Ama-metabolite e-immunomodulatory ayisici esibalulekile se-tumor microenvironment (TME), kodwa ngaphandle kwezimbalwa, ubunikazi bawo abukaziwa kakhulu. Lapha, sihlaziye ama-tumors nama-T cell avela kuma-tumors nama-ascites eziguli ezine-high-grade serous carcinoma (HGSC) ukuze siveze i-metabolome yalezi zingxenye ezahlukene ze-TME. Ama-Ascites nama-tumors anomehluko omkhulu we-metabolite. Uma kuqhathaniswa nama-ascites, ama-T cell angena ema-tumors acebile kakhulu ku-1-methylnicotinamide (MNA). Nakuba izinga le-MNA kuma-T cells liphakeme, ukuvezwa kwe-nicotinamide N-methyltransferase (i-enzyme ekhuthaza ukudluliselwa kwamaqembu e-methyl kusuka ku-S-adenosylmethionine kuya ku-nicotinamide) kunqunyelwe kuma-fibroblast nama-tumors. Ngokusebenza kahle, i-MNA ikhuthaza ama-T cells ukuthi akhiphe i-cytokine tumor necrosis factor alpha ekhuthaza isimila. Ngakho-ke, i-MNA etholakala ku-TME inegalelo ekulawulweni kwamasosha omzimba kwama-T cells futhi imelela inhloso engaba khona ye-immunotherapy yokwelapha umdlavuza womuntu.
Ama-metabolite atholakala esimila angaba nomthelela omkhulu ekuvimbeleni ukuzivikela komzimba ekulweni nesimila, futhi ubufakazi obuningi bubonisa ukuthi angasebenza njengamandla abalulekile okuqhuba ukuqhubekela phambili kwesifo (1). Ngaphezu komphumela weWarburg, umsebenzi wakamuva usuqalile ukuchaza isimo se-metabolic samaseli esimila kanye nobudlelwano baso nesimo sokuzivikela komzimba se-tumor microenvironment (TME). Izifundo kumamodeli egundane namaseli e-T abantu zikhombisile ukuthi i-glutamine metabolism (2), i-oxidative metabolism (3) kanye ne-glucose metabolism (4) ingasebenza ngokuzimela emaqenjini ahlukahlukene amaseli omzimba. Ama-metabolite amaningana kulezi zindlela avimbela umsebenzi we-anti-tumor wamaseli e-T. Kuye kwafakazelwa ukuthi ukuvinjelwa kwe-coenzyme tetrahydrobiopterin (BH4) kungalimaza ukwanda kwamaseli e-T, futhi ukwanda kwe-BH4 emzimbeni kungathuthukisa impendulo ye-anti-tumor eqondiswa yi-CD4 ne-CD8. Ngaphezu kwalokho, umphumela wokucindezela umzimba we-kynurenine ungasindiswa ngokuphathwa kwe-BH4 (5). Ku-isocitrate dehydrogenase (IDH) mutant glioblastoma, ukukhiqizwa kwe-enantiometabolic (R)-2-hydroxyglutarate (R-2-HG) kuvimbela ukusebenza kweseli le-T, ukwanda kanye nomsebenzi we-cytolysis (6). Muva nje, kuboniswe ukuthi i-methylglyoxal, umkhiqizo ophuma ku-glycolysis, ikhiqizwa amaseli okucindezela avela ku-myeloid, kanye nokudluliswa kwamaseli e-T kwe-methylglyoxal kungavimbela ukusebenza kweseli le-T elisebenzayo. Ekwelapheni, ukungathathi hlangothi kwe-methylglyoxal kunganqoba umsebenzi wamaseli okucindezela avela ku-myeloid (MDSC) futhi kuthuthukise ukwelashwa kokuvinjelwa kwe-checkpoint kumamodeli egundane (7). Lezi zifundo zigcizelela ndawonye indima ebalulekile yama-metabolites avela ku-TME ekulawuleni umsebenzi kanye nomsebenzi wamaseli e-T.
Ukungasebenzi kahle kwamaseli e-T kubikwe kabanzi kumdlavuza wesibeletho (8). Lokhu kungenxa yezinye izici ze-metabolic ezitholakala ku-hypoxia kanye ne-tumor vasculature engavamile (9), okuholela ekuguqulweni kwe-glucose ne-tryptophan kube yimikhiqizo efana ne-lactic acid ne-kynurenine. I-lactate eningi kakhulu yangaphandle inciphisa ukukhiqizwa kwe-interferon-γ (IFN-γ) futhi iqhubekisela phambili ukuhlukaniswa kwamaqembu amancane e-myelosuppressive (10, 11). Ukusetshenziswa kwe-tryptophan kuvimbela ngqo ukwanda kwamaseli e-T futhi kuvimbela ukusayinwa kwe-T cell receptor (12-14). Naphezu kwalokhu okubonwe, umsebenzi omningi ozungeze i-metabolic yomzimba wenziwa ekukhuleni kwamaseli e-T in vitro kusetshenziswa imidiya elungiselelwe, noma kukhawulelwe kumamodeli egundane afanayo in vivo, okungekho kuwo okubonisa ngokugcwele ukungafani komdlavuza wabantu kanye nendawo yemvelo ye-macro ne-micro.
Isici esivamile somdlavuza wesibeletho ukusabalala kwe-peritoneal kanye nokuvela kwama-ascites. Ukuqongelela koketshezi lwamaseli kuma-ascites kuhlotshaniswa nesifo esithuthukile kanye nokubikezela okubi (15). Ngokusho kwemibiko, le ndawo eyingqayizivele ayinayo i-hypoxic, inamazinga aphezulu e-vascular endothelial growth factor (VEGF) kanye ne-indoleamine 2,3-dioxygenase (IDO), futhi ingena ngamaseli alawulayo e-T kanye namaseli avimbela i-myeloid (15-18). Indawo yokusebenza kwe-metabolic yama-ascites ingase ihluke kweyesimila ngokwaso, ngakho-ke ukuhlelwa kabusha kwamaseli e-T esikhaleni se-peritoneal akucaci. Ngaphezu kwalokho, umehluko obalulekile kanye nokungafani phakathi kwama-ascites nama-metabolites akhona endaweni yesimila kungavimbela ukungena kwamaseli omzimba kanye nomsebenzi wawo kumathumba, futhi kudingeka ucwaningo olwengeziwe.
Ukuze sixazulule lezi zinkinga, saklama indlela yokuhlukaniswa kwamaseli ebucayi kanye ne-liquid chromatography tandem mass spectrometry (LC-MS/MS) yokufunda izinhlobo ezahlukene zamaseli (kufaka phakathi amaseli e-CD4 + kanye ne-CD8 + T) kanye nangaphakathi naphakathi kwamathumba. Ama-metabolites ayo ahlanganisa amaseli endaweni efanayo yama-ascites kanye nendawo yamathumba yesiguli. Sisebenzisa le ndlela ngokubambisana ne-high-dimensional flow cytometry kanye ne-single-cell RNA sequencing (scRNA-seq) ukuze sinikeze isithombe esixazululiwe kakhulu sesimo se-metabolic salezi zinhlobo ezibalulekile. Le ndlela yembule ukwanda okukhulu kwezinga le-1-methylnicotinamide (MNA) kumaseli e-tumor T, kanti izivivinyo ze-in vitro zikhombisile ukuthi umphumela we-immunomodulatory we-MNA emsebenzini weseli le-T wawungaziwa ngaphambilini. Ngokuvamile, le ndlela yembula ukusebenzisana kwe-metabolic phakathi kwamathumba namaseli omzimba, futhi inikeza ukuqonda okuhlukile kuma-metabolites okulawula amasosha omzimba, okungaba usizo ekwelapheni amathuba okwelashwa komdlavuza we-ovarian osekelwe kumaseli e-T.
Sisebenzise i-high-dimensional flow cytometry ukuze silinganise ngesikhathi esisodwa ukuthathwa kwe-glucose [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)] (7, 19, 20) ziyizimpawu ezivamile eceleni ezihlukanisa amangqamuzana omzimba kanye nenani lamaseli esimila (Ithebula S2 kanye nesithombe S1A). Lokhu kuhlaziywa kubonise ukuthi uma kuqhathaniswa namaseli e-T, ama-ascites namaseli esimila anamazinga aphezulu okuthathwa kwe-glucose, kodwa anomehluko omncane emsebenzini we-mitochondrial. Ukuthathwa kwe-glucose okumaphakathi kwamaseli esimila [CD45-EpCAM (EpCAM)+] kuphindwe kathathu kuya kane kunamaseli e-T, kanti ukuthathwa kwe-glucose okumaphakathi kwamaseli e-CD4 + T kuphindwe ka-1.2 kunamaseli e-CD8 + T, okubonisa ukuthi ama-lymphocyte angena ngesimila (TIL) anezidingo ezahlukene ze-metabolic ngisho naku-TME efanayo (Isithombe 1A). Ngokuphambene nalokho, umsebenzi we-mitochondrial kumaseli esimila ufana nowamangqamuzana e-CD4 + T, kanti umsebenzi we-mitochondrial wazo zombili izinhlobo zamaseli uphakeme kunowamangqamuzana e-CD8 + T (Isithombe 1B). Ngokuvamile, le miphumela yembula izinga lokuguqulwa kwe-metabolic. Umsebenzi wokuguqulwa kwe-metabolic wamaseli esimila uphakeme kunowamangqamuzana e-CD4 + T, kanti umsebenzi wokuguqulwa kwe-metabolic wamaseli e-CD4 + T uphakeme kunowamangqamuzana e-CD8 + T. Naphezu kwale miphumela kuzo zonke izinhlobo zamaseli, akukho mehluko oqhubekayo esimweni sokuguqulwa kwe-metabolic samaseli e-CD4 + kanye ne-CD8 + T noma izilinganiso zawo ezihambisanayo kuma-ascites uma kuqhathaniswa nama-tumors (Isithombe 1C). Ngokuphambene nalokho, engxenyeni yamaseli e-CD45, isilinganiso samaseli e-EpCAM + esimila sanda uma kuqhathaniswa nama-ascites (Isithombe 1D). Siphinde sabona umehluko ocacile we-metabolic phakathi kwezingxenye ze-EpCAM + kanye ne-EpCAM- cell. Amaseli e-EpCAM + (isimila) anomuncwa ophezulu we-glucose kanye nomsebenzi we-mitochondrial kune-EpCAM- cells, okuphakeme kakhulu kunomsebenzi wokuguqulwa kwe-metabolic wama-fibroblast kumaseli esimila ku-TME (Isithombe 1, E kanye no-F).
(A no-B) Ukuqina kwe-fluorescence okumaphakathi (i-MFI) kokuthathwa kwe-glucose (2-NBDG) (A) kanye nomsebenzi we-mitochondrial wamaseli e-CD4 + T (ubomvu obumnyama be-MitoTracker) (B) Amagrafu amele (kwesobunxele) kanye nedatha ebekwe etafuleni (Kwesokudla), amaseli e-CD8 + T kanye namaseli e-EpCAM + CD45-tumor avela kuma-ascites kanye ne-tumor. (C) Isilinganiso samaseli e-CD4 + kanye ne-CD8 + (amaseli e-CD3 + T) kuma-ascites kanye ne-tumor. (D) Isilinganiso samaseli e-EpCAM + tumor kuma-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 (ubomvu obumnyama be-MitoTracker) (F) amagrafu amele (kwesobunxele) kanye nedatha ebekwe etafuleni (Kwesokudla) Ama-Ascites namaseli e-tumor. (G) Amagrafu amele ukubonakaliswa kwe-CD25, CD137 kanye ne-PD1 nge-flow cytometry. (H kanye no-I) Ukubonakaliswa kwe-CD25, i-CD137 kanye ne-PD1 kumaseli e-CD4 + T (H) kanye namaseli e-CD8 + T (I). (J kanye no-K) I-Naive, inkumbulo ephakathi (Tcm), i-effector (Teff) kanye ne-effector memory (Tem) phenotypes ngokusekelwe ekubonakalisweni kwe-CCR7 kanye ne-CD45RO. Izithombe ezimele (kwesobunxele) kanye nedatha yethebula (kwesokudla) yamaseli e-CD4 + T (J) kanye namaseli e-CD8 + T (K) kuma-ascites kanye nama-tumors. Amanani e-P anqunywa yi-t-test ebhangqiwe (*P<0.05, **P<0.01 kanye ***P<0.001). Umugqa umele iziguli ezifanayo (n = 6). I-FMO, i-fluorescence minus eyodwa; i-MFI, ukuqina kwe-fluorescence okumaphakathi.
Ukuhlaziywa okwengeziwe kwembule eminye umehluko obalulekile phakathi kwesimo se-phenotypic samaseli e-T esixazululiwe kakhulu. Imemori esebenzayo (Isithombe 1, G kuya ku-I) kanye nememori ye-effector (Isithombe 1, J kanye no-K) kuma-tumors ivame kakhulu kune-ascites (isilinganiso samaseli e-CD3 + T). Ngokufanayo, ukuhlaziya i-phenotype ngokubonakaliswa kwama-activation markers (CD25 kanye ne-CD137) kanye nama-depletion markers [iphrotheni yokufa kwamaseli ehlelwe 1 (PD1)] kubonise ukuthi yize izici ze-metabolic zalezi zinhlobo zihlukile (Isithombe S1, B kuya ku-E), Kodwa akukho mehluko obalulekile we-metabolic owabonwa njalo phakathi kwama-naive, ama-effector noma ama-memory subsets (Isithombe S1, F kuya ku-I). Le miphumela yaqinisekiswa ngokusebenzisa izindlela zokufunda komshini ukuze kwabelwe ngokuzenzakalelayo ama-phenotypes amaseli (21), okwaveza nokuba khona kwenani elikhulu lamaseli omnkantsha (CD45 + / CD3- / CD4 + / CD45RO +) kuma-ascites esiguli (Isithombe S2A). Phakathi kwazo zonke izinhlobo zamaseli ezitholiwe, leli nani lamaseli e-myeloid libonise ukumuncwa okuphezulu kakhulu kwe-glucose kanye nomsebenzi we-mitochondrial (Isithombe S2, B kuya ku-G). Le miphumela iqokomisa umehluko omkhulu we-metabolic phakathi kwezinhlobo zamaseli amaningi ezitholakala kuma-ascites kanye nama-tumors ezigulini ze-HGSC.
Inselele enkulu ekuqondeni izici ze-metabonomic ze-TIL yisidingo sokuhlukanisa amasampula e-T cell ahlanzekile ngokwanele, ikhwalithi kanye nobuningi obuvela ezimilanjeni. Izifundo zamuva zibonise ukuthi izindlela zokuhlunga kanye nokucebisa ubuhlalu ezisekelwe ku-flow cytometry zingaholela ezinguqukweni kumaphrofayili e-metabolite eseli (22-24). Ukuze sinqobe le nkinga, senze ngcono indlela yokucebisa ubuhlalu ukuze ihlukanise futhi ihlukanise i-TIL kumdlavuza wesibeletho womuntu osuswe ngokuhlinzwa ngaphambi kokuhlaziywa yi-LC-MS/MS (bheka Izinto Nezindlela; Umfanekiso 2A). Ukuze sihlole umthelela ophelele walesi simiso ekushintsheni kwe-metabolite, siqhathanise amaphrofayili e-metabolite amaseli e-T asebenze ngabanikeli abanempilo ngemuva kwesinyathelo sokuhlukaniswa kobuhlalu esingenhla namaseli angahlukaniswanga ubuhlalu kodwa ahlala eqhweni. Lokhu kuhlaziywa kokulawula ikhwalithi kuthole ukuthi kukhona ukuhlobana okuphezulu phakathi kwalezi zimo ezimbili (r = 0.77), futhi ukuphindaphindeka kobuchwepheshe kweqembu lama-metabolite angu-86 kunokuphindaphindeka okuphezulu (Isithombe 2B). Ngakho-ke, lezi zindlela zingenza ukuhlaziywa kwe-metabolite okunembile kumaseli athuthukiswa uhlobo lwamaseli, ngaleyo ndlela zinikeze ipulatifomu yokuqala enesinqumo esiphezulu sokuhlonza ama-metabolite athile ku-HGSC, ngaleyo ndlela zenze abantu bakwazi ukuthola ukuqonda okujulile ngokucaciswa kwamaseli Uhlelo lokusebenzisa umzimba ngokocansi.
(A) Umdwebo weskimu wokucebisa ubuhlalu be-magnetic. Ngaphambi kokuhlaziywa yi-LC-MS/MS, amaseli azodlula emizuliswaneni emithathu elandelanayo yokucebisa ubuhlalu be-magnetic noma ahlale eqhweni. (B) Umphumela wohlobo lokucebisa ebuningini bama-metabolites. Isilinganiso sezilinganiso ezintathu zohlobo ngalunye lokucebisa ± SE. Umugqa ompunga umele ubudlelwano obungu-1:1. Ubudlelwano bangaphakathi kwekilasi (ICC) bokulinganisa okuphindaphindwayo okuboniswe kulebula ye-axis. I-NAD, i-nicotinamide adenine dinucleotide. (C) Umdwebo weskimu wokusebenza kokuhlaziywa kwe-metabolite yesiguli. Ama-Ascites noma amathumba aqoqwa ezigulini futhi agcinwe. Ingxenye encane yesampula ngayinye yahlaziywa nge-flow cytometry, kuyilapho amasampula asele edlula emizuliswaneni emithathu yokucebisa amaseli e-CD4+, CD8+ kanye ne-CD45. Lezi zingxenyana zamaseli zahlaziywa kusetshenziswa i-LC-MS/MS. (D) Imephu yokushisa yobuningi be-metabolite obujwayelekile. I-dendrogram imelela ukuhlanganiswa kukaWard kwamabanga e-Euclidean phakathi kwamasampula. (E) Ukuhlaziywa kwengxenye eyinhloko (i-PCA) yemephu yesampula ye-metabolite, ekhombisa amakhophi amathathu esampula ngayinye, amasampula avela esigulini esifanayo axhunywe ngomugqa. (F) I-PCA yephrofayili ye-metabolite yesampula ebekwe esigulini (okungukuthi, kusetshenziswa ukuphindaphinda okungaphelele); uhlobo lwesampula lunqunyelwe yi-convex hull. I-PC1, ingxenye eyinhloko 1; I-PC2, ingxenye eyinhloko 2.
Okulandelayo, sisebenzise le ndlela yokucebisa ukuhlaziya ama-metabolite angu-99 kuma-CD4+, CD8+ kanye nama-CD45-cell fractions kuma-ascites ayinhloko kanye nama-tumors eziguli eziyisithupha ze-HGSC (Isithombe 2C, Umfanekiso S3A kanye neThebula S3 kanye ne-S4). Inani labantu abanesithakazelo libalelwa ku-2% kuya ku-70% wesampula enkulu yokuqala yamaseli aphilayo, kanti inani lamaseli liyahlukahluka kakhulu phakathi kweziguli. Ngemva kokuhlukanisa ama-beads, ingxenye ecebile yesithakazelo (i-CD4+, i-CD8+ noma i-CD45-) ibalwa ngaphezu kuka-85% wawo wonke amaseli aphilayo kusampula ngokwesilinganiso. Le ndlela yokucebisa isivumela ukuthi sihlaziye inani lamaseli kusuka kumetabolism yezicubu zomdlavuza womuntu, okungenakwenzeka ukwenza kumasampula amakhulu. Sisebenzisa le protocol, sithole ukuthi i-l-kynurenine kanye ne-adenosine, lawa ma-metabolite amabili acacile okucindezela amasosha omzimba aphakanyisiwe kumaseli e-T omdlavuza noma kumaseli omdlavuza (Isithombe S3, B kanye no-C). Ngakho-ke, le miphumela ibonisa ukwethembeka kanye nekhono lobuchwepheshe bethu bokuhlukaniswa kwamaseli kanye ne-mass spectrometry lokuthola ama-metabolite abalulekile ngokwebhayoloji ezicutshini zeziguli.
Ukuhlaziywa kwethu kuphinde kwembule ukuhlukaniswa okunamandla kwezinhlobo zamaseli ngaphakathi naphakathi kweziguli (Isithombe 2D kanye nesithombe S4A). Ikakhulukazi, uma kuqhathaniswa nezinye iziguli, isiguli esingu-70 sibonise izici ezahlukene ze-metabolic (Isithombe 2E kanye nesithombe S4B), okubonisa ukuthi kungaba nokungafani okukhulu kwe-metabolic phakathi kweziguli. Kubalulekile ukuqaphela ukuthi uma kuqhathaniswa nezinye iziguli (1.2 kuya ku-2 amalitha; Ithebula S1), inani eliphelele lama-ascites aqoqwe esigulini esingu-70 (80 ml) lalilincane. Ukulawulwa kokungafani phakathi kweziguli ngesikhathi sokuhlaziywa kwezingxenye eziyinhloko (isibonelo, kusetshenziswa ukuhlaziywa kokuphindaphinda okungaphelele) kubonisa izinguquko eziqhubekayo phakathi kwezinhlobo zamaseli, kanti izinhlobo zamaseli kanye/noma imvelo encane zihlanganiswa ngokucacile ngokwephrofayili ye-metabolite (Isithombe 2F). Ukuhlaziywa kwama-metabolite angawodwa kugcizelele le miphumela futhi kwembulwe umehluko omkhulu phakathi kwezinhlobo zamaseli kanye nemvelo encane. Kubalulekile ukuqaphela ukuthi umehluko omkhulu kakhulu obonwe yi-MNA, evame ukucebiswa kumaseli e-CD45 kanye namaseli e-CD4+ kanye ne-CD8+ angena ngaphakathi kwesimila (Isithombe 3A). Kumaseli e-CD4 +, lo mphumela usobala kakhulu, kanti i-MNA kumaseli e-CD8 + nayo ibonakala ithinteka kakhulu yindawo ezungezile. Kodwa-ke, lokhu akubalulekile, ngoba iziguli ezintathu kuphela kweziyisithupha ezingahlolwa ukuze kutholakale amaphuzu e-CD8 + esimila. Ngaphezu kwe-MNA, ezinhlotsheni ezahlukene zamaseli kuma-ascites namathumba, amanye ama-metabolite angachazwanga kahle ku-TIL nawo acebile ngokuhlukile (Izibalo S3 kanye ne-S4). Ngakho-ke, le datha yembula isethi ethembisayo yama-metabolite okulungisa amasosha omzimba ukuze kwenziwe ucwaningo olwengeziwe.
(A) Okuqukethwe okujwayelekile kwe-MNA kumaseli e-CD4+, CD8+ kanye ne-CD45- avela kuma-ascites kanye ne-tumor. Isakhiwo sebhokisi sibonisa ububanzi obuphakathi (umugqa), ububanzi be-interquartile (i-frame hinge) kanye nobubanzi bedatha, kufika ku-1.5 izikhathi ze-interquartile range (uhlaka lwe-whisker). Njengoba kuchaziwe ku-Patient Materials and Methods, sebenzisa inani le-limma lesiguli ukunquma inani le-P (*P<0.05 kanye ne-**P<0.01). (B) Umdwebo we-schematic we-metabolism ye-MNA (60). Ama-Metabolites: I-S-adenosyl-1-methionine; I-SAH, i-S-adenosine-1-homocysteine; I-NA, i-nicotinamide; I-MNA, i-1-methylnicotinamide; I-2-PY, i-1-methyl-2-pyridone-5-carboxamide; I-4-PY, i-1-methyl-4-pyridone-5-carboxamide; I-NR, i-nicotinamide ribose; I-NMN, i-nicotinamide mononucleotide. Ama-enzyme (aluhlaza): i-NNMT, i-nicotinamide N-methyltransferase; i-SIRT, ama-sirtuin; i-NAMPT, i-nicotinamide phosphoribosyl transferase; i-AOX1, i-aldehyde oxidase 1; i-NRK, i-nicotinamide riboside kinase; i-NMNAT, i-nicotinamide mono Nucleotide adenylate transferase; i-Pnp1, i-purine nucleoside phosphorylase. (C) i-t-SNE ye-scRNA-seq yama-ascites (grey) kanye ne-tumor (ebomvu; n = iziguli ezingu-3). (D) Ukuvezwa kwe-NNMT emiphakathini ehlukene yamaseli ekhonjwe kusetshenziswa i-scRNA-seq. (E) Ukuvezwa kwe-NNMT kanye ne-AOX1 kumaseli e-SK-OV-3, i-human embryonic kidney (HEK) 293T, amaseli e-T kanye namaseli e-T aphathwe nge-MNA. Ukuvezwa okugoqiwe kuboniswa maqondana ne-SK-OV-3. Iphethini yokubonakaliswa ene-SEM iboniswa (n = abanikeli abangu-6 abanempilo). Amanani e-Ct angaphezu kuka-35 abhekwa njengangatholakali (UD). (F) Ukuvezwa kwe-SLC22A1 kanye ne-SLC22A2 kumaseli e-SK-OV-3, HEK293T, T kanye namaseli e-T aphathwe nge-8mM MNA. Ukuvezwa okugoqiwe kuboniswa uma kuqhathaniswa ne-SK-OV-3. Iphethini yokuvezwa ene-SEM iboniswa (n = 6 abanikeli abaphilile). Amanani e-Ct angaphezu kuka-35 abhekwa njengangatholakali (UD). (G) Okuqukethwe kwe-MNA yamaseli kumaseli e-T abanikeli abaphilile asebenzayo ngemuva kwamahora angu-72 okufuthwa nge-MNA. Iphethini yokuvezwa ene-SEM iboniswa (n = 4 abanikeli abaphilile).
I-MNA ikhiqizwa ngokudlulisa iqembu le-methyl kusuka ku-S-adenosyl-1-methionine (SAM) kuya ku-nicotinamide (NA) yi-nicotinamide N-methyltransferase (NNMT; Isithombe 3B). I-NNMT ivame kakhulu ezinhlobonhlobo zomdlavuza womuntu futhi ihlotshaniswa nokwanda, ukuhlasela kanye nokwanda kwe-metastasis (25-27). Ukuze siqonde kangcono umthombo we-MNA kumaseli e-T ku-TME, sisebenzise i-scRNA-seq ukuchaza ukuvezwa kwe-NNMT kuzo zonke izinhlobo zamaseli kuma-ascites nama-tumors eziguli ezintathu ze-HGSC (Ithebula S5). Ukuhlaziywa kwamaseli angaba ngu-6,500 kubonise ukuthi ezindaweni zama-ascites nama-tumors, ukubonakaliswa kwe-NNMT kwakukhawulelwe kumaseli e-fibroblast namaseli e-tumors acatshangelwayo (Isithombe 3, C no-D). Kubalulekile ukuqaphela ukuthi akukho ukubonakaliswa kwe-NNMT okusobala kunoma yiliphi inani labantu eliveza i-PTPRC (CD45 +) (Isithombe 3D kanye nesithombe S5A), okubonisa ukuthi i-MNA etholakale ku-metabolite spectrum ingeniswe kumaseli e-T. Ukuvezwa kwe-aldehyde oxidase 1 (AOX1) kuguqula i-MNA ibe yi-1-methyl-2-pyridone-5-carboxamide (2-PYR) noma i-1-methyl-4-pyridone-5-carboxamide (4-PYR); Isithombe 3B) sikhawulelwe futhi kubantu abaningi be-fibroblast abaveza i-COL1A1 (Isithombe S5A), okubonisa ukuthi amaseli e-T awanamandla okusebenza kwe-metabolism ye-MNA evamile. Iphethini yokubonakaliswa kwalezi zakhi zofuzo ezihlobene ne-MNA yaqinisekiswa kusetshenziswa isethi yesibili yedatha yamaseli azimele evela kuma-ascites avela ezigulini ze-HGSC (Isithombe S5B; n = 6) (16). Ngaphezu kwalokho, ukuhlaziywa kwe-quantitative polymerase chain reaction (qPCR) kwamaseli e-T anikelayo anempilo aphathwe nge-MNA kubonise ukuthi uma kuqhathaniswa namaseli okulawula i-SK-OV-3 ovarian tumor, i-NNMT noma i-AOX1 cishe ayivezwanga (Isithombe 3E). Le miphumela engalindelekile ikhombisa ukuthi i-MNA ingase ikhishwe kuma-fibroblast noma emathunjini iye kumaseli e-T aseduze ku-TME.
Nakuba abantu abazongenela ukhetho bahlanganisa umndeni wabathuthi be-organic cation 1 kuya ku-3 (OCT1, OCT2 kanye no-OCT3) abafakwe ikhodi ngumndeni we-soluble carrier 22 (SLC22) (SLC22A1, SLC22A2 kanye no-SLC22A3), abathuthi abangaba khona be-MNA basalokhu bengachazwanga (28). I-QPCR ye-mRNA evela kumaseli e-T anikelayo anempilo ibonise amazinga aphansi okuvezwa kwe-SLC22A1 kodwa amazinga angabonakali e-SLC22A2, okuqinisekisile ukuthi ibike ngaphambilini ezincwadini (Isithombe 3F) (29). Ngokuphambene nalokho, umugqa weseli lesimila se-ovarian SK-OV-3 uveze amazinga aphezulu wabo bobabili abathuthi (Isithombe 3F).
Ukuze kuhlolwe ukuthi kungenzeka yini ukuthi amaseli e-T anekhono lokumunca i-MNA yangaphandle, amaseli e-T anikelayo anempilo akhuliswa amahora angama-72 lapho kukhona amazinga ahlukene e-MNA. Uma kungekho i-MNA yangaphandle, okuqukethwe kweseli kwe-MNA akukwazi ukutholakala (Isithombe 3G). Kodwa-ke, amaseli e-T asebenzayo aphathwe nge-MNA yangaphandle abonise ukwanda okuncike kumthamo kokuqukethwe kwe-MNA emaseli, kufika ku-6 mM MNA (Isithombe 3G). Lo mphumela ubonisa ukuthi naphezu kwezinga eliphansi lokubonakaliswa kwe-transporter kanye nokuntuleka kwe-enzyme eyinhloko ebangela i-metabolism ye-MNA yangaphakathi kweseli, i-TIL isengabamba i-MNA.
Ububanzi bama-metabolites kumaseli e-T eziguli kanye nokuhlolwa kokumuncwa kwe-MNA ngaphakathi kwesivitho kwandisa amathuba okuthi ama-fibroblast ahlobene nomdlavuza (CAF) akhiphe i-MNA kanye namaseli esimila angalawula i-phenotype kanye nomsebenzi we-TIL. Ukuze kutholakale umphumela we-MNA kumaseli e-T, amaseli e-T anikelayo anempilo avuselelwa ngaphakathi kwesivitho lapho kukhona noma kungekho khona i-MNA, futhi ukwanda kwawo kanye nokukhiqizwa kwe-cytokine kwahlolwa. Ngemva kwezinsuku eziyi-7 zokwengeza i-MNA kumthamo ophezulu kakhulu, inani lokuphindwa kabili kwabantu lancishiswa ngokulinganisela, kuyilapho amandla agcinwa kuzo zonke izilinganiso (Isithombe 4A). Ngaphezu kwalokho, ukwelashwa kwe-MNA yangaphandle kwaholela ekwandeni kwesilinganiso samaseli e-CD4 + kanye ne-CD8 + T aveza i-tumor necrosis factor-α (TNFα; Isithombe 4B). Ngokuphambene nalokho, ukukhiqizwa kwe-IFN-γ ngaphakathi kweseli kwancishiswa kakhulu kumaseli e-CD4 + T, kodwa hhayi kumaseli e-CD8 + T, futhi akukho shintsho olubalulekile ku-interleukin 2 (IL-2; Isithombe 4, C kanye no-D). Ngakho-ke, ukuhlolwa kwe-immunosorbent okuxhunywe yi-enzyme (ELISA) kwama-supernatant avela kula ma-T cell cultures aphathwe yi-MNA kubonise ukwanda okukhulu kwe-TNFα, ukwehla kwe-IFN-γ, kanye nokungabikho koshintsho ku-IL-2 (Isithombe 4, E kuya ku-G). . Ukwehla kwe-IFN-γ kubonisa ukuthi i-MNA ingadlala indima ekuvimbeleni umsebenzi wokulwa nomdlavuza wamaseli e-T. Ukuze kulingiswe umphumela we-MNA ku-cytotoxicity ebangelwa yi-T cell, amaseli e-chimeric antigen receptor T (FRα-CAR-T) aqondisa i-folate receptor α kanye namaseli e-CAR-T (GFP) alawulwa amaseli e-green fluorescent protein (GFP) -CAR-T akhiqizwa ngamaseli e-peripheral blood mononuclear anempilo (PBMC). Amaseli e-CAR-T akhuliswa amahora angama-24 lapho kukhona i-MNA, abese ekhuliswa namaseli e-ovarian tumor e-SK-OV-3 yomuntu aveza i-folate receptor α ku-effector kuya ku-target ratio engu-10:1. Ukwelashwa kwe-MNA kuholele ekunciphiseni okukhulu komsebenzi wokubulala wamaseli e-FRα-CAR-T, okwakufana namaseli e-FRα-CAR-T aphathwe nge-adenosine (Isithombe 4H).
(A) Inani lamaseli asebenzayo kanye nokuphindaphinda kwenani labantu (PD) ngqo kusuka ekukhuleni ngosuku lwesi-7. Igrafu yebha imelela isilinganiso + SEM sabanikeli abayisithupha abaphilile. Imelela idatha evela okungenani ku-n = 3 ukuhlolwa okuzimele. (B kuya ku-D) I-CD3/CD28 kanye ne-IL-2 kwasetshenziswa ukwenza amaseli e-T asebenze ekugxilweni kwawo kwe-MNA izinsuku eziyi-7. Ngaphambi kokuhlaziywa, amaseli avuselelwa nge-PMA/ionomycin nge-GolgiStop amahora ama-4. Ukubonakaliswa kwe-TNFα (B) kumaseli e-T. Isithombe sesibonelo (kwesobunxele) kanye nedatha yethebula (kwesokudla) yokubonakaliswa kwe-TNFα kumaseli aphilayo. Ukubonakaliswa kwe-IFN-γ (C) kanye ne-IL-2 (D) kumaseli e-T. Ukubonakaliswa kwama-cytokine kwalinganiswa nge-flow cytometry. Igrafu yebha imelela isilinganiso (n = 6 abanikeli abaphilile) + SEM. Sebenzisa ukuhlaziywa kwendlela eyodwa kokuhlukahluka kanye nezilinganiso eziphindaphindwayo (*P<0.05 kanye **P<0.01) ukunquma inani le-P. Imelela idatha evela okungenani ku-n = 3 ukuhlolwa okuzimele. (E kuya ku-G) i-CD3/CD28 kanye ne-IL-2 zisetshenziswe ukwenza amaseli e-T asebenze ekugxilweni kwawo kwe-MNA izinsuku eziyi-7. Le medium yaqoqwa ngaphambi nangemva kwamahora ama-4 okukhuthazwa kwe-PMA/ionomycin. Ukugxilwa kwe-TNFα (E), i-IFN-γ (F) kanye ne-IL-2 (G) kwalinganiswa yi-ELISA. Igrafu yebha imelela isilinganiso (n = 5 abanikeli abanempilo) + i-SEM. Inani le-P linqunywe kusetshenziswa ukuhlaziywa kwendlela eyodwa kokuhlukahluka kanye nokulinganisa okuphindaphindiwe (*P<0.05). Umugqa onamachashazi ukhombisa umkhawulo wokutholwa kokutholwa. (H) Ukuhlolwa kwe-cell lysis. Amaseli e-FRα-CAR-T noma e-GFP-CAR-T alungiswe nge-adenosine (250μM) noma i-MNA (10 mM) amahora angama-24, noma ashiywa engalashwanga (Ctrl). Ukubulawa kwamaphesenti kwamaseli e-SK-OV-3 kwalinganiswa. Inani le-P linqunywe ukuhlolwa kwe-Welch t (*P<0.5 kanye ne-**P<0.01).
Ukuze kutholakale ukuqonda okusebenzayo kokulawulwa kokubonakaliswa kwe-TNFα okuncike ku-MNA, kuhlolwe izinguquko ku-TNFα mRNA yamaseli e-T aphathwe nge-MNA (Isithombe 5A). Amaseli e-T anikelayo anempilo aphathwe nge-MNA abonise ukwanda okuphindwe kabili kwamazinga okubhalwa kwe-TNFα, okubonisa ukuthi i-MNA incike kukulawulwa kokubhalwa kwe-TNFα. Ukuze kuhlolwe le ndlela yokulawula engenzeka, izici ezimbili zokubhalwa ezaziwayo ezilawula i-TNFα, okungukuthi i-activated T cell nuclear factor (NFAT) kanye neprotheni ethile 1 (Sp1), zahlolwa ngokuphendula ukubopha kwe-MNA kumgqugquzeli we-TNFα oseduze (30). Umgqugquzeli we-TNFα uqukethe izindawo ezi-6 zokubopha ze-NFAT ezikhonjwe kanye nezindawo ezi-2 zokubopha ze-Sp1, ezihambisana endaweni eyodwa [-55 base pairs (bp) kusuka ku-5'cap] (30). I-Chromatin immunoprecipitation (ChIP) ibonise ukuthi lapho iphathwa nge-MNA, ukubopha kwe-Sp1 kumgqugquzeli we-TNFα kwanda kathathu. Ukufakwa kwe-NFAT nakho kwanda futhi kwasondela ekubalulekeni (Isithombe 5B). Le datha ikhombisa ukuthi i-MNA ilawula ukuvezwa kwe-TNFα ngokubhalwa kwe-Sp1, kanye nokuvezwa kwe-NFAT ngezinga elincane.
(A) Uma kuqhathaniswa namaseli e-T akhuliswe ngaphandle kwe-MNA, ushintsho lokugoqa kokubonakaliswa kwe-TNFα kumaseli e-T aphathwe nge-MNA. Iphethini yokubonakaliswa nge-SEM iyaboniswa (n = 5 abanikeli abaphilile). Imelela idatha evela okungenani ku-n = 3 ukuhlolwa okuzimele. (B) Umthuthukisi we-TNFα wamaseli e-T aphathwe nge-8 mM MNA noma ngaphandle kwayo ngemuva kwe-NFAT ne-Sp1 kuhlanganiswe ne-(Ctrl) kanye ne-PMA/ionomycin stimulation amahora ama-4. I-Immunoglobulin G (IgG) kanye ne-H3 kusetshenziswe njengezilawuli ezingezinhle nezinhle zokuvikela amasosha omzimba, ngokulandelana. Ukulinganiswa kwe-ChIP kubonise ukuthi ukubopha kwe-Sp1 kanye ne-NFAT kumthuthukisi we-TNFα kumaseli aphathwe nge-MNA kukhuphuke izikhathi eziningana uma kuqhathaniswa nokulawula. Imelela idatha evela okungenani ku-n = 3 ukuhlolwa okuzimele. Inani le-P elinqunywe yizivivinyo eziningi ze-t (*** P <0.01). (C) Uma kuqhathaniswa nama-ascites e-HGSC, amaseli e-T (angewona ama-cytotoxic) abonise ukubonakaliswa okwandisiwe kwe-TNF ku-tumor. Imibala imelela iziguli ezahlukene. Amaseli abonisiwe athathwe amasampula ngokungahleliwe abe ngu-300 futhi ajikijelwa ukuze kuncishiswe ukudonswa ngokweqile (** Padj = 0.0076). (D) Imodeli ephakanyisiwe ye-MNA yomdlavuza wesibeletho. I-MNA ikhiqizwa kumaseli esimila kanye nama-fibroblast ku-TME futhi ithathwa amaseli e-T. I-MNA ikhulisa ukubopha kwe-Sp1 kumgqugquzeli we-TNFα, okuholela ekwandeni kokubhalwa kwe-TNFα kanye nokukhiqizwa kwe-cytokine ye-TNFα. I-MNA ibangela nokwehla kwe-IFN-γ. Ukuvinjelwa komsebenzi wamaseli e-T kuholela ekunciphiseni ikhono lokubulala kanye nokukhula kwesimila okusheshayo.
Ngokusho kwemibiko, i-TNFα inemiphumela yokulwa nomdlavuza kanye nokulwa nomdlavuza encike ngaphambili nangemuva, kodwa inendima eyaziwayo ekukhuthazeni ukukhula kanye nokusabalala komdlavuza wesibeletho (31-33). Ngokusho kwemibiko, ukuhlushwa kwe-TNFα kuma-ascites kanye nezicubu zesibeletho ezigulini ezinomdlavuza wesibeletho kuphakeme kunalokho okusezicutshini ezingengozini (34-36). Ngokuphathelene nendlela esebenza ngayo, i-TNFα ingalawula ukusebenza, ukusebenza kanye nokwanda kwamaseli amhlophe egazi, futhi ishintshe uhlobo lwamaseli omdlavuza (37, 38). Ngokuhambisana nalokhu okutholakele, ukuhlaziywa kokubonakaliswa kwezakhi zofuzo okuhlukile kubonise ukuthi i-TNF yayilawulwa kakhulu kumaseli e-T ezicutshini zesibeletho uma kuqhathaniswa nama-ascites (Isithombe 5C). Ukwanda kokubonakaliswa kwe-TNF kwabonakala kuphela kubantu bamaseli e-T abane-phenotype engeyona i-cytotoxic (Isithombe S5A). Ngamafuphi, le datha isekela umbono wokuthi i-MNA inemiphumela emibili yokucindezela amasosha omzimba kanye nokukhuthaza isibeletho ku-HGSC.
Ukufakwa kwelebula kwe-fluorescent okusekelwe ku-flow cytometry sekuyindlela eyinhloko yokufunda i-metabolism ye-TIL. Lezi zifundo zibonise ukuthi uma kuqhathaniswa nama-lymphocyte egazi angaphandle noma amaseli e-T avela ezithweni ze-lymphoid yesibili, i-murine kanye ne-TIL yabantu inomkhuba omkhulu wokudonsa i-glucose (4, 39) kanye nokulahlekelwa kancane kancane komsebenzi we-mitochondrial (19, 40). Nakuba sibonile imiphumela efanayo kulolu cwaningo, intuthuko eyinhloko ukuqhathanisa i-metabolism yamaseli esimila kanye ne-TIL evela ezicutshini ezifanayo zesimila ezisusiwe. Ngokuvumelana neminye yale mibiko yangaphambilini, amaseli esimila (CD45-EpCAM +) avela kuma-ascites namathumba anokungenisa i-glucose okuphezulu kunamaseli e-CD8 + kanye ne-CD4 + T, okusekela ukuthi ukungenisa i-glucose ephezulu kwamaseli esimila kungaqhathaniswa namaseli e-T. Umqondo wokuncintisana kwamaseli e-T. I-TME. Kodwa-ke, umsebenzi we-mitochondrial wamaseli esimila uphakeme kunowamaseli e-CD8 + T, kodwa umsebenzi we-mitochondrial ufana nowamaseli e-CD4 + T. Le miphumela iqinisa ingqikithi evelayo yokuthi i-metabolism ye-oxidative ibalulekile kumaseli esimila (41, 42). Baphinde basikisele ukuthi amaseli e-CD8 + T angase abe sengozini enkulu yokungasebenzi kahle kwe-oxidative kunamaseli e-CD4 + T, noma ukuthi amaseli e-CD4 + T angasebenzisa imithombo yekhabhoni ngaphandle kwe-glucose ukuze alondoloze umsebenzi we-mitochondrial (43, 44). Kufanele kuqashelwe ukuthi asibonanga mehluko ekuthathweni kwe-glucose noma emsebenzini we-mitochondrial phakathi kwama-effector e-CD4 + T, inkumbulo ye-T effector kanye namaseli enkumbulo ephakathi e-T kuma-ascites. Ngokufanayo, isimo sokwehlukanisa samaseli e-CD8 + T kuma-tumors asinalutho oluhlobene noshintsho ekuthathweni kwe-glucose, okugqamisa umehluko obalulekile phakathi kwamaseli e-T akhuliswe ku-vitro kanye ne-TIL yomuntu ku-vivo (22). Lokhu okubonwe kwaqinisekiswa futhi ngokusetshenziswa kokwabiwa kwenani lamaseli okuzenzakalelayo okungachemile, okwaveza futhi ukuthi amaseli e-CD45 + / CD3- / CD4 + / CD45RO + anokudonswa kwe-glucose okuphezulu kanye nomsebenzi we-mitochondrial kunamaseli esimila avamile kodwa anenani lamaseli asebenzayo e-Metabolic. Leli nani lingase limelele inani elincane lamaseli e-myeloid suppressor noma amaseli e-plasmacytoid dendritic atholakale ekuhlaziyweni kwe-scRNA-seq. Nakuba zombili lezi zibikwe ezimilanjeni zesibeletho somuntu [45], zisadinga umsebenzi owengeziwe ukuchaza lokhu kuhlanganiswa kwe-myeloid.
Nakuba izindlela ezisekelwe ku-flow cytometry zingacacisa umehluko ojwayelekile ku-glucose kanye ne-oxidative metabolism phakathi kwezinhlobo zamaseli, ama-metabolite aqondile akhiqizwa yi-glucose noma eminye imithombo yekhabhoni ye-metabolism ye-mitochondrial ku-TME awakatholakali okwamanje. Ukunikeza ubukhona noma ukungabikho kwama-metabolite ku-TIL subset ethile kudinga ukuhlanzwa kwenani lamaseli kusuka ezicutshini ezikhishwe. Ngakho-ke, indlela yethu yokucebisa amaseli ehlanganiswe ne-mass spectrometry inganikeza ukuqonda ngama-metabolite acebiswe ngokuhlukile kumaseli e-T kanye nenani lamaseli esimila kumasampula eziguli afanayo. Nakuba le ndlela inezinzuzo ngaphezu kokuhlunga kwamaseli asebenze nge-fluorescence, amanye amalabhulali e-metabolite angathinteka ngenxa yokuzinza okungokwemvelo kanye/noma izinga lokujika okusheshayo (22). Noma kunjalo, indlela yethu yakwazi ukuhlonza ama-metabolite amabili aqashelwayo okucindezela amasosha omzimba, i-adenosine ne-kynurenine, ngoba ahluka kakhulu phakathi kwezinhlobo zamasampula.
Ukuhlaziywa kwethu kwe-metabonomic kwama-tumors kanye nezinhlobo ze-TIL kunikeza ukuqonda okwengeziwe ngendima yama-metabolites ku-TME yama-ovari. Okokuqala, sisebenzisa i-flow cytometry, sithole ukuthi akukho mehluko emsebenzini we-mitochondrial phakathi kwama-tumors nama-CD4 + T cells. Kodwa-ke, ukuhlaziywa kwe-LC-MS/MS kwembule izinguquko ezibalulekile ebuningini bama-metabolites phakathi kwalaba bantu, okubonisa ukuthi iziphetho mayelana ne-metabolism ye-TIL kanye nomsebenzi wayo wonke we-metabolism kudinga ukuchazwa ngokucophelela. Okwesibili, i-MNA iyi-metabolite enomehluko omkhulu phakathi kwama-CD45-cell nama-T cells kuma-ascites, hhayi ama-tumors. Ngakho-ke, ukuhlukanisa kanye nendawo yama-tumors kungaba nemiphumela ehlukene ku-metabolism ye-TIL, okugqamisa ukungafani okungenzeka endaweni encane ethile. Okwesithathu, ukubonakaliswa kwe-enzyme ekhiqiza i-MNA i-NNMT kukhawulelwe kakhulu ku-CAF, okungama-tumors ngokwezinga elincane, kodwa amazinga e-MNA atholakalayo abonwa kuma-T cells atholakala kuma-tumors. Ukuvezwa ngokweqile kwe-NNMT ku-CAF yama-ovari kunomphumela owaziwayo wokukhuthaza umdlavuza, ngokwengxenye ngenxa yokukhuthazwa kwe-metabolism ye-CAF, ukuhlasela kwama-tumors kanye ne-metastasis (27). Nakuba izinga lilonke le-TIL lilinganiselwe, ukuvezwa kwe-NNMT ku-CAF kuhlobene eduze ne-Cancer Genome Atlas (TCGA) mesenchymal subtype, ehlotshaniswa nokubikezela okubi (27, 46, 47). Okokugcina, ukuvezwa kwe-enzyme AOX1 ebangela ukuwohloka kwe-MNA nakho kukhawulelwe kubantu be-CAF, okubonisa ukuthi amaseli e-T awakwazi ukugaya i-MNA. Le miphumela isekela umbono wokuthi nakuba kudingeka umsebenzi owengeziwe ukuqinisekisa lokhu okutholakele, amazinga aphezulu e-MNA kumaseli e-T angabonisa ukuba khona kwe-CAF microenvironment ecindezela amasosha omzimba.
Njengoba izinga lokuvezwa eliphansi lama-transporter e-MNA kanye namazinga angabonakali amaprotheni abalulekile ahilelekile ekusetshenzisweni kwe-MNA, ukuba khona kwe-MNA kumaseli e-T akulindelekile. I-NNMT noma i-AOX1 azitholakalanga ngokuhlaziywa kwe-scRNA-seq kanye ne-qPCR eqondiwe yamaqoqo amabili azimele. Le miphumela ikhombisa ukuthi i-MNA ayikhiqizwanga amaseli e-T, kodwa imuncwa yi-TME ezungezile. Ukuhlolwa kwe-in vitro kubonisa ukuthi amaseli e-T avame ukuqongelela i-MNA yangaphandle.
Izifundo zethu ze-in vitro zibonise ukuthi i-MNA yangaphandle ibangela ukuvezwa kwe-TNFα kumaseli e-T futhi ithuthukisa ukubopha kwe-Sp1 kumgqugquzeli we-TNFα. Nakuba i-TNFα inomsebenzi wokulwa nomdlavuza kanye nowokulwa nomdlavuza, kumdlavuza wesibeletho, i-TNFα ingakhuthaza ukukhula komdlavuza wesibeletho (31-33). Ukungapheleli kwe-TNFα ekukhuleni kwamaseli esibeletho sesibeletho sesibeletho noma ukuqedwa kwesignali ye-TNFα kumamodeli egundane kungathuthukisa ukukhiqizwa kwe-cytokine yokuvuvukala okubangelwa yi-TNFα futhi kuvimbele ukukhula kwesibeletho (32, 35). Ngakho-ke, kulokhu, i-MNA etholakala ku-TME ingasebenza njenge-metabolite ekhuthaza ukuvuvukala ngokusebenzisa indlela encike ku-TNFα ngokusebenzisa i-autocrine loop, ngaleyo ndlela ikhuthaze ukwenzeka nokusabalala komdlavuza wesibeletho (31). Ngokusekelwe kulokhu kungenzeka, ukuvinjelwa kwe-TNFα kufundwa njenge-ejenti yokwelapha engaba khona yomdlavuza wesibeletho (37, 48, 49). Ngaphezu kwalokho, i-MNA iphazamisa ukuqina kwamangqamuzana e-CAR-T kumaseli esibeletho sesibeletho esibangelwa yi-MNA, okunikeza ubufakazi obengeziwe bokucindezelwa kokuzivikela komzimba okubangelwa yi-MNA. Ngokubambisana, le miphumela iphakamisa imodeli lapho izimila kanye namaseli e-CAF ekhipha khona i-MNA ku-TME yangaphandle. Ngokusebenzisa (i) ukukhuthaza ukukhula komdlavuza wesibeletho okubangelwa yi-TNF kanye (ii) nokuvimbela umsebenzi we-cytotoxic weseli le-T okubangelwa yi-MNA, lokhu kungaba nomphumela we-tumor ophindwe kabili (Isithombe 5D).
Ekuphetheni, ngokusebenzisa inhlanganisela yokucebisa ngokushesha amaseli, ukulandelana kwamaseli elilodwa kanye nokuhlola i-metabolic, lolu cwaningo lwembule umehluko omkhulu we-immunometabolomic phakathi kwamaseli ezimila kanye namaseli e-ascites ezigulini ze-HGSC. Lokhu kuhlaziywa okuphelele kubonise ukuthi kunomehluko ekuthathweni kwe-glucose kanye nomsebenzi we-mitochondrial phakathi kwamaseli e-T, futhi kwahlonza i-MNA njenge-metabolite elawula amasosha omzimba engeyona iseli. Le datha inomthelela endleleni i-TME ethinta ngayo i-metabolism yamaseli e-T emdlavuza wabantu. Nakuba ukuncintisana okuqondile kwezakhamzimba phakathi kwamaseli e-T namaseli omdlavuza kubikiwe, ama-metabolite angasebenza njengabalawuli abangaqondile ukukhuthaza ukuqhubeka kwesimila futhi mhlawumbe ukucindezela izimpendulo zomzimba ezingokwemvelo. Incazelo eyengeziwe yendima yokusebenza yala ma-metabolite alawulayo ingavula amasu ahlukile okuthuthukisa impendulo yomzimba yokulwa nesimila.
Amasampula eziguli kanye nedatha yezokwelapha kutholakale ngokusebenzisa indawo yokugcina izicubu ze-tumor yomdlavuza ye-BC eqinisekiswe yi-Canadian Tissue Repository Network. Ngokusho kwephrothokholi evunyiwe yi-BC Cancer Research Ethics Committee kanye ne-University of British Columbia (H07-00463), wonke amasampula eziguli kanye nedatha yezokwelapha athole imvume ebhaliwe enolwazi noma ayeke ngokusemthethweni imvume yawo. Amasampula agcinwa ku-BioBank eqinisekisiwe (BRC-00290). Izici zesiguli ezinemininingwane ziboniswe kuThebula S1 kanye ne-S5. Ukuze kugcinwe i-cryopreservation, kusetshenziswa i-scalpel ukubola isampula ye-tumor yesiguli ngomshini bese uyisunduza ngesihlungi se-100-micron ukuze kutholakale ukumiswa kweseli elilodwa. Ama-ascites esiguli afakwe i-centrifuge ku-1500 rpm imizuzu eyi-10 ku-4°C ukuze kufakwe amaseli futhi kususwe i-supernatant. Amaseli atholwe esimila kanye nama-ascites agcinwa ku-50% we-serum ye-AB yomuntu engavuselelwanga ngokushisa (i-Sigma-Aldrich), i-40% RPMI-1640 (i-Thermo Fisher Scientific) kanye ne-10% dimethyl sulfoxide. Lokhu kumiswa kweseli elilodwa okugciniwe kwancibilikiswa futhi kwasetshenziswa ekunqumeni i-metabolomics kanye ne-metabolite echazwe ngezansi.
I-medium ephelele iqukethe i-0.22 μm ehlungiwe engu-50:50 engeziwe i-RPMI 1640: AimV. RPMI 1640 + 2.05 mM l-glutamine (Thermo Fisher Scientific) engezwe nge-10% human-inactivated heat-inactivated serum (Sigma-Aldrich), i-12.5 mM Hepes (Thermo Fisher Scientific), i-2 mM l-glutamine (Thermo Fisher Scientific) Fisher Scientific), isixazululo esingu-1 se-Penicillin Streptomycin (PenStrep) (Thermo Fisher Scientific) kanye ne-50 μMB-mercaptoethanol. I-AimV (Invitrogen) ingezwe nge-20 mM Hepes (Thermo Fisher Scientific) kanye ne-2 mM l-glutamine (Thermo Fisher Scientific). I-flow cytometer staining buffer yayiqukethe i-0.22μm filtered phosphate buffered saline (PBS; Invitrogen) engezwe nge-3% heat-inactivated AB human serum (Sigma). I-buffer yokucebisa amaseli yakhiwe yi-PBS ehlungiwe engu-0.22μm futhi ihlanganiswe ne-0.5% ye-serum ye-AB yomuntu engavuthisi ukushisa (i-Sigma-Aldrich).
Ku-medium ephelele engu-37°C, amaseli agcotshwa nge-10 nM MT DR kanye ne-100 μM 2-NBDG imizuzu engama-30. Okulandelayo, amaseli agcotshwa nge-viability dye eF506 ku-4°C imizuzu eyi-15. Phinda umise amaseli ku-FC Block (eBioscience) kanye ne-Brilliant Stain Buffer (BD Biosciences), unciphise i-flow cytometer staining buffer (ngokwemiyalelo yomenzi), bese ubeka i-incubator imizuzu eyi-10 ekushiseni kwegumbi. Faka i-stain kumaseli ngesethi yama-antibodies (Ithebula S2) ku-flow cytometry staining buffer ku-4°C imizuzu engama-20. Phinda umise amaseli ku-flow cytometry staining buffer (Cytek Aurora; 3L-16V-14B-8R configuration) ngaphambi kokuhlaziya. Sebenzisa i-SpectroFlo kanye ne-FlowJo V10 ukuhlaziya idatha yokubalwa kwamaseli, bese usebenzisa i-GraphPad Prism 8 ukudala idatha. Ukuqina kwe-median fluorescence (MFI) kwe-2-NBDG kanye ne-MT DR kwenziwa kwaba ngokwejwayelekile, kwabe sekusetshenziswa ukuhlolwa kwe-t okubhangqiwe ekuhlaziyweni kwezibalo ukuze kubhekwe iziguli ezifanayo. Susa zonke izakhamuzi ezinezehlakalo ezingaphansi kuka-40 ekuhlaziyweni; faka inani le-MFI elingu-1 kunoma yimaphi amanani angemahle ngaphambi kokwenza ukuhlaziywa kwezibalo kanye nokuboniswa kwedatha.
Ukuze sigcwalise isu lokufaka igethi ngesandla lephaneli yenqubo engenhla, sisebenzise isichasiselo esigcwele ngesihlahla sokuvimbela ukuma (FAUST) (21) ukuze sinikeze amaseli ngokuzenzakalelayo kubantu ngemuva kokususa amaseli afile ku-FlowJo. Siphatha umkhiqizo ngesandla ukuze sihlanganise abantu ababonakala besabelwe kabi (ukuhlanganisa i-PD1+ namaseli e-PD1-tumor) kanye nabantu abagciniwe. Isampula ngayinye iqukethe isilinganiso samaseli angaphezu kuka-2%, kubantu abangu-11 sebebonke.
I-Ficoll gradient density centrifugation yasetshenziswa ukuhlukanisa i-PBMC nemikhiqizo yokuhlukaniswa kwe-leukocyte (STEMCELL Technologies). Amaseli e-CD8 + T ahlukaniswa ku-PBMC kusetshenziswa ama-CD8 MicroBeads (Miltenyi) futhi anwetshwa endaweni ephelele kusetshenziswa i-TransAct (Miltenyi) amasonto ama-2 ngokwemiyalelo yomenzi. Amaseli avunyelwe ukuma izinsuku ezi-5 endaweni ephelele equkethe i-IL-7 (10 ng/ml; PeproTech), bese evuselelwa kabusha nge-TransAct. Ngosuku lwesi-7, ngokwemiyalelo yomenzi, ama-CD45 MicroBeads (Miltenyi) abantu asetshenziswa ukucebisa amaseli ngemijikelezo emithathu elandelanayo. Amaseli ahlanganiswa ukuze kuhlaziywe i-flow cytometry (njengoba kuchaziwe ngenhla), kanti amaseli ayisigidi ahlanganiswa kathathu ukuze kuhlaziywe i-LC-MS/MS. Amasampula acutshungulwa yi-LC-MS/MS njengoba kuchaziwe ngezansi. Silinganise inani le-metabolite elilahlekile ngenombolo ye-ion engu-1,000. Isampula ngayinye ilungiswa ngenombolo ye-ion iyonke (i-TIC), iguqulwe ngokwe-logarithmic futhi ilungiswe ngokuzenzakalelayo ku-MetaboAnalystR ngaphambi kokuhlaziywa.
Ukumiswa kweseli elilodwa lesiguli ngasinye kuncibilikisiwe futhi kwahlungwa ngesihlungi esingu-40 μm sibe yindawo ephelele (njengoba kuchaziwe ngenhla). Ngokusho kwephrothokholi yomenzi, imijikelezo emithathu elandelanayo yokukhetha okuhle ngokuhlukaniswa kwama-magnetic bead kusetshenziswa ama-MicroBeads (Miltenyi) yasetshenziswa ukucebisa amasampula amaseli e-CD8+, CD4+ kanye ne-CD45 (eqhweni). Ngamafuphi, amaseli aphinde axhunywe ku-buffer yokucebisa amaseli (njengoba kuchaziwe ngenhla) futhi abalwa. Amaseli afakwe ama-CD8 beads abantu, ama-CD4 beads abantu noma ama-CD45 abantu (Miltenyi) ku-4°C imizuzu eyi-15, bese egezwa nge-buffer yokucebisa amaseli. Isampula idluliswa kukholomu ye-LS (Miltenyi), bese kuqoqwa izingxenyana ezinhle nezimbi. Ukuze kuncishiswe isikhathi futhi kukhuliswe isinyathelo sokubuyiselwa kwamaseli, ingxenyana ye-CD8 isetshenziswa emzuliswaneni wesibili wokucebisa amaseli, bese ingxenyana ye-CD4 isetshenziselwa ukucebisa ama-CD45 okulandelayo. Gcina isixazululo eqhweni kuyo yonke inqubo yokuhlukaniswa.
Ukuze kulungiselelwe amasampula okuhlaziywa kwe-metabolite, amaseli ahlanzwa kanye ngesisombululo sikasawoti obandayo, kwathi i-1 ml ye-80% methanol yafakwa kusampula ngayinye, yabe isifakwa i-vortex futhi yaqandisiwe ku-nitrogen ewuketshezi. Amasampula afakwa emijikelezweni emithathu yokuqandisa-ukuncibilikisa futhi afakwa i-centrifuge ku-14,000 rpm imizuzu eyi-15 ku-4°C. I-supernatant equkethe ama-metabolite iyahwamuka kuze kube yilapho yomile. Ama-metabolite aphinde ancibilikiswa ku-50 μl ye-0.03% formic acid, afakwa i-vortex ukuze axutshwe, bese efakwa i-centrifuge ukuze kususwe udoti.
Khipha ama-metabolite njengoba kuchaziwe ngenhla. Dlulisa i-supernatant ebhodleleni le-chromatography eliwuketshezi elisebenza kahle kakhulu ukuze kwenziwe ucwaningo lwe-metabolomics. Sebenzisa inqubo yokwelapha engahleliwe ukwelapha isampula ngayinye ngenani elifanayo lamaseli ukuvimbela imiphumela ye-batch. Senze ukuhlolwa kwekhwalithi kwama-metabolite omhlaba wonke okushicilelwe ngaphambilini ku-AB SCIEX QTRAP 5500 Triple Quadrupole Mass Spectrometer (50). Ukuhlaziywa kwe-Chromatographic kanye nokuhlanganiswa kwendawo ephakeme kwenziwa kusetshenziswa isofthiwe ye-MultiQuant version 2.1 (Applied Biosystems SCIEX).
Kusetshenziswe inani lama-ion angu-1000 ukulinganisa inani le-metabolite elingekho, kanti i-TIC yesampula ngayinye yasetshenziswa ukubala indawo ephakeme ejwayelekile ye-metabolite ngayinye etholakele ukuze kulungiswe izinguquko ezethulwe ukuhlaziywa kwezinsimbi kusukela ekucutshungulweni kwesampula. Ngemva kokuthi i-TIC ijwayelekile, i-MetaboAnalystR(51) (ipharamitha ezenzakalelayo) isetshenziselwa ukuguqulwa kwe-logarithmic kanye nokukala umugqa ozenzakalelayo. Sisebenzise i-PCA enephakheji ye-vegan R ukwenza ukuhlaziywa kokuhlola komehluko we-metabolome phakathi kwezinhlobo zamasampula, futhi sasebenzisa ukuhlaziywa kwe-redundancy engaphelele ukuhlaziya iziguli. Sebenzisa indlela ye-Ward ukwakha i-dendrogram yemephu yokushisa ukuhlanganisa ibanga le-Euclidean phakathi kwamasampula. Sisebenzise i-limma (52) ngobuningi be-metabolite obujwayelekile ukuhlonza ama-metabolite amaningi ngokuhlukile kulo lonke uhlobo lweseli kanye ne-microenvironment. Ukuze kube lula ukuchaza, sisebenzisa ipharamitha yesilinganiso seqembu ukucacisa imodeli, futhi sicabangele izinhlobo zamaseli endaweni encane njengeqembu ngalinye (n = amaqembu angu-6); ekuhlolweni kokubaluleka, senze izilinganiso ezintathu eziphindaphindwayo ze-metabolite ngayinye Ukuze sigweme ukuphindaphinda okungamanga, isiguli safakwa njengesithiyo ekwakhiweni kwe-limma. Ukuze sihlole umehluko kuma-metabolite phakathi kweziguli ezahlukene, silungise imodeli ye-limma kufaka phakathi iziguli ngendlela eqondile. Sibika ukubaluleka komehluko ochazwe ngaphambilini phakathi kohlobo lweseli kanye nendawo encane ye-Padj <0.05 (ukulungiswa kwe-Benjamini-Hochberg).
Ngemva kokunothisa amandla kusetshenziswa i-Miltenyi Dead Cell Removal Kit (>80% viability), ukulandelelana kwe-transcriptome yeseli elilodwa kwenziwa kuma-ascites aqandisiwe aphilayo kanye namasampula esimila kusetshenziswa iphrothokholi yokubonakaliswa kwe-10x 5′gene. Amacala amahlanu anezimila ezifanayo kanye nama-ascites ahlaziyiwe, yize ukulingana okuphansi okuvela kusampula eyodwa yesimila kuvimbele ukufakwa kwayo. Ukuze kufezwe ukukhetha okuningi kweziguli, sihlanganise amasampula esiguli ngasinye emigqeni yomlawuli we-10x chromium, futhi sahlaziya ama-ascites kanye nezindawo zesimila ngokwehlukana. Ngemva kokulandelelana [Illumina HiSeq 4000 28×98 bp paired end (PE), i-Quebec genome; isilinganiso sama-reads angu-73,488 kanye no-41,378 ngeseli ngayinye ye-tumor kanye ne-ascites ngokulandelana]], sisebenzise i-CellSNP kanye ne-Vireo (53) (ngokusekelwe ku-CellSNP njengoba i-SNP evamile yomuntu (i-VCF) enikezwe yi-GRCh38 inikezwa ubunikazi bomnikeli. Sisebenzisa i-SNPRelate ukunquma ubunikazi obuseduze (i-IBS) besimo se-genotype yesiguli (i-IBS), ngaphandle kwamaseli angabelwe namaseli ahlonzwe njengama-duplex kanye nabaxhasi abafanisayo phakathi kwama-ascites namasampula e-tumor (54). Ngokusekelwe kulo msebenzi, sigcine amacala amathathu anokumelwa okuningi kwamaseli ku-tumor kanye ne-ascites ukuze kuhlaziywe phansi. Ngemva kokwenza isinyathelo sokuhlunga ngobuningi ku-scater (55) kanye ne-scran (56) BioConductor packaging, lokhu kukhiqize amaseli angu-6975 (amaseli angu-2792 kanye no-4183 avela ku-tumor kanye ne-ascites, ngokulandelana) ukuze kuhlaziywe. Sisebenzisa i-igraph's (57) Louvain clustering yenethiwekhi yomakhelwane eseduze (i-SNN) ehlanganyelwe ngokusekelwe ku-Jaccard ibanga ukuya kumaseli eqembu ngokwendlela yokuvezwa. Amaqoqo achazwe ngesandla ngezinhlobo zamaseli ezifakazelwe ngokusekelwe ekubonakalisweni kwezakhi zofuzo ze-marker futhi abonakala nge-t-SNE. Amaseli e-T ane-cytotoxic achazwa ngokuvezwa kwe-CD8A kanye ne-GZMA, ngaphandle kwama-subclusters anokubonakaliswa kwephrotheni ye-ribosomal ephansi. Sifinyelele idatha eshicilelwe ka-Izar et al. (16), kufaka phakathi ukushumeka kwawo kwe-t-SNE, kungalawula ukugqagqana kokubonakaliswa phakathi kwama-immune cell markers kanye nokubonakaliswa kwe-NNMT.
Ama-PBMC ahlukaniswe nemikhiqizo yokuhlukaniswa kwama-leukocyte (STEMCELL Technologies) nge-Ficoll gradient density centrifugation. Amaseli e-CD3 + ahlukaniswe ne-PBMC kusetshenziswa ama-CD3 beads (Miltenyi). Lapho kukhona noma kungekho khona i-MNA, amaseli e-CD3+ asebenza nge-plate-bound CD3 (5μg/ml), i-soluble CD28 (3μg/ml) kanye ne-IL-2 (300 U/ml; Proleukin). Ngosuku lokugcina lokwandiswa, i-viability (Fixable Viability Dye eFluor450, eBioscience) kanye ne-proliferation (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 amahora ama-4, bese uqapha i-CD8-PerCP (RPA-T8, BioLegend), i-CD4-AF700 (RPA-T4), i-BioLegend) kanye ne-TNFα-fluorescein isothiocyanate (FITC) (MAb11, BD). Vuselela amaseli e-qPCR kanye ne-ChIP nge-PMA (20 ng/ml) kanye ne-ionomycin (1μg/ml) amahora ama-4. I-ELISA supernatant yaqoqwa ngaphambi nangemva kokuvuselela nge-PMA (20 ng/ml) kanye ne-ionomycin (1 μg/ml) amahora ama-4.
Landela inqubo yomenzi ukuze uhlukanise i-RNA usebenzisa i-RNeasy Plus Mini Kit (QIAGEN). Sebenzisa i-QIAshredder (QIAGEN) ukuze uhlanganise isampula ngendlela efanayo. Sebenzisa i-RNA enamandla aphezulu kuya ku-cDNA kit (Thermo Fisher Scientific) ukuze uhlanganise i-DNA ehambisanayo (cDNA). Sebenzisa i-TaqMan Rapid Advanced Master Mix (Thermo Fisher Scientific) ukuze ulinganise ukubonakaliswa kwezakhi zofuzo (ngokusho kwenqubo yomenzi) ngama-probe alandelayo: Hs00196287_m1 (NNMT), Hs00154079_m1 (AOX1), Hs00427552_m1 (SLC22A1), Hs02786624_g1 [glyceraldehyde-3-phosphate off Hydrogen (GAPDH)] kanye ne-Hs01010726_m1 (SLC22A2). Amasampula aqhutshwa ohlelweni lwe-StepOnePlus real-time PCR (Applied Biosystems) (Applied Biosystems) ku-MicroAmp fast optical 96-well reaction plate (Applied Biosystems) enefilimu ye-MicroAmp optical. Noma yiliphi inani le-Ct elidlula u-35 libhekwa njengelingaphezu komkhawulo wokuthola futhi limakwe njengelingabonakali.
Yenza i-ChIP njengoba kuchaziwe ngaphambilini (58). Ngamafuphi, amaseli aphathwe nge-formaldehyde (ukuhlushwa kokugcina okungu-1.42%) futhi afakwa ekushiseni kwegumbi imizuzu eyi-10. Sebenzisa i-buffer yokuvuvukala eyengeziwe (25 mM Hepes, 1.5 mM MgCl2, 10 mM KCl kanye ne-0.1% NP-40) eqhweni imizuzu eyi-10, bese uphinda uxhunywe ku-immunoprecipitation buffer njengoba kuchaziwe (58). Isampula yabe isifakwa nge-sonication ngemijikelezo elandelayo: imijikelezo eyi-10 (ama-pulse angu-20 esekhondi eli-1) kanye nesikhathi esingaguquki semizuzwana engama-40. Faka i-immunoglobulin G (Cell Signaling Technology; 1μl), i-histone H3 (Cell Signaling Technology; 3μl), i-NFAT (Invitrogen; 3μl) kanye nama-antibodies e-SP1 (Cell Signaling Technology; 3μl) ngesampula ku-4°CC shake ubusuku bonke. Faka ubuhlalu beprotheyini A (Thermo Fisher Scientific) ngesampula ku-4°C ngokushukumisa kancane ihora eli-1, bese usebenzisa ubuhlalu be-chelex (Bio-Rad) ukuze ucebise i-DNA, bese usebenzisa i-proteinase K (Thermo Fisher) ukuze ugaye amaprotheni. Umgqugquzeli we-TNFα utholwe yi-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). Izithombe zakhiqizwa yi-Image Lab (Bio-Rad) futhi zalinganiswa kusetshenziswa isofthiwe ye-ImageJ.
I-supernatant ye-cell culture yaqoqwa njengoba kuchaziwe ngenhla. Ukunqunywa kwenziwa ngokwezinqubo zomenzi ze-human TNFα ELISA kit (Invitrogen), human IL-2 ELISA kit (Invitrogen) kanye ne-human IFN-γ ELISA kit (Abcam). Ngokwephrothokholi yomenzi, i-supernatant yancishiswa ngo-1:100 ukuthola i-TNFα kanye ne-IL-2, kanye no-1:3 ukuthola i-IFN-γ. Sebenzisa i-EnVision 2104 Multilabel Reader (PerkinElmer) ukukala ukumuncwa ku-450 nm.
Ama-PBMC ahlukaniswe nemikhiqizo yokuhlukaniswa kwama-leukocyte (STEMCELL Technologies) nge-Ficoll gradient density centrifugation. Amaseli e-CD3 + ahlukaniswe ne-PBMC kusetshenziswa ama-CD3 beads (Miltenyi). Uma kukhona noma kungekho i-MNA, amaseli e-CD3+ asebenza nge-plate-bound CD3 (5μg/ml), i-soluble CD28 (3μg/ml) kanye ne-IL-2 (300 U/ml; Proleukin) izinsuku ezi-3. Ngemva kwezinsuku ezi-3, amaseli aqoqwa futhi agezwa nge-saline engu-0.9%, futhi i-pellet yaqandiswa. Inani lamaseli lenziwa nge-flow cytometry (Cytek Aurora; 3L-16V-14B-8R configuration) kusetshenziswa ama-eBeads angu-123count.
Ama-metabolite okukhipha njengoba kuchaziwe ngenhla. Ukukhishwa okomisiwe kwaphinde kwahlanganiswa ekugxilweni kwama-cell equivalents angu-4000/μl. Hlaziya isampula nge-reversed-phase chromatography (1290 Infinity II, Agilent Technologies, Santa Clara, CA) kanye nekholomu ye-CORTECS T3 (2.1×150 mm, usayizi wezinhlayiya 1.6-μm, usayizi we-pore 120-Å; #186008500, Amanzi). I-Polar mass spectrometer (6470, Agilent), lapho i-electrospray ionization isebenza khona ngendlela enhle. Isigaba esiphathwayo A siyi-0.1% formic acid (ku-H2O), isigaba esiphathwayo B siyi-90% acetonitrile, i-0.1% formic acid. I-LC gradient ingu-0 kuya ku-2 imizuzu engu-100% A, imizuzu emi-2 kuya ku-7.1 ku-99% B, kanye nemizuzu engu-7.1 kuya ku-8 ku-99% B. Bese ulinganisa kabusha ikholomu nesigaba esihambayo A ngesilinganiso sokugeleza esingu-0.6 ml/min imizuzu emi-3. . Izinga lokugeleza lingu-0.4ml/min, bese igumbi lekholomu lishiswa ku-50°C. Sebenzisa indinganiso yamakhemikhali emsulwa ye-MNA (M320995, Toronto Research Chemical Company, North York, Ontario, Canada) ukusungula isikhathi sokugcina (RT) kanye nokuguqulwa (RT = 0.882 imizuzu, ukuguqulwa 1 = 137→94.1, ukuguqulwa 2 = 137→92, Ukuguqulwa 3 = 137→78). Lapho zonke izinguquko ezintathu zenzeka ngesikhathi esifanele sokugcina, ushintsho 1 lusetshenziselwa ukulinganisa ukuqinisekisa ukucaca. Ijika elijwayelekile le-MNA (iToronto Research Chemical Company) lakhiqizwa ngokuxutshwa okuyisithupha okulandelanayo kwesisombululo sesitoko (1 mg/ml) ukuze kutholakale amazinga ka-0.1, 1.0, 10 kanye no-100 ng/ml kanye no-1.0 kanye no-10μg/ml ngokulandelana koketshezi. Umkhawulo wokuthola ungu-1 ng/ml, kanti impendulo eqondile iphakathi kuka-10 ng/ml no-10μg/ml. Ukujova ngakunye kwama-microliter amabili esampula kanye nejwayelekile kusetshenziselwa ukuhlaziywa kwe-LC/MS, futhi isampula yokulawula ikhwalithi exubile iqhutshwa njalo ngemijovo eyisishiyagalombili ukuqinisekisa ukuzinza kwepulatifomu yokuhlaziya. Izimpendulo ze-MNA zazo zonke izibonelo zamaseli aphathwe yi-MNA zazingaphakathi kobubanzi obuqondile bokuhlolwa. Ukuhlaziywa kwedatha kwenziwe kusetshenziswa isofthiwe yokuhlaziya eqondile ye-MassHunter (v9.0, Agilent).
Ukwakhiwa kwesizukulwane sesibili se-αFR-CAR kuthathwe kuSong et al. (59). Ngamafuphi, ukwakhiwa kuqukethe okuqukethwe okulandelayo: Uchungechunge lomholi we-CD8a, isiqephu esiguquguqukayo se-single-chain fragment yomuntu, i-CD8a hinge kanye nesifunda se-transmembrane, isizinda sangaphakathi se-CD27 kanye nesizinda sangaphakathi se-CD3z. Uchungechunge oluphelele lwe-CAR lwenziwa yi-GenScript, lwabe seluhlanganiswa ku-vector yokubonakaliswa kwe-lentiviral yesizukulwane sesibili phezulu kwekhasethi yokubonakaliswa kwe-GFP esetshenziselwa ukuhlola ukusebenza kahle kokudluliselwa.
I-Lentivirus ikhiqizwa ngokufakelwa kwamaseli e-HEK293T [Iqoqo Le-American Type Culture (ATCC); ikhuliswe ku-Dulbecco's modified Eagle medium equkethe i-10% ye-fetal bovine serum (FBS) kanye ne-1% PenStrep, futhi kusetshenziswe i-CAR-GFP vector kanye nama-plasmids okupakisha (psPAX2 kanye ne-pMD2.G, i-Addgene) asebenzisa i-lipofection amine (Sigma-Aldrich). I-supernatant equkethe igciwane yaqoqwa emahoreni angu-48 kanye nama-72 ngemva kokufakelwa, yahlungwa, futhi yaqoqwa nge-ultracentrifugation. Gcina i-supernatant ehlanganisiwe yegciwane ku--80°C kuze kube yilapho idluliselwa.
Ama-PBMC ahlukaniswe nemikhiqizo yokuhlukaniswa kwama-leukocyte anikelayo anempilo (i-STEMCELL Technologies) nge-Ficoll gradient density centrifugation. Sebenzisa ama-microbead e-CD8 akhethiwe (i-Miltenyi) ukuze uhlukanise amaseli e-CD8+ ku-PBMC. Vuselela amaseli e-T nge-TransAct (i-Miltenyi) kanye ne-TexMACS medium [i-Miltenyi; engezwe nge-3% ye-human serum engasebenzi kahle, i-1% ye-PenStrep kanye ne-IL-2 (300 U/ml)]. Amahora angamashumi amabili nane ngemva kokukhuthazwa, amaseli e-T adluliselwa nge-lentivirus (10 μl concentrated virus supernatant ngamaseli angu-106). Ezinsukwini ezi-1 kuya kwezi-3 ngemva kokudluliswa ku-Cytek Aurora (ku-FSC (Forward Scatter)/SSC (Side Scatter), Singlet, GFP+), hlola ukuvezwa kwe-GFP kwamaseli ukuze kuboniswe ukusebenza kahle kokudluliswa okungenani okungu-30%.
Amaseli e-CAR-T akhuliswa amahora angama-24 ku-Immunocult (STEMCELL Technologies; agcwaliswe nge-1% PenStrep) ngaphansi kwezimo ezilandelayo: angelashwanga, aphathwa nge-250 μM adenosine noma i-10 mM MNA. Ngemva kokwelashwa kwangaphambili, amaseli e-CAR-T ahlanzwa nge-PBS futhi ahlanganiswa namaseli e-SK-OV-3 angu-20,000 [ATCC; ku-McCoy 5A medium (Sigma-Aldrich) agcwaliswe nge-10% FBS kanye ne-1% PenStrep ku-10: Isilinganiso se-effector kuya ku-target esingu-1 sandiswa nge-triplicate ku-Immunocult medium egcwalisiwe. Amaseli e-SK-OV-3 namaseli e-SK-OV-3 agcwaliswe nge-digitalis saponin (0.5mg/ml; Sigma-Aldrich) asetshenziswa njengezilawuli ezingezinhle nezingezinhle, ngokulandelana. Ngemva kwamahora angama-24 okulima ndawonye, ​​i-supernatant yaqoqwa futhi i-lactate dehydrogenase (LDH) yalinganiswa ngokwemiyalelo yomkhiqizi (i-LDH Glo Cytotoxicity Assay Kit, i-Promega). I-LDH supernatant yancishiswa ngo-1:50 ku-LDH buffer. Iphesenti lokubulala lalinganiswa kusetshenziswa ifomula elandelayo: iphesenti lokubulala = iphesenti lokulungisa / izinga lokubulala eliphezulu x 100%, lapho iphesenti lokulungisa = amaseli e-co-culture-T kuphela, kanye nezinga lokubulala eliphezulu = ukulawula okuhle nokubi.
Njengoba kuchaziwe embhalweni noma ezintweni nasezindleleni, sebenzisa i-GraphPad Prism 8, i-Microsoft Excel noma i-R v3.6.0 ukuze kuhlaziywe izibalo. Uma kuqoqwa amasampula amaningi esigulini esifanayo (njenge-ascites kanye ne-tumor), sisebenzisa ukuhlolwa kwe-t okubhangqiwe noma sifake isiguli njengomphumela ongahleliwe kumodeli eqondile noma ejwayelekile njengoba kufanele. Ekuhlaziyweni kwe-metabolomics, ukuhlolwa kokubaluleka kwenziwa kathathu.
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Lo mbuzo usetshenziselwa ukuhlola ukuthi uyisivakashi yini futhi uvimbele ukuthunyelwa kogaxekile okuzenzakalelayo.
Marisa K. Kilgour (Marisa K. Kilgour), Sarah MacPherson (Sarah MacPherson), Lauren G. Zacharias (Lauren G. Zacharias), Abigail Eli Aris G. Watson (H. Watson), John Stagg (John Stagg), Brad H. Nelson (Brad H. Nelson), Ralph J. De Bellar Jones (G.Russell. G. Jones), Phineas T. Hamilton (Phineas T.
I-MNA inegalelo ekucindezelweni kwamasosha omzimba kwamaseli e-T futhi imelela umgomo ongaba khona wokwelashwa kwamasosha omzimba wokwelapha umdlavuza womuntu.
Marisa K. Kilgour (Marisa K. Kilgour), Sarah MacPherson (Sarah MacPherson), Lauren G. Zacharias (Lauren G. Zacharias), Abigail Eli Aris G. Watson (H. Watson), John Stagg (John Stagg), Brad H. Nelson (Brad H. Nelson), Ralph J. De Bellar Jones (G.Russell. G. Jones), Phineas T. Hamilton (Phineas T.
I-MNA inegalelo ekucindezelweni kwamasosha omzimba kwamaseli e-T futhi imelela umgomo ongaba khona wokwelashwa kwamasosha omzimba wokwelapha umdlavuza womuntu.
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