CN118272527A - Application of tumor-derived ILT3 in prognosis and treatment of lung adenocarcinoma - Google Patents
Application of tumor-derived ILT3 in prognosis and treatment of lung adenocarcinoma Download PDFInfo
- Publication number
- CN118272527A CN118272527A CN202410261076.3A CN202410261076A CN118272527A CN 118272527 A CN118272527 A CN 118272527A CN 202410261076 A CN202410261076 A CN 202410261076A CN 118272527 A CN118272527 A CN 118272527A
- Authority
- CN
- China
- Prior art keywords
- ilt3
- tumor
- cells
- product
- tams
- 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.)
- Pending
Links
- 101000984186 Homo sapiens Leukocyte immunoglobulin-like receptor subfamily B member 4 Proteins 0.000 title claims abstract description 171
- 102100025578 Leukocyte immunoglobulin-like receptor subfamily B member 4 Human genes 0.000 title claims abstract description 158
- 206010028980 Neoplasm Diseases 0.000 title claims abstract description 118
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 title claims abstract description 65
- 201000005249 lung adenocarcinoma Diseases 0.000 title claims abstract description 65
- 238000004393 prognosis Methods 0.000 title claims abstract description 13
- 238000011282 treatment Methods 0.000 title abstract description 10
- 230000000694 effects Effects 0.000 claims abstract description 31
- 230000004614 tumor growth Effects 0.000 claims abstract description 31
- 230000010287 polarization Effects 0.000 claims abstract description 27
- 238000011247 total mesorectal excision Methods 0.000 claims abstract description 21
- 208000037956 transmissible mink encephalopathy Diseases 0.000 claims abstract description 21
- 230000001506 immunosuppresive effect Effects 0.000 claims abstract description 12
- 230000003472 neutralizing effect Effects 0.000 claims abstract description 12
- 230000006052 T cell proliferation Effects 0.000 claims abstract description 10
- 239000003814 drug Substances 0.000 claims abstract description 7
- 230000014509 gene expression Effects 0.000 claims description 86
- 210000004027 cell Anatomy 0.000 claims description 72
- 210000004881 tumor cell Anatomy 0.000 claims description 30
- 230000002401 inhibitory effect Effects 0.000 claims description 23
- 230000007115 recruitment Effects 0.000 claims description 22
- 229940076838 Immune checkpoint inhibitor Drugs 0.000 claims description 20
- 102000037984 Inhibitory immune checkpoint proteins Human genes 0.000 claims description 20
- 108091008026 Inhibitory immune checkpoint proteins Proteins 0.000 claims description 20
- 239000012274 immune-checkpoint protein inhibitor Substances 0.000 claims description 20
- 230000005012 migration Effects 0.000 claims description 16
- 238000013508 migration Methods 0.000 claims description 16
- 230000017188 evasion or tolerance of host immune response Effects 0.000 claims description 15
- 239000000126 substance Substances 0.000 claims description 15
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 12
- 201000005202 lung cancer Diseases 0.000 claims description 12
- 208000020816 lung neoplasm Diseases 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 10
- 230000003013 cytotoxicity Effects 0.000 claims description 9
- 231100000135 cytotoxicity Toxicity 0.000 claims description 9
- 238000003209 gene knockout Methods 0.000 claims description 9
- 108090000623 proteins and genes Proteins 0.000 claims description 9
- 230000005764 inhibitory process Effects 0.000 claims description 8
- 230000004083 survival effect Effects 0.000 claims description 8
- 230000000259 anti-tumor effect Effects 0.000 claims description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
- 238000011156 evaluation Methods 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- 239000003795 chemical substances by application Substances 0.000 claims description 6
- 230000002708 enhancing effect Effects 0.000 claims description 6
- 208000015181 infectious disease Diseases 0.000 claims description 6
- 239000003112 inhibitor Substances 0.000 claims description 6
- 230000001737 promoting effect Effects 0.000 claims description 6
- 239000012269 PD-1/PD-L1 inhibitor Substances 0.000 claims description 5
- 108091027967 Small hairpin RNA Proteins 0.000 claims description 5
- 229940121653 pd-1/pd-l1 inhibitor Drugs 0.000 claims description 5
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 claims description 4
- 108020004459 Small interfering RNA Proteins 0.000 claims description 4
- 239000000074 antisense oligonucleotide Substances 0.000 claims description 4
- 238000012230 antisense oligonucleotides Methods 0.000 claims description 4
- 230000015572 biosynthetic process Effects 0.000 claims description 4
- 238000003501 co-culture Methods 0.000 claims description 4
- 230000009368 gene silencing by RNA Effects 0.000 claims description 4
- 239000004055 small Interfering RNA Substances 0.000 claims description 4
- 150000003384 small molecules Chemical class 0.000 claims description 4
- 238000011144 upstream manufacturing Methods 0.000 claims description 4
- 108020000948 Antisense Oligonucleotides Proteins 0.000 claims description 3
- 239000002924 silencing RNA Substances 0.000 claims description 3
- 239000004480 active ingredient Substances 0.000 claims description 2
- 229940079593 drug Drugs 0.000 claims description 2
- 230000009467 reduction Effects 0.000 claims description 2
- 108091034117 Oligonucleotide Proteins 0.000 claims 1
- 101150037123 APOE gene Proteins 0.000 abstract description 25
- 102100029470 Apolipoprotein E Human genes 0.000 abstract description 25
- 108010074708 B7-H1 Antigen Proteins 0.000 abstract description 19
- 102000008096 B7-H1 Antigen Human genes 0.000 abstract description 19
- 238000001727 in vivo Methods 0.000 abstract description 9
- 230000033540 T cell apoptotic process Effects 0.000 abstract description 5
- 239000003446 ligand Substances 0.000 abstract description 5
- 238000011160 research Methods 0.000 abstract description 5
- 230000002441 reversible effect Effects 0.000 abstract description 3
- 206010061309 Neoplasm progression Diseases 0.000 abstract description 2
- 230000022534 cell killing Effects 0.000 abstract description 2
- 230000007365 immunoregulation Effects 0.000 abstract description 2
- 238000009169 immunotherapy Methods 0.000 abstract description 2
- 230000005751 tumor progression Effects 0.000 abstract description 2
- 210000004981 tumor-associated macrophage Anatomy 0.000 description 63
- 210000001744 T-lymphocyte Anatomy 0.000 description 53
- 210000001519 tissue Anatomy 0.000 description 28
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 22
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 22
- 238000011740 C57BL/6 mouse Methods 0.000 description 20
- 238000010186 staining Methods 0.000 description 20
- 241000699670 Mus sp. Species 0.000 description 18
- 238000000034 method Methods 0.000 description 17
- 230000008595 infiltration Effects 0.000 description 16
- 238000001764 infiltration Methods 0.000 description 16
- 108010009992 CD163 antigen Proteins 0.000 description 15
- 102100025831 Scavenger receptor cysteine-rich type 1 protein M130 Human genes 0.000 description 15
- 238000000684 flow cytometry Methods 0.000 description 14
- 230000002018 overexpression Effects 0.000 description 14
- 210000002540 macrophage Anatomy 0.000 description 13
- 210000000952 spleen Anatomy 0.000 description 12
- 210000002865 immune cell Anatomy 0.000 description 11
- 241000699666 Mus <mouse, genus> Species 0.000 description 10
- 238000004458 analytical method Methods 0.000 description 10
- 239000003636 conditioned culture medium Substances 0.000 description 10
- 230000003247 decreasing effect Effects 0.000 description 10
- 230000001105 regulatory effect Effects 0.000 description 10
- 101100236065 Mus musculus Lilrb4a gene Proteins 0.000 description 9
- 230000002596 correlated effect Effects 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 9
- 101000934372 Homo sapiens Macrosialin Proteins 0.000 description 8
- 241000713666 Lentivirus Species 0.000 description 8
- 102100025136 Macrosialin Human genes 0.000 description 8
- 230000003993 interaction Effects 0.000 description 8
- 229930192851 perforin Natural products 0.000 description 8
- 208000031261 Acute myeloid leukaemia Diseases 0.000 description 7
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 7
- 102000004503 Perforin Human genes 0.000 description 7
- 108010056995 Perforin Proteins 0.000 description 7
- KHGNFPUMBJSZSM-UHFFFAOYSA-N Perforine Natural products COC1=C2CCC(O)C(CCC(C)(C)O)(OC)C2=NC2=C1C=CO2 KHGNFPUMBJSZSM-UHFFFAOYSA-N 0.000 description 7
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 7
- 230000000903 blocking effect Effects 0.000 description 7
- 239000003550 marker Substances 0.000 description 7
- 210000005259 peripheral blood Anatomy 0.000 description 7
- 239000011886 peripheral blood Substances 0.000 description 7
- 230000035755 proliferation Effects 0.000 description 7
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 239000012091 fetal bovine serum Substances 0.000 description 6
- 230000002147 killing effect Effects 0.000 description 6
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 5
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 238000003757 reverse transcription PCR Methods 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 102000013918 Apolipoproteins E Human genes 0.000 description 3
- 108010025628 Apolipoproteins E Proteins 0.000 description 3
- -1 EDTA) Chemical class 0.000 description 3
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 description 3
- 239000012980 RPMI-1640 medium Substances 0.000 description 3
- 210000000424 bronchial epithelial cell Anatomy 0.000 description 3
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 230000000670 limiting effect Effects 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 210000001616 monocyte Anatomy 0.000 description 3
- 239000012679 serum free medium Substances 0.000 description 3
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 description 2
- 102100021723 Arginase-1 Human genes 0.000 description 2
- 101710129000 Arginase-1 Proteins 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 102100027581 Forkhead box protein P3 Human genes 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 101000861452 Homo sapiens Forkhead box protein P3 Proteins 0.000 description 2
- 101000946889 Homo sapiens Monocyte differentiation antigen CD14 Proteins 0.000 description 2
- 102100037850 Interferon gamma Human genes 0.000 description 2
- 108010074328 Interferon-gamma Proteins 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- 208000007433 Lymphatic Metastasis Diseases 0.000 description 2
- 102000007651 Macrophage Colony-Stimulating Factor Human genes 0.000 description 2
- 108010046938 Macrophage Colony-Stimulating Factor Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 2
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 2
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 2
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 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
- 230000005975 antitumor immune response Effects 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 231100000504 carcinogenesis Toxicity 0.000 description 2
- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical compound OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000003197 gene knockdown Methods 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 230000001024 immunotherapeutic effect Effects 0.000 description 2
- 239000005414 inactive ingredient Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 210000001165 lymph node Anatomy 0.000 description 2
- RLSSMJSEOOYNOY-UHFFFAOYSA-N m-cresol Chemical compound CC1=CC=CC(O)=C1 RLSSMJSEOOYNOY-UHFFFAOYSA-N 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- AQIXEPGDORPWBJ-UHFFFAOYSA-N pentan-3-ol Chemical compound CCC(O)CC AQIXEPGDORPWBJ-UHFFFAOYSA-N 0.000 description 2
- 239000000092 prognostic biomarker Substances 0.000 description 2
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- GHMLBKRAJCXXBS-UHFFFAOYSA-N resorcinol Chemical compound OC1=CC=CC(O)=C1 GHMLBKRAJCXXBS-UHFFFAOYSA-N 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 230000002103 transcriptional effect Effects 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 238000009020 BCA Protein Assay Kit Methods 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- GHXZTYHSJHQHIJ-UHFFFAOYSA-N Chlorhexidine Chemical compound C=1C=C(Cl)C=CC=1NC(N)=NC(N)=NCCCCCCN=C(N)N=C(N)NC1=CC=C(Cl)C=C1 GHXZTYHSJHQHIJ-UHFFFAOYSA-N 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- 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 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 101100044298 Drosophila melanogaster fand gene Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 101150064015 FAS gene Proteins 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 101710087079 Gene 49 protein Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 241000282575 Gorilla Species 0.000 description 1
- 102000001398 Granzyme Human genes 0.000 description 1
- 108060005986 Granzyme Proteins 0.000 description 1
- 208000002250 Hematologic Neoplasms Diseases 0.000 description 1
- 101001002657 Homo sapiens Interleukin-2 Proteins 0.000 description 1
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 208000006552 Lewis Lung Carcinoma Diseases 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 206010027459 Metastases to lymph nodes Diseases 0.000 description 1
- 102100035877 Monocyte differentiation antigen CD14 Human genes 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 101100335198 Pneumocystis carinii fol1 gene Proteins 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 102100040678 Programmed cell death protein 1 Human genes 0.000 description 1
- 101710089372 Programmed cell death protein 1 Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 239000012083 RIPA buffer Substances 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 238000010240 RT-PCR analysis Methods 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 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 1
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 1
- 210000000662 T-lymphocyte subset Anatomy 0.000 description 1
- 102100023935 Transmembrane glycoprotein NMB Human genes 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 102100031988 Tumor necrosis factor ligand superfamily member 6 Human genes 0.000 description 1
- 108050002568 Tumor necrosis factor ligand superfamily member 6 Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 101100514842 Xenopus laevis mtus1 gene Proteins 0.000 description 1
- SAZUGELZHZOXHB-UHFFFAOYSA-N acecarbromal Chemical compound CCC(Br)(CC)C(=O)NC(=O)NC(C)=O SAZUGELZHZOXHB-UHFFFAOYSA-N 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 229940024606 amino acid Drugs 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000005809 anti-tumor immunity Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 238000003782 apoptosis assay Methods 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 239000003899 bactericide agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229960000686 benzalkonium chloride Drugs 0.000 description 1
- 229960001950 benzethonium chloride Drugs 0.000 description 1
- UREZNYTWGJKWBI-UHFFFAOYSA-M benzethonium chloride Chemical compound [Cl-].C1=CC(C(C)(C)CC(C)(C)C)=CC=C1OCCOCC[N+](C)(C)CC1=CC=CC=C1 UREZNYTWGJKWBI-UHFFFAOYSA-M 0.000 description 1
- CADWTSSKOVRVJC-UHFFFAOYSA-N benzyl(dimethyl)azanium;chloride Chemical compound [Cl-].C[NH+](C)CC1=CC=CC=C1 CADWTSSKOVRVJC-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 238000002619 cancer immunotherapy Methods 0.000 description 1
- 230000005773 cancer-related death Effects 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000000546 chi-square test Methods 0.000 description 1
- 229960003260 chlorhexidine Drugs 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000002716 delivery method Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 210000003162 effector t lymphocyte Anatomy 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 230000017214 establishment of T cell polarity Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012997 ficoll-paque Substances 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 229940093915 gynecological organic acid Drugs 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000009033 hematopoietic malignancy Effects 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 102000046699 human CD14 Human genes 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 229920001477 hydrophilic polymer Polymers 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000037451 immune surveillance Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 238000011532 immunohistochemical staining Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 201000005243 lung squamous cell carcinoma Diseases 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 235000010270 methyl p-hydroxybenzoate Nutrition 0.000 description 1
- 239000004292 methyl p-hydroxybenzoate Substances 0.000 description 1
- 238000010232 migration assay Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 230000000242 pagocytic effect Effects 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000013610 patient sample Substances 0.000 description 1
- 210000004976 peripheral blood cell Anatomy 0.000 description 1
- WVDDGKGOMKODPV-ZQBYOMGUSA-N phenyl(114C)methanol Chemical compound O[14CH2]C1=CC=CC=C1 WVDDGKGOMKODPV-ZQBYOMGUSA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 229940068965 polysorbates Drugs 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000005522 programmed cell death Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 235000010232 propyl p-hydroxybenzoate Nutrition 0.000 description 1
- 239000004405 propyl p-hydroxybenzoate Substances 0.000 description 1
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 1
- 229950010131 puromycin Drugs 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000002271 resection Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 206010041823 squamous cell carcinoma Diseases 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- SFVFIFLLYFPGHH-UHFFFAOYSA-M stearalkonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCCCC[N+](C)(C)CC1=CC=CC=C1 SFVFIFLLYFPGHH-UHFFFAOYSA-M 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 230000008093 supporting effect Effects 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 238000012385 systemic delivery Methods 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 239000008181 tonicity modifier Substances 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 108091007466 transmembrane glycoproteins Proteins 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 230000005748 tumor development Effects 0.000 description 1
- 210000003171 tumor-infiltrating lymphocyte Anatomy 0.000 description 1
- 102000009816 urokinase plasminogen activator receptor activity proteins Human genes 0.000 description 1
- 108040001269 urokinase plasminogen activator receptor activity proteins Proteins 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 238000011816 wild-type C57Bl6 mouse Methods 0.000 description 1
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention belongs to the technical fields of biological medicine and molecular biology, and provides application of tumor-derived ILT3 in prognosis and treatment of lung adenocarcinoma. The invention researches the immunoregulation effect of tumor-derived ILT3 in LUAD, and results show that ILT3 and ligand APOE thereof can induce TAMs to recruit and M2-like polarization, inhibit T cell proliferation and killing function, promote T cell apoptosis, and reverse APOE effect when neutralizing antibody blocks ILT 3. In vivo studies have found that ILT3 induces immunosuppressive TMEs and promotes tumor progression, whereas knockout of ILT3 in combination with PD-L1 better inhibits tumor growth. The invention first discusses the influence of tumor-derived ILT3 on the immune microenvironment of the LUAD, and proposes that ILT3 is a potential immunotherapy target and prognosis predictor of the LUAD, thereby having good practical application value.
Description
Technical Field
The invention belongs to the technical fields of biological medicine and molecular biology, and particularly relates to application of tumor-derived ILT3 in prognosis and treatment of lung adenocarcinoma.
Background
The information disclosed in the background of the invention is only for enhancement of understanding of the general background of the invention and is not necessarily to be taken as an admission or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.
Lung cancer is one of the most common malignancies and is also the leading cause of cancer-related death worldwide. Non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancer cases, mainly including lung adenocarcinoma (LUAD), which is the most common type of NSCLC, and squamous cell carcinoma (lung squamous cell carcinomas, luc). However, more than 40% of NSCLC patients are already in stage IIIB or IV at the time of diagnosis, thus missing the best opportunity to surgically resect the tumor. Recently, immune checkpoint inhibitors (immune checkpoint inhibitors, ICIs) targeting programmed cell death protein 1 (pd-1) and its ligand programmed cell death ligand 1 (pd-L1) revolutionarily alter the therapeutic pattern of NSCLC. However, the objective remission rate of PD-1/PD-L1 inhibitor alone to NSCLC patients is about 20% -30%. Development of new immunotherapeutic targets, in particular immunomodulatory molecules against other immune cells in TME, is a current research hotspot in the field of tumor therapy.
ILT3, also known as LILRB4, CD85k, LIR-5 or gp49B (homologue of ILT3 in mice), belongs to the leukoig-like receptor superfamily, comprising type I transmembrane glycoproteins with an extracellular Ig-like domain and two intracellular immunoreceptor tyrosine-inhibiting groups (immunoreceptor tyrosine-based inhibition motif, ITIM). ILT3 is expressed in a variety of immune cell types. The expression level of ILT3 in monocytic acute myeloid leukemia (acute myeloid leukaemia, AML) cells is significantly higher than normal cells and is inversely related to the Overall Survival (OS) of AML patients. ILT3 promotes tissue infiltration of leukemia cells and inhibits T cell activity in AML cells via the ApoE/ILT3/SHP 2/NFkB/uPAR/ARG 1 axis, thus supporting tumor development. In addition to hematopoietic malignancies, ILT3 is also expressed in solid cancer cells (such as NSCLC). However, the effect of ILT3 expression in NSCLC on immune cells in TME is not clear.
T cells and tumor-associated macrophages (TAMs) are the most common and important immune cell components used as anti-tumor immune responses in the tumor microenvironment (tumor microenvironment, TME). T cells are the primary effector of anti-tumor immune responses. Among the myriad receptors expressed by T cells, CD3 is a unique molecule that converts the presence of a specific antigen into the intracellular signal required to trigger an immune response to a tumor. As effector T cells, cd8+ T cells recognize the antigen CD3 molecule and eliminate tumors mainly by inducing cell death via perforin, granzyme and Fas/Fas ligand pathways. In addition to T cells, TAMs are another major immune cell component in TMEs, which, like replacing activated macrophages (M2), lack phagocytic activity, promoting tumor cells to evade immune surveillance and metastasize to other tissues and organs. However, the effect of ILT3 on T cell proliferation and polarization in LUAD cells has not been reported.
Disclosure of Invention
In view of the deficiencies in the prior art, it is an object of the present invention to provide the use of tumour derived ILT3 in LUAD prognosis and treatment. Specifically, the invention researches the immunoregulation effect of tumor-derived ILT3 in LUAD, and the result shows that ILT3 and ligand APOE thereof can induce TAMs to recruit and M2-like polarization, inhibit T cell proliferation and killing function, promote T cell apoptosis, and reverse the APOE effect when neutralizing antibody blocks ILT 3. In vivo studies have found that ILT3 induces immunosuppressive TMEs and promotes tumor progression, whereas knockout of ILT3 in combination with PD-L1 better inhibits tumor growth. Based on the above results, the present invention has been completed.
In a first aspect of the invention, there is provided the use of a reagent for detecting the gene encoding ILT3 and its expression products in the manufacture of a product for the prognosis evaluation of LUAD.
In a second aspect of the invention there is provided the use of a substance which inhibits the ILT3 gene and its expression products and/or reduces its activity in any one or more of the following a 1) to a 8):
a1 Inhibition of TAM recruitment and TAM M2-like polarization or preparation of products that inhibit TAM recruitment and TAM M2-like polarization;
a2 Inhibit migration of TAMs or preparing a product that inhibits migration of TAMs;
a3 Promoting T cell proliferation and cytotoxicity or preparing a product that promotes T cell proliferation and cytotoxicity;
a4 A product that inhibits or prepares the formation of immunosuppressive TMEs;
a5 Inhibit tumor growth or preparing a product that inhibits tumor growth;
a6 Inhibiting tumor immune escape or preparing a product for inhibiting tumor immune escape;
a7 Inhibiting tumor resistance to an immune checkpoint inhibitor (enhancing immune checkpoint inhibitor anti-tumor activity) or preparing a product that inhibits tumor resistance to an immune checkpoint inhibitor (enhancing immune checkpoint inhibitor anti-tumor activity);
a8 A product for treating tumor.
In a third aspect of the invention there is provided the use of the above composition in any one or more of the following:
b1 Preparing a product that normalizes immunosuppressive TME;
b2 Preparing a product for inhibiting tumor growth;
b3 Preparing a product for inhibiting tumor immune escape;
b4 A product for treating tumor.
In a fourth aspect of the invention, there is provided a method of tumour therapy, the method comprising: administering to the subject a substance or composition described above that inhibits the ILT3 gene and its expression product and/or reduces its activity.
The above-described technical scheme is equally effective for gp49B, which is a homologous molecule of mouse ILT 3.
The beneficial technical effects of one or more of the technical schemes are as follows:
the technical proposal reports that the expression of ILT3 in lung cancer cells is obviously higher than that of normal lung epithelial cells for the first time. In LUAD, high expression of ILT3 is significantly associated with poor clinical prognosis in patients. Tumor-derived ILT3 promotes recruitment of TAMs and M2-like polarization, inhibiting proliferation and killing capacity of T cells. The ILT3 ligand APOE promotes recruitment of TAMs and M2-like polarization by binding to ILT 3. In vivo, gp49b promotes tumor growth and M2-like polarization of TAMs, and inhibits T cell proportion and killing function. Inhibition of gp49b binding to PD-L1 in tumor cells can better inhibit tumor growth and immune escape.
The above protocol suggests that tumor-derived ILT3 induces immunosuppressive TMEs and is significantly associated with a poor prognosis for patients. ILT3 is a potential novel immunotherapeutic target for LUAD patients.
In conclusion, the technical scheme provides a new mechanism research for the occurrence and development of the LUAD and a promising treatment strategy for the LUAD patient, so that the method has good potential practical application value.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 shows that increased ILT3 expression in LUAD patients is indicative of poor OS in the examples of the present invention.
(A) RT-PCR analysis showed that ILT3 expression was up-regulated in most NSCLC cell lines (H1299, H226, H827, H1650, H520, H11975 and A549) compared to the bronchial epithelial cell line BEAS-2B. The expression level of ILT3 was normalized to GAPDH.
(B) The expression level of ILT3 in lung cancer is significantly higher than that of paracancerous normal tissues.
(C) In 156 patients, elevated ILT3 expression in tumor cells correlated with late stage tumor, no correlation with tumor size and lymph node metastasis.
(D) The expression level of ILT3 in human LUAD tissue and NSCLC tissue was inversely related to the patient's OS. The transcriptional expression level of ILT3 and patient survival information were from the Kaplan-Meier plotter online database. 1308 LUAD and 931 LUSC patients were included in the OS analysis, and the online tool of the KM-plotter database automatically selected the best threshold (ALL p=0.0027, LUAD p=3e-06, LUSC p=0.47).
High expression of ILT3 in (E, F) LUAD tissue (n=105) correlated with larger tumor, increased number of lymph node metastases and late TNM staging. In contrast, expression of ILT3 in the luc tissue (n=51) was not correlated with these clinical pathological parameters. * p <0.05, < p <0.01, < p <0.001.
FIG. 2 is a graph showing that ILT3 promotes TAM recruitment and M2-like polarization in the LUAD in an embodiment of the invention.
(A, B) by immunofluorescence (immunofluorescence, IF) analysis, ILT3 expression in LUAD correlated positively with CD163+ TAM and CD68+ TAM levels. (A) Representative images of ILT3, CD163 and CD68 co-localize staining; green represents ILT3 positive staining, orange represents CD163 positive staining, and red represents CD68 positive staining. (B) statistics of 105 patients. Scale bar: 100um.
(C) High levels of ILT3 are significantly positively correlated with TAM infiltration of type M2. Correlation between the level of immune cell infiltration in ILT3 and TME was analyzed using TIMER 2.0.
(D-E) collecting tumor cells CM with different ILT3 expression levels for macrophage culture and TAM induction, and the tumor cells induced TAM has stronger migration capacity compared with the parent macrophages. Knocking out ILT3 in a549 cells inhibited tumor-induced TAM migration, whereas overexpression of ILT3 in H1299 and H1650 promoted tumor-induced TAM migration. (D) an image of the migrating cells; (E) average results of 3 independent experiments. Scale bar: 100 μm.
(F-H) knock-out of ILT3 in A549 cells by RT-PCR (F) and flow cytometry (G-H) decreased the expression levels of the M2-type markers (CD 163, CD206, IL-10, CD209 and Arg 1) in the induced TAMs, and increased the expression levels of the M1-type markers (CD 80, CD86, IL-12 and TNFα). Over-expression of ILT3 in H1299 and H1650 cells increased expression levels of the M2-type marker in the induced TAMs and decreased expression levels of the M1-type marker compared to knockout of ILT 3. * p <0.05, < p <0.01, < p <0.001.
FIG. 3 shows that ILT3 directly affects proliferation and cytotoxicity of T cells in LUAD cells in examples of the present invention.
(A-B) by IF analysis, the number of tumor-infiltrating T cells was lower in patients with high ILT3 expression than in patients with low ILT3 expression in the LUAD tissue. (A) Representative images of CD3, CD45 and panCK co-localized staining; green represents CD3 positive staining, orange represents CD45 positive staining, and red represents panCK positive staining. (B) statistics of 105 patients. Scale bar: 100 μm.
(C-D) proliferation of T cells co-cultured with A549 cells down-regulated by ILT3 was increased, while proliferation of T cells co-cultured with H1299 and H1650 cells up-regulated by ILT3 was decreased. Tumor cells expressing different ILT3 were first co-cultured with CD3+ T cells at a ratio of 1:2 for 48 hours. Proliferation capacity of T cells was assessed by CFSE proliferation assay. (C) representative flow cytometry results of T cell proliferation; (D) statistical results of 3 independent experiments.
(E-F) ILT3 knockdown inhibited T cell apoptosis in A549 cells compared to control. Overexpression of ILT3 in H1299 and H1650 cells promotes T cell apoptosis. Apoptotic T cells were evaluated following co-culture with different tumor cells using flow cytometry. (E) representative flow cytometry results of T cell apoptosis; (F) statistics of 3 independent experiments.
(G-H) knockout of ILT3 in A549 cells increased perforin expression levels on co-cultured T cells. Overexpression of ILT3 in H1299 and H1650 inhibited expression levels of co-cultured T cell perforins. T cells co-cultured with tumor cells were collected and expression levels of perforin were assessed by flow cytometry. (G) flow cytometry representative results; (H) statistics of 3 independent experiments.
FIG. 4 is a graph showing that APOE-ILT3 interactions promote TAM recruitment and class M2 polarization in an embodiment of the invention.
(A-B) expression of APOE in LUAD was positively correlated with levels of CD163+ TAM and CD68+ TAM by IF analysis. (A) Representative images of ApoE, CD163 and CD68 co-localized staining; green represents ApoE positive staining, orange represents CD163 positive staining, and red represents CD68 positive staining. (B) statistics of 105 patients. Scale bar: 100um.
(C) The expression level of APOE in human LUAD tissue is inversely related to patient OS. The transcriptional expression levels and patient survival information for APOE were from Kaplan-Meier plotter online databases. 1308 LUAD and 931 LUSC patients were included in the OS analysis, and the online tool of the KM-plotter database automatically selected the best threshold (ALL p=0.35, LUAD p=0.0059, and LUSC p=0.7).
(D) Timer2.0 analysis showed that the level of APOE expression in LUAD was positively correlated with infiltration of type M2 TAMs.
(E-F) RhApoE increased recruitment of TAMs by H1650 and H1299 cells with high ILT3 expression. First pre-treating H1650-ILT3 and H1299-ILT3 cells with anti-ILT 3 (1. Mu.g/ml) or isotype antibodies for 6 hours, then stimulating the cells with rhaoE (500 ng/ml) for 24 hours, it was found that ILT3 neutralizing antibodies reversed APOE-induced recruitment of TAMs. (E) representative images of migrating cells. (F) statistics of 3 independent experiments. Scale bar: 100 μm.
(G-I) the addition of rhAPOE to H1299-ILT3 and H1650-ILT3 cells increased the expression of the M2-type markers (CD 163, CD206, IL-10, CD209 and Arg 1) in TAMs and decreased the expression of the M1-type markers (CD 80, CD86, IL-12 and TNFα) as detected by RT-PCR (G) and flow cytometry (H-I). Addition of ILT3 neutralizing antibodies reversed rhAPOE changes in expression of the M1 and M2 type marker molecules in TAMs.
FIG. 5 shows that Gp49b promotes tumor growth and immune escape in vivo in an embodiment of the invention.
(A-B) Gp49B can obviously promote the growth of transplanted tumors of C57BL/6 mice. Mice lung cancer cell line LLC (2X 10 5 cells/mouse) infected with gp49b over-expressed and control lentiviruses were subcutaneously injected into male C57BL/6 mice (6/group) for 6-8 weeks. Tumor size was measured every 3 days and expressed as mean ± SD. (a) tumor growth curves for each group; (B) tumor image of mice.
(C) The tumor weight of Gp49 b-overexpressed group was significantly greater than that of the control group (6/group).
(D-F) overexpression of gp49b in LLC can promote tumor growth and increase tumor size and weight in NSG mice. Cell preparation, cell injection number and procedure were the same as described in (a). Tumor size was measured once every 3 days (6/group). (D) tumor growth curves for each group; (E) tumor image of mice. (F) the tumor weight of gp49b overexpressing group was greater than that of the control group. The data in (D) and (F) are expressed as mean.+ -. SD.
(G) Upon overexpression of gp49b in LLC cells, the number of CD163+ and CD206+ macrophages in tumor tissue of C57BL/6 mice was significantly increased and the number of CD80+ macrophages was significantly decreased.
(H) When gp49b was overexpressed in LLC, the proportion of CD3+ T cells in CD45+ lymphocytes was significantly reduced in tumors, spleen and peripheral blood of C57BL/6 mice.
(I) When gp49b was overexpressed in LLC, the proportion of FOXP3+ T cells in tumor, spleen and peripheral blood of C57BL/6 mice was significantly increased.
The expression level of perforin and IFN-gamma on T cells in tumor, spleen and blood of C57BL/6 mice in the (J-K) gp49b overexpression group is low.
(L-N) knockout of gp49b in LLC can inhibit tumor growth in C57BL/6 mice, and reduce tumor size and tumor weight. LLC cells from gp49b knockout or control lentiviruses were inoculated subcutaneously into C57BL/6 mice. The number and procedure of cell injections were the same as in (A). Tumor size was measured every 3 days (6/group). (L) tumor growth rate curves for each group; (M) tumor image per mouse. (N) the tumor weight of gp49b knockout group was less than that of the control group. The data in (L) and (N) are expressed as mean.+ -. SD.
(O) when gp49b in LLC cells was knocked out, the number of CD163+ and CD206+ TAMs in the C57BL/6 mouse tumor was significantly reduced, while the number of CD80+ TAMs was significantly increased.
(P) after gp49b was knocked out in LLC, the proportion of CD3+ T cells in CD45+ lymphocytes in tumors, spleen and peripheral blood of C57BL/6 mice was significantly increased.
(Q) flow cytometry showed a significant decrease in the proportion of FOXP3+ T cells in C57BL/6 mice tumors, spleen and peripheral blood when gp49b was knocked out in LLC.
The expression of perforin and IFN-gamma on T cells in tumor, spleen and blood of (R-S) gp49b gene knockout mice is higher. * p <0.05, < p <0.01, < p <0.001.
FIG. 6 shows that Gp49b and PD-L1 blockade synergistically prevents tumor growth and immune escape in vivo in embodiments of the invention.
(A-B) Gp49B knockout or PD-L1 neutralizing antibodies significantly inhibited tumor growth in C57BL/6 mice, whereas combined blocking of Gp49B and PD-L1 resulted in the slowest tumor growth rate. Mouse lung cancer cell line LLC (2X 10 5 cells/mouse) infected with gp49b knockout or control lentivirus was inoculated subcutaneously into C57BL/6 mice. PD-L1 inhibitory antibodies or control IgG (200 μg) were intraperitoneally injected every 4 days from day 7 post tumor inoculation into tumor mice. Tumor size was measured every 3 days (6/group). (a) tumor growth curves for each group; (B) tumor image of mice. (C) The tumor weight of the Gp49b knockout group or the PD-L1 suppressed group was significantly less than that of the control group, while the tumor weight of the Gp49b and PD-L1 combined blocking group was minimal. The histogram shows the mean ± SD of the tumor weights of each group at the end of the experiment.
(D) When gp49b or PD-L1 is blocked using a specific knockout lentivirus or neutralizing antibody, the number of CD163+ and CD206+ TAMs is significantly reduced, but the number of CD80+ TAMs is increased. The combined blocking of these two molecules minimizes the number of CD163+ and CD206+ TAMs and maximizes the number of CD80+ TAMs in tumor tissue.
(E) When gp49b and/or PD-L1 was inhibited, the trend of decrease in the proportion of cd3+ T cells in cd45+ lymphocytes was significantly reversed compared to the control group, with the highest proportion of cd3+ T cells in tumor tissue, spleen and blood of the combined blocking group mice. * p <0.05, < p <0.01, < p <0.001.
FIG. 7 shows that ILT3 was knocked out in A549, and ILT3 was overexpressed in H1299 and H1650 by lentiviral transfection technique, and that stable transfected strains were successfully constructed by PCR (A) and WB (B).
FIG. 8 shows that gp49B is knocked out and overexpressed in LLC by lentiviral transfection technique, and that PCR (A) and WB (B) confirm successful construction of stable transfected strain with gp49B over-expressed and knocked out in examples of the present invention.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the present invention. As used herein, the singular is also intended to include the plural unless the context clearly indicates otherwise, and furthermore, it is to be understood that the terms "comprises" and/or "comprising" when used in this specification are taken to specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof.
The invention will now be further illustrated with reference to specific examples, which are given for the purpose of illustration only and are not intended to be limiting in any way. If experimental details are not specified in the examples, it is usually the case that the conditions are conventional or recommended by the reagent company; reagents, consumables, etc. used in the examples described below are commercially available unless otherwise specified.
In an exemplary embodiment of the invention, there is provided the use of a reagent for detecting the ILT3 encoding gene and its expression product in the preparation of a prognostic evaluation product for lung adenocarcinoma.
The product is capable of predicting the progression of LUAD by detecting the expression level of the ILT3 encoding gene and/or the expression product of the ILT3 encoding gene (e.g., ILT3 protein). The present study found that increased expression of ILT3 in LUAD tumor cells is predictive of advanced disease and poor patient prognosis. ILT3 may be a potential driver of LUAD progression and prognostic biomarker.
Specifically, the prognostic evaluation includes an evaluation of OS; LUAD patients with high ILT3 expression had larger tumors, later regional lymph node involvement, later TNM staging, and worse OS than those with low ILT3 expression.
Wherein the product comprises a substance that detects transcription of ILT3 in a lung adenocarcinoma sample based on a high throughput sequencing method and/or based on a quantitative PCR method and/or based on a probe hybridization method; or substances for detecting the condition of the ILT3 expression product in the lung adenocarcinoma sample based on an immunodetection method, are not particularly limited herein.
In yet another embodiment of the present invention, there is provided the use of a substance that inhibits the ILT3 gene and its expression products and/or reduces its activity in any one or more of the following a 1) -a 8):
a1 Inhibition of TAM recruitment and TAM M2-like polarization or preparation of products that inhibit TAM recruitment and TAM M2-like polarization;
a2 Inhibit migration of TAMs or preparing a product that inhibits migration of TAMs;
a3 Promoting T cell proliferation and cytotoxicity or preparing a product that promotes T cell proliferation and cytotoxicity;
a4 A product that inhibits or prepares the formation of immunosuppressive TMEs;
a5 Inhibit tumor growth or preparing a product that inhibits tumor growth;
a6 Inhibiting tumor immune escape or preparing a product for inhibiting tumor immune escape;
a7 Inhibiting tumor resistance to an immune checkpoint inhibitor (enhancing immune checkpoint inhibitor anti-tumor activity) or preparing a product that inhibits tumor resistance to an immune checkpoint inhibitor (enhancing immune checkpoint inhibitor anti-tumor activity);
a8 A product for treating tumor.
The product may be a drug or an experimental reagent that may be used for basic research. For example, the product can be used for in vitro induced regulation of macrophage polarization, thereby establishing an efficient and economical macrophage polarization experimental model.
In yet another embodiment of the present invention, the tumor may be lung adenocarcinoma and the tumor cell may be a lung adenocarcinoma cell.
Thus, in a further embodiment of the invention, the application a 1) is: use of a substance that inhibits ILT3 gene and its expression products and/or reduces its activity in the preparation of a product for inhibiting TAM recruitment and TAMs M2-like polarization in a lung adenocarcinoma tumor cell-macrophage co-culture system.
Wherein the substances inhibiting ILT3 gene and its expression products and/or activity reduction include, but are not limited to, RNA interference molecules or antisense oligonucleotides, small molecule inhibitors, shRNA, siRNA, substances for carrying out lentiviral infection or gene knockout and specific antibodies against ILT3 itself or molecules upstream and downstream thereof, including ILT3 neutralizing antibodies.
In the a 7), the immune checkpoint inhibitor comprises a PD-1/PD-L1 inhibitor.
According to the invention, when the product is a medicament, the medicament further comprises at least one pharmaceutically inactive ingredient.
The pharmaceutically inactive ingredient may be a pharmaceutically acceptable carrier. Further, the composition can be formulated into various dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc., for oral administration, external use, suppositories, and sterile injectable solutions according to a usual method.
The pharmaceutically acceptable carrier dose should be harmless to the subject, specifically including but not limited to buffers (such as acetate, tris, phosphate, citrate and other organic acids), antioxidants (such as ascorbic acid and methionine), preservatives (such as octadecyldimethylbenzyl ammonium chloride, butanol or benzyl alcohol, methyl or propyl parahydroxybenzoate, catechol, resorcinol, 3-pentanol and m-cresol), bactericides (such as chlorhexidine, benzalkonium chloride, benzethonium chloride), proteins (such as serum proteins, gelatin or immunoglobulins), hydrophilic polymers (such as polyvinylpyrrolidone), amino acids (such as glycine, glutamine, asparagine, histidine, arginine or lysine) monosaccharides, disaccharides and other carbohydrates (including glucose, mannose or dextrin), chelating agents (such as EDTA), tonicity modifiers (such as trehalose and sodium chloride), sugars (such as sucrose, mannitol, trehalose or sorbitol), surfactants (such as polysorbates), salt forming counterions (such as sodium), metal complexes (such as Zn-protein complexes) and/or non-polyethylene glycol surfactants (such as not specifically defined herein.
In yet another embodiment of the invention, the medicament of the invention may be administered to the body in a known manner. For example, by intravenous systemic delivery or local injection into the tissue of interest. Alternatively via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration may be via single or multiple doses. It will be appreciated by those skilled in the art that the actual dosage to be administered in the present invention may vary greatly depending on a variety of factors, such as the target cell, the type of organism or tissue thereof, the general condition of the subject to be treated, the route of administration, the mode of administration, and the like.
In yet another embodiment of the present invention, the subject to be administered can be human or non-human mammal, such as mice, rats, guinea pigs, rabbits, dogs, monkeys, gorillas, etc.
In yet another embodiment of the present invention, a composition is provided whose active ingredients include at least an agent that inhibits and/or reduces the activity of the ILT3 gene and its expression products and an immune checkpoint inhibitor.
Such substances that inhibit the decrease in the ILT3 gene and its expression products and/or activity include, but are not limited to, RNA interference molecules or antisense oligonucleotides directed against ILT3, small molecule inhibitors, shRNAs, siRNAs, substances that effect lentiviral infection or gene knockout, and specific antibodies directed against ILT3 itself or molecules upstream and downstream thereof, including ILT3 neutralizing antibodies.
The immune checkpoint inhibitor comprises a PD-1/PD-L1 inhibitor.
In yet another embodiment of the present invention, there is provided the use of the above composition in any one or more of the following:
b1 Preparing a product that normalizes immunosuppressive TME;
b2 Preparing a product for inhibiting tumor growth;
b3 Preparing a product for inhibiting tumor immune escape;
b4 A product for treating tumor.
Wherein the tumor is solid tumor, further lung cancer, and further lung adenocarcinoma.
The product may also be a pharmaceutical or experimental agent.
In yet another embodiment of the present invention, there is provided a method of tumor treatment, the method comprising: administering to the subject a substance or composition described above that inhibits the ILT3 gene and its expression product and/or reduces its activity.
In yet another embodiment of the present invention, the tumor is a solid tumor, further lung cancer, further lung adenocarcinoma.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The following examples are test methods in which specific conditions are noted, and are generally conducted under conventional conditions.
Examples
Experimental method
Tumor patient samples: a total of 156 tumor specimens from patients with NSCLC undergoing surgical resection were collected by the affiliated tumor hospital at the first medical university in the Shandong, 1 month to 12 month in 2016. These patients did not receive any treatment prior to surgery, including radiation, chemotherapy, or immunotherapy. Patients were classified according to the UICC/AJCC NSCLC staging system (version 8). The study was approved by the ethical committee of the oncology hospital, shandong province, and informed consent was obtained for each patient.
Obtaining and culturing tumor cell lines: human NSCLC cell lines (A549, H1299, H1650, H1975, PC9, H520, H226 and H827) and bronchial epithelial cell line BEAS-2B were purchased from the national academy of sciences cell resource center (Beijing, china). NSCLC cell lines were cultured in RPMI-1640 medium (Hyclone, logan, calif., USA) supplemented with 10% fetal bovine serum (FBS; gibco, carlsbad, calif., USA). BEAS-2B cells were cultured in BEGM BulletKit medium (Lonza, walkersville, md., USA) and 10% FBS was added. Cells were cultured in a humidified incubator at 37℃with 5% CO 2.
Conditioned Medium (CM) was prepared: cells H1650 and H1299 expressing ILT3 were treated with rhAPOE (500 ng/ml)/anti-ILT 3 (1 ug/ml) and cultured in serum-free medium for 48 hours. ILT3 high expressing/knockout cells were also cultured in serum-free medium for 48 hours. CM was then centrifuged at 2000×g for 10min and harvested to induce TAM polarization and migration.
Human TAMs and T cell production: human Peripheral Blood Mononuclear Cells (PBMCs) were isolated from fresh whole blood donated from healthy volunteers using Ficoll-Paque density centrifugation. Human monocytes were isolated from PBMCs using the EasySep human CD14 positive selection kit (StemCell Technologies; cat No. 17858) and human naive CD3+ T cells were isolated using the EasySep T cell enrichment kit (StemCell Technologies; cat No. 19751). CD14+ monocytes were cultured in RPMI-1640 supplemented with 10% FBS and 100ng/ml recombinant human macrophage colony-stimulating factor (M-CSF) (Biolegend; cat No. 574802) was added for 7 days to generate macrophages. To induce TAMs, macrophages were seeded in 12-well plates and stimulated with 1ml CM and FBS-containing mixed medium (9:1) for 48 hours. The 6-well plate was first pre-coated with 2ug/ml of anti-human CD3 (Biolegend; cat No. 317326) to activate human naive CD3+ T, and then cultured with RPMI-1640 containing 10% FBS, 1ug/ml of anti-human CD28 (Biolegend; cat No. 302914) and 100IU/ml of recombinant human IL-2 (PeproTech; cat No. 2000250) was continued.
IF staining and quantification: human LUAD tissue was first paraffin embedded and then cut into 4 μm slices. IF staining was performed according to the method of absin four-color multiple fluorescence immunohistochemical staining kit (abs 50012). Tissue sections were incubated with one of the following antibodies for 1 hour at room temperature: anti-ILT 3 antibodies (1:200;Abcam;Cat No.ab182422), anti-CD 3 (1:300;Abcam;Cat No.ab5690), anti-CD 45 (1:150;Abcam;Cat No.ab40763), anti-pan-CK (1:500;Abcam;Cat No.ab86734), anti-APOE (1:200;Abcam;Cat No.ab52607), anti-CD 68 (1:400;Abcam;Cat No.ab955). At least three fields per slide were counted randomly at a magnification of x 200.
Lentiviral transfection: ILT3/gp49b over-expression or gene knockout viruses were purchased from GeneCopoeia. Prior to infection, NSCLC cells were inoculated into 6-well plates overnight and 2 ml of fresh medium containing lentivirus (MOI: 5-10) was added to each well. After 72 hours, the effect of infection was observed under a fluorescence microscope, and transfected cells were screened with 2. Mu.g/mL puromycin.
RNA extraction and RT-PCR: RNA from tumor cells and TAMs was extracted using SPARKEASY cell RNA flash extraction kit (SparkJade Cat No. AC025-B). RT-PCR was performed on an ABI 7500Real-Time PCR system (Applied Biosystem, carlsbad, calif., USA) using Maxima SYBR GREEN QPCR MASTER Mix (Thermo FISHER SCIENTIFIC) under the following reaction conditions: 50℃for 2 minutes and 94℃for 10 minutes (initial denaturation), followed by a cycle of 40 times at 94℃for 15 seconds and 60℃for 60 seconds. mRNA expression of ILT3 and gp49b and TAMs markers in tumor cells were determined using specific primers, analyzed using the comparative Ct method, and normalized to GAPDH levels.
Western Blot: cell whole lysates were prepared and total protein was extracted with RIPA buffer (baiyou days) containing protease and phosphatase inhibitors. Protein concentration of cell lysates was determined using BCA protein assay kit (hundred days) and then solubilized with loading buffer (hundred days). Samples were transferred to PVDF membrane after SDS-PAGE gel separation. The membrane was incubated with primary antibody overnight at 4 ℃, then washed with water, and incubated with HRP-conjugated secondary antibody for 1 hour at room temperature. The primary antibodies used were as follows: anti-ILT 3 (1:1000; abcam; ab229747), anti-GAPDH (1:1000; abcam; ab8245), anti-gp 49b (1:1000; abcam; ab231813). ILT3 was normalized to GAPDH after automatic exposure on a chemiluminescent gel imaging system (FluorChem M, proteinsimple, USA).
Flow cytometry analysis: human tumor cells, human and mouse TAMs cells, and T cell markers were surface or intracellular stained with different fluorescent conjugated specific antibodies and detected by flow cytometry. For flow staining of mice, red blood cells in blood, spleen and tumor tissue were excluded using red blood cell lysate (Solarbio, cat No. r 1010) and then flow stained. For IFN-. Gamma.staining, treatment with Cell Stimulation Cocktail plus protein transport inhibitors (eBioscience; cat No. 4975-03) was first performed, incubation at 37℃for 5 hours at 5% CO 2, and staining was then performed. All antibodies were purchased from Biolegend. The human antibodies used were as follows PE-anti-ILT3, PE-anti-CD163, PE-anti-CD80, PE-anti-CD86, PE-anti-CD206. The following mouse antibodies ：FITC-anti-CD11b,Percp5.5-anti-F4/80,PE-anti-CD163,APC-anti-CD80,PE-anti-CD86,Alexa FlourTM700-anti-CD206,APC/CY7-anti-CD45,Percp5.5-anti-CD3,FITC-anti-CD4,APC-anti-CD8,Alexa FlourTM700-anti-FOXP3,PE-anti-perforin, and PE-anti-IFN-gamma were used. The analysis was performed on a FACS Calibur flow cytometer (BD Bioscience) using FlowJo10 software (Tree Star, inc).
And (5) letter generation analysis: the correlation of ILT3 and APOE expression levels with prognosis of NSCLC patients was analyzed using the online tool Kaplan-Meier plotter (https:// kmpilot. The Timer2.0 database (http:// timer. Cistrom. Org /) was used to analyze the correlation of ILT3 and APOE expression levels with immune cells in TME.
Cell migration assay: to test for the migration capacity of TAM, 1X 10 5 macrophages were resuspended in 100ul serum-free medium and added to the upper chamber of a 24-well transwell plate. 600ul CM was added to the lower chamber. After 24 hours of incubation, the upper luminal cells were fixed with 4% paraformaldehyde for 15 minutes followed by staining with 0.1% crystal violet for 20 minutes. Cells not migrated in the upper chamber were erased with a cotton swab. The migrated cells in 3 fields were photographed and counted randomly under a microscope.
In vivo study: c57BL/6 mice were purchased from SPF Biotechnology Co., ltd (Beijing, china) and bred under specific pathogen-free conditions. All animal experiments are approved by animal health committee of Chinese medical academy of sciences, and accord with the current use rules and standards of experimental animals in China. The effect of gp49b on tumor growth and TME was studied in C57BL/6 mice. Mice Lewis lung carcinoma cells (LLC) were transfected with gp49b over-expressed and lentiviruses knocked out. 2×10 5 different treated LLC cells were subcutaneously injected to the right of C57BL/6 mice (n=6). Tumor volumes were measured every 3 days using digital calipers and the formula was 0.5 x length x width 2. When the tumor grows to the limit size (2 cm), the mice are killed, the tumor isolated and weighed. Peripheral blood cells, spleen cells and tumor tissue were isolated and analyzed by flow cytometry.
Statistical analysis: mapping and analysis were performed using GRAPHPAD PRISM 8.0.0 software (GraphPad Software inc.). The relationship between the expression of ILT3 and the expression level of APOE, the proportion of M2-like TAMs and the proportion of T cell subsets was analyzed using T-test. Correlation between ILT3 expression and clinical pathology was analyzed using the Chi-square test or Fisher precision test depending on sample size. The survival rate curve is drawn by using a Kaplan-Meier method. P < 0.05 is statistically significant for the differences.
Experimental results
Increased ILT3 expression in LUAD patients is indicative of poor Overall Survival (OS)
To determine whether ILT3 is present in large amounts in NSCLC cells, we first analyzed the expression of ILT3 in 8 human NSCLC cell lines (PC 9, H1299, H226, H827, H1650, H520, H1975 and a 549). Almost all tumor cell lines showed higher expression levels of ILT3 compared to the bronchial epithelial cell line bees-2B (fig. 1A). We collected tumor tissue from NSCLC patients and stained IF. The results showed that ILT3 was expressed more strongly in tumor tissue than in paracancerous normal tissue (fig. 1B). Subsequently, we analyzed the correlation of ILT3 expression with patient clinical pathology. TNM staging was later in the ILT 3-high-expression group compared to the ILT 3-low-expression group (FIG. 1C).
Correlation between ILT3 expression in tumor tissue and patient clinical prognosis was further retrospectively analyzed using Kaplan-Meier database. We observed that high expression of ILT3 indicated that NSCLC patients had shortened OS (hr=1.2, 95% CI: 1.07-1.35), that LUAD patients had more significant differences in OS (hr=1.5, 95% CI: 1.27-1.78), whereas lucc patients had no differences in OS (hr=0.93, 95% CI: 0.77-1.13) (fig. 1D). We again analyzed 156 NSCLC patients collected and found that the ILT3 high-expressing group had larger tumors, regional lymph node involvement was later, TNM staging was later than the ILT3 low-expressing group in LUAD, with no differences in LUSC (fig. 1E-F). In summary, increased expression of ILT3 in LUAD tumor cells is predictive of advanced disease and poor patient prognosis. ILT3 may be a potential driver of LUAD progression and prognostic biomarker.
ILT3 in LUAD promotes TAM recruitment and M2-like polarization
The regulatory role of ILT3 in the LUAD tumor immune microenvironment remains unclear. Macrophages are the most abundant immune cells in the lung (about 70% of immune cells). TAMs can be divided into two classes, one class being M1, which has a phenotype of killing tumor cells, and the other class being M2, which has a phenotype of promoting tumors. In view of the high abundance of TAMs in TMEs, we further explored whether ILT3 on tumor cells would affect TAMs, thereby mediating tumor immune escape.
To confirm the effect of specific ILT3 expression on M2-type TAMs infiltration in LUAD, a total of 105 human LUAD samples were studied and IF stained. The LUAD tissues were stained with ILT3, CD163 (M2-like TAMs marker molecule) and CD68 (TAMs marker molecule) (fig. 2A-B). The timer2.0 database was searched for the association of ILT3 with immunoinfiltrating cells and found to be significantly positively correlated with TAMs of type M2 in LUAD (fig. 2C). Next, we studied the effect of ILT3 on TAMs recruitment and polarization in LUAD. From our previous studies, we selected H1650 and H1299 cells with low expression of endogenous ILT3, up-regulated ILT3 by transfection of ILT3 lentivirus, and down-regulated expression of ILT3 in a549 cells with high expression of endogenous ILT3 with ILT3 shRNA(shILT3-1:5'-GGGAGTACCGTTGGATAAAG-3',SEQ ID NO.1;shILT3-2:5'-GACAGGAGCCTACAGTAAACC-3',SEQ ID NO.2) (fig. 7). We also purified cd14+ monocytes from PBMCs of healthy volunteers and induced macrophages to produce TAMs using CM of a different tumor cell line expressing ILT 3. The migration ability of TAMs was evaluated by a Transwell test. Not shown, CM cultured TAMs of a549, H1299 and H1650 cells showed stronger migration ability than macrophages cultured in normal medium. Down-regulated portions of ILT3 in tumor cells prevented migration of TAMs, whereas overexpression of ILT3 promoted migration of TAMs (fig. 2D-E).
We collected CMs from tumor cells with different levels of ILT3 expression to detect changes in different CM-induced M2-like TAM markers (CD 163, CD206, CD209, IL-10 and arginase 1) and M1-like TAM markers (CD 80, CD86, IL-12 and TNF- α). We observed that ILT3 gene knockout in tumor cells decreased M2-like markers including CD163, CD206, CD209, IL-10 and arginase 1, but increased M1-like markers including CD80, CD86, IL-12 and TNF- α. However, when ILT3 was overexpressed, the changes in the M1-like and M2-like markers were opposite to ILT3 knockouts (fig. 2F-H). Taken together, these results indicate that ILT3 in the LUAD promotes aggregation of TAMs and M2-like polarization.
ILT3 directly affects proliferation and cytotoxicity of T cells in LUAD cells
Given that T cell immunity is the most important component of anti-tumor immunity, we assessed the infiltration of ILT 3-regulated T cells into TME by IF. In our patient cohort, we first demonstrated a correlation between ILT3 levels and T cell infiltration by co-staining ILT3, CD45 and pan-CK in serial tissue sections. Expression of ILT3 in tumor cells was inversely related to the density of cd3+ T cells (fig. 3A-B).
Next, we established a tumor-T cell co-culture system to assess the effect of ILT3 on T cell survival and cytotoxicity. We found that when T cells were co-cultured with a549 cells with down-regulated ILT3 expression, their proliferative capacity was significantly enhanced (fig. 3C-D), but apoptosis was significantly reduced (fig. 3E-F). When T cells were co-cultured with H1299 and H1650 cells with upregulated ILT3 expression, the changes in proliferation and apoptosis were opposite to a549 cells (fig. 3C-F). Tumor cells with different ILT3 expression levels were co-cultured with T cells, and the expression levels of perforin on T cells were examined, as a result, it was found that ILT3 inhibited the expression levels of perforin on T cells (FIG. 3G-H). These results indicate that ILT3 in tumor cells directly inhibits T cell survival and killing ability.
APOE-ILT3 interactions promote TAM recruitment and M2-like polarization
In our previous studies, ILT3 can up-regulate the expression level of endogenous APOE, thereby inducing expression of ILT3 in NSCLC, promoting migration and invasion of tumor cells. The interaction between ILT3 and APOE mediates infiltration of acute myeloid leukemia cells. Disruption of the ILT3/APOE interaction by potent humanized antibodies can reverse T cell inhibition and prevent AML progression. This result prompted us to determine whether the ApoE-ILT3 interaction on the LUAD is involved in recruitment and polarization of TAMs.
The association between them was confirmed in patients by IF staining. As a result, it was found that infiltration of CD68+ TAM and CD163+ TAM was significantly increased in patients with high expression of APOE (FIGS. 4A-B). Searching the timer2.0 database found that APOE correlated positively with infiltration of TAM type M2 in the LUAD tissue (fig. 4C). Analysis of the Kaplan-Meier plotter online database showed that the APOE high expression group LUAD patients had better OS (hr=1.27, 95% CI: 1.07-1.51) than the APOE low expression group (fig. 4D). Next, we studied whether there is a direct interplay between APOE and ILT 3. Our previous studies have shown that APOE promotes expression of ILT3 and demonstrate that APOE and ILT3 co-localize in the cell membranes and cytoplasm of H1299 and H1650 cells.
To determine whether APOE-ILT3 interactions would promote the recruitment of TAMs, the effect of rhaoe on TAMs recruitment was first examined. We found that rhApoE significantly enhanced the ability of ILT3 overexpressing tumor cells to recruit TAMs. Notably, APOE-induced TAMs recruitment was significantly reversed when ILT3 was blocked with specific neutralizing antibodies (fig. 4E-F). We also assessed the effect of APOE-ILT3 interaction on polarization of TAMs. Likewise, CM from rhepo e stimulated tumor cells increased the M2-type marker in TAMs, decreased the M1-type marker in TAMs, while ILT3 neutralizing antibody treatment almost completely restored the changes in the M1 and M2-type markers induced by rhepo e pretreatment (fig. 2G-H). Taken together, our findings indicate that APOE promotes TAMs recruitment and M2-type polarization in LUAD by binding ILT 3.
Gp49b induces an immunosuppressive T cell/TAM internal environment
To verify the regulation of TME by ILT3 in vivo, we established C57BL/6 and NSG mouse lung cancer plantation models using LLC cells. 2X 10 5 LLC cells transfected with gp49b over-expression or control lentiviruses were subcutaneously injected into C57BL/6 and NSG mice and the gp49b over-expression efficiency was analyzed (FIG. 8). When the tumor reached the limit size (longest diameter of 2 cm), the mice were euthanized in real time, and then the tumor, spleen and mouse peripheral blood were isolated for subsequent experiments. We found that in C57BL/6 mice, gp49B overexpression accelerated tumor growth compared to the control group (FIGS. 5A-B). The final tumor weights for each group showed similar results (fig. 5C). Gp49b overexpressing LLC cells (2X 10 5) were implanted subcutaneously (6/group) in NSG mice in order to exclude the effects of immune microenvironment. Although the tumor growth rate was higher in the over-expressed group than in the control group, the difference in tumor growth rate was smaller than in the C57BL/6 mice (FIGS. 5D-F). Immune cell subsets in tumor, spleen and peripheral blood were determined by flow cytometry analysis, gp49b overexpression increased the proportion of cd163+ and cd206+ TAMs in tumor, but decreased the proportion of cd80+ TAMs (fig. 5G). Gp49b overexpression decreased the proportion of cd3+, ifnγ+ and performin+ T cells in tumor, spleen and peripheral blood, but increased the proportion of foxp3+ T cells (fig. 5H-K).
To further explore the anti-tumor effect of inhibiting ILT3, a C57BL/6 mouse tumor treatment model was established with gp49b gene knockout LLC cells and gp49b knockout efficiency was determined (fig. 8). Gp49b gene knockout reduced the growth rate of tumor cells compared to the control group (FIG. 5L-N). Flow cytometry analysis showed that gp49B knockout increased the infiltration rate and killing capacity of T cells and decreased the proportion of M2-type TAMs (fig. 5O-S). These results indicate that ILT3 inhibits T cell infiltration ratio and killing ability, induces M2-like polarization of TAMs, promotes formation of immunosuppressive TMEs, and promotes tumor growth. ILT3 may be a promising target for lung cancer immunotherapy.
Gp49b and PD-L1 blockade can synergistically prevent tumor growth and immune escape in vivo
Our studies indicate that ILT3 results in infiltration of M2-like TAMs and low reactivity of T cells. We next examined whether the combined blockade of ILT3 and PD-L1 has a synergistic effect on tumorigenesis, M2-like TAM infiltration, and T cell dysfunction. We subcutaneously injected 2X 10 5 gp49b down-regulated LLC cells into wild-type C57BL/6 mice. After 7 days, tumor mice were intraperitoneally injected with anti-PD-L1 (200 ug/4 days) or control IgG every 4 days and tumor sizes were measured. FIG. 6A shows that gp49b gene knockdown (shgp b: CAATGGAACATTCAGATGCTATCAAGAGTAGCATCTGAATGTTCCATTG,
Both SEQ ID NO. 3) and PD-L1 blockade slowed tumor growth, whereas the combined blockade of both molecules had a synergistic effect on tumor inhibition. These results were confirmed by measuring tumor size and tumor weight (FIGS. 6B-C). Next, we determined whether gp49b and PD-L1 blockade would remodel the infiltration and function of TAMs and T cells. Immune cells in mouse tumor tissue, spleen and blood were isolated and analyzed for the number, phenotype and subpopulation of TAMs and T cells using flow cytometry. We examined CD206/CD163/CD80 positive cells in tumor tissue and analyzed the M2-like phenotype of TAM. Inhibition of gp49b or PD-L1 was found to reduce the proportion of CD206+ and CD163+ TAMs in tumor tissue, but to increase the proportion of CD80+ TAMs, the change in the combined blocking group was most pronounced (FIG. 6D). In addition to the effect on TAM polarization, T cells were also inhibited, blocking gp49b or PD-L1 in tumors partially restored T cell infiltration, with the combined blocking effect being best (fig. 6E). Taken together, these results indicate that gp49b and PD-L1 blockade synergistically normalize immunosuppressive TMEs, prevent tumor growth and immune escape in vivo, and thus make the combined use of ILT3 and PD-L1 blockade in lung cancer therapy more rational.
The above embodiments are provided to illustrate the technical concept and features of the present invention and are intended to enable those skilled in the art to understand the content of the present invention and implement the same, and are not intended to limit the scope of the present invention. All equivalent changes or modifications made in accordance with the spirit of the present invention should be construed to be included in the scope of the present invention.
Claims (10)
1. The application of a reagent for detecting ILT3 coding genes and expression products thereof in preparing lung adenocarcinoma prognosis evaluation products.
2. The use of claim 1, wherein the prognostic evaluation comprises an evaluation of total survival of a lung adenocarcinoma patient.
3. Use of a substance that inhibits the ILT3 gene and its expression products and/or reduces its activity in any one or more of the following a 1) -a 8):
a1 Inhibition of TAM recruitment and TAM M2-like polarization or preparation of products that inhibit TAM recruitment and TAM M2-like polarization;
a2 Inhibit migration of TAMs or preparing a product that inhibits migration of TAMs;
a3 Promoting T cell proliferation and cytotoxicity or preparing a product that promotes T cell proliferation and cytotoxicity;
a4 A product that inhibits or prepares the formation of immunosuppressive TMEs;
a5 Inhibit tumor growth or preparing a product that inhibits tumor growth;
a6 Inhibiting tumor immune escape or preparing a product for inhibiting tumor immune escape;
a7 Inhibiting tumor resistance to an immune checkpoint inhibitor (enhancing immune checkpoint inhibitor anti-tumor activity) or preparing a product that inhibits tumor resistance to an immune checkpoint inhibitor (enhancing immune checkpoint inhibitor anti-tumor activity);
a8 A product for treating tumor.
4. The use according to claim 3, wherein the product is a pharmaceutical or experimental agent.
5. The use of claim 3, wherein the tumor is lung adenocarcinoma and the tumor cells are lung adenocarcinoma cells.
6. The application of claim 3, wherein the application a 1) is: use of a substance that inhibits ILT3 gene and its expression products and/or reduces its activity in the preparation of a product for inhibiting TAM recruitment and TAMs M2-like polarization in a lung adenocarcinoma tumor cell-macrophage co-culture system;
Or, the substances inhibiting ILT3 gene and its expression products and/or activity reduction include RNA interference molecules or antisense oligonucleotides, small molecule inhibitors, shRNA, siRNA of ILT3, substances for carrying out lentiviral infection or gene knockout and specific antibodies against ILT3 itself or upstream and downstream molecules thereof, including ILT3 neutralizing antibodies;
or, the immune checkpoint inhibitor comprises a PD-1/PD-L1 inhibitor.
7. A composition comprising, as active ingredients, at least an agent that inhibits and/or reduces the activity of the ILT3 gene and its expression products and an immune checkpoint inhibitor.
8. The composition of claim 7, wherein the agent that inhibits the decrease in the ILT3 gene and its expression products and/or activity comprises an RNA interference molecule or antisense oligonucleotide, a small molecule inhibitor, shRNA, siRNA against ILT3, an agent that performs lentiviral infection or gene knockout, and a specific antibody against ILT3 itself or a molecule upstream and downstream thereof, including an ILT3 neutralizing antibody;
The immune checkpoint inhibitor comprises a PD-1/PD-L1 inhibitor.
9. Use of a composition according to claim 7 or 8 in any one or more of the following:
b1 Preparing a product that normalizes immunosuppressive TME;
b2 Preparing a product for inhibiting tumor growth;
b3 Preparing a product for inhibiting tumor immune escape;
b4 A product for treating tumor.
10. The use of claim 9, wherein the tumor is a solid tumor, further lung cancer, further lung adenocarcinoma;
The product is a drug or an experimental reagent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410261076.3A CN118272527A (en) | 2024-03-07 | 2024-03-07 | Application of tumor-derived ILT3 in prognosis and treatment of lung adenocarcinoma |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410261076.3A CN118272527A (en) | 2024-03-07 | 2024-03-07 | Application of tumor-derived ILT3 in prognosis and treatment of lung adenocarcinoma |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN118272527A true CN118272527A (en) | 2024-07-02 |
Family
ID=91633215
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410261076.3A Pending CN118272527A (en) | 2024-03-07 | 2024-03-07 | Application of tumor-derived ILT3 in prognosis and treatment of lung adenocarcinoma |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN118272527A (en) |
-
2024
- 2024-03-07 CN CN202410261076.3A patent/CN118272527A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR102081567B1 (en) | Biomarkers and combination therapies using oncolytic virus and immunomodulation | |
| EP3374497B1 (en) | Modified macrophages for use in the treatment of cancer | |
| EP3411063B1 (en) | Methods and pharmaceutical compositions for enhancing cd8+ t cell-dependent immune responses in subjects suffering from cancer | |
| EP4069284A1 (en) | Tumor cell vaccines | |
| WO2019083971A1 (en) | Methods of treating cancer using lsd1 inhibitors in combination with immunotherapy | |
| US20110184045A1 (en) | Silencng and rig-i activation by dual function oligonucleotides | |
| US20220155303A1 (en) | Use of tctp as biomarker for predicting efficacy, prognosis of immunotherapy or resistance thereto, and target of immunotherapy for enhancing efficacy | |
| WO2020146345A1 (en) | Methods of treating cancer using lsd1 inhibitors and/or tgf-beta inhibitors in combination with immunotherapy | |
| US20210386780A1 (en) | Methods for treating cancer with double stranded rna sensor activators and adoptive cell therapy | |
| JP2023082128A (en) | Methods of treating neoplastic diseases | |
| US20230000963A1 (en) | Tumor cell vaccines | |
| CN118272527A (en) | Application of tumor-derived ILT3 in prognosis and treatment of lung adenocarcinoma | |
| CN116474095A (en) | Application of inhibitor of small G protein RBJ | |
| US20220016205A1 (en) | Methods of overcoming resistance to immune checkpoint inhibitors | |
| US20210396737A1 (en) | Nanoplexed poly(i:c) formulations and uses thereof | |
| Kong et al. | CDYL knockdown reduces glioma development through an antitumor immune response in the tumor microenvironment | |
| US20210169913A1 (en) | Fcrl6 and its uses related to cancer | |
| CN106554962B (en) | Prevention, diagnosis and treatment of GPR160 overexpressing cancers | |
| US20230340127A1 (en) | Methods and compositions for cancer treatment by inhibition of fbxo44 | |
| US20240252533A1 (en) | Methods and compositions for pancreatic cancer evaluation and treatment | |
| CN112294968B (en) | Application of ILT4 in EGFR activated non-small cell lung cancer treatment | |
| US20230250433A1 (en) | Methods and compositions for treatment of apc-deficient cancer | |
| Nakata et al. | Autophagy inhibition in pancreatic cancer cells synergizes with immunotherapy via DC activation due to increased antigenicity and adjuvanticity | |
| Lin et al. | Targeting ZDHHC9 enhances anti-PD-L1 immunotherapy efficacy by reprogramming the tumour microenvironment | |
| JP2023539436A (en) | Methods and compositions for the evaluation and treatment of pancreatic cancer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination |