＵＶ励起及び青色の発光）における蛍光観察により、有核細胞を同定し（赤血球の除外）、ＣＤ４５及びＣＫの発現の有無を同定する。このようにして同定された、ＤＡＰＩ陽性、ＣＫ陽性およびＣＤ４５陰性の細胞を腫瘍細胞（ＣＴＣ）と、ＤＡＰＩ陽性、ＣＫ陰性、ＣＤ４５陽性の細胞を白血球と、ＤＡＰＩ陽性、ＣＫ陽性およびＣＤ４５陽性もしくはＤＡＰＩ陰性、ＣＫ陽性の細胞をゴミなどに代表されるノイズシグナルとして区別を可能にする。またＤＡＰＩ陽性、ＣＫ陰性およびＣＤ４５陰性の細胞は、明視野観察において、赤血球や白血球等の正常細胞と細胞形態や大きさを比較する、もしくはパパニコロウ染色やギムザ染色を実施することで細胞内の核、細胞質等の形態的特徴によりＣＴＣを特定することもできる。検出工程において、腫瘍細胞（CTC）を同定する場合、その同定は、蛍光顕微鏡により、撮影された蛍光標識画像を、手動で又はソフトウェアにより自動で行うことができる。 In a general detection step, the action part existing in the tumor cells may be labeled with a labeling substance to be detected, and at the same time, another labeling substance may be used or the bright field observation may be performed to identify the tumor cells. As an example, tumor cells may be fluorescently labeled using an antibody labeled with FITC or PE, for example, an anti-CK antibody, or a fluorescent labeling substance for identifying cells other than tumor cells, for example, an anti-CD45 antibody or a nuclear staining substance. After fluorescent labeling with (DAPI etc.), observation with a fluorescence microscope or the like can be mentioned. More specifically, in the present invention, in order to detect CTC, nucleated cells are identified (excluding red blood cells) by fluorescence observation at a DAPI wavelength (UV excitation and blue emission), and the presence or absence of expression of CD45 and CK. Identify. The DAPI-positive, CK-positive and CD45-negative cells thus identified are tumor cells (CTC), DAPI-positive, CK-negative and CD45-positive cells are leukocytes, and DAPI-positive, CK-positive and CD45-positive or DAPI Negative and CK positive cells can be distinguished as a noise signal typified by dust. In addition, DAPI-positive, CK-negative, and CD45-negative cells are compared with normal cells such as red blood cells and white blood cells in bright field observation by comparing the cell morphology and size, or by performing Papanicolaou staining or Giemsa staining to obtain intracellular nuclei. CTCs can also be specified by morphological characteristics such as cytoplasm. When a tumor cell (CTC) is identified in the detection step, the identification can be performed manually by a fluorescence microscope, by a fluorescence-labeled image taken, or automatically by software.
評価工程
検出工程において、腫瘍細胞に存在する作用部の光強度を検出後に、検出された光強度に基づき、抗癌剤の効果を決定することができる。作用部に結合する標識物質は、抗癌剤の該作用部への結合性に応じて、作用部への結合できなくなり、標識物質からの光強度が変化するからである。例えば、抗癌剤が、腫瘍細胞表面に存在する作用部に作用（結合）することで、作用部がマスキングされると、標識物質が作用部に結合できず光強度が低下する。また、抗癌剤が、腫瘍細胞表面に存在する作用部に作用（結合）することで、特定物質が内在化（Ｉｎｔｅｒｎａｌｉｚａｔｉｏｎ）し、増殖シグナルを抑制するような薬効を示す場合も、抗癌剤適用後に標識物質は作用部に作用できず、光強度は減少する。したがって、抗癌剤の適用後に、腫瘍細胞の特定物質に結合した標識物質からの光強度が減少していれば、抗癌剤が作用部に作用可能であり、それにより薬効が高いとと判断することができる。
Evaluation step In the detection step, after detecting the light intensity of the action site existing in the tumor cell, the effect of the anticancer agent can be determined based on the detected light intensity. This is because the labeling substance that binds to the action site cannot bind to the action site depending on the binding property of the anticancer agent to the action site, and the light intensity from the labeling substance changes. For example, when the anti-cancer agent acts (binds) on the action site existing on the surface of the tumor cell to mask the action site, the labeling substance cannot bind to the action site and the light intensity decreases. In addition, even when the anticancer agent acts (bonds) on the action site existing on the surface of the tumor cell to internalize the specific substance (Internalization) and exhibits a drug effect of suppressing the growth signal, the labeling substance after application of the anticancer agent Cannot act on the action part, and the light intensity decreases. Therefore, after the application of the anti-cancer agent, if the light intensity from the labeling substance bound to the specific substance of the tumor cells is decreased, the anti-cancer agent can act on the action site, and thus it can be determined that the drug efficacy is high. ..
抗癌剤未適用の生物試料について、本発明と同じ濃縮、展開、検出工程を行って検出した光強度と比較することにより、光強度の変化を決定することができ、それにより抗癌剤の効果を決定することができる。したがって、評価工程に代えて、又は追加かして、比較工程を含んでもよい。また、予め癌の種類に応じて、対照や検量線を作っておき、測定した光強度と比較することで、抗癌剤の効果を決定してもよい。薬剤未適用の生物試料との比較を行う場合、薬剤未適用の生物試料と比較して、光強度が７０％以下に低下した場合、好ましくは５０％以下に低下した場合、さらに好ましくは３０％以下に低下した場合、試験した抗癌剤が有効であると判断することができ、一方で光強度の変化がほとんど無い、例えば変化が２０％以下、好ましくは１０％以下、より好ましくは５％以下である場合には、抗癌剤が有効ではないと判断することができる。 By comparing the light intensity detected by performing the same concentration, development, and detection steps as those of the present invention on the biological sample to which the anticancer agent is not applied, the change in the light intensity can be determined, thereby determining the effect of the anticancer agent be able to. Therefore, a comparison step may be included instead of or in addition to the evaluation step. Further, the effect of the anticancer agent may be determined by preparing a control or a calibration curve in advance according to the type of cancer and comparing it with the measured light intensity. When compared with a biological sample to which no drug is applied, the light intensity is reduced to 70% or less, preferably to 50% or less, and more preferably 30% as compared with the biological sample to which the drug is not applied. When it decreases below, it can be judged that the tested anticancer agent is effective, while there is almost no change in light intensity, for example, the change is 20% or less, preferably 10% or less, more preferably 5% or less. In some cases, it can be determined that the anticancer drug is not effective.
抗癌剤が投与された患者における抗癌剤の薬効を評価する方法では、抗癌剤投与前に取得した生物試料について、本発明と同じ濃縮、展開、検出工程を行って検出した光強度と、抗癌剤投与後に取得した生物試料について検出された光強度とを比較することにより、光強度の変化を決定することができ、それにより抗癌剤の効果を決定することができる。したがって、評価工程に代えて、又は追加して、比較工程を含んでもよい。生物試料の取得が、抗癌剤の投与後、経時的に行われている場合、そのようにして得られた生物試料間で、光強度を比較することで、抗癌剤に対する薬効の変化を評価することができる。また、予め癌の種類に応じて、対照や検量線を作っておき、測定した光強度と比較することで、投与された抗癌剤の効果を決定してもよい。抗癌剤投与前の生物試料との比較を行う場合、抗癌剤投与前の生物試料と比較して、光強度が７０％以下に低下した場合、好ましくは５０％以下に低下した場合、さらに好ましくは３０％以下に低下した場合、試験した抗癌剤が有効であると判断することができる。一方で光強度の変化がほとんど無い、例えば変化が２０％以下、好ましくは１０％以下、より好ましくは５％以下である場合には、抗癌剤が有効ではないと判断することができる。 In the method for evaluating the efficacy of an anticancer agent in a patient to which an anticancer agent is administered, the biological sample obtained before the administration of the anticancer agent is the same concentration as that of the present invention, the light intensity detected by performing the detection step, and the obtained after the administration of the anticancer agent. By comparing the light intensity detected for the biological sample, the change in light intensity can be determined, and thereby the effect of the anti-cancer agent. Therefore, a comparison step may be included instead of or in addition to the evaluation step. When the biological sample is obtained over time after the administration of the anticancer drug, the change in the efficacy of the anticancer drug can be evaluated by comparing the light intensities among the biological samples thus obtained. it can. Further, the effect of the administered anticancer agent may be determined by preparing a control or a calibration curve in advance according to the type of cancer and comparing it with the measured light intensity. When comparing with a biological sample before administration of an anticancer agent, as compared with a biological sample before administration of an anticancer agent, when the light intensity is reduced to 70% or less, preferably to 50% or less, more preferably 30% The tested anti-cancer agent can be judged to be effective if it falls below. On the other hand, when there is almost no change in the light intensity, for example, when the change is 20% or less, preferably 10% or less, more preferably 5% or less, it can be determined that the anticancer agent is not effective.
本明細書において言及される全ての文献はその全体が引用により本明細書に取り込まれる。以下、本発明を実施例に基づいて更に詳細に説明するが、本発明は実施例に限定されるものではない。 All documents mentioned in this specification are incorporated herein by reference in their entirety. Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to the examples.
実施例１ ＨＥＲ２発現株（ＳＫＢＲ３）の検出方法
血液試料は、インフォームドコンセントを得て取得した健常者血液３ｍｌに約３０００個のヒト乳癌細胞（ＳＫＢＲ３）を添加した懸濁液を、抗癌剤未適用試料として用いた。同様にして、健常者血液３ｍｌに１０μｇ／ｍＬのトラスツマブ（ハーセプチン）で３時間処理した約３０００個のヒト乳癌細胞（ＳＫＢＲ３）を混合させた懸濁液を、抗癌剤適用試料として用いた。
Example 1 Method for detecting HER2-expressing strain (SKBR3) As a blood sample, a suspension prepared by adding about 3000 human breast cancer cells (SKBR3) to 3 ml of blood of a healthy person obtained by obtaining informed consent was prepared by applying no anticancer agent. It was used as a sample. Similarly, a suspension prepared by mixing about 3000 human breast cancer cells (SKBR3) treated with 10 μg/mL trastuzumab (Herceptin) for 3 hours in 3 ml of blood of a healthy person was used as a sample for anticancer drug application.
図１に示した態様の分離構造体を使用して、これらの試料から腫瘍細胞を濃縮した。分離構造体は、詳細には、筒状部材２は内径Φ１８ｍｍ、縦７０ｍｍ、容量１５ｍＬの円筒状のポリプロピレン製部材である。また筒状部材２の先細り形状の部分の傾斜角度は３０°であり、筒状部材２との連通開口はΦ２ｍｍである。筒状部材３は、内径Φ１０ｍｍ、縦５４ｍｍ、容量３ｍＬのポリプロピレン製部材である。 Tumor cells were enriched from these samples using the isolation construct of the embodiment shown in FIG. In detail, in the separation structure, the cylindrical member 2 is a cylindrical polypropylene member having an inner diameter of 18 mm, a length of 70 mm, and a capacity of 15 mL. The inclination angle of the tapered portion of the tubular member 2 is 30°, and the communication opening with the tubular member 2 is Φ2 mm. The tubular member 3 is a polypropylene member having an inner diameter of 10 mm, a length of 54 mm, and a volume of 3 mL.
図５に模式的に示したように、分離構造体の下側の筒状部材３に、密度が１．０８４ｇ/ｍＬの密度勾配溶液２５を３ｍＬ注入した（図中下部の白抜き部分が密度勾配溶液で満たした部分である）。詳しくは密度勾配溶液の液面高さが、上側の筒状部材２の連通開口６よりも約１ｍｍ高くなるように（従って液面は、上側筒状部材の内部に位置する）注入した。続いて密度勾配溶液の上に、３ｍＬの血液試料と３ｍＬの生理食塩水と７５μＬの結合剤（商標ＲｏｓｅｔｔｅＳｅｐ、ＳｔｅｍＣｅｌｌ Ｔｅｃｈｎｏｌｏｇｉｅｓ Ｉｎｃ）の混合液２６を重層した（図中、黒塗部分が重層した混合液の部分である）。 As schematically shown in FIG. 5, 3 mL of the density gradient solution 25 having a density of 1.084 g/mL was injected into the tubular member 3 on the lower side of the separation structure (the white portion in the lower part of the figure represents the density. Part filled with gradient solution). Specifically, the solution was injected so that the liquid level of the density gradient solution was higher than the communication opening 6 of the upper tubular member 2 by about 1 mm (the liquid surface is therefore located inside the upper tubular member). Subsequently, a mixed solution 26 of 3 mL of blood sample, 3 mL of physiological saline, and 75 μL of a binder (trademark RosetteSep, StemCell Technologies Inc) was overlaid on the density gradient solution (mixing in which black-painted portion was overlaid in the figure). Part of the liquid).
試料注入後、分離構造体の開口をキャップ４（ポリプロピレン製）で密閉し、２０００×ｇで１０分間、室温にて遠心分離した。遠心分離操作により、図６左に示したように、密度勾配溶液と試料の界面２７に細胞は維持された。キャップを取り外すことなく分離構造体を構成する筒状部材２及び３を分離部で分離した後、図６右に示したようにキャップをはずして密閉を開放することで、上側の筒状部材２の連通開口６より密度勾配溶液の一部とその上に維持された細胞を流出させ、下方に設置した５０ｍＬチューブで回収するとともに、上側筒状部材の内壁を洗浄し、壁に付着した細胞も同時に回収した。 After injecting the sample, the opening of the separation structure was sealed with a cap 4 (made of polypropylene), and the mixture was centrifuged at 2000×g for 10 minutes at room temperature. By the centrifugation operation, the cells were maintained at the interface 27 between the density gradient solution and the sample, as shown on the left side of FIG. After the tubular members 2 and 3 constituting the separation structure are separated at the separating portion without removing the cap, the cap is removed to open the seal as shown on the right side of FIG. A part of the density gradient solution and the cells maintained thereon were allowed to flow out from the communication opening 6 of the above, and were collected with a 50 mL tube installed below, and the inner wall of the upper tubular member was washed to remove the cells attached to the wall. Collected at the same time.
回収した細胞の懸濁液に塩化アンモニウムを主成分とする赤血球破砕液を添加して３０ｍＬまでメスアップし、３００×ｇで１０分間室温にて遠心分離した。遠心分離後、ペレットの頂部の液体をピペットで取り出し、ペレット中の細胞を３００ｍＭのマンニトール溶液３０ｍＬに再懸濁し、３００×ｇで５分間室温にて遠心分離した。この遠心分離操作は、細胞破片及び血小板を除去し、腫瘍細胞を濃縮するためのものである。なお、腫瘍細胞とともに混入する正常白血球は３０〜５０万個であり、結合剤で腫瘍細胞外の細胞（赤血球、白血球）を互いに結合することで密度を高くし、腫瘍細胞との密度差を大きくしたうえで、分離構造体を用いた分離を行うことにより、高い回収率と選択性をもって腫瘍細胞を分離できる。 An erythrocyte lysate containing ammonium chloride as a main component was added to the collected cell suspension to make up to 30 mL, and the mixture was centrifuged at 300×g for 10 minutes at room temperature. After centrifugation, the liquid at the top of the pellet was pipetted off, the cells in the pellet were resuspended in 30 mL of 300 mM mannitol solution and centrifuged at 300 xg for 5 minutes at room temperature. This centrifugation operation is for removing cell debris and platelets and for concentrating tumor cells. The number of normal leukocytes mixed with the tumor cells is 300,000 to 500,000, and the density is increased by binding cells outside the tumor cells (erythrocytes and leukocytes) with a binder to increase the density difference with the tumor cells. Then, by performing the separation using the separation structure, the tumor cells can be separated with high recovery and selectivity.
回収した細胞の測定は、スライドへの塗布もしくはウェルへ捕捉した細胞を顕微鏡観察する手法やフローサイトメトリー法などを用いることができる。本実施例１では、分離した細胞を誘電泳動力によって基板上に備えた孔径Φ３０μｍ、深さ４０μｍの保持孔（約３０万個）に捕捉して測定する手法を採用した。 For the measurement of the collected cells, a method such as coating on a slide or observing cells captured in a well under a microscope or a flow cytometry method can be used. In the present Example 1, a method was adopted in which the separated cells were captured by the dielectrophoretic force in the holding holes (about 300,000) having a hole diameter of Φ30 μm and a depth of 40 μm, which were provided on the substrate.
図２に示した構造体の収容部に細胞懸濁液を導入し、基板間に交流電圧（電圧２０Ｖｐｐ、周波数１ＭＨｚ、矩形波）を印加し、誘電泳動力により細胞を保持孔に捕捉した。
前記交流電圧を印加しながら、１個の保持孔に概ね１個の細胞が捕捉された収容部に０．０１％ポリ−Ｌ−リジンを含む３００ｍＭマンニトール水溶液を導入し、３分静置後、前記交流電圧の印加を停止し、収容部の溶液を吸引除去した。続いて、収容部へ細胞膜透過試薬を導入し、１０分静置することで細胞膜を透過させた。その後、収容部の溶液を吸引除去し、ＰＢＳを導入し、収容部内に残留した細胞膜透過試薬を洗浄した。
The cell suspension was introduced into the accommodating portion of the structure shown in FIG. 2, an AC voltage (voltage 20 Vpp, frequency 1 MHz, rectangular wave) was applied between the substrates, and the cells were trapped in the holding holes by the dielectrophoretic force.
While applying the AC voltage, a 300 mM mannitol aqueous solution containing 0.01% poly-L-lysine was introduced into a container in which approximately one cell was trapped in one holding hole, and after standing for 3 minutes, The application of the alternating voltage was stopped, and the solution in the container was removed by suction. Then, a cell membrane permeation reagent was introduced into the accommodation part, and the cell membrane was permeated by leaving still for 10 minutes. Then, the solution in the container was removed by suction, PBS was introduced, and the cell membrane permeation reagent remaining in the container was washed.
次に収容部へ８００μＬの細胞染色液（ＦＩＴＣ標識抗ＣＫ抗体（Ｍｉｌｔｅｎｙｉ Ｂｉｏｔｅｃ）、ＰＥ標識抗ＨＥＲ２抗体（Ｒ＆Ｄ ｓｙｓｔｅｍｓ）、ＡＰＣ標識抗ＣＤ４５抗体（Ｂｅｃｋｍａｎ−Ｃｏｕｌｔｅｒ）、ＤＡＰＩ（０．５μｇ／ｍＬ）（株式会社同仁化学研究所）を混合した細胞染色液）を送液し、細胞標識を行った後（２５℃、３０分）、マンニトール水溶液にて洗浄し、ヒト乳癌細胞を検出した。腫瘍細胞および正常な白血球細胞はともに核を有しているためＤＡＰＩで染色され、赤血球や死細胞片などの核を有していないものは検出ソフトで排除した。また、腫瘍細胞はＣＤ４５を発現していないが、ＣＫを発現している細胞、発現していない細胞が存在するため、ＤＡＰＩ陽性、ＣＤ４５陰性、ＣＫ陽性および陰性の細胞を画像処理により抽出した。続いて、前記画像処理により抽出した１細胞毎のヒト乳癌細胞においてＰＥ標識抗ＨＥＲ２による光強度を算出した。なお正常な白血球細胞はＣＤ４５を発現しているが、ＣＫを発現していないため、ＤＡＰＩ陽性、ＣＫ陰性、ＣＤ４５陽性細胞を検出することで腫瘍細胞と識別した。 Next, 800 μL of cell stain (FITC-labeled anti-CK antibody (Miltenyi Biotec), PE-labeled anti-HER2 antibody (R&D systems), APC-labeled anti-CD45 antibody (Beckman-Coulter), DAPI (0.5 μg/mL) A cell staining solution mixed with (Dojindo Laboratories Inc.) was sent to carry out cell labeling (25° C., 30 minutes), followed by washing with an aqueous mannitol solution to detect human breast cancer cells. Since both tumor cells and normal white blood cells have nuclei, they were stained with DAPI, and those without nuclei such as red blood cells and dead cell debris were excluded with detection software. Further, although tumor cells do not express CD45, but there are cells expressing CK and cells not expressing CK, DAPI-positive, CD45-negative, CK-positive and negative cells were extracted by image processing. Then, the light intensity by PE-labeled anti-HER2 was calculated for each human breast cancer cell extracted by the image processing. Since normal white blood cells express CD45 but not CK, they were identified as tumor cells by detecting DAPI-positive, CK-negative, and CD45-positive cells.
複数の保持孔に捕捉した全ての細胞を観察するために保持部全体の撮像を行った。これにはコンピューター制御式電動ステージ、電子増倍型冷却ＣＣＤカメラ（ＥＭＣＣＤ；ＦＬＯＶＥＬ， ＡＤＴ-１００）を装備した蛍光顕微鏡（ＩＸ７１; Ｏｌｙｍｐｕｓ）を用いた。画像取得及び解析ソフトウェアにはＬａｂＶＩＥＷ（Ｎａｔｉｏｎａｌ Ｉｎｓｔｒｕｍｅｎｔｓ）を用いた。処理後に保持孔に捕捉された正常白血球数は１０〜５０万個ほどであったが、ヒト乳癌細胞の検出に際しては妨げにならない量であった。また、ヒト乳癌細胞は保持孔に捕捉されているため、保持部のスキャニングで全細胞数をカウントできた。血液試料に添加したヒト乳癌細胞（ＳＫＢＲ３）はＣＫ、ＨＥＲ２ともに高発現の細胞株であるため、抗癌剤未適用試料では、図７Ａに示すようにＣＫ、ＨＥＲ２ともに陽性の細胞（ＤＡＰＩ陽性、ＣＤ４５陰性）を検出することができた。
一方で、抗癌剤適用試料では、図７Ｂに示すようにＣＫは陽性であるが、ＨＥＲ２標識による光強度が著しく低下した細胞が検出された。
An image of the entire holding part was taken in order to observe all the cells captured in the plurality of holding holes. For this, a fluorescence microscope (IX71; Olympus) equipped with a computer-controlled motorized stage and an electron multiplying cooled CCD camera (EMCCD; FLOVEL, ADT-100) was used. LabVIEW (National Instruments) was used as image acquisition and analysis software. The number of normal white blood cells trapped in the holding holes after the treatment was about 100,000 to 500,000, which was an amount that did not interfere with the detection of human breast cancer cells. Moreover, since human breast cancer cells were trapped in the holding holes, the total number of cells could be counted by scanning the holding part. Since human breast cancer cells (SKBR3) added to the blood sample are cell lines in which both CK and HER2 are highly expressed, in the sample to which the anticancer agent is not applied, as shown in FIG. 7A, cells positive for both CK and HER2 (DAPI positive, CD45 negative ) Could be detected.
On the other hand, in the sample to which the anticancer agent was applied, as shown in FIG. 7B, cells in which CK was positive but the light intensity due to HER2 labeling was remarkably reduced were detected.
ＨＥＲ２標識陽性の腫瘍細胞について、その光強度と存在率との関係をそれぞれ測定し、グラフに示した（図８）。トラスツズマブ処理によるＨＥＲ２標識の光強度は、低輝度側へ大きくシフトしていたことから、トラスツズマブが前記ヒト乳癌細胞に対して作用していることが確認できた。 The relationship between the light intensity and the abundance of each HER2-labeled tumor cell was measured and shown in the graph (FIG. 8). Since the light intensity of the HER2 label treated with trastuzumab was largely shifted to the low brightness side, it was confirmed that trastuzumab acts on the human breast cancer cells.
実施例２ ＥＧＦＲ発現株（ＷｉＤr）の標準検出法
実施例１と同様にして、健常者血液３ｍｌに約１０００個のヒト大腸癌細胞（ＷｉＤｒ）を混合させた試料を、抗癌剤未適用試料とし、健常者血液３ｍｌに、２０μｇ／ｍＬのパニツムマブ（ベクティビックス）で２時間処理した約１０００個のヒト大腸癌細胞（ＷｉＤｒ）を混合させた試料を、抗癌剤適用試料として用いた。
Example 2 Standard Detection Method for EGFR-Expressing Strain (WiDr) In the same manner as in Example 1, a sample prepared by mixing approximately 1000 human colon cancer cells (WiDr) with 3 ml of blood of a healthy subject was used as a sample to which an anticancer agent was not applied, A sample prepared by mixing 3 ml of blood of a healthy subject with about 1000 human colon cancer cells (WiDr) treated with panitumumab (Vectivix) at 20 μg/mL for 2 hours was used as an anticancer drug application sample.
実施例１と同様に、抗癌剤未適用試料及び抗癌剤適用試料を、それぞれ、生理食塩水、結合剤の混合液を密度勾配溶液の上へ重層し、遠心分離、回収操作を実施した。 In the same manner as in Example 1, the anticancer agent-untreated sample and the anticancer agent-applied sample were overlaid with a mixed solution of physiological saline and a binder on the density gradient solution, followed by centrifugation and recovery operations.
図２に示した構造体の収容部に細胞懸濁液を導入し、基板間に交流電圧（電圧２０Ｖｐｐ、周波数１ＭＨｚ、矩形波）を印加し、誘電泳動力により細胞を保持孔に捕捉した。 The cell suspension was introduced into the accommodating portion of the structure shown in FIG. 2, an AC voltage (voltage 20 Vpp, frequency 1 MHz, rectangular wave) was applied between the substrates, and the cells were trapped in the holding holes by the dielectrophoretic force.
前記交流電圧を印加しながら、１個の保持孔に概ね１個の細胞が捕捉された収容部に０．０１％ポリ−Ｌ−リジンを含む３００ｍＭマンニトール水溶液を導入し、３分静置後、前記交流電圧の印加を停止し、収容部の溶液を吸引除去した。続いて、収容部へ細胞膜透過試薬を導入し、１０分静置することで細胞膜を透過させた。その後、収容部の溶液を吸引除去し、ＰＢＳを導入し、収容部内に残留した細胞膜透過試薬を洗浄した。 While applying the AC voltage, a 300 mM mannitol aqueous solution containing 0.01% poly-L-lysine was introduced into a container in which approximately one cell was trapped in one holding hole, and after standing for 3 minutes, The application of the alternating voltage was stopped, and the solution in the container was removed by suction. Then, a cell membrane permeation reagent was introduced into the accommodation part, and the cell membrane was permeated by leaving still for 10 minutes. Then, the solution in the container was removed by suction, PBS was introduced, and the cell membrane permeation reagent remaining in the container was washed.
次に収容部へ８００μＬの細胞染色液（ＦＩＴＣ標識抗ＣＫ抗体、ＰＥ標識抗ＥＧＦＲ抗体、ＡＰＣ標識抗ＣＤ４５抗体、ＤＡＰＩ（０．５μｇ／ｍＬ）を混合した細胞染色液）を送液し、細胞標識を行った後（２５℃、３０分）、マンニトール水溶液にて洗浄し、ヒト大腸癌細胞を検出した。腫瘍細胞および正常な白血球細胞はともに核を有しているためＤＡＰＩで染色され、赤血球や死細胞片などの核を有していないものは検出ソフトで排除した。また、腫瘍細胞はＣＤ４５を発現していないが、ＣＫを発現している細胞、発現していない細胞が存在するため、ＤＡＰＩ陽性、ＣＤ４５陰性、ＣＫ陽性および陰性の細胞を画像処理により抽出した。続いて、前記画像処理により抽出した１細胞毎のヒト大腸癌細胞においてＰＥ標識抗ＥＧＦＲによる光強度を算出した。なお正常な白血球細胞はＣＤ４５を発現しているが、ＣＫを発現していないため、ＤＡＰＩ陽性、ＣＫ陰性、ＣＤ４５陽性細胞を検出することで腫瘍細胞と識別した。 Next, 800 μL of cell stain (FITC-labeled anti-CK antibody, PE-labeled anti-EGFR antibody, APC-labeled anti-CD45 antibody, DAPI (0.5 μg/mL)-mixed cell stain) was delivered to the container, and the cells were transferred. After labeling (25° C., 30 minutes), the cells were washed with an aqueous mannitol solution to detect human colon cancer cells. Since both tumor cells and normal white blood cells have nuclei, they were stained with DAPI, and those without nuclei such as red blood cells and dead cell debris were excluded with detection software. Further, although tumor cells do not express CD45, but there are cells expressing CK and cells not expressing CK, DAPI-positive, CD45-negative, CK-positive and negative cells were extracted by image processing. Then, the light intensity by the PE-labeled anti-EGFR was calculated for each human colon cancer cell extracted by the image processing. Since normal white blood cells express CD45 but not CK, they were identified as tumor cells by detecting DAPI-positive, CK-negative, and CD45-positive cells.
複数の保持孔に捕捉した全ての細胞を観察するために保持部全体の撮像を行った。これにはコンピューター制御式電動ステージ、電子増倍型冷却ＣＣＤカメラ（ＥＭＣＣＤ；ＦＬＯＶＥＬ， ＡＤＴ-１００）を装備した蛍光顕微鏡（ＩＸ７１; Ｏｌｙｍｐｕｓ）を用いた。画像取得及び解析ソフトウェアにはＬａｂＶＩＥＷ（Ｎａｔｉｏｎａｌ Ｉｎｓｔｒｕｍｅｎｔｓ）を用いた。処理後に保持孔に捕捉された正常白血球数は１０〜５０万個ほどであったが、ヒト大腸癌細胞の検出に際しては妨げにならない量であった。また、ヒト大腸癌細胞は保持孔に捕捉されているため、保持部のスキャニングで全細胞数をカウントできた。実施例２で使用したヒト大腸癌細胞（ＷｉＤｒ）はＣＫ高発現、ＥＧＦＲ中発現の細胞株であるため、抗癌剤未適用試料は、図９Ａに示すようにＣＫは良好に標識でき、ＥＧＦＲも光強度は低かったが検出可能な程度に標識することができた。一方で、抗癌剤適用試料では、図９Ｂに示すようにＣＫは陽性であるが、ＥＧＦＲ標識による光強度が低下した細胞が検出された。パニツムマブ処理の有無によるＥＧＦＲ標識の光強度変化を図１０に示した。パニツムマブを曝露していないヒト大腸癌細胞に対するＥＧＦＲ標識の光強度が低かったにも関わらず、パニツムマブ処理区分では低強度側へシフトしていることが光強度解析で判別できたことから、薬剤の腫瘍細胞への作用を高感度に検出できることが確認できた。 An image of the entire holding part was taken in order to observe all the cells captured in the plurality of holding holes. For this, a fluorescence microscope (IX71; Olympus) equipped with a computer-controlled motorized stage and an electron multiplying cooled CCD camera (EMCCD; FLOVEL, ADT-100) was used. LabVIEW (National Instruments) was used as image acquisition and analysis software. The number of normal white blood cells trapped in the holding holes after the treatment was about 100,000 to 500,000, which was not an obstacle to the detection of human colon cancer cells. Moreover, since human colon cancer cells were trapped in the holding holes, the total number of cells could be counted by scanning the holding part. Since the human colon cancer cell (WiDr) used in Example 2 is a cell line that highly expresses CK and expresses in EGFR, the sample to which the anticancer agent is not applied can be labeled well with CK as shown in FIG. Although the intensity was low, it could be detectably labeled. On the other hand, in the sample to which the anticancer agent was applied, as shown in FIG. 9B, cells in which the CK was positive but the light intensity was decreased by the EGFR labeling were detected. FIG. 10 shows the change in the light intensity of the EGFR label with and without the panitumumab treatment. Although the light intensity of the EGFR-labeled human colon cancer cells not exposed to panitumumab was low, the light intensity analysis showed that the panitumumab treatment group shifted to the low intensity side. It was confirmed that the effect on tumor cells can be detected with high sensitivity.
１ 分離構造体
２ 筒状部材（上側）
３ 筒状部材（下側）
４ キャップ
５ 底部
６ 連通開口端（分離部）
７ 保持孔（貫通孔）
８ 構造体
９ 基板
１０ 上蓋基板
１１ 絶縁膜
１２ 遮光膜
１３ 収容部
１４ 導入口
１５ 排出口
１６ 電極（＋）
１７ 電極（−）
１８ 櫛状電極
１９ 交流電源
２０ 光
２１ 検出部
２２ 導電線
２３ 細胞
２４ 誘電泳動力
２５ 密度勾配溶液
２６ 混合液
２７ 界面
1 Separation structure 2 Cylindrical member (upper side)
3 Cylindrical member (lower side)
4 Cap 5 Bottom 6 Communication opening end (separation part)
7 Holding hole (through hole)
8 structure 9 substrate 10 upper lid substrate 11 insulating film 12 light-shielding film 13 accommodating portion 14 inlet port 15 outlet port 16 electrode (+)
17 electrodes (-)
18 Comb-shaped electrode 19 AC power supply 20 Light 21 Detection part 22 Conductive wire 23 Cell 24 Dielectrophoretic force 25 Density gradient solution 26 Mixed liquid 27 Interface
Claims (9)
患者から取得された生物試料に抗癌剤を適用する抗癌剤適用工程と、
前記生物試料から腫瘍細胞を濃縮する濃縮工程と、
前記濃縮工程で得られた前記濃縮液に含まれる細胞を基板上に展開する展開工程と、
前記基板上に展開した腫瘍細胞に存在する抗癌剤の作用部を標識して、光強度を検出する検出工程と、
検出された光強度に基づき、抗癌剤の薬効を決定する評価工程と、
を含む、前記方法。 A method of assessing the efficacy of an anti-cancer agent in a patient, comprising:
An anticancer agent applying step of applying an anticancer agent to a biological sample obtained from a patient,
A concentrating step of concentrating tumor cells from the biological sample,
A developing step of developing cells contained in the concentrated solution obtained in the concentrating step on a substrate,
A detection step of detecting the light intensity by labeling the action part of the anticancer agent present in the tumor cells spread on the substrate,
Based on the detected light intensity, an evaluation step of determining the efficacy of the anticancer agent,
The method comprising:
抗癌剤を投与された患者から得られた生物試料から腫瘍細胞を濃縮する濃縮工程と、
前記濃縮工程で得られた前記濃縮液に含まれる生体試料を基板上に展開する展開工程と、
前記基板上に展開した腫瘍細胞に存在する抗癌剤の作用部を標識して、光強度を検出する検出工程と、
検出された光強度に基づき、抗癌剤の効果を決定する評価工程、
を含む、前記方法。 A method of assessing the efficacy of an anti-cancer agent in a patient, comprising:
An enrichment step of enriching tumor cells from a biological sample obtained from a patient administered an anti-cancer agent,
A development step of developing a biological sample contained in the concentrated liquid obtained in the concentration step on a substrate,
A detection step of detecting the light intensity by labeling the action part of the anticancer agent present in the tumor cells spread on the substrate,
An evaluation step of determining the effect of the anticancer agent based on the detected light intensity,
The method comprising:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2016008264A JP6700048B2 (en) | 2016-01-19 | 2016-01-19 | Anti-cancer drug evaluation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2016008264A JP6700048B2 (en) | 2016-01-19 | 2016-01-19 | Anti-cancer drug evaluation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2017129425A JP2017129425A (en) | 2017-07-27 |
| JP6700048B2 true JP6700048B2 (en) | 2020-05-27 |
Family
ID=59394590
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2016008264A Active JP6700048B2 (en) | 2016-01-19 | 2016-01-19 | Anti-cancer drug evaluation method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP6700048B2 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2018116040A (en) * | 2017-01-19 | 2018-07-26 | 東ソー株式会社 | Centrifuge tube and method of using the same |
| JP7306618B2 (en) * | 2017-09-26 | 2023-07-11 | 東ソー株式会社 | Factors Involved in Metastasis Formation of Cancer and Method for Predicting Prognosis of Cancer Patients Using Said Factors |
| JP2019101021A (en) * | 2017-11-28 | 2019-06-24 | 東ソー株式会社 | Biological material retainer and method for detecting biological material |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7776612B2 (en) * | 2001-04-13 | 2010-08-17 | Chugai Seiyaku Kabushiki Kaisha | Method of quantifying antigen expression |
| US20040171091A1 (en) * | 2003-02-27 | 2004-09-02 | Cell Work, Inc. | Standardized evaluation of therapeutic efficacy based on cellular biomarkers |
| JP2012237586A (en) * | 2011-05-10 | 2012-12-06 | Fujirebio Inc | Diagnostic reagent for selecting effective treatment method of anticancer agent |
| JP2014020930A (en) * | 2012-07-18 | 2014-02-03 | Kanazawa Univ | Pancreatic cancer diagnosis and biomarker for treatment effect prediction determination |
| JP6364946B2 (en) * | 2013-05-31 | 2018-08-01 | 東ソー株式会社 | Separation structure and separation method |
-
2016
- 2016-01-19 JP JP2016008264A patent/JP6700048B2/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| JP2017129425A (en) | 2017-07-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10082508B2 (en) | Automated analysis of circulating tumor cells | |
| KR101716555B1 (en) | Methods for detecting 5t4-positive circulating tumor cells and methods of diagnosis of 5t4-positive cancer in a mammalian subject | |
| US9506927B2 (en) | Method for detecting low concentrations of specific cell from high concentrations of cell populations, and method for collecting and analyzing detected cell | |
| ES2970741T3 (en) | Use of circulating cellular biomarkers in the blood for the detection and diagnosis of diseases and methods to isolate them | |
| JP6639906B2 (en) | Biological sample detection method | |
| JP2015505966A (en) | Apparatus, system and method for identifying circulating tumor cells | |
| JP6936984B2 (en) | How to Predict the Prognosis of Cancer Patients Using Rare Cells | |
| Choi et al. | Oncogenic regulation of extracellular vesicle proteome and heterogeneity | |
| CN110914690B (en) | Method and apparatus for analyzing protein-protein interactions | |
| JP6259828B2 (en) | Method for identifying treatment-responsive non-small cell lung cancer using anaplastic lymphoma kinase (ALK) as a marker | |
| JP6700048B2 (en) | Anti-cancer drug evaluation method | |
| JPWO2008041594A1 (en) | Antibody drug sensitivity test method | |
| JP6324381B2 (en) | Histological marker for identifying patients with non-small cell lung cancer for treatment with anti-EGFR drugs | |
| KR20240033068A (en) | Method for predicting and/or monitoring cancer treatment response using changes in circulating cancer-related macrophage-like cells (CAML) | |
| JP5307426B2 (en) | Complement activity test method | |
| JP2018044950A (en) | Method for detecting cell contained in sample | |
| Labrosse et al. | Protocol for quantifying drug sensitivity in 3D patient-derived ovarian cancer models | |
| Townsend | A study of CD4+ follicular helper T cells in the follicular lymphoma microenvironment and normal germinal centres | |
| Singh | Selection and high-throughput immunofluorescence detection of cell lines with a hybrid epithelial/mesenchymal phenotype: towards improved characterization of Circulating Tumor Cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A80 | Written request to apply exceptions to lack of novelty of invention |
Free format text: JAPANESE INTERMEDIATE CODE: A80 Effective date: 20160216 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20170609 |
|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20190107 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20191010 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20191029 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20200407 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20200430 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 6700048 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| S111 | Request for change of ownership or part of ownership |
Free format text: JAPANESE INTERMEDIATE CODE: R313117 |
|
| R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |