Ama-immunomodulatory metabolites ayisici esiqakathekileko se-tumor microenvironment (TME), kodwana ngaphandle kokumbalwa, ubunikazi bawo buhlala bungaziwa. Lapha, sihlaziye amathumba namaseli we-T avela emathunjini nama-ascites wezigulani ezine-high-grade serous carcinoma (HGSC) ukuveza i-metabolome yamagumbi we-TME ahlukileko. I-ascites namaseli wesimila anomehluko omkhulu we-metabolite. Nayiqathaniswa ne-ascites, amaseli we-T angena nge-tumor anothiswe khulu nge-1-methylnicotinamide (MNA). Nanyana izinga le-MNA kumaseli we-T liphakeme, ukuvezwa kwe-nicotinamide N-methyltransferase (i-enzayimu ekhuthaza ukudluliselwa kwamaqembu we-methyl ukusuka ku-S-adenosylmethionine ukuya ku-nicotinamide) kukhawulelwe kuma-fibroblasts namaseli we-tumor. Ngokusebenza, i-MNA ikhuthaza amaseli we-T bona akhiphe i-cytokine ekhuthaza i-tumor tumor necrosis factor alpha. Ngalokho-ke, i-MNA evela ku-TME ifaka isandla ekulawuleni amasotja womzimba wamaseli we-T begodu ijamele umnqopho ongaba khona we-immunotherapy yokwelapha umdlavuza wabantu.
Ama-metabolites avela esimila angaba nomthelela omkhulu wokuvimbela ukuzivikela kwe-anti-tumor, begodu ubufakazi obunengi butjengisa bona angaba mtjhini oqakathekileko wokuragela phambili kwesifo (1). Ngaphezu komphumela we-Warburg, umsebenzi wakamuva sele uqalile ukuhlathulula ubujamo bokugaya ukudla kwamaseli wesimila kanye nobudlelwano bawo nobujamo bamasotja womzimba we-tumor microenvironment (TME). Iimfundo eziphathelene namamodeli wegundane namaseli we-T wabantu zitjengise bona ukusebenza kwe-glutamine (2), ukusebenza kwe-oxidative (3) kanye nokusebenza kwe-glucose (4) kungasebenza ngokuzijamela emaqenjini ahlukahlukeneko wamaseli wamasotja womzimba. Ama-metabolites amanengi eendleleni lezi avimbela umsebenzi wokulwisana nesimila wamaseli we-T. Kufakazelwe bona ukuvinjwa kwe-coenzyme tetrahydrobiopterin (BH4) kungalimaza ukwanda kwamaseli we-T, begodu ukwanda kwe-BH4 emzimbeni kungakhuphula ukuphendula kwamasotja womzimba okulwa ne-tumor okulawulwa yi-CD4 ne-CD8. Ukungezelela, umphumela wokuvikela amasotja womzimba we-kynurenine ungasindiswa ngokuphathwa kwe-BH4 (5). Ku-isocitrate dehydrogenase (IDH) mutant glioblastoma, ukukhiqizwa kwe-enantiometabolic (R)-2-hydroxyglutarate (R-2-HG) kuvimbela ukusebenza kwamaseli we-T, ukwanda kanye nomsebenzi we-cytolysis (6). Mhlapha nje, kutjengiswe bona i-methylglyoxal, umkhiqizo we-glycolysis, ukhiqizwa maseli we-suppressor we-myeloid origin, begodu ukudluliselwa kwe-T cell kwe-methylglyoxal kungavimbela ukusebenza kwe-T cell. Ekwelapheni, ukuqedwa kwe-methylglyoxal kungahlula umsebenzi wamaseli we-myeloid-derived suppressor (MDSC) begodu kuthuthukise ngokubambisana ukwelatjhwa kokuvala i-checkpoint kumamodeli wegundane (7). Iimfundo lezi ngokuhlanganyela zigandelela indima eqakathekileko yama-metabolites avela ku-TME ekulawuleni ukusebenza kwamaseli we-T.
Ukungasebenzi kuhle kwamaseli we-T kubikwe khulu emdlavuzeni wesibeletho (8). Lokhu kungenxa yezimpawu zokugaya ukudla ezikhona ekutlhogekeni kwe-oksijini kanye nemithambo engakajayeleki ye-tumor (9), okurholela ekuguqulweni kwe-glucose ne-tryptophan zibe ziinsetjenziswa ezifana ne-lactic acid ne-kynurenine. I-lactate engaphezu kwamaseli yehlisa ukukhiqizwa kwe-interferon-γ (IFN-γ) begodu yenza ukuhlukaniswa kwama-subgroups we-myelosuppressive (10, 11). Ukusetjenziswa kwe-tryptophan kuvimbela ngqo ukwanda kwamaseli we-T begodu kuvimbele ukuthunyelwa kwe-T cell receptor (12-14). Nanyana kunjalo, umsebenzi omnengi ophathelene nokusebenza kwamasotja womzimba wenziwa nge-in vitro T cell culture kusetjenziswa imidiya ehlelekileko, nofana ilinganiselwe kumamodeli wegundane afanako in vivo, okungekho nanye kizo etjengisa ngokuzeleko ukuhlukahluka kwekankere yabantu kanye nebhoduluko elikhulu nelincani le-Physiological.
Isici esivamileko somdlavuza we-ovari kukurhatjheka kwe-peritoneal nokuvela kwe-ascites. Ukubuthelela uketshezi lwamaseli ku-ascites kuhlotjaniswa nesifo esithuthukileko kanye nokubikezela okumbi (15). Ngokuya kwemibiko, indawo le ehlukileko ine-hypoxian, inamazinga aphezulu we-vascular endothelial growth factor (VEGF) kanye ne-indoleamine 2,3-dioxygenase (IDO), begodu ingena ngamaseli we-T regulatory kanye namaseli wokuvimbela i-myeloid (15-18). Indawo yokugaya ukudla ye-ascites ingahluka kweyesimila ngokwayo, ngalokho ukuhlelwa kabutjha kwamaseli we-T esikhaleni se-peritoneal akucaci. Ukungezelela, umehluko oqakathekileko nokungafani hlangana nama-ascites nama-metabolites akhona endaweni yesimila kungavimbela ukungena kwamaseli wamasotja womzimba nomsebenzi wawo emathunjini, begodu irhubhululo elinabileko liyatlhogeka.
Ukurarulula imiraro le, saklama indlela yokuhlukaniswa kwamaseli kanye ne-liquid chromatography tandem mass spectrometry (LC-MS/MS) ukufunda imihlobo ehlukileko yamaseli (okufaka hlangana amaseli we-CD4 + kanye ne-CD8 + T) kanye nangaphakathi naphakathi kwamathumba. Sisebenzisa indlela le ngokubambisana ne-high-dimensional flow cytometry kanye ne-single-cell RNA sequencing (scRNA-seq) ukunikela isithombe esihle khulu sesimo sokugaya ukudla sabantu abaqakathekileko. Indlela le iveze ukwanda okukhulu kwezinga le-1-methylnicotinamide (MNA) kumaseli we-T we-tumor, begodu ukuhlolwa kwe-in vitro kutjengise bona umphumela wokuvikela umzimba we-MNA emsebenzini wamaseli we-T bewungaziwa ngaphambilini. Ngokuvamileko, indlela le iveza ukusebenzisana kwe-metabolic hlangana namathumba namaseli wamasotja womzimba, begodu inikela ukuzwisisa okuhlukileko ngama-metabolites wokulawula amasotja womzimba, okungaba lisizo ekwelapheni amathuba wokwelapha umdlavuza wesibeletho osekelwe kumaseli we-T.
Sisebenzise i-high-dimensional flow cytometry ukulinganisa ukuthathwa kwe-glucose ngesikhathi sinye [2-(N-(7-nitrophenyl-2-oxa-1,3-diaza-4-yl)amino)-2-deoxyglucose (2-NBDG) kanye nomsebenzi we-mitochondrial [MitoTracker Deep Red (MT DR)] (2017, 2019) ziseceleni amatshwayo ajayelekileko ahlukanisa amaseli wamasotja womzimba kanye namaseli wesimila (Ithebula S2 kanye nomfanekiso S1A). Ukuhlaziywa lokhu kutjengise bona nakuqathaniswa namaseli we-T, amaseli we-ascites namaseli we-tumor anamazinga aphezulu wokuthatha i-glucose, kodwana anomehluko omncani emsebenzini we-mitochondrial. Ukuthathwa kwe-glucose okulinganiselweko kwamaseli we-tumor [CD45-EpCAM (EpCAM)+] kuphindwe kathathu ukuya kane kwamaseli we-T, begodu ukuthathwa kwe-glucose okuphakathi kwamaseli we-CD4 + T kuphindwe ka-1.2 kwamaseli we-CD8 + T, okutjengisa bona ama-lymphocyte angenako we-Tumor (TIL) aneemfuneko ezihlukileko zokugaya ukudla ngitjho naku-TME efanako (Umfanekiso 1). Ngokuphikisako, umsebenzi we-mitochondrial kumaseli we-tumor uyafana nowe-CD4 + T cells, begodu umsebenzi we-mitochondrial wemihlobo yomibili yamaseli uphezulu kunalowo we-CD8 + T cells (Umfanekiso 1B). Ngokuvamileko, imiphumela le iveza izinga lokugaya ukudla. Umsebenzi wokugaya ukudla wamaseli we-tumor uphezulu kunalawo wamaseli we-CD4 + T, begodu umsebenzi wokugaya ukudla wamaseli we-CD4 + T uphezulu kunalawo wamaseli we-CD8 + T. Nanyana kunemiphumela le kiyo yoke imihlobo yamaseli, akukho mehluko ongaguqukiko ebujameni bokugaya ukudla kwamaseli we-CD4 + kanye ne-CD8 + T nofana ukulinganisa kwawo okuhlobeneko ku-ascites nayiqathaniswa namathumba (Umfanekiso 1C). Ngokuphikisako, engcenyeni yamaseli we-CD45, inani lamaseli we-EpCAM+ esimila lakhula nayiqathaniswa ne-ascites (Umfanekiso 1D). Sibuye sabona umehluko ocacileko wokugaya ukudla hlangana namalunga we-EpCAM+ kanye ne-EpCAM-. Amaseli we-EpCAM+ (isimila) ane-glucose uptake ephezulu kanye nomsebenzi we-mitochondrial ukudlula amaseli we-EpCAM-, okuphezulu khulu kunomsebenzi we-metabolic wama-fibroblasts kumaseli we-tumor ku-TME (Umfanekiso 1, E no-F).
(A no-B) Ukukhanya okuphakathi (MFI) kokuthathwa kwe-glucose (2-NBDG) (A) nomsebenzi we-mitochondrial wamaseli we-CD4 + T (i-MitoTracker ebomvu khulu) (B) Amagrafu ajameleko (ngesinceleni) kanye nedatha ethebuliweko (ngesidle), amaseli we-CD8 + T kanye namaseli we-EpCAM + CD45 we-tumor kanye namaseli we-tumor njengama-tumor cites. (C) Isilinganiso samaseli we-CD4 + kanye ne-CD8 + (wamaseli we-CD3 + T) ku-ascites kanye nethumba. (D) Inani lamaseli we-EpCAM + we-tumor ku-ascites kanye ne-tumor (CD45−). (E no-F) I-EpCAM + CD45-tumor kanye ne-EpCAM-CD45-matrix glucose uptake (2-NBDG) (E) kanye nomsebenzi we-mitochondrial (i-MitoTracker ebomvu khulu) (F) amagrafu ajameleko (kwesokunxele) kanye nedatha ethebuliweko (Ngesokudla) Ama-ascites namaseli we-tumor. (G) Amagrafu ajameleko we-CD25, CD137 kanye ne-PD1 ukuvezwa nge-flow cytometry. (H no-I) ukuvezwa kwe-CD25, CD137 kanye ne-PD1 kumaseli we-CD4 + T (H) kanye namaseli we-CD8 + T (I). (J no-K) Ama-phenotypes we-Naive, central memory (Tcm), effector (Teff) kanye ne-effector memory (Tem) ngokuya ngokuvezwa kwe-CCR7 ne-CD45RO. Iinthombe ezijameleko (ngesinceleni) kanye nedatha yethebula (kwesokudla) yamaseli we-CD4 + T (J) kanye namaseli we-CD8 + T (K) kuma-ascites namathumba. Amanani we-P atholakala nge-t-test ehlangeneko (*P<0.05, **P<0.01 kanye ne-***P<0.001). Umuda ujamele iingulani ezifanako (n = 6). I-FMO, ukukhanya kwe-fluorescence ukususa eyodwa; I-MFI, ukuqina kokukhanya okuphakathi.
Ukuhlaziywa okungeziweko kwaveza eminye imihluko eqakathekileko hlangana nobujamo be-T cell obuhle khulu. Iinkumbulo ezisebenzako (Umfanekiso 1, G ukuya ku-I) kanye ne-effector inkumbulo (Umfanekiso 1, J no-K) emathunjini zivame khulu ukudlula i-ascites (isilinganiso samaseli we-CD3 + T). Ngokufanako, ukuhlaziya i-phenotype ngokuvezwa kwamatshwayo wokusebenza (CD25 kanye ne-CD137) kanye namatshwayo wokuphelelwa [amaphrotheyini wokufa kwamaseli ahlelelwe 1 (PD1)] kutjengise bona nanyana ubujamo bokugaya ukudla bemihlobo le buhlukile (Umfanekiso S1, B ukuya ku-E), Kodwana akukho mehluko obonakalako wokugaya ukudla owabonwa ngokungaguqukiko hlangana nemiphumela yememori engakajayeleki, namkha isethi encani (Umfanekiso S1, F1 MINA). Imiphumela le yaqinisekiswa ngokusebenzisa iindlela zokufunda ngomtjhini ukwabela ngokuzenzakalelayo ama-phenotypes wamaseli (21), okubuye kwaveza ukuba khona kwenani elikhulu lamaseli womnkantsha wethambo (CD45 + / CD3- / CD4 + / CD45RO +) ku-ascites yesiguli (Umfanekiso S2A ). Hlangana nayo yoke imihlobo yamaseli akhonjiweko, inani lamaseli we-myeloid litjengise ukuthathwa kwe-glucose okuphezulu khulu nomsebenzi we-mitochondrial (Umfanekiso S2, B ukuya ku-G). Imiphumela le iveza umehluko oqinileko wokugaya ukudla hlangana nemihlobo eminengi yamaseli atholakala kuma-ascites kanye namathumba eengulini ze-HGSC.
Inselele ekulu ekuzwisiseni ubujamo be-TIL kusidingo sokuhlukanisa amasampula wamaseli we-T wobumsulwa, ikhwalithi nobunengi obulingeneko emathunjini. Iimfundo zamva nje zitjengise bona iindlela zokuhlunga nokunothisa ubuhlalu ezisekelwe ku-flow cytometry zingadosela ekutjhugulukeni kwamaphrofayili we-metabolite yamaseli (22-24). Ukuze sinqobe umraro lo, senze ngcono indlela yokunothisa ubuhlalu ukuhlukanisa nokuhlukanisa i-TIL emdlavuzeni wesibeletho womuntu okhishwe ngokuhlinzwa ngaphambi kokuhlaziywa nge-LC-MS / MS (bona izinto nezindlela; Umfanekiso 2A). Ukuze sihlole umthelela opheleleko wale phrothokholi ematjhugulukweni we-metabolite, simadanise amaphrofayili we-metabolite wamaseli we-T asebenza ngabanikeli abaphilileko ngemva kwesinyathelo sokuhlukaniswa kwama-bead angenhla namaseli ebekangahlukaniswanga kodwana ahlala eqhweni. Ukuhlaziywa kokulawula ikhwalithi kuthole bona kunokuhlobana okuphezulu hlangana nobujamo obubili (r = 0.77), begodu ukuphindaphindeka kwezobuchwepheshe kweqembu lama-metabolites angama-86 kunokuphindaphindwa okuphezulu (Umfanekiso 2B). Ngalokho-ke, iindlela lezi zingenza ukuhlaziywa okunembileko kwe-metabolite emaseli enza ukunothiswa kwemihlobo yamaseli, ngaleyo ndlela kunikelwe iplatifomu yokuthoma ephezulu yokukhomba ama-metabolite athileko ku-HGSC, ngaleyindlela kwenza abantu bakwazi ukuzwisisa okudephileko kwehlelo lokukhetha amaseli.
(A) Umdwebo wesikimu wokunothisa ubuhlalu bamagnethi. Ngaphambi kokuhlaziywa nge-LC-MS/MS, amaseli azokudlula emizuliswaneni emithathu elandelanako yokucebisa ubuhlalu bamagnethi nofana ahlale eqhweni. (B) Umphumela womhlobo wokunothisa ebunengini bama-metabolites. Isilinganiso seenlinganiso ezintathu zohlobo ngalunye lokunothisa ± SE. Umugqa ompunga umele ubuhlobo obungu-1:1. Ukuhlobana kwangaphakathi kweklasi (ICC) kweenlinganiso eziphindaphindiweko ezitjengiswe elebulini ye-eksisi. I-NAD, i-nicotinamide adenine dinucleotide. (C) Umdwebo wesikimu wokugeleza komsebenzi wokuhlaziywa kwe-metabolite yesiguli. I-ascites nofana iimvukuzane zibuthelelwa eengulini bese zibulungwa. Ingcenye encani yesampula ngayinye yahlaziywa nge-flow cytometry, lokha amasampula aseleko enza imijikelezo emithathu yokucebisa amaseli we-CD4+, CD8+ kanye ne-CD45-. Amafraktjhini wamaseli la ahlaziywa kusetjenziswa i-LC-MS/MS. (D) Imephu yokutjhisa yobunengi be-metabolite ejwayelekileko. I-dendrogram ijamele ukuhlanganiswa kwaka-Ward kwamabanga we-Euclidean hlangana namasampula. (E) Ukuhlaziywa kwengcenye ekulu (i-PCA) yemephu yesampula ye-metabolite, etjengisa ukuphindaphindwa kwesampula ngayinye, amasampula avela esigulini esifanako ahlanganiswe ngomugqa. (F) I-PCA yephrofayili ye-metabolite yesampula ebekwe esigulini (okutjho, ukusebenzisa ukungabi khona okuyingcenye); umhlobo wesampula ulinganiselwe yi-hull evulekileko. I-PC1, ingcenye ekulu 1; I-PC2, ingcenye ekulu 2.
Okulandelako, sisebenzise indlela yokunothisa ukuhlaziya ama-metabolites ama-99 ku-CD4 +, CD8 + kanye namaseli we-CD45 kuma-ascites aphambili kanye namathumba wezigulani ezisithandathu ze-HGSC (Umfanekiso 2C, Umfanekiso S3A kanye neThebula S3 no-S4). Inani eliqakathekileko libalelwa ku-2% ukuya ku-70% wesampula ekulu yokuthoma yamaseli aphilako, begodu inani lamaseli lihluka khulu hlangana nezigulani. Ngemva kokuhlukanisa ubuhlalu, ingcenye ecebileko (i-CD4+, i-CD8+ nofana i-CD45-) ibalelwa ngaphezu kwama-85% wawo woke amaseli aphilako esampula ngokwesilinganiso. Indlela yokunothisa le isivumela bona sihlaziye inani lamaseli avela ekusebenzeni kwezicubu zethumba lomuntu, okuyinto engenakwenzeka ukuyenza emasampula amakhulu. Ngokusebenzisa iphrothokholi le, sathola bona i-l-kynurenine ne-adenosine, ama-metabolites amabili la aphakanyisiweko we-immunosuppressive gade aphakanyisiwe kumaseli we-T we-tumor nofana kumaseli we-tumor (Umfanekiso S3, B no-C). Ngalokho-ke, imiphumela le itjengisa ukuthembeka nekghono lokuhlukaniswa kwamaseli kanye nethekhnoloji ye-mass spectrometry ukuthola ama-metabolites aqakathekileko wezinto eziphilako eencutjhweni zesigulani.
Ukuhlaziywa kwethu kwaveza nokuhlukaniswa okuqinileko kwemihlobo yamaseli ngaphakathi naphakathi kwezigulani (Umfanekiso 2D noMfanekiso S4A). Khulukhulu, nayiqathaniswa nezinye iingulani, isigulani 70 satjengisa ubujamo obuhlukileko bokugaya ukudla (Umfanekiso 2E noMfanekiso S4B), okutjengisa bona kungaba nokuhlukahluka okukhulu kokugaya ukudla hlangana nezigulani. Kuqakathekile ukutjheja bona nayiqathaniswa nezinye iingulani (1.2 ukuya ku-2 yamalitha; Ithebula S1), inani elipheleleko lama-ascites abuthelelwe esigulini 70 (80 ml) belilincani. Ukulawulwa kokungafani hlangana nezigulani ngesikhathi sokuhlaziywa kwengcenye ekulu (ngokwesibonelo, ukusebenzisa ukuhlaziywa kokungabi khona okuyingcenye) kutjengisa amatjhuguluko angaguqukiko hlangana nemihlobo yamaseli, begodu imihlobo yamaseli kanye/nofana ibhoduluko elincani lihlanganiswe ngokucacileko ngokuya ngephrofayili ye-metabolite (Umfanekiso 2F ). Ukuhlaziywa kwama-metabolites ahlukileko kwagandelela imiphumela le begodu kwaveza umehluko oqakathekileko hlangana nemihlobo yamaseli kanye nendawo encani. Kuqakathekile ukutjheja bona umehluko omkhulu khulu obonwako yi-MNA, evamise ukunothiswa kumaseli we-CD45- kanye namaseli we-CD4+ kanye ne-CD8+ angena esimila (Umfanekiso 3A). Emaseli we-CD4 +, umphumela lo ubonakala khulu, begodu i-MNA kumaseli we-CD8 + nayo ibonakala ithinteka khulu yibhoduluko. Kodwana, lokhu akukaqakatheki, ngombana ziingulani ezintathu kwezisithandathu ezingahlolwa ukuthola amaphuzu we-CD8+ we-tumor. Ngaphezu kwe-MNA, emihlobeni ehlukileko yamaseli we-ascites kanye namathumba, amanye ama-metabolites angabonakali kuhle ku-TIL nawo anothileko ngokuhlukileko (Imifanekiso S3 no-S4). Ngalokho-ke, idatha le iveza isethi ethembisako yama-immunomodulatory metabolites ukwenzela irhubhululo elinabileko.
(A) Okuqukethwe okujayelekileko kwe-MNA ku-CD4+, CD8+ kanye namaseli we-CD45- avela ku-ascites kanye nethumba. Iplothi yebhokisi itjengisa ibanga eliphakathi (umugqa), ububanzi be-interquartile (i-hinge yefreyimu) kanye nebanga ledatha, ukuya ku-1.5 yebanga le-interquartile (ifreyimu ye-whisker). Njengombana kuchaziwe ku-Patient Materials and Methods, sebenzisa inani le-limma lesigulani ukuthola inani le-P (*P<0.05 kanye ne-**P<0.01). (B) Umdwebo we-MNA (60). Ama-metabolites: S-adenosyl-1-methionine; I-SAH, i-S-adenosine-1-homocysteine; NA, i-nicotinamide; I-MNA, 1-methylnicotinamide; 2-PY, 1-methyl- 2-pyridone-5-carboxamide; 4-PY, 1-methyl-4-pyridone-5-carboxamide; NR, i-nicotinamide ribose; I-NMN, i-nicotinamide mononucleotide. Ama-enzayimu (aluhlaza): NNMT, nicotinamide N-methyltransferase; SIRT, ama-sirtuins; I-NAMPT, i-nicotinamide phosphoribosyl transferase; AOX1, i-aldehyde oxidase 1; NRK, nicotinamide riboside kinase; I-NMNAT, i-nicotinamide mono Nucleotide adenylate transferase; I-Pnp1, i-purine nucleoside phosphorylase. (C) t-SNE ye-scRNA-seq ye-ascites (grey) kanye ne-tumor (ebomvu; n = 3 iziguli). (D) Ukuvezwa kwe-NNMT emaseli ahlukileko akhonjwe ngokusebenzisa i-scRNA-seq. (E) Ukuvezwa kwe-NNMT ne-AOX1 ku-SK-OV-3, izinso zomntwana (HEK) 293T, amaseli we-T kanye namaseli we-T aphathwe nge-MNA. Ukuvezwa okugoqiweko kutjengiswa ngokuhlobene ne-SK-OV-3. Iphetheni yokuvezwa nge-SEM itjengiswa (n = 6 abanikeli abaphilileko). Amanani we-Ct angaphezu kwama-35 athathwa njengalawo angabonakaliko (UD). (F) Ukuvezwa kwe-SLC22A1 ne-SLC22A2 ku-SK-OV-3, HEK293T, amaseli we-T kanye namaseli we-T aphathwe nge-8mM MNA. Ukuvezwa okugoqiweko kutjengiswa ngokuhlobene ne-SK-OV-3. Iphetheni yokuvezwa nge-SEM itjengiswa (n = 6 abanikeli abaphilileko). Amanani we-Ct angaphezu kwama-35 athathwa njengalawo angabonakaliko (UD). (G) Okuqukethwe kwe-MNA yamaseli kumaseli we-T wokunikela aphilileko ngemva kwama-awa ama-72 wokufukamela nge-MNA. Iphetheni yokuvezwa nge-SEM itjengiswa (n = 4 abanikeli abaphilileko).
I-MNA yenziwa ngokudlulisa isiqhema se-methyl ukusuka ku-S-adenosyl-1-methionine (SAM) ukuya ku-nicotinamide (NA) nge-nicotinamide N-methyltransferase (NNMT; Umfanekiso 3B). I-NNMT ivezwa khulu emihlobeni ehlukahlukeneko yomdlavuza wabantu begodu ihlotjaniswa nokwanda, ukuhlasela kanye nokusabalala (25-27). Ukuze sizwisise ngcono umthombo we-MNA kumaseli we-T ku-TME, sisebenzise i-scRNA-seq ukuveza ukuvezwa kwe-NNMT kiyo yoke imihlobo yamaseli kuma-ascites kanye namathumba wezigulani ezintathu ze-HGSC (Ithebula S5). Ukuhlaziywa kwamaseli angaba ma-6,500 kutjengise bona eendaweni ezine-ascites kanye nezimila, ukuvezwa kwe-NNMT bekukhawulelwe kuma-fibroblast kanye namaseli wethumba (Umfanekiso 3, C no-D). Kuqakathekile ukutjheja bona akukho ukuvezwa okusobala kwe-NNMT kinoma ngiliphi isiqhema esiveza i-PTPRC (CD45 +) (Umfanekiso 3D noMfanekiso S5A), okutjengisa bona i-MNA etholakele ku-metabolite spectrum ingeniswe kumaseli we-T. Ukuvezwa kwe-aldehyde oxidase 1 (AOX1) kutjhugulula i-MNA ibe yi-1-methyl-2-pyridone-5-carboxamide (2-PYR) nofana i-1-methyl-4-pyridone-5-carboxamide (4-PYR); Umfanekiso 3B) nawo ukhawulelwe esibalweni sama-fibroblasts aveza i-COL1A1 (Umfanekiso S5A), okutjengisa bona amaseli we-T awanakghono lokusebenza kwe-MNA. Iphetheni yokuvezwa kwamajini la ahlobene ne-MNA yaqinisekiswa kusetjenziswa isethi yedatha yamaseli yesibili ezijameleko evela ku-ascites evela eengulini ze-HGSC (Umfanekiso S5B; n = 6) (16). Ukungezelela, ukuhlaziywa kwe-quantitative polymerase chain reaction (qPCR) yamaseli we-T we-donor aphilileko aphathwe nge-MNA kutjengise bona nayiqathaniswa namaseli we-SK-OV-3 we-ovarian tumor, i-NNMT nofana i-AOX1 beyingakavezwa (Umfanekiso 3E). Imiphumela le engakalindelwa itjengisa bona i-MNA ingakhishwa ema-fibroblasts nofana emathunjini iye kumaseli we-T aseduze ku-TME.
Nanyana abakhethiweko bafaka hlangana umndeni wabathuthi be-organic cation 1 ukuya ku-3 (OCT1, OCT2 kanye ne-OCT3) abakhodiwe mthwali oncibilikako 22 (SLC22) umndeni (SLC22A1, SLC22A2 kanye ne-SLC22A3), abathuthi abangaba khona be-MNA abakachazwa (28) . I-QPCR ye-mRNA evela kumaseli we-T anikelako aphilileko itjengise amazinga aphasi wokuvezwa kwe-SLC22A1 kodwana amazinga angabonakaliko we-SLC22A2, okuqinisekisa bona bekubikwe ngaphambilini emitlolweni (Umfanekiso 3F) (29). Ngokuphikisako, i-SK-OV-3 ye-ovarian tumor cell line iveze amazinga aphezulu womabili abathuthi (Umfanekiso 3F).
Ukuhlola amathuba wokuthi amaseli we-T angaba nekghono lokumunya i-MNA yangaphandle, amaseli we-T anikelako aphilileko akhuliswa ama-awa ama-72 phambi kwamazinga ahlukileko we-MNA. Nakungabikho i-MNA yangaphandle, okuqukethwe kwamaseli we-MNA angeze kwatholakala (Umfanekiso 3G). Kodwana, amaseli we-T asebenzako aphathwe nge-MNA yangaphandle atjengisa ukwanda okuthembele emthamo we-MNA emaseli, ukuya ku-6 mM MNA (Umfanekiso 3G). Umphumela lo utjengisa bona nanyana kunezinga eliphasi lokuvezwa kwe-transporter nokutlhogeka kwe-enzayimu ekulu enesibopho sokusebenza kwe-MNA ngaphakathi kwamaseli, i-TIL isakghona ukuthatha i-MNA.
I-spectrum yama-metabolites kumaseli we-T wezigulani kanye nokuhlolwa kokumuncwa kwe-MNA kwe-in vitro kwandisa amathuba wokuthi ama-fibroblasts ahlotjaniswa nomdlavuza (CAF) akhiphe i-MNA begodu amaseli wesimila angalawula ubujamo nomsebenzi we-TIL. Ukuthola umphumela we-MNA kumaseli we-T, amaseli we-T anikelako aphilileko asebenza nge-in vitro ebukhoneni nofana ukungabi khona kwe-MNA, begodu ukwanda kwawo nokukhiqizwa kwama-cytokine kwahlolwa. Ngemva kwamalanga ali-7 wokungezelela i-MNA ngomthamo ophezulu, inani lokuphinda kabili labantu lehliswe ngokulinganisela, ngesikhathi amandla agcinwa kiwo woke amadosi (Umfanekiso 4A). Ukungezelela, ukwelatjhwa kwe-MNA yangaphandle kwabangela ukwanda kwenani lamaseli we-CD4 + kanye ne-CD8 + T aveza i-tumor necrosis factor-α (TNFα; Umfanekiso 4B). Ngokuphikisako, ukukhiqizwa kwe-IFN-γ ngaphakathi kwamaseli kwehla khulu kumaseli we-CD4 + T, kodwana ingasi kumaseli we-CD8 + T, begodu bekunganatjhuguluko elikhulu ku-interleukin 2 (IL-2; Umfanekiso 4, C no-D). Ngalokho-ke, i-enzyme-linked immunosorbent assay (ELISA) yama-supernatants avela e-MNA-treated T cell cultures itjengise ukwanda okukhulu kwe-TNFα, ukwehla kwe-IFN-γ, begodu akukho ukutjhuguluka ku-IL-2 (Umfanekiso 4, E ukuya ku-G). . Ukwehla kwe-IFN-γ kutjengisa bona i-MNA ingadlala indima ekuvimbeleni umsebenzi wokulwa nesimila wamaseli we-T. Ukulingisa umphumela we-MNA ku-T cell-mediated cytotoxicity, amaseli we-chimeric antigen receptor T (FRα-CAR-T) ahlose i-folate receptor α kanye ne-CAR-T (GFP) alawulwa maseli we-green fluorescent protein (GFP) -CAR-T) akhiqizwa ngamaseli weengazi eziphilileko (PBMC). Amaseli we-CAR-T akhuliswa ama-awa ama-24 phambi kwe-MNA, bese akhuliswa ndawonye namaseli we-SK-OV-3 womuntu we-ovarian tumor aveza i-folate receptor α nge-effector ukuya ku-target ratio ye-10:1. Ukwelatjhwa nge-MNA kwabangela ukwehla okukhulu komsebenzi wokubulala wamaseli we-FRα-CAR-T, ebegade afana namaseli we-FRα-CAR-T aphathwe nge-adenosine (Umfanekiso 4H).
(A) Inani elipheleleko lamaseli aphilako nokuphinda kabili inani labantu (PD) ngqo ukusuka esikweni ngelanga lesi-7. Igrafu yebha imele i-mean + SEM yabanikeli abasithandathu abaphilileko. Imele idatha evela okungenani ku-n = 3 ukuhlolwa okuzijameleko. (B ukuya ku-D) I-CD3/CD28 ne-IL-2 zasetjenziselwa ukusebenzisa amaseli we-T emazingeni wawo we-MNA amalanga ali-7. Ngaphambi kokuhlaziywa, amaseli akhuthazwa nge-PMA/ionomycin nge-GolgiStop ama-awa ama-4. Ukuvezwa kwe-TNFα (B) kumaseli we-T. Isibonelo sesithombe (kwesokunxele) nedatha yethebula (kwesokudla) yokuvezwa kwe-TNFα kumaseli aphilako. Ukuvezwa kwe-IFN-γ (C) kanye ne-IL-2 (D) kumaseli we-T. Ukuvezwa kwama-cytokines kwalinganiswa nge-flow cytometry. Igrafu yebha imele isilinganiso (n = 6 abanikeli abaphilileko) + SEM. Sebenzisa ukuhlaziywa kwendlela eyodwa yokuhlukahluka kanye nokulinganisa okuphindaphindiweko (*P<0.05 kanye no-**P<0.01) ukuthola inani le-P. Imele idatha evela okungenani ku-n = 3 ukuhlolwa okuzijameleko. (E ukuya ku-G) I-CD3/CD28 ne-IL-2 zasetjenziselwa ukusebenzisa amaseli we-T emazingeni wawo we-MNA amalanga ali-7. I-medium yabuthelelwa ngaphambi nangemva kwama-awa ama-4 wokuvuselelwa kwe-PMA/ionomycin. Ukugxila kwe-TNFα (E), IFN-γ (F) kanye ne-IL-2 (G) kwalinganiswa nge-ELISA. Igrafu yebha imele isilinganiso (n = 5 abanikeli abaphilileko) + SEM. Inani le-P elitholwe ngokusebenzisa ukuhlaziywa kwendlela eyodwa yokuhlukahluka nokulinganisa okuphindaphindiweko (*P<0.05). Umugqa onamachashazi utjengisa umkhawulo wokutholwa kokuthola. (H) Ukuhlolwa kokuhlukaniswa kwamaseli. Amaseli we-FRα-CAR-T nofana we-GFP-CAR-T alungiswa nge-adenosine (250μM) nofana i-MNA (10 mM) ama-awa ama-24, nofana atjhiywa angalatjhwa (Ctrl). Iphesenteji yokubulawa kwamaseli we-SK-OV-3 yalinganiswa. Inani le-P elitholwe yi-Welch t test (*P<0.5 kanye ne-**P<0.01).
Ukuze sithole ukuzwisisa kwendlela yokulawula ukuvezwa kwe-TNFα okuthembele ku-MNA, amatjhuguluko ku-TNFα mRNA yamaseli we-T aphathwe nge-MNA ahlolwa (Umfanekiso 5A). Amaseli we-T we-donor aphilileko aphathwe nge-MNA atjengisa ukwanda okuphindwe kabili kwamazinga wokutloliswa kwe-TNFα, okutjengisa bona i-MNA ithembele emthethweni wokutloliswa kwe-TNFα. Ukuphenya indlela yokulawula, izinto ezimbili ezaziwako ezilawula i-TNFα, okuyi-activated T cell nuclear factor (NFAT) kanye nephrotheyini ethileko 1 (Sp1), zahlolwa ngokuphendula ukubophelela kwe-MNA kumkhuthazi we-TNFα oseduze ( 30). Umkhuthazi we-TNFα uneendawo ezi-6 zokubophelela i-NFAT ezikhonjiweko kanye neendawo ezi-2 zokubopha i-Sp1, ezihlangana endaweni eyodwa [-55 ama-base pairs (bp) avela ku-5'cap] (30). I-Chromatin immunoprecipitation (ChIP) itjengise bona lokha nayiphathwa nge-MNA, ukubophelela kwe-Sp1 kumkhuthazi we-TNFα kwanda kathathu. Ukufakwa kwe-NFAT nakho kwanda begodu kwasondela ekuqakathekeni (Umfanekiso 5B). Idatha le itjengisa bona i-MNA ilawula ukuvezwa kwe-TNFα ngokusebenzisa ukutlolwa kwe-Sp1, begodu ngezinga elincani ukuvezwa kwe-NFAT.
(A) Nakuqathaniswa namaseli we-T akhuliswe ngaphandle kwe-MNA, ukutjhuguluka okuphindaphindiweko kokuvezwa kwe-TNFα kumaseli we-T aphathwe nge-MNA. Iphetheni yokuvezwa nge-SEM itjengiswa (n = 5 abanikeli abaphilileko). Imele idatha evela okungenani ku-n = 3 ukuhlolwa okuzijameleko. (B) Umkhuthazi we-TNFα wamaseli we-T aphathwe nge-8 mM MNA nofana ngaphandle kwayo ngemva kokuthi i-NFAT ne-Sp1 zihlanganiswe ne-(Ctrl) kanye nokuvuselelwa kwe-PMA/ionomycin ama-awa ama-4. I-Immunoglobulin G (IgG) kanye ne-H3 zasetjenziswa njengezilawuli ezimbi nezihle zokuvikela umzimba, ngokulandelana. Ukulinganisa kwe-ChIP kutjengise bona ukubophelela kwe-Sp1 ne-NFAT kumkhuthazi we-TNFα kumaseli aphathwe nge-MNA kwanda kanengi nayiqathaniswa nokulawula. Imele idatha evela okungenani ku-n = 3 ukuhlolwa okuzijameleko. Inani le-P eliqunywe ziimhloliso ezinengi ze-t (*** P <0.01). (C) Nayiqathaniswa ne-ascites ye-HGSC, amaseli we-T (angasiwo ama-cytotoxic) atjengisa ukuvezwa okukhulu kwe-TNF esimila. Imibala ijamele iingulani ezihlukileko. Amaseli atjengisiweko akhethwe ngokungakahleleki abe ma-300 begodu atjhugululwa ukukhandela ukudweba okudluleleko (** Padj = 0.0076). (D) Imodeli ehlongozwayo ye-MNA yomdlavuza wesibeletho. I-MNA ikhiqizwa ngamaseli wesimila kanye nama-fibroblasts ku-TME begodu ithathwa ngamaseli we-T. I-MNA ikhuphula ukubophelela kwe-Sp1 kumkhuthazi we-TNFα, okurholela ekukhuphukeni kokutlolwa kwe-TNFα kanye nokukhiqizwa kwama-cytokine we-TNFα. I-MNA nayo ibangela ukwehla kwe-IFN-γ. Ukuvinjelwa komsebenzi wamaseli we-T kurholela ekwehliseni ikghono lokubulala nokukhula kwesimila okurhabako.
Ngokuya kwemibiko, i-TNFα inemiphumela yokulwisana nethumba nokulwa nethumba, kodwana inendima eyaziwako ekukhuthazeni ukukhula nokusabalala komdlavuza wesibeletho (31-33). Ngokuya kwemibiko, ukugxila kwe-TNFα ku-ascites kanye nezicubu zesimila ezigulini ezinomdlavuza wesibeletho kuphezulu kunalokho okusezicutshini ezingenabungozi (34-36). Ngokuya ngendlela, i-TNFα ingalawula ukusebenza, ukusebenza nokwanda kwamaseli weengazi amhlophe, begodu itjhugulule ubujamo bamaseli womdlavuza (37, 38). Ngokuvumelana nalokhu okutholakeleko, ukuhlaziywa kokuvezwa kwezakhi zofuzo okuhlukileko kutjengise bona i-TNF beyilawulwa khulu kumaseli we-T emathitjhu wethumba nayiqathaniswa ne-ascites (Umfanekiso 5C). Ukwanda kokuvezwa kwe-TNF kwabonakala kwaphela eendaweni ze-T cell ezine-non-cytotoxic phenotype (Umfanekiso S5A). Ngokufingqa, idatha le isekela umbono wokobana i-MNA inemiphumela emibili yokugandelela amasotja womzimba nokukhuthaza isimila ku-HGSC.
Ukulebula ngokukhanya okusekelwe ku-flow cytometry sekuyindlela ekulu yokufunda ukusebenza kwe-TIL. Iimfundo zitjengise bona nayiqathaniswa nama-lymphocyte weengazi ezingaphandle nofana amaseli we-T avela eenthweni ze-lymphoid zesibili, i-murine ne-human TIL inomkhuba ophezulu wokuthatha i-glucose (4, 39) nokulahlekelwa kancanikancani komsebenzi we-mitochondrial (19, 40). Nanyana sibone imiphumela efanako esifundweni lesi, ukuthuthukiswa okuqakathekileko kukumadanisa ukusebenza kwamaseli wesimila kanye ne-TIL evela esicutshini esifanako sesimila esisusiweko. Ngokuvumelana neminye yemibiko edlulileko, amaseli we-tumor (CD45-EpCAM +) avela ku-ascites kanye nama-tumor ane-glucose uptake ephezulu ukudlula ama-CD8 + kanye nama-CD4 + T cells, okusekela bona ukuthathwa kwe-glucose okuphezulu kwamaseli we-tumor kungamadaniswa nama-T cells. Umqondo wokuncintisana kwamaseli we-T. TME. Kodwana, umsebenzi we-mitochondrial wamaseli we-tumor uphezulu kunalowo wamaseli we-CD8 + T, kodwana umsebenzi we-mitochondrial uyafana newe-CD4 + T cells. Imiphumela le iqinisa isihloko esivelako sokuthi ukugaya ukudla kwe-oxidative kuqakathekile kumaseli wesimila (41, 42). Baveza nokuthi amaseli we-CD8 + T angaba sengozini yokungasebenzi kuhle kwe-oxidative ukudlula amaseli we-CD4 + T, nofana amaseli we-CD4 + T angasebenzisa imithombo yekhabhoni ngaphandle kwe-glucose ukugcina umsebenzi we-mitochondrial (43, 44). Kufanele kuqashelwe bona asizange sibone umehluko ekuthathweni kwe-glucose nofana emsebenzini we-mitochondrial hlangana nama-CD4 + T effectors, i-T effector memory kanye nama-T central memory cells ku-ascites. Ngokufanako, ubujamo bokuhlukaniswa kwamaseli we-CD8 + T emathunjini abuhlangene nokutjhuguluka ekuthathweni kwe-glucose, okuveza umehluko oqakathekileko hlangana namaseli we-T akhuliswe ku-vitro kanye ne-TIL yomuntu ku-vivo (22). Lokhu okubonwako kwaqinisekiswa ngokusetjenziswa kokwabiwa kwamaseli okungathathi ihlangothi, okubuye kwaveza bona amaseli we-CD45 + / CD3- / CD4 + / CD45RO + ane-glucose uptake ephezulu kanye nomsebenzi we-mitochondrial ukudlula amaseli we-tumor avamile kodwana ane-Metabolic active cell population. Isibalo lesi singajamela isibalo esincani samaseli we-myeloid suppressor nofana amaseli we-plasmacytoid dendritic akhonjwe ekuhlaziyweni kwe-scRNA-seq. Nanyana kokubili lokhu kubikwe emathunjini wabantu [45], kusatlhogeka.
Nanyana iindlela ezisekelwe ku-flow cytometry zingacacisa umehluko jikelele we-glucose kanye ne-oxidative metabolism hlangana nemihlobo yamaseli, ama-metabolites aqondileko akhiqizwa yi-glucose nofana eminye imithombo yekhabhoni ye-mitochondrial metabolism ku-TME awakatholwa. Ukunikela ubukhona nofana ukungabi khona kwama-metabolites esigabeni esithileko se-TIL kutlhoga ukuhlanzwa kwenani lamaseli ukusuka esicutshini esikhitshiweko. Ngalokho-ke, indlela yethu yokunothisa amaseli ehlanganiswe ne-mass spectrometry inganikela ilwazi ngama-metabolites anothiswe ngokuhlukileko kumaseli we-T kanye namaseli we-tumor ekufaniseni amasampula wesigulani. Nanyana indlela le inezinzuzo phezu kokuhlunga amaseli asebenza ngokukhanya, amanye amalayibhrari we-metabolite angathinteka ngebanga lokuzinza okungokwemvelo kanye/nofana izinga lokujika elirhabako (22). Nanyana kunjalo, indlela yethu yakghona ukukhomba ama-metabolites amabili aqakathekileko, i-adenosine ne-kynurenine, ngombana ziyahluka khulu hlangana nemihlobo yamasampula.
Ukuhlaziywa kwethu kwe-metabonomic yamathumba kanye nama-TIL subtypes kunikela ukuzwisisa okungeziweko ngendima yama-metabolites ku-ovarian TME. Kokuthoma, ngokusebenzisa i-flow cytometry, sathola bona bekunganamehluko emsebenzini we-mitochondrial hlangana namathumba namaseli we-CD4 + T. Kodwana, ukuhlaziywa kwe-LC-MS/MS kuveze amatjhuguluko aqakathekileko ebunengini bama-metabolites hlangana nabantu, okutjengisa bona iimpendulo eziphathelene nokugaya ukudla kwe-TIL nomsebenzi wayo woke wokugaya ukudla zitlhoga ukuhlathulula ngokutjheja. Kwesibili, i-MNA yi-metabolite enomehluko omkhulu hlangana namaseli we-CD45 namaseli we-T ku-ascites, ingasi amathumba. Ngalokho-ke, ukuhlukaniswa kanye nendawo yesimila kungaba nemiphumela ehlukileko ekusebenzeni kwe-TIL, okuveza ukuhlukahluka okungenzeka endaweni encani. Okwesithathu, ukuvezwa kwe-enzyme ekhiqiza i-MNA i-NNMT kukhawulelwe khulu ku-CAF, okumaseli wesimila ngezinga elincani, kodwana amazinga we-MNA abonakalako kumaseli we-T avela esimweni. Ukuvezwa ngokweqileko kwe-NNMT ku-CAF ye-ovarian kunomphumela owaziwako wokukhuthaza umdlavuza, ngokwengcenye ngebanga lokukhuthazwa kwe-CAF, ukuhlasela kwesimila kanye nokusabalala (27). Nanyana izinga elipheleleko le-TIL liphakathi, ukuvezwa kwe-NNMT ku-CAF kuhlobana khulu ne-Cancer Genome Atlas (TCGA) mesenchymal subtype, ehlotjaniswa nokubikezela okumbi (27, 46, 47). Ekugcineni, ukuvezwa kwe-enzyme AOX1 enesibopho sokuwohloka kwe-MNA nakho kukhawulelwe ebantwini be-CAF, okutjengisa bona amaseli we-T awanakghono lokugaya i-MNA. Imiphumela le isekela umbono wokobana nanyana umsebenzi ongezelelweko uyatlhogeka ukuqinisekisa lokhu okutholweko, amazinga aphezulu we-MNA kumaseli we-T angatjengisa ubukhona bendawo encani ye-CAF evikela amasotja womzimba.
Njengombana kunikelwe izinga eliphasi lokuvezwa kwabathuthi be-MNA kanye namazinga angabonakaliko wamaphrotheyini aqakathekileko abandakanyeka ekusebenzeni kwe-MNA, ukuba khona kwe-MNA kumaseli we-T akukalindelwa. I-NNMT nofana i-AOX1 azange itholakale ngokuhlaziywa kwe-scRNA-seq kanye ne-qPCR ehlosiweko yamaqoqo amabili azijameleko. Imiphumela le itjengisa bona i-MNA ayikhiqizwa maseli we-T, kodwana idonswa yi-TME ezungezeko. Iimhloliso ze-in vitro zitjengisa bona amaseli we-T avamise ukubuthelela i-MNA yangaphandle.
Iimfundo zethu ze-in vitro zitjengise bona i-MNA yangaphandle ikhuthaza ukuvezwa kwe-TNFα kumaseli we-T begodu ikhulisa ukubophelela kwe-Sp1 kumkhuthazi we-TNFα. Nanyana i-TNFα inemisebenzi yokulwisana nethumba, emdlavuzeni we-ovari, i-TNFα ingakhuthaza ukukhula komdlavuza we-ovari (31-33). Ukuqedwa kwe-TNFα esikolweni samaseli wesimila se-ovarian nofana ukuqedwa kwesiginali ye-TNFα emamodelini wegundane kungenza ngcono ukukhiqizwa kwama-cytokine wokuvuvuka okuphakathi kwe-TNFα begodu kuvimbele ukukhula kwesimila (32, 35). Ngalokho-ke, endabeni le, i-MNA evela ku-TME ingasebenza njenge-metabolite ekhuthaza ukuvuvuka ngendlela ethembele ku-TNFα nge-autocrine loop, ngaleyo ndlela ikhuthaze ukwenzeka nokurhatjheka komdlavuza wesibeletho (31). Ngokuya ngalokhu, ukuvinjwa kwe-TNFα kuyafundwa njengendlela yokwelapha umdlavuza wesibeletho (37, 48, 49). Ukungezelela, i-MNA iphazamisa ubuthi bamaseli we-CAR-T kumaseli wesimila sesibeletho, okunikela ubufakazi obungezelelweko bokugandeleleka kwamasotja womzimba okulawulwa yi-MNA. Ngokuhlanganyela, imiphumela le iveza imodeli lapho amathumba namaseli we-CAF akhipha i-MNA ibe yi-TME yangaphandle kwamaseli. Ngokusebenzisa (i) ukuvuselela ukukhula komdlavuza wesibeletho okubangelwa yi-TNF kanye (ii) nokuvinjelwa komsebenzi we-cytotoxic weseli ye-T okubangelwa yi-MNA, lokhu kungaba nomphumela wethumba ophindwe kabili (Umfanekiso 5D).
Ekuphetheni, ngokusebenzisa inhlanganisela yokunothisa amaseli okurhabako, ukulandelana kweseli elilodwa kanye nokuphrofayila kwe-metabolic, irhubhululo leli liveze umehluko omkhulu we-immunometabolomic hlangana namathumba namaseli we-ascites eengulini ze-HGSC. Ukuhlaziywa okupheleleko kutjengise bona kunomehluko ekuthathweni kwe-glucose nomsebenzi we-mitochondrial hlangana namaseli we-T, begodu kwaveza i-MNA njenge-metabolite elawula amasotja womzimba. Idatha le inomthelela ekutheni i-TME ithinta njani ukusebenza kwamaseli we-T emdlavuzeni wabantu. Nanyana ukuncintisana okunqophileko kwezakhamzimba hlangana namaseli we-T namaseli womdlavuza kubikwe, ama-metabolites angasebenza njengabalawuli abangakaqondi ukukhuthaza ukuragela phambili kwesimila begodu mhlamunye ancindezele ukuphendula kwamasotja womzimba. Ihlathululo eragela phambili yendima esebenzako yama-metabolites la alawulako ingavula amanye amaqhinga wokuthuthukisa ukuphendula kwamasotja womzimba.
Iimbonelo zesigulani nedatha yezokwelapha zitholwe nge-BC cancer tumor tissue repository eqinisekiswe yi-Canadian Tissue Repository Network. Ngokuya kwephrothokholi evunywe yi-BC Cancer Research Ethics Committee kanye ne-University of British Columbia (H07-00463), zoke iinthombe zezigulani kanye nedatha yezokwelapha zithole imvumo etlolwe phasi namkha ziyeke ngokusemthethweni imvumo yazo. Amasampula agcinwa ku-BioBank eqinisekisiweko (BRC-00290). Iimfanelo zesiguli ezineenkcukacha zitjengiswe kumaThebuli S1 no-S5. Ukulondolozwa kwe-cryopreservation, i-scalpel isetjenziselwa ukubola isampula yesimila sesigulani bese iyayifaka ngesihlungi esiyi-100-micron ukuthola ukumiswa kweseli elilodwa. I-ascites yesigulani yatjhugululwa nge-1500 rpm imizuzu eli-10 ku-4°C ukukhipha amaseli bese kususa amanzi. Amaseli atholakala esimila kanye ne-ascites agcinwe e-cryopreserved ku-50% heat-inactivated human AB serum (Sigma-Aldrich), 40% RPMI-1640 (Thermo Fisher Scientific) kanye ne-10% dimethyl sulfoxide. Ama-suspension weseli elilodwa agcinweko la ancibilika begodu asetjenziselwa i-metabolomics nokuqunta i-metabolite okuchazwe ngenzasi.
Indawo epheleleko iqukethe i-0.22 μm ehlungwe 50:50 enezelelwe i-RPMI 1640: AimV. I-RPMI 1640 + 2.05 mM l-glutamine (Thermo Fisher Scientific) enezwe nge-10% ye-heat-inactivated human AB serum (Sigma-Aldrich), 12.5 mM Hepes (Thermo Fisher Scientific), 2 mM l-glutamine (Thermo Fisher Scientific) x1 Isisombululo se-Penicillin Streptomycin (PenStrep) (i-Thermo Fisher Scientific) kanye ne-50 μMB-mercaptoethanol. I-AimV (Invitrogen) ingezelelwa nge-20 mM Hepes (Thermo Fisher Scientific) kanye ne-2 mM l-glutamine (Thermo Fisher Scientific). I-flow cytometer staining buffer beyinama-0.22μm ahlungiweko we-phosphate buffered saline (PBS; Invitrogen) enezwe nge-3% heat-inactivated AB human serum (Sigma). Ibhafa yokunothisa amaseli yakhiwe yi-0.22μm ye-PBS ehlungiweko begodu ingezelelwe nge-0.5% ye-human AB serum (Sigma-Aldrich).
Ngaphakathi kwe-37°C epheleleko, amaseli ahlanjululwe nge-10 nM MT DR kanye ne-100 μM 2-NBDG imizuzu ema-30. Okulandelako, amaseli afakwa ngedayi ye-eF506 ku-4°C imizuzu eli-15. Faka kabutjha amaseli ku-FC Block (eBioscience) kanye ne-Brilliant Stain Buffer (BD Biosciences), uhlanjululwe nge-flow cytometer staining buffer (ngokuya kwemiyalo yomenzi), bese uwafukamela imizuzu eli-10 emazingeni womtjhiso wegumbi. Faka amaseli ngesethi yama-antibodies (Ithebula S2) ku-flow cytometry staining buffer ku-4°C imizuzu ema-20. Faka kabutjha amaseli ku-flow cytometry staining buffer (Cytek Aurora; 3L-16V-14B-8R configuration) ngaphambi kokuhlaziywa. Sebenzisa i-SpectroFlo ne-FlowJo V10 ukuhlaziya idatha yokubala amaseli, begodu usebenzise i-GraphPad Prism 8 ukwenza idatha. I-median fluorescence intensity (MFI) ye-2-NBDG ne-MT DR beyi-log-normalized, bese kuthi i-t test ehlangeneko isetjenziselwe ukuhlaziywa kwezibalo ukulandisa iingulani ezifanako. Susa zoke iintjhaba ezinezehlakalo ezingaphasi kwama-40 ekuhlaziyweni; faka inani le-MFI elingu-1 kiwo woke amanani angakalungi ngaphambi kokwenza ukuhlaziywa kweenbalo nokubonwa kwedatha.
Ukuze singezelele iqhinga lokuvala ngesandla lephaneli yenqubo engehla, sisebenzise ukuhlathulula okupheleleko ngomuthi wokuvinjelwa kwejamo (FAUST) (21) ukwabela amaseli ngokuzenzakalelako esiqhemeni ngemva kokuqeda amaseli ahlongakeleko ku-FlowJo. Siphatha ngesandla umphumela ukuhlanganisa iinqhema ezibonakala zingahlukaniswa kuhle (ukuhlanganisa i-PD1+ namaseli we-PD1-tumor) kanye neenqhema ezigciniweko. Isampula ngayinye iqukethe isilinganiso samaseli angaphezu kwama-2%, inani elipheleleko labantu abali-11.
I-Ficoll gradient density centrifugation yasetjenziselwa ukuhlukanisa i-PBMC emikhiqizweni yokuhlukanisa ama-leukocyte (i-STEMCELL Technologies). Amaseli we-CD8 + T ahlukaniswe ku-PBMC kusetjenziswa i-CD8 MicroBeads (Miltenyi) begodu anwetshwa emkhakheni opheleleko kusetjenziswa i-TransAct (Miltenyi) amaviki ama-2 ngokuya kwemilayo yomkhiqizi. Amaseli avunyelwa bona ajame amalanga ama-5 endaweni epheleleko ene-IL-7 (10 ng/ml; PeproTech), bese avuselelwa nge-TransAct. Ngelanga lesi-7, ngokuya kwemilayo yomkhiqizi, ama-CD45 MicroBeads womuntu (i-Miltenyi) asetjenziselwa ukunothisa amaseli ngemizuliswano emithathu elandelanako. Amaseli ahlukaniswa ukuhlaziywa kwe-flow cytometry (njengombana kuchaziwe ngehla), begodu amaseli ayi-1 miliyoni ahlukaniswa kathathu ukuhlaziywa kwe-LC-MS/MS. Amasampula asetjenziswe nge-LC-MS/MS njengombana kuchaziwe ngenzasi. Silinganise inani le-metabolite elilahlekileko ngenani le-ion elingu-1,000. Isampula ngayinye ijwayelekile ngenombolo ye-ion epheleleko (TIC), itjhugululwe nge-logarithm begodu ijwayelekile ngokuzenzakalelako ku-MetaboAnalystR ngaphambi kokuhlaziywa.
Ukumiswa kweseli elilodwa lesiguli ngasinye kwancibilikiswa kwahlungwa ngesihlungi esingu-40 μm kwaba yindawo epheleleko (njengombana kuchaziwe ngehla). Ngokuya kwephrothokholi yomkhiqizi, imijikelezo emithathu elandelanako yokukhetha okuhle ngokuhlukaniswa kwama-magnetic bead ngokusebenzisa i-MicroBeads (Miltenyi) yasetjenziselwa ukunothisa amasampula we-CD8+, CD4+ kanye namaseli we-CD45- (eqhweni). Ngamafuphi, amaseli abuyiselwa emuva esivikelweni sokunothisa amaseli (njengombana kuchaziwe ngehla) bese abalwa. Amaseli afukanywa ngama-CD8 womuntu, ama-CD4 womuntu nofana ama-CD45 womuntu (i-Miltenyi) ku-4°C imizuzu eli-15, bese ahlanzwa nge-buffer yokunothisa amaseli. Isampula idluliselwa ngekholomu ye-LS (i-Miltenyi), begodu amafraktjhini amahle namambi ayabuthelelwa. Ukuze kwehliswe isikhathi begodu kukhuliswe igadango lokubuyiselwa kwamaseli, ingcenye ye-CD8 isetjenziselwa umjikelezo wesibili wokunothisa i-CD4+, bese ingcenye ye-CD4 isetjenziselwa ukunothisa okulandelako kwe-CD45. Gcina isisombululo phezu kweqhwa kiyo yoke ikambiso yokuhlukanisa.
Ukulungiselela amasampula wokuhlaziywa kwe-metabolite, amaseli ahlanzwa kanye ngesisombululo setshwayi esibanda njengeqhwa, begodu i-1 ml ye-80% ye-methanol yengezwa esampuleni ngayinye, bese yafakwa i-vortex begodu yaqhwazwa nge-nitrogen ewuketshezi. Amasampula afakwa ngaphasi kwemijikelezo emithathu yokuqanda-ukuncibilikisa begodu ahlanganiswa nge-14,000 rpm imizuzu eli-15 ku-4°C. I-supernatant equkethe ama-metabolites iyahwamuka ize yomile. Ama-metabolites aphinda ancibilikiswa ku-50 μl ye-0.03% ye-formic acid, ahlanganiswa ukuze ahlanganise, bese ahlanganiswa ukususa iimvuthuluka.
Khipha ama-metabolites njengombana kuchaziwe ngehla. Dlulisela i-supernatant ebhodleleni elisebenza kuhle khulu le-liquid chromatography ukwenzela irhubhululo le-metabolomics. Sebenzisa iphrothokholi yokwelapha engakahleleki ukwelapha isampula ngayinye ngenani elifanako lamaseli ukukhandela imiphumela ye-batch. Senze ukuhlolwa kwekhwalithi yama-metabolites wephasi loke ebegade agadangiswe ku-AB SCIEX QTRAP 5500 Triple Quadrupole Mass Spectrometer (50). Ukuhlaziywa kwe-chromatographic nokuhlanganiswa kwendawo ephezulu kwenziwa kusetjenziswa isofthiwe ye-MultiQuant version 2.1 (i-Applied Biosystems SCIEX).
Inani le-ion eliyi-1000 lasetjenziselwa ukulinganisa inani le-metabolite elilahlekileko, begodu i-TIC yesampula ngayinye yasetjenziselwa ukubala indawo ejwayelekileko ye-metabolite ngayinye etholakeleko ukulungisa amatjhuguluko angeniswe kuhlaziywa kwethulusi elivela ekusetjenzisweni kwesampula. Ngemva kokuthi i-TIC ibe yinto ejayelekileko, i-MetaboAnalystR(51) (ipharamitha ejwayelekileko) isetjenziselwa ukuguqulwa kwe-logarithmic nokulinganisa okuzenzakalelayo. Sisebenzise i-PCA enephakheji ye-vegan R ukwenza ukuhlaziywa kokuhlola umehluko we-metabolome hlangana nemihlobo yamasampula, begodu sasebenzisa ukuhlaziywa kokungabi khona okuyingcenye ukuhlaziya iingulani. Sebenzisa indlela ka-Ward ukwakha i-dendrogram yebalazwe lokutjhisa ukuhlanganisa ibanga le-Euclidean hlangana namasampula. Sisebenzise i-limma (52) ngobunengi be-metabolite ukukhomba ama-metabolite amanengi ahlukileko kiyo yoke imihlobo yamaseli kanye nendawo encani. Ukuze senze lula ihlathululo, sisebenzisa ipharamitha yesiqhema ukucacisa imodeli, begodu sicabangele imihlobo yamaseli endaweni encani njengesiqhema ngasinye (n = amaqembu ama-6); ukuhlolwa kokuqakatheka, senza iinlinganiso eziphindaphindiweko ezintathu ze-metabolite ngayinye Ukuze sigweme ukuphindaphindwa kwamanga, isiguli safakwa njengesithiyo emklamweni we-limma. Ukuze sihlole umehluko wama-metabolites hlangana nezigulani ezihlukileko, silungise imodeli ye-limma efaka hlangana izigulani ngendlela engaguqukiko. Sibika ukuqakatheka kokuhlukana okubekwe ngaphambilini hlangana nomhlobo weseli kanye nendawo encani ye-Padj <0.05 (ukulungiswa kwe-Benjamini-Hochberg).
Ngemva kokunothiswa kwamandla kusetjenziswa i-Miltenyi Dead Cell Removal Kit (>80% viability), ukulandelana kwe-single-cell transcriptome kwenziwa kumasampula we-ascites aphilako aphilako kanye namasampula we-tumor kusetjenziswa iphrothokholi yokuvezwa kwe-10x 5′gene. Amacala amahlanu anezimila ezifanako nama-ascites ahlaziywa, nanyana ukuphila okuphasi kwesampula eyodwa yesimila kwavimbela ukufakwa kwayo. Ukuze sifinyelele ukukhetha okunengi kwezigulani, sihlanganise amasampula wesiguli ngasinye emigqeni ye-10x chromium controller, begodu sahlaziya ama-ascites nama-tumor sites ngokwehlukana. Ngemva kokulandelana [Illumina HiSeq 4000 28×98 bp paired end (PE), Quebec genome; isilinganiso sama-73,488 kanye nama-41,378 wokufundwa ngeseli ngalinye le-tumor kanye ne-ascites ngokulandelana]], sisebenzise i-CellSNP ne-Vireo (53) (ngokusekelwe ku-CellSNP njengoba I-SNP yomuntu ejayelekileko (VCF) enikelwa yi-GRCh38 inikelwa ubunikazi bomnikeli. Sisebenzisa i-SNPRelate ukuthola ubunikazi obuseduze (IBS) yesiguli (IBS), ubujamo besiguli (IBS), ngaphandle kwamaseli angakabelwa kanye namaseli akhonjwe njengama-duplexes kanye nama-donor afanako hlangana namasampula we-ascites kanye ne-tumor (54). (amaseli ama-2792 kanye nama-ascites, ngokulandelana) ukuhlaziywa Sisebenzisa i-igraph's (57) ukuhlanganiswa kwenethiwekhi eseduze (SNN) ngokuya ngebanga le-Jaccard ukuya kumaseli we-cluster ngokuvezwa. amaseli ahlathululwa ngokuvezwa kwe-CD8A ne-GZMA, ngaphandle kwama-subclusters anokuvezwa kwamaphrotheyini we-ribosomal aphasi.
I-PBMC yahlukaniswa nemikhiqizo yokuhlukaniswa kwe-leukocyte (i-STEMCELL Technologies) nge-Ficoll gradient density centrifugation. Amaseli we-CD3 + ahlukaniswe ku-PBMC kusetjenziswa ubuhlalu be-CD3 (Miltenyi). Ebukhoneni nofana ukungabi khona kwe-MNA, amaseli we-CD3+ asebenza nge-CD3 (5μg/ml), i-CD28 encibilikako (3μg/ml) kanye ne-IL-2 (300 U/ml; Proleukin). Ngelanga lokugcina lokunwetshwa, ukusebenza (Fixable Viability Dye eFluor450, eBioscience) nokwanda (123count eBeads, Thermo Fisher Scientific) kwahlolwa nge-flow cytometry. Hlola umsebenzi we-effector ngokuvuselela amaseli nge-PMA (20 ng/ml) kanye ne-ionomycin (1μg/ml) nge-GolgiStop ama-awa ama-4, begodu ugade i-CD8-PerCP (RPA-T8, BioLegend), i-CD4-AF700 (RPA-T4), i-BioLegend) kanye ne-TNFα-fluoresce isothiyanate (TCFIMA1) BD). Khuthaza amaseli we-qPCR ne-ChIP nge-PMA (20 ng/ml) kanye ne-ionomycin (1μg/ml) ama-awa ama-4. I-ELISA supernatant yaqoqwa ngaphambi nangemva kokuvuselelwa nge-PMA (20 ng/ml) kanye ne-ionomycin (1 μg/ml) ama-awa ama-4.
Landela iphrothokholi yomkhiqizi ukuhlukanisa i-RNA ngokusebenzisa i-RNeasy Plus Mini Kit (QIAGEN). Sebenzisa i-QIAshredder (QIAGEN) ukuhlanganisa isampula. Sebenzisa i-RNA enekhono eliphezulu ukuya ku-cDNA kit (i-Thermo Fisher Scientific) ukwenza i-DNA (cDNA). Sebenzisa i-TaqMan Rapid Advanced Master Mix (Thermo Fisher Scientific) ukulinganisa ukuvezwa kwezakhi zofuzo (ngokuya ngephrothokholi yomkhiqizi) ngama-probe alandelako: Hs00196287_m1 (NNMT), Hs00154079_m1 (AOX1), Hs0042755m1 (AOX1), Hs02786624_g1 [glyceraldehyde-3-phosphate off Hydrogen (GAPDH)] kanye ne-Hs01010726_m1 (SLC22A2). Amasampula asetjenziswe ku-StepOnePlus real-time PCR system (Applied Biosystems) (Applied Biosystems) ku-MicroAmp fast optical 96-well reaction plate (Applied Biosystems) nge-MicroAmp optical film. Nanyana ngiliphi inani le-Ct elidlula ama-35 lithathwa njengelingaphezulu komkhawulo wokutholwa begodu litshwaywe njengelingabonakaliko.
Yenza i-ChIP njengombana kuchaziwe ngaphambilini (58). Ngamafuphi, amaseli alatjhwa nge-formaldehyde (ukugxila kokugcina 1.42%) begodu afukamela emazingeni womtjhiso wegumbi imizuzu eli-10. Sebenzisa i-buffer yokuvuvuka eyengeziweko (25 mM Hepes, 1.5 mM MgCl2, 10 mM KCl kanye ne-0.1% NP-40) phezu kweqhwa imizuzu eli-10, bese ubuyisela emuva ku-immunoprecipitation buffer njengombana kuchaziwe (58 ). Isampula yabe seyitjhugululwa ngemijikelezo elandelako: imijikelezo eli-10 (ama-pulse ama-20 we-1-second) kanye nesikhathi esingatjhugulukiko semizuzwana ema-40. Fukamela ama-antibodies we-ChIP-grade immunoglobulin G (Cell Signaling Technology; 1μl), i-histone H3 (Cell Signaling Technology; 3μl), i-NFAT (Invitrogen; 3μl) kanye ne-SP1 (Cell Signaling Technology; 3μl) ngesampula ku-4°CC shake ubusuku boke. Faka ama-protein A beads (i-Thermo Fisher Scientific) nesampula ku-4°C ngokunyakazisa kancani i-awa eli-1, bese usebenzisa ama-chelex beads (i-Bio-Rad) ukunothisa i-DNA, bese usebenzisa i-proteinase K (i-Thermo Fisher) ukugayeka kwamaphrotheyini. I-TNFα umkhuthazi watholakala nge-PCR: phambili, i-GGG TAT CCT TGA TGC TTG TGT; ngokuphambene nalokho, i-GTG CCA ACA ACT GCC TTT ATA TG (umkhiqizo we-207-bp). Iinthombe zakhiqizwa yi-Image Lab (Bio-Rad) begodu zalinganiswa kusetjenziswa isofthiwe ye-ImageJ.
I-cell culture supernatant yabuthelelwa njengombana kuchaziwe ngehla. Ukuqunta kwenziwa ngokuya ngeenkambiso zomkhiqizi we-human TNFα ELISA kit (Invitrogen), human IL-2 ELISA kit (Invitrogen) kanye ne-human IFN-γ ELISA kit (Abcam). Ngokuya kwephrothokholi yomkhiqizi, i-supernatant yahlanjululwa nge-1:100 ukuthola i-TNFα ne-IL-2, kanye ne-1:3 ukuthola i-IFN-γ. Sebenzisa i-EnVision 2104 Multilabel Reader (PerkinElmer) ukulinganisa ukumuncwa ku-450 nm.
I-PBMC yahlukaniswa nemikhiqizo yokuhlukaniswa kwama-leukocyte (i-STEMCELL Technologies) nge-Ficoll gradient density centrifugation. Amaseli we-CD3 + ahlukaniswe ku-PBMC kusetjenziswa ubuhlalu be-CD3 (Miltenyi). Ebukhoneni nofana ukungabi khona kwe-MNA, amaseli we-CD3+ asebenza nge-CD3 (5μg/ml), i-CD28 encibilikako (3μg/ml) kanye ne-IL-2 (300 U/ml; Proleukin) amalanga ama-3. Ngemva kwamalanga ama-3, amaseli abuthelelwa begodu ahlanzwa nge-0.9% ye-saline, begodu i-pellet yaqhwazwa msinyana. Ukubalwa kwamaseli kwenziwa nge-flow cytometry (Cytek Aurora; 3L-16V-14B-8R configuration) kusetjenziswa i-123count eBeads.
Khipha ama-metabolites njengombana kuchaziwe ngehla. I-extract eyomileko yabuyiselwa ngezinga lama-4000 amaseli alingana/μl. Hlaziya isampula nge-reversed-phase chromatography (1290 Infinity II, Agilent Technologies, Santa Clara, CA) kanye nekholomu ye-CORTECS T3 (2.1×150 mm, ubukhulu beenhlayiya 1.6-μm, ubukhulu be-pore 120-Å; #186008500, Waters). I-polar mass spectrometer (6470, Agilent), lapho i-electrospray ionization isebenza ngendlela ehle. I-mobile phase A yi-0.1% ye-formic acid (ku-H2O), i-mobile phase B yi-90% ye-acetonitrile, 0.1% ye-formic acid. I-LC gradient yi-0 ukuya ku-2 imizuzu ye-100% A, 2 ukuya ku-7.1 imizuzu ye-99% B, kanye nemizuzu engu-7.1 ukuya ku-8 ye-99% B. Bese ubuyisela ikholomu ngesigaba esihambako A ngezinga lokugeleza le-0.6 ml/min imizuzu emi-3. . Izinga lokugeleza lingu-0.4ml/min, begodu ikamelo lekholomu lifuthunyelwa libe ngu-50°C. Sebenzisa izinga lamakhemikhali ahlanzekileko we-MNA (M320995, Toronto Research Chemical Company, North York, Ontario, Canada) ukusungula isikhathi sokugcina (RT) nokutjhuguluka (RT = 0.882 imizuzu, ukutjhuguluka 1 = 137→94.1, ukutjhuguluka 2 = 137→92 , Ukutjhuguluka = ​​17→77). Lokha zoke iintjhuguluko ezintathu zenzeka ngesikhathi esifaneleko sokugcina, itjhuguluko 1 lisetjenziselwa ukulinganisa ukuqinisekisa ukuqakatheka. Ijika elijwayelekileko le-MNA (i-Toronto Research Chemical Company) lakhiwa ngokuhlanjululwa okusithandathu kwesisombululo sesitokwe (1 mg/ml) ukuthola amazinga we-0.1, 1.0, 10 kanye ne-100 ng/ml kanye ne-1.0 kanye ne-10μg/ml ngokulandelana kwamanzi. Umkhawulo wokuthola ngu-1 ng/ml, begodu ukuphendula okulandelanako kuphakathi kwe-10 ng/ml kanye ne-10μg/ml. Umjovo ngamunye wama-microliter amabili wesampula nezinga lisetjenziselwa ukuhlaziywa kwe-LC/MS, begodu isampula yokulawula ikhwalithi ehlangeneko yenziwa qobe ngemva kokujova okusibhozo ukuqinisekisa ukuzinza kweplatifomu yokuhlaziya. Iimpendulo ze-MNA zawo woke amasampula wamaseli aphathwe nge-MNA bezingaphakathi kwebanga eliqondileko lokuhlolwa. Ukuhlaziywa kwedatha kwenziwa kusetjenziswa i-MassHunter quantitative analysis software (v9.0, Agilent).
Isizukulwana sesibili se-αFR-CAR sathathwa ku-Song et al. (59). Ngamafuphi, ukwakhiwa kuqukethe okuqukethwe okulandelako: ukulandelana komdosiphambili we-CD8a, isiqetjhana esihlukileko se-αFR-eqondene nomuntu, i-CD8a hinge kanye ne-transmembrane region, i-CD27 intracellular domain kanye ne-CD3z intracellular domain. Ukulandelana okupheleleko kwe-CAR kwakhiwe yi-GenScript, bese kwakhiwa ku-lentiviral expression vector yesizukulwana sesibili ngaphezulu kwekhasethi yokuvezwa kwe-GFP esetjenziselwa ukuhlola ukusebenza kuhle kokudlulisa.
I-lentivirus yenziwa ngokudluliselwa kwamaseli we-HEK293T [i-American Type Culture Collection (ATCC); ikhuliswe ku-Dulbecco's modified Eagle medium ene-10% fetal bovine serum (FBS) kanye ne-1% PenStrep, begodu yasebenzisa i-CAR-GFP vector kanye namaplasmidi wokupaka (psPAX2 kanye ne-pMD2.G, Addgene) asebenzisa i-lipofection amine (Sigma-Aldrich). I-supernatant enegciwane yaqoqwa ngemva kwama-awa ama-48 nama-72 ngemva kokudluliselwa, yahlungwa, begodu yahlanganiswa ngokusebenzisa i-ultracentrifugation. Beka i-viral supernatant ehlanganisiweko ku--80°C bekube kulapho idluliselwa khona.
I-PBMC ihlukaniswe nemikhiqizo yokuhlukaniswa kwe-leukocyte ye-donor ephilileko (i-STEMCELL Technologies) nge-Ficoll gradient density centrifugation. Sebenzisa ama-CD8 microbeads (Miltenyi) wokukhetha okuhle ukuhlukanisa amaseli we-CD8+ ku-PBMC. Khuthaza amaseli we-T nge-TransAct (Miltenyi) kanye ne-TexMACS medium [Miltenyi; enezwe nge-3% ye-serum yabantu engasebenzi ngokutjhisa, i-1% ye-PenStrep kanye ne-IL-2 (300 U/ml)]. Ngemva kwama-awa amatjhumi amabili nane ngemva kokuvuselelwa, amaseli we-T atjhugululwa nge-lentivirus (10 μl yegciwane eliphezulu ngamaseli ali-106). 1 ukuya kwama-3 amalanga ngemva kokudluliselwa ku-Cytek Aurora (ku-FSC (Forward Scatter)/SSC (Side Scatter), Singlet, GFP+), hlola ukuvezwa kwe-GFP kwamaseli ukutjengisa ukusebenza kuhle kokudluliselwa okungenani kwama-30%.
Amaseli we-CAR-T akhuliswe ama-awa ama-24 ku-Immunocult (STEMCELL Technologies; angezelelwe nge-1% PenStrep) ngaphasi kwemibandela elandelako: angakaphathwa, aphathwe nge-250 μM adenosine nofana i-10 mM MNA. Ngemva kokwelatjhwa ngaphambilini, amaseli we-CAR-T ahlanzwa nge-PBS begodu ahlanganiswa namaseli we-SK-OV-3 ayi-20,000 [ATCC; ku-McCoy 5A medium (Sigma-Aldrich) enezwe nge-10% FBS kanye ne-1% PenStrep ku-10: Isilinganiso se-effector ukuya kwelithagethi se-1 sakhuliswa kathathu emkhakheni we-Immunocult ongezelelweko. Amaseli we-SK-OV-3 namaseli we-SK-OV-3 ahlanjululwe nge-digitalis saponin (0.5mg/ml; Sigma-Aldrich) asetjenziswa njengezilawuli ezimbi nezihle, ngokulandelana. Ngemva kwama-awa ama-24 wokutjala ndawonye, ​​i-supernatant yabuthelelwa begodu i-lactate dehydrogenase (LDH) yalinganiswa ngokuya kwemiyalo yomkhiqizi (LDH Glo Cytotoxicity Assay Kit, Promega). I-LDH supernatant yahlanjululwa ngo-1:50 ku-LDH buffer. Iphesenti lokubulala lalinganiswa ngokusebenzisa ifomula elandelako: iphesenti lokubulala = iphesenti lokulungiswa / izinga eliphezulu lokubulala x 100%, lapho iphesenti lokulungiswa = amaseli we-T wokukhula ndawonye kwaphela, begodu izinga eliphezulu lokubulala = ukulawula okuhle-ukulawula okumbi.
Njengombana kuchaziwe emtlolweni nofana eentweni neendlela, sebenzisa i-GraphPad Prism 8, i-Microsoft Excel nofana i-R v3.6.0 ukuhlaziywa kweenbalo. Nangabe amasampula amanengi abuthelelwe esigulini sinye (njenge-ascites nesimila), sisebenzisa i-t test ehlangeneko nofana sifake isiguli njengomphumela ongahleleki emfanekisweni oqondileko nofana ojwayelekileko njengombana kufanele. Ukuhlaziywa kwe-metabolomics, ukuhlolwa kokuqakatheka kwenziwa kathathu.
Ukuthola izinto ezingezelelweko ze-atikili le, sibawa ubone i-http://advances.sciencemag.org/cgi/content/full/7/4/eabe1174/DC1
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Umbuzo lo usetjenziselwa ukuhlola bona usivakatjhi begodu uvimbele ukuthunyelwa kwe-spam okuzenzakalelayo.
U-Marisa K. Kilgour (u-Marisa K. Kilgour), u-Sarah MacPherson (u-Sarah MacPherson), u-Lauren G. Zacharias (u-Lauren G. Zacharias), u-Abigail Eli Aris G. Watson (H. Watson), u-John Stagg (u-John Stagg), u-Brad H. Nelson (u-Brad H. Nelson), u-Ralph J. Dephrini (R.R.R.R.R.R.R.R.R.R.G.). Jones (Russell G. Jones), Phineas T. Hamilton (Phineas T.
I-MNA inesandla ekugandelelekeni kwamasotja womzimba wamaseli we-T begodu ijamele umnqopho wokuvikela umzimba wokwelapha umdlavuza wabantu.
U-Marisa K. Kilgour (u-Marisa K. Kilgour), u-Sarah MacPherson (u-Sarah MacPherson), u-Lauren G. Zacharias (u-Lauren G. Zacharias), u-Abigail Eli Aris G. Watson (H. Watson), u-John Stagg (u-John Stagg), u-Brad H. Nelson (u-Brad H. Nelson), u-Ralph J. Dephrini (R.R.R.R.R.R.R.R.R.R.G.). Jones (Russell G. Jones), Phineas T. Hamilton (Phineas T.
I-MNA inesandla ekugandelelekeni kwamasotja womzimba wamaseli we-T begodu ijamele umnqopho wokuvikela umzimba wokwelapha umdlavuza wabantu.
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