Ii-metabolites ze-immunomodulatory zizinto eziphambili kwi-tumor microenvironment (TME), kodwa ngaphandle kweminye embalwa, ubuwena bazo abukaziwa kangako. Apha, sihlalutye ii-tumor kunye nee-T cells ezivela kwii-tumor kunye nee-ascites zezigulane ezine-high-grade serous carcinoma (HGSC) ukuze sityhile i-metabolome yezi compartments zahlukeneyo ze-TME. Ii-Ascites kunye nee-tumor cells zinezahluko ezinkulu ze-metabolite. Xa kuthelekiswa nee-ascites, ii-T cells ezifaka i-tumor zityebile kakhulu kwi-1-methylnicotinamide (MNA). Nangona inqanaba le-MNA kwii-T cells liphezulu, ukubonakaliswa kwe-nicotinamide N-methyltransferase (i-enzyme ekhuthaza ukudluliselwa kwamaqela e-methyl ukusuka kwi-S-adenosylmethionine ukuya kwi-nicotinamide) kunqunyelwe kwi-fibroblasts kunye nee-tumor cells. Ngokusebenzayo, i-MNA ikhuthaza ii-T cells ukuba zikhuphe i-cytokine tumor necrosis factor alpha ekhuthaza i-tumor. Ke ngoko, i-MNA evela kwi-TME inegalelo kulawulo lomzimba lwee-T cells kwaye imele injongo ye-immunotherapy enokubakho yokunyanga umhlaza womntu.
Iimetabolites ezivela kwi-tumor zinokuba nefuthe elikhulu ekuthinteleni ukhuseleko lomzimba oluchasene ne-tumor, kwaye ubungqina obuninzi bubonisa ukuba zinokusebenza njengamandla aphambili okuqhuba ukuqhubela phambili kwesifo (1). Ukongeza kwisiphumo seWarburg, umsebenzi wakutshanje uqalile ukuchaza imeko ye-metabolic yeeseli ze-tumor kunye nolwalamano lwayo nemeko ye-immune ye-tumor microenvironment (TME). Izifundo kwiimodeli zegundane kunye neeseli ze-T zabantu zibonise ukuba i-glutamine metabolism (2), i-oxidative metabolism (3) kunye ne-glucose metabolism (4) zinokusebenza ngokuzimeleyo kumaqela ahlukeneyo eeseli ze-immune. Iimetabolites ezininzi kwezi ndlela zithintela umsebenzi we-anti-tumor weeseli ze-T. Kuye kwabonakaliswa ukuba ukuvinjwa kwe-coenzyme tetrahydrobiopterin (BH4) kunokonakalisa ukwanda kweeseli ze-T, kwaye ukwanda kwe-BH4 emzimbeni kunokunyusa impendulo ye-anti-tumor immune ebangelwa yi-CD4 kunye ne-CD8. Ukongeza, isiphumo se-immunosuppressive se-kynurenine sinokuhlangulwa ngokunikezelwa kwe-BH4 (5). Kwi-isocitrate dehydrogenase (IDH) mutant glioblastoma, ukukhutshwa kwe-enantiometabolic (R)-2-hydroxyglutarate (R-2-HG) kuthintela ukusebenza kweeseli ze-T, ukwanda kwazo kunye nomsebenzi we-cytolysis (6). Kutshanje, kuboniswe ukuba i-methylglyoxal, imveliso ephuma kwi-glycolysis, iveliswa ziiseli ezithintelayo ezivela kwi-myeloid, kwaye ukudluliselwa kweeseli ze-T ze-methylglyoxal kunokuthintela ukusebenza kweeseli ze-T ezichaphazelayo. Kunyango, ukunganyamezeli kwe-methylglyoxal kunokoyisa umsebenzi weeseli ezithintelayo ezivela kwi-myeloid (MDSC) kwaye kuphucule unyango lokuthintela i-checkpoint kwiimodeli zeempuku (7). Ezi zifundo zigxininisa indima ephambili yee-metabolites ezivela kwi-TME ekulawuleni umsebenzi weeseli ze-T kunye nomsebenzi wazo.
Ukungasebenzi kakuhle kweeseli ze-T kuye kwaxelwa kakhulu kumhlaza wesibeleko (8). Oku kungenxa yeempawu zemetabolism ezifumaneka kwi-hypoxia kunye ne-tumor vasculature engaqhelekanga (9), nto leyo ekhokelela ekuguqulweni kweglucose kunye ne-tryptophan zibe ziimveliso ezifana ne-lactic acid kunye ne-kynurenine. I-lactate engaphezulu kwe-extracellular inciphisa imveliso ye-interferon-γ (IFN-γ) kwaye iqhubela phambili umahluko we-myelosuppressive subgroups (10, 11). Ukusetyenziswa kwe-tryptophan kuthintela ngokuthe ngqo ukwanda kweeseli ze-T kwaye kuthintele i-T cell receptor signaling (12-14). Ngaphandle kwezi ngqwalasela, umsebenzi omninzi ojikeleze i-immune metabolism wenziwa kwi-in vitro T cell culture kusetyenziswa i-optimized media, okanye kunqunyelwe kwiimodeli zegundane ezihambelanayo kwi-vivo, kwaye akukho nanye kuzo ebonisa ngokupheleleyo ukungafani komhlaza womntu kunye ne-Physiological macro kunye ne-micro environment.
Uphawu oluqhelekileyo lomhlaza wesibeleko kukusasazeka kwe-peritoneal kunye nokubonakala kwe-ascites. Ukuqokelelana kolwelo lweseli kwi-ascites kunxulunyaniswa nesifo esiqhubekileyo kunye nokubikezela okubi (15). Ngokweengxelo, eli candelo likhethekileyo alina-hypoxic, linamanqanaba aphezulu e-vascular endothelial growth factor (VEGF) kunye ne-indoleamine 2,3-dioxygenase (IDO), kwaye lingena kwiiseli ezilawulayo ze-T kunye neeseli ezithintela i-myeloid (15-18). Indawo ye-metabolic ye-ascites inokwahluka kweye-tumor ngokwayo, ngoko ke ukuhlelwa kwakhona kweeseli ze-T kwindawo ye-peritoneal akucaci. Ukongeza, umahluko ophambili kunye nokungafani phakathi kwe-ascites kunye ne-metabolites ezikhoyo kwindawo ye-tumor kunokuthintela ukungena kweeseli zomzimba kunye nomsebenzi wazo kwii-tumor, kwaye kufuneka uphando olongezelelweyo.
Ukuze sisombulule ezi ngxaki, siyile indlela yokwahlulwa kweeseli ezibuthathaka kunye ne-liquid chromatography tandem mass spectrometry (LC-MS/MS) yokufunda iintlobo ezahlukeneyo zeeseli (kubandakanya iiseli ze-CD4 + kunye ne-CD8 + T) kunye nangaphakathi naphakathi kwee-tumor. Ii-metabolites zayo zijikeleza iiseli kwindawo efanayo ye-ascites kunye nendawo ye-tumor yesigulana. Sisebenzisa le ndlela kunye ne-high-dimensional flow cytometry kunye ne-single-cell RNA sequencing (scRNA-seq) ukubonelela ngomfanekiso ochanekileyo wesimo se-metabolic sale mimandla ibalulekileyo. Le ndlela ityhile ukwanda okukhulu kwinqanaba le-1-methylnicotinamide (MNA) kwiiseli ze-tumor T, kwaye uvavanyo lwe-in vitro lubonise ukuba isiphumo se-immunomodulatory se-MNA kumsebenzi weeseli ze-T sasingaziwa ngaphambili. Ngokubanzi, le ndlela ityhila ukusebenzisana kwe-metabolic phakathi kwee-tumor kunye neeseli zomzimba, kwaye ibonelela ngolwazi olukhethekileyo kwi-immune regulation metabolites, ezinokuba luncedo kunyango lwe-T cell-based ovarian cancer immunotherapy amathuba onyango.
Sisebenzise i-high-dimensional flow cytometry ukuze ngaxeshanye silinganise ukuthathwa kweglucose [2-(N-(7-nitrophenyl-2-oxa-1,3-diaza-4-yl)amino)-2-deoxyglucose (2-NBDG) kunye nomsebenzi we-mitochondrial [MitoTracker Deep Red (MT DR)] (7, 19, 20) ziimpawu eziqhelekileyo ecaleni kwelinye ezihlukanisa iiseli zomzimba kunye nabemi beeseli ze-tumor (Itheyibhile S2 kunye noMfanekiso S1A). Olu hlalutyo lubonise ukuba xa kuthelekiswa neeseli ze-T, ii-ascites kunye neeseli ze-tumor zinamanqanaba aphezulu okuthathwa kweglucose, kodwa zinomahluko omncinci kumsebenzi we-mitochondrial. Ukuthathwa kweglucose okuqhelekileyo kweeseli ze-tumor [CD45-EpCAM (EpCAM)+] kuphindwe kathathu ukuya kane kuneeseli ze-T, kwaye ukuthathwa kweglucose okuqhelekileyo kweeseli ze-CD4 + T kuphindwe ka-1.2 kuneeseli ze-CD8 + T, nto leyo ebonisa ukuba ii-lymphocytes ezingene kwi-Tumor (TIL) zineemfuno ezahlukeneyo ze-metabolic nakwi-TME efanayo (Umfanekiso 1A). Ngokwahlukileyo koko, umsebenzi we-mitochondrial kwiiseli ze-tumor ufana noweeseli ze-CD4 + T, kwaye umsebenzi we-mitochondrial weentlobo zombini zeeseli uphezulu kunoweeseli ze-CD8 + T (Umfanekiso 1B). Ngokubanzi, ezi ziphumo zibonisa inqanaba le-metabolic. Umsebenzi we-metabolic weeseli ze-tumor uphezulu kunoweeseli ze-CD4 + T, kwaye umsebenzi we-metabolic weeseli ze-CD4 + T uphezulu kunoweeseli ze-CD8 + T. Nangona ezi ziphumo kwiintlobo zeeseli, akukho mahluko ungaguqukiyo kwisimo se-metabolic seeseli ze-CD4 + kunye ne-CD8 + T okanye umlinganiselo wazo kwi-ascites xa kuthelekiswa nee-tumor (Umfanekiso 1C). Ngokwahlukileyo koko, kwi-fraction yeseli ye-CD45, umlinganiselo weeseli ze-EpCAM + kwi-tumor unyuke xa kuthelekiswa nee-ascites (Umfanekiso 1D). Sikwabone umahluko ocacileyo we-metabolic phakathi kweenxalenye ze-EpCAM + kunye ne-EpCAM- cell. Iiseli ze-EpCAM + (tumor) zinomthamo ophezulu we-glucose kunye nomsebenzi we-mitochondrial kuneeseli ze-EpCAM, ophakamileyo kakhulu kunomsebenzi we-metabolic we-fibroblasts kwiiseli ze-tumor kwi-TME (Umfanekiso 1, E kunye no-F).
(A kunye no-B) Ubunzulu be-median fluorescence (MFI) yokufunxwa kweglucose (2-NBDG) (A) kunye nomsebenzi we-mitochondrial weeseli ze-CD4 + T (ubomvu obumnyama beMitoTracker) (B) Iigrafu ezimeleyo (ekhohlo) kunye nedatha edwelisiweyo (Ekunene), iiseli ze-CD8 + T kunye neeseli ze-EpCAM + CD45-tumor ezivela kwi-ascites kunye ne-tumor. (C) Umlinganiselo weeseli ze-CD4 + kunye ne-CD8 + (zeeseli ze-CD3 + T) kwi-ascites kunye ne-tumor. (D) Ubungakanani beeseli ze-EpCAM + tumor kwi-ascites kunye ne-tumor (CD45−). (E kunye no-F) I-EpCAM + CD45-tumor kunye ne-EpCAM-CD45-matrix glucose uptake (2-NBDG) (E) kunye nomsebenzi we-mitochondrial (ubomvu obumnyama beMitoTracker) (F) iigrafu ezimeleyo (ekhohlo) kunye nedatha edwelisiweyo (Ekunene) Ii-Ascites kunye neeseli ze-tumor. (G) Iigrafu ezimeleyo ze-CD25, CD137 kunye ne-PD1 expression nge-flow cytometry. (H kunye no-I) Ukubonakaliswa kwe-CD25, i-CD137 kunye ne-PD1 kwiiseli ze-CD4 + T (H) kunye neeseli ze-CD8 + T (I). (J kunye no-K) I-Naive, imemori ephakathi (Tcm), i-effector (Teff) kunye ne-effector memory (Tem) phenotypes ngokusekelwe ekubonakalisweni kwe-CCR7 kunye ne-CD45RO. Imifanekiso emeleyo (ekhohlo) kunye nedatha yetafile (ekunene) yeeseli ze-CD4 + T (J) kunye neeseli ze-CD8 + T (K) kwi-ascites kunye neethumba. Amaxabiso e-P amiselwe ngovavanyo lwe-t oludibeneyo (*P<0.05, **P<0.01 kunye ***P<0.001). Umgca umele izigulane ezifanisiweyo (n = 6). I-FMO, i-fluorescence minus enye; i-MFI, i-median fluorescence intensity.
Uhlalutyo olongezelelweyo lutyhile eminye imiba ebalulekileyo phakathi kwesimo se-T cell esilungisiweyo kakhulu. I-activated (Umfanekiso 1, G ukuya ku-I) kunye ne-effector memory (Umfanekiso 1, J kunye no-K) kwi-tumor zixhaphake kakhulu kune-ascites (umlinganiselo wee-CD3 + T cells). Ngokufanayo, ukuhlalutya i-phenotype ngokubonakaliswa kweempawu zokuqalisa (CD25 kunye ne-CD137) kunye neempawu zokuphelelwa ngamandla [iproteni yokufa kweseli ecwangcisiweyo 1 (PD1)] kubonise ukuba nangona iimpawu ze-metabolic zala maqela zahlukile (Umfanekiso S1, B ukuya ku-E), kodwa akukho mahluko abalulekileyo e-metabolic abonwa rhoqo phakathi kwe-naive, effector okanye i-memory subsets (Umfanekiso S1, F ukuya ku-I). Ezi ziphumo ziqinisekiswe ngokusebenzisa iindlela zokufunda koomatshini ukunikezela ngokuzenzekelayo ii-phenotypes zeseli (21), nto leyo etyhile ukubakho kwenani elikhulu lee-bone marrow cells (CD45 + / CD3- / CD4 + / CD45RO +) kwi-ascites yesigulane (Umfanekiso S2A). Phakathi kwazo zonke iintlobo zeeseli ezichongiweyo, olu luhlu lweeseli ze-myeloid lubonise ukufunxwa okuphezulu kweglucose kunye nomsebenzi we-mitochondrial (Umfanekiso S2, B ukuya ku-G). Ezi ziphumo zibonisa umahluko omkhulu we-metabolic phakathi kweentlobo ezininzi zeeseli ezifumaneka kwi-ascites kunye neethumba kwizigulane ze-HGSC.
Umngeni ophambili ekuqondeni iimpawu ze-metabonomic ze-TIL kukufuneka ukwahlula iisampuli zeeseli ze-T ezinobumsulwa obaneleyo, umgangatho kunye nobungakanani kwiithumba. Izifundo zakutshanje zibonise ukuba iindlela zokwahlula kunye nokukhulisa iintsimbi ezisekelwe kwi-flow cytometry zinokukhokelela kutshintsho kwiiprofayili ze-metabolite zeseli (22-24). Ukuze soyise le ngxaki, siphucule indlela yokukhulisa iintsimbi ukuze sahlukanise kwaye sahlukanise i-TIL kumhlaza wesibeleko somntu osuswe ngotyando ngaphambi kokuba sihlalutywe yi-LC-MS/MS (jonga izixhobo kunye neendlela; Umfanekiso 2A). Ukuze sivavanye impembelelo iyonke yale protocol kutshintsho lwe-metabolite, sithelekise iiprofayili ze-metabolite zeeseli ze-T ezivuselelwe ngabanikeli abaphilileyo emva kwenyathelo lokwahlula iintsimbi elingentla kunye neeseli ezazingahlukaniswanga iintsimbi kodwa zahlala kwiqhwa. Olu hlalutyo lolawulo lomgangatho lufumanise ukuba kukho ulwalamano oluphezulu phakathi kwezi meko zimbini (r = 0.77), kwaye ukuphindaphinda kobugcisa kweqela lee-metabolite ezingama-86 kunokuphindaphinda okuphezulu (Umfanekiso 2B). Ke ngoko, ezi ndlela zinokwenza uhlalutyo oluchanekileyo lwe-metabolite kwiiseli eziphuculwa ngohlobo lweeseli, ngaloo ndlela zibonelela ngeqonga lokuqala elinesisombululo esiphezulu sokuchonga ii-metabolites ezithile kwi-HGSC, ngaloo ndlela zivumela abantu ukuba bafumane ukuqonda okunzulu malunga nokucaciswa kweeseli Inkqubo ye-metabolism yesondo.
(A) Umzobo weskematiki wokuhluzwa kweentsimbi zemagnethi. Ngaphambi kohlalutyo lwe-LC-MS/MS, iiseli ziya kudlula kwimijikelo emithathu elandelelanayo yokuhluzwa kweentsimbi zemagnethi okanye zihlale kwiqhwa. (B) Impembelelo yohlobo lokuhluzwa kubuninzi beemetabolites. Umyinge wemilinganiselo emithathu yohlobo ngalunye lokuhluzwa ± SE. Umgca ongwevu umele ubudlelwane be-1:1. Ulwalamano lwangaphakathi kweklasi (ICC) lokulinganisa okuphindaphindiweyo okuboniswe kwileyibhile ye-axis. I-NAD, i-nicotinamide adenine dinucleotide. (C) Umzobo weskematiki womsebenzi wohlalutyo lwe-metabolite yesigulane. Ii-Ascites okanye ii-tumor ziqokelelwa kwizigulana kwaye zigcinwe. Inxalenye encinci yesampuli nganye ihlalutywe yi-flow cytometry, ngelixa iisampuli eziseleyo zidlule kwimijikelo emithathu yokuhluzwa kweeseli ze-CD4+, CD8+ kunye ne-CD45. Ezi nxalenye zeeseli zihlalutywe kusetyenziswa i-LC-MS/MS. (D) Imephu yobushushu yobuninzi be-metabolite esemgangathweni. I-dendrogram imele ukuqokelelwa kukaWard komgama we-Euclidean phakathi kweesampuli. (E) Uhlalutyo lwecandelo eliphambili (i-PCA) lemephu yesampulu ye-metabolite, ebonisa iikopi ezintathu zesampulu nganye, iisampulu ezivela kwisigulana esinye ziqhagamshelwe ngomgca. (F) I-PCA yeprofayili ye-metabolite yesampulu exhomekeke kwisigulana (oko kukuthi, kusetyenziswa i-redundancy engaphelelanga); uhlobo lwesampulu lunqunyelwe yi-convex hull. I-PC1, icandelo eliphambili 1; I-PC2, icandelo eliphambili 2.
Okulandelayo, sisebenzise le ndlela yokutyebisa ukuhlalutya ii-metabolites ezingama-99 kwi-CD4+, CD8+ kunye nee-CD45-cell fractions kwi-ascites yokuqala kunye nee-tumor zezigulane ezintandathu ze-HGSC (Umfanekiso 2C, Umfanekiso S3A kunye neTheyibhile S3 kunye ne-S4). Inani labantu abanomdla libalelwa kwi-2% ukuya kwi-70% yesampulu enkulu yokuqala yeeseli eziphilayo, kwaye umlinganiselo weeseli uyahluka kakhulu phakathi kwezigulane. Emva kokwahlula ii-beads, i-fraction etyebileyo yentshisekelo (CD4+, CD8+ okanye CD45-) ibalwa ngaphezu kwe-85% yazo zonke iiseli eziphilayo kwisampulu ngokomyinge. Le ndlela yokutyebisa isivumela ukuba sihlalutye amaqela eeseli kwi-metabolism yezicubu zomhlaza womntu, into engenakwenzeka kwiisampulu ezinkulu. Sisebenzisa le protocol, sigqibe kwelokuba i-l-kynurenine kunye ne-adenosine, ezi metabolites zimbini eziphawulwe kakuhle ze-immunosuppressive ziphakanyisiwe kwiiseli ze-tumor T okanye kwiiseli ze-tumor (Umfanekiso S3, B kunye no-C). Ke ngoko, ezi ziphumo zibonisa ukuthembeka kunye nokukwazi kokwahlukana kweeseli zethu kunye netekhnoloji ye-mass spectrometry yokufumana ii-metabolites ezibalulekileyo kwizicubu zesigulane.
Uhlalutyo lwethu lukwabonise ukwahlulwa okunamandla kwe-metabolic kwiintlobo zeeseli ngaphakathi naphakathi kwezigulane (Umfanekiso 2D kunye noMfanekiso S4A). Ngokukodwa, xa kuthelekiswa nezinye izigulane, isigulane 70 sibonise iimpawu ezahlukeneyo ze-metabolic (Umfanekiso 2E kunye noMfanekiso S4B), nto leyo ebonisa ukuba kunokubakho ukungafani okukhulu kwe-metabolic phakathi kwezigulane. Kubalulekile ukuqaphela ukuba xa kuthelekiswa nezinye izigulane (1.2 ukuya kwi-2 ilitha; Itheyibhile S1), inani lilonke le-ascites eliqokelelwe kwisigulane 70 (80 ml) lalincinci. Ulawulo lwe-heterogeneity phakathi kwezigulane ngexesha lohlalutyo lwecandelo eliphambili (umzekelo, kusetyenziswa uhlalutyo lwe-redundancy oluyinxenye) lubonisa utshintsho oluqhubekayo phakathi kweentlobo zeeseli, kwaye iintlobo zeeseli kunye/okanye i-microenvironment zihlanganiswe ngokucacileyo ngokweprofayili ye-metabolite (Umfanekiso 2F). Uhlalutyo lwee-metabolites ezizodwa lugxininise ezi ziphumo kwaye lubonise umahluko omkhulu phakathi kweentlobo zeeseli kunye ne-microenvironment. Kubalulekile ukuqaphela ukuba umahluko omkhulu obonwe yi-MNA, edla ngokutyetyiswa kwiiseli ze-CD45 nakwiiseli ze-CD4+ kunye ne-CD8+ ezingena kwi-tumor (Umfanekiso 3A). Kwiiseli ze-CD4 +, esi siphumo sibonakala kakhulu, kwaye i-MNA kwiiseli ze-CD8 + nayo ibonakala ichaphazeleka kakhulu yimeko-bume. Nangona kunjalo, oku akubalulekanga, kuba zithathu kuphela kwizigulane ezithandathu ezinokuvavanywa amanqaku e-CD8 + yethumba. Ukongeza kwi-MNA, kwiintlobo ezahlukeneyo zeeseli kwi-ascites kunye neethumba, ezinye ii-metabolites ezingachazwanga kakuhle kwi-TIL nazo zizityebi ngokwahlukileyo (Imifanekiso S3 kunye ne-S4). Ke ngoko, ezi datha zityhila iseti ethembisayo yee-metabolites ze-immunomodulatory zophando olongezelelweyo.
(A) Umxholo oqhelekileyo we-MNA kwiiseli ze-CD4+, CD8+ kunye ne-CD45 ezivela kwi-ascites kunye ne-tumor. Iploti yebhokisi ibonisa i-median (umgca), uluhlu lwe-interquartile (i-frame hinge) kunye noluhlu lwedatha, ukuya kuthi ga kwi-1.5 kuluhlu lwe-interquartile (i-frame whisker). Njengoko kuchaziwe kwiZixhobo zeSigulana kunye neendlela, sebenzisa ixabiso le-limma yesigulane ukumisela ixabiso le-P (*P<0.05 kunye ne-**P<0.01). (B) Umzobo we-Schematic we-metabolism ye-MNA (60). IiMetabolites: S-adenosyl-1-methionine; SAH, S-adenosine-1-homocysteine; NA, nicotinamide; MNA, 1-methylnicotinamide; 2-PY, 1-methyl- 2-pyridone-5-carboxamide; 4-PY, 1-methyl-4-pyridone-5-carboxamide; NR, nicotinamide ribose; NMN, nicotinamide mononucleotide. Ii-enzymes (eziluhlaza): i-NNMT, i-nicotinamide N-methyltransferase; i-SIRT, ii-sirtuins; 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 ye-ascites (engwevu) kunye ne-tumor (ebomvu; n = izigulane ezi-3). (D) Ukubonakaliswa kwe-NNMT kumaqela ahlukeneyo eeseli achongiweyo kusetyenziswa i-scRNA-seq. (E) Ukubonakaliswa kwe-NNMT kunye ne-AOX1 kwi-SK-OV-3, i-human embryonic kidney (HEK) 293T, ii-T cells kunye nee-T cells eziphathwe yi-MNA. Ukubonakaliswa okugoqiweyo kuboniswe ngokumalunga ne-SK-OV-3. Ipatheni yokubonakaliswa ene-SEM ibonisiwe (n = abanikeli abaphilileyo abayi-6). Amaxabiso e-Ct angaphezu kwama-35 athathwa njengangabonwayo (UD). (F) Ukubonakaliswa kwe-SLC22A1 kunye ne-SLC22A2 kwi-SK-OV-3, HEK293T, iiseli ze-T kunye neeseli ze-T ezinyangwa nge-8mM MNA. Ukubonakaliswa okugoqiweyo kuboniswe ngokumalunga ne-SK-OV-3. Ipateni yokubonakaliswa ene-SEM ibonisiwe (n = 6 abanikeli abasempilweni). Amaxabiso e-Ct angaphezu kwama-35 athathwa njengangabonakaliyo (UD). (G) Umxholo we-MNA yeseli kwiiseli ze-T zabanikeli abasempilweni ezivuselelweyo emva kweeyure ezingama-72 zokufunxwa nge-MNA. Ipateni yokubonakaliswa ene-SEM ibonisiwe (n = 4 abanikeli abasempilweni).
I-MNA iveliswa ngokudlulisela iqela le-methyl ukusuka kwi-S-adenosyl-1-methionine (SAM) ukuya kwi-nicotinamide (NA) yi-nicotinamide N-methyltransferase (NNMT; Umfanekiso 3B). I-NNMT ibonakaliswa kakhulu kwiintlobo ngeentlobo zomhlaza zabantu kwaye inxulunyaniswa nokwanda, ukuhlasela kunye nokwanda kwe-metastasis (25-27). Ukuze siqonde ngcono umthombo we-MNA kwiiseli ze-T kwi-TME, sisebenzise i-scRNA-seq ukuchaza ukubonakaliswa kwe-NNMT kwiintlobo zeeseli kwi-ascites kunye neethumba zezigulane ezintathu ze-HGSC (Itheyibhile S5). Uhlalutyo lweeseli ezimalunga nama-6,500 lubonise ukuba kwiindawo ze-ascites kunye neethumba, ukubonakaliswa kwe-NNMT kuthintelwe kwi-fibroblast ecingelwayo kunye neeseli zethumba (Umfanekiso 3, C kunye no-D). Kubalulekile ukuqaphela ukuba akukho ukubonakaliswa kwe-NNMT okucacileyo kulo naliphi na inani labantu eliveza i-PTPRC (CD45 +) (Umfanekiso 3D kunye nomfanekiso S5A), nto leyo ebonisa ukuba i-MNA efunyenwe kwi-metabolite spectrum ingeniswe kwiiseli ze-T. Ukubonakaliswa kwe-aldehyde oxidase 1 (AOX1) kuguqula i-MNA ibe yi-1-methyl-2-pyridone-5-carboxamide (2-PYR) okanye i-1-methyl-4-pyridone-5-carboxamide (4-PYR); Umfanekiso 3B) ukwakhawulelwe kubemi be-fibroblasts eziveza i-COL1A1 (Umfanekiso S5A), ezibonisa ukuba ii-T cells azinawo amandla e-metabolism ye-MNA eqhelekileyo. Ipatheni yokubonakaliswa kwezi genes ezinxulumene ne-MNA iqinisekisiwe kusetyenziswa iseti yesibini yedatha yeseli ezimeleyo evela kwi-ascites evela kwizigulane ze-HGSC (Umfanekiso S5B; n = 6) (16). Ukongeza, uhlalutyo lwe-quantitative polymerase chain reaction (qPCR) lwee-T cells eziphilileyo ezinyangwe nge-MNA lubonise ukuba xa kuthelekiswa nee-ovarian tumor cells ze-SK-OV-3 control, i-NNMT okanye i-AOX1 phantse ayivezwanga (Umfanekiso 3E). Ezi ziphumo zingalindelekanga zibonisa ukuba i-MNA inokukhutshwa kwi-fibroblasts okanye kwi-tumor kwii-T cells ezikufutshane kwi-TME.
Nangona abaviwa bequka usapho lwee-organic cation transporters 1 ukuya kwi-3 (OCT1, OCT2 kunye ne-OCT3) ezifakwe kwi-soluble carrier 22 (SLC22) family (SLC22A1, SLC22A2 kunye ne-SLC22A3), ii-MNA transporters ezinokubakho azikachazwa (28). I-QPCR ye-mRNA evela kwiiseli ze-T ezinikelayo eziphilileyo ibonise amanqanaba aphantsi okubonakaliswa kwe-SLC22A1 kodwa amanqanaba angafumanekiyo e-SLC22A2, nto leyo eqinisekisile ukuba ibike yaxelwa ngaphambili kwiincwadi (Umfanekiso 3F) (29). Ngokwahlukileyo koko, umgca weseli ye-SK-OV-3 ovarian tumor cell ubonakalise amanqanaba aphezulu kuzo zombini ii-transporters (Umfanekiso 3F).
Ukuze kuvavanywe ukuba kungenzeka na ukuba ii-T cells zikwazi ukufunxa i-MNA yangaphandle, ii-T cells eziphilileyo zakhuliswa iiyure ezingama-72 xa kukho amazinga ahlukeneyo e-MNA. Xa kungekho MNA yangaphandle, umxholo weseli we-MNA awunakubonwa (Umfanekiso 3G). Nangona kunjalo, ii-T cells ezisebenzayo ezinyangwe nge-MNA yangaphandle zibonise ukwanda okuxhomekeke kumthamo kumxholo we-MNA kwiiseli, ukuya kuthi ga kwi-6 mM MNA (Umfanekiso 3G). Esi siphumo sibonisa ukuba nangona inqanaba eliphantsi lokubonakaliswa kwe-transporter kunye nokungabikho kwe-enzyme ephambili enoxanduva lwe-metabolism ye-MNA yangaphakathi kwiseli, i-TIL isenokuyithatha i-MNA.
Ububanzi bee-metabolites kwiiseli ze-T zezigulane kunye novavanyo lwe-MNA yokufunxwa kwi-in vitro bonyusa amathuba okuba ii-fibroblasts ezinxulumene nomhlaza (CAF) zikhuphe i-MNA kwaye iiseli ze-tumor zinokulawula i-phenotype kunye nomsebenzi we-TIL. Ukufumanisa impembelelo ye-MNA kwiiseli ze-T, iiseli ze-T ezinikelayo ezisempilweni zenziwe zasebenza kwi-vitro xa kukho okanye kungekho MNA, kwaye ukwanda kwazo kunye nemveliso ye-cytokine kwavavanywa. Emva kweentsuku ezi-7 zokongeza i-MNA kwidosi ephezulu, inani eliphindwe kabini labantu lancitshiswa ngokuphakathi, ngelixa amandla agcinwa kuzo zonke iidosi (Umfanekiso 4A). Ukongeza, unyango lwe-MNA yangaphandle lubangele ukwanda kwenani leeseli ze-CD4 + kunye ne-CD8 + T ezibonisa i-tumor necrosis factor-α (TNFα; Umfanekiso 4B). Ngokwahlukileyo koko, ukuveliswa kwe-IFN-γ ngaphakathi kweseli kwancitshiswa kakhulu kwiiseli ze-CD4 + T, kodwa kungekhona kwiiseli ze-CD8 + T, kwaye akukho tshintsho lubalulekileyo kwi-interleukin 2 (IL-2; Umfanekiso 4, C kunye no-D). Ngoko ke, uvavanyo lwe-immunosorbent oludityaniswe ne-enzyme (ELISA) lwee-supernatants ezivela kwezi nkcubeko zeeseli ze-T eziphathwe yi-MNA lubonise ukwanda okukhulu kwi-TNFα, ukwehla kwe-IFN-γ, kwaye akukho tshintsho kwi-IL-2 (Umfanekiso 4, E ukuya ku-G). . Ukwehla kwe-IFN-γ kubonisa ukuba i-MNA inokudlala indima ekuthinteleni umsebenzi we-anti-tumor weeseli ze-T. Ukuze kulinganiswe isiphumo se-MNA kwi-cytotoxicity ebangelwa yi-T cell, iiseli ze-chimeric antigen receptor T (FRα-CAR-T) ezijolise kwi-folate receptor α kunye neeseli ze-CAR-T (GFP) ezilawulwa ziiseli ze-green fluorescent protein (GFP) -CAR-T) ziveliswa ziiseli ze-peripheral blood mononuclear eziphilileyo (PBMC). Iiseli ze-CAR-T zakhuliswa iiyure ezingama-24 phambi kwe-MNA, zaze zakhuliswa kunye neeseli ze-ovarian tumor ze-SK-OV-3 zabantu eziveza i-folate receptor α kwi-effector to target ratio ye-10:1. Unyango lwe-MNA lubangele ukwehla okukhulu komsebenzi wokubulala weeseli ze-FRα-CAR-T, nto leyo efana neeseli ze-FRα-CAR-T ezinyangwe nge-adenosine (Umfanekiso 4H).
(A) Inani elipheleleyo leeseli ezisebenzayo kunye nokuphindaphinda kabini kwenani labantu (PD) ngokuthe ngqo kwinkcubeko ngomhla wesi-7. Igrafu yebha imele i-avareji + SEM yabanikeli abathandathu abasempilweni. Imela idatha evela ubuncinane kwizilingo ezizimeleyo ze-n = 3. (B ukuya ku-D) I-CD3/CD28 kunye ne-IL-2 zisetyenziselwe ukwenza iiseli ze-T zisebenze kumanqanaba azo e-MNA kangangeentsuku ezi-7. Ngaphambi kohlalutyo, iiseli zavuselelwa nge-PMA/ionomycin ngeGolgiStop iiyure ezi-4. Ukubonakaliswa kwe-TNFα (B) kwiiseli ze-T. Umfanekiso womzekelo (ekhohlo) kunye nedatha yetafile (ekunene) yokubonakaliswa kwe-TNFα kwiiseli eziphilayo. Ukubonakaliswa kwe-IFN-γ (C) kunye ne-IL-2 (D) kwiiseli ze-T. Ukubonakaliswa kwee-cytokines kulinganiswe nge-flow cytometry. Igrafu yebha imele i-avareji (n = 6 abanikeli abasempilweni) + SEM. Sebenzisa uhlalutyo lwendlela enye yokwahluka kunye nemilinganiselo ephindaphindwayo (*P<0.05 kunye **P<0.01) ukumisela ixabiso le-P. Imela idatha evela ubuncinane kwizilingo ezizimeleyo ze-n = 3. (E ukuya ku-G) i-CD3/CD28 kunye ne-IL-2 zisetyenziselwe ukwenza iiseli ze-T zisebenze kumanqanaba azo e-MNA kangangeentsuku ezi-7. Le medium iqokelelwe ngaphambi nasemva kweeyure ezi-4 zokukhuthaza i-PMA/ionomycin. Amanqanaba e-TNFα (E), i-IFN-γ (F) kunye ne-IL-2 (G) alinganiswe yi-ELISA. Igrafu yebha imele i-avareji (n = 5 abanikeli abasempilweni) + i-SEM. Ixabiso le-P limiselwe kusetyenziswa uhlalutyo lwendlela enye lokwahluka kunye nokulinganisa okuphindaphindiweyo (*P<0.05). Umgca onamachaphaza ubonisa umda wokufumanisa. (H) Uvavanyo lwe-cell lysis. Iiseli ze-FRα-CAR-T okanye ze-GFP-CAR-T zilungisiwe nge-adenosine (250μM) okanye i-MNA (10 mM) kangangeeyure ezingama-24, okanye zinganyangwa (Ctrl). Ukubulawa kwepesenti kweeseli ze-SK-OV-3 kulinganiswe. Ixabiso le-P limiselwe luvavanyo lwe-Welch t (*P<0.5 kunye ne-**P<0.01).
Ukuze kufunyanwe ukuqonda okusemgangathweni kokulawulwa kokubonakaliswa kwe-TNFα exhomekeke kwi-MNA, utshintsho kwi-TNFα mRNA yeeseli ze-T eziphathwe yi-MNA luvavanyiwe (Umfanekiso 5A). Iiseli ze-T ezinikelayo eziphilileyo eziphathwe nge-MNA zibonise ukwanda okuphindwe kabini kumanqanaba okubhalwa kwe-TNFα, okubonisa ukuba i-MNA ixhomekeke kumgaqo wokubhalwa kwe-TNFα. Ukuphanda le ndlela yokulawula enokwenzeka, izinto ezimbini ezaziwayo zokubhalwa kwe-TNFα, ezizezi: i-activated T cell nuclear factor (NFAT) kunye neproteni ethile 1 (Sp1), zivavanyiwe ukuphendula kwi-MNA ebopha kwi-proximal TNFα promoter (30). I-promoter ye-TNFα iqulethe iindawo ezi-6 ezichongiweyo zokubopha i-NFAT kunye neendawo ezi-2 zokubopha ze-Sp1, ezihambelana kwindawo enye [-55 base pairs (bp) ukusuka kwi-5'cap] (30). I-Chromatin immunoprecipitation (ChIP) ibonise ukuba xa inyangwa nge-MNA, ukubopha kwe-Sp1 kwi-promoter ye-TNFα kwanda kathathu. Ukufakwa kwe-NFAT nako kwanda kwaye kwasondela ekubalulekeni (Umfanekiso 5B). Ezi datha zibonisa ukuba i-MNA ilawula ukubonakaliswa kwe-TNFα ngokubhalwa kwe-Sp1, kwaye kancinci ilawula ukubonakaliswa kwe-NFAT.
(A) Xa kuthelekiswa neeseli ze-T ezikhuliswe ngaphandle kwe-MNA, utshintsho oluphindaphindeneyo lokubonakaliswa kwe-TNFα kwiiseli ze-T ezinyangwe nge-MNA. Ipatheni yokubonakaliswa nge-SEM ibonisiwe (n = 5 abanikeli abasempilweni). Imela idatha evela ubuncinane kwizilingo ezizimeleyo ze-n = 3. (B) Umkhuthazi we-TNFα weeseli ze-T ezinyangwe nge-8 mM MNA okanye ngaphandle kwayo emva kwe-NFAT kunye ne-Sp1 zidibene ne-(Ctrl) kunye ne-PMA/ionomycin stimulation kangangeeyure ezi-4. I-Immunoglobulin G (IgG) kunye ne-H3 zisetyenziswe njengolawulo olubi nolulungileyo lwe-immunoprecipitation, ngokulandelelana. Ukulinganiswa kwe-ChIP kubonise ukuba ukubopha kwe-Sp1 kunye ne-NFAT kwi-promoter ye-TNFα kwiiseli eziphathwe nge-MNA kwanda amaxesha amaninzi xa kuthelekiswa nolawulo. Imela idatha evela ubuncinane kwizilingo ezizimeleyo ze-n = 3. Ixabiso le-P limiselwe zii-t-tests ezininzi (*** P <0.01). (C) Xa kuthelekiswa ne-ascites ye-HGSC, iiseli ze-T (ezingeyo-cytotoxic) zibonise ukwanda kokubonakaliswa kwe-TNF kwithumba. Imibala imele izigulane ezahlukeneyo. Iiseli ezibonisiweyo zithathwe iisampulu ngokungacwangciswanga ukuya kwi-300 kwaye zaxutywa ukuze kuthintelwe ukutsalwa kakhulu (** Padj = 0.0076). (D) Imodeli ecetywayo ye-MNA yomhlaza wesibeleko. I-MNA iveliswa kwiiseli zethumba kunye ne-fibroblasts kwi-TME kwaye ithathwa ziiseli ze-T. I-MNA yonyusa ukubopha kwe-Sp1 kwi-promoter ye-TNFα, okukhokelela ekwandeni kokubhalwa kwe-TNFα kunye nemveliso ye-cytokine ye-TNFα. I-MNA ikwabangela ukwehla kwe-IFN-γ. Ukuthintelwa komsebenzi weeseli ze-T kukhokelela ekunciphiseni amandla okubulala kunye nokukhula ngokukhawuleza kwethumba.
Ngokweengxelo, i-TNFα ineempembelelo zokulwa nethumba kunye ne-anti-tumor ezixhomekeke ngaphambili nangasemva, kodwa inendima eyaziwayo ekukhuthazeni ukukhula kunye nokusasazeka komhlaza wesibeleko (31-33). Ngokweengxelo, uxinaniso lwe-TNFα kwi-ascites kunye nezicubu zethumba kwizigulane ezinomhlaza wesibeleko luphezulu kunolo lukwizicubu ezingengobungozi (34-36). Ngokwendlela esebenza ngayo, i-TNFα inokulawula ukusebenza, ukusebenza kunye nokwanda kweeseli ezimhlophe zegazi, kwaye itshintshe uhlobo lweeseli zomhlaza (37, 38). Ngokuhambelana nezi ziphumo, uhlalutyo lokubonakaliswa kwezakhi zofuzo ezahlukeneyo lubonise ukuba i-TNF inyuswe kakhulu kwiiseli ze-T kwizicubu zethumba xa kuthelekiswa ne-ascites (Umfanekiso 5C). Ukwanda kokubonakaliswa kwe-TNF kwabonakala kuphela kubemi beeseli ze-T abane-phenotype engeyiyo i-cytotoxic (Umfanekiso S5A). Ngamafutshane, ezi datha zixhasa umbono wokuba i-MNA ineempembelelo ezimbini zokucinezela amasosha omzimba kunye nokukhuthaza ithumba kwi-HGSC.
Ukubhala iilebhile ze-fluorescent ngokusekelwe kwi-flow cytometry kuye kwaba yindlela ephambili yokufunda imetabolism ye-TIL. Ezi zifundo zibonise ukuba xa kuthelekiswa nee-lymphocytes zegazi ezingaphandle okanye iiseli ze-T ezivela kwizitho ze-lymphoid yesibini, i-murine kunye ne-TIL yomntu zinotyekelo oluphezulu lokufunxa i-glucose (4, 39) kunye nokulahleka kancinci komsebenzi we-mitochondrial (19, 40). Nangona sibonile iziphumo ezifanayo kolu phononongo, uphuhliso oluphambili kukuthelekisa imetabolism yeeseli ze-tumor kunye ne-TIL ezivela kwizicubu ze-tumor ezifanayo ezisusiweyo. Ngokuhambelana nezinye zezi ngxelo zangaphambili, iiseli ze-tumor (CD45-EpCAM +) ezivela kwi-ascites kunye nee-tumor zifunxa i-glucose ephezulu kuneeseli ze-CD8 + kunye ne-CD4 + T, ezixhasa ukuba ukufunxa i-glucose ephezulu kwiiseli ze-tumor kunokuthelekiswa neeseli ze-T. Ingcamango yokhuphiswano lweeseli ze-T. TME. Nangona kunjalo, umsebenzi we-mitochondrial weeseli ze-tumor uphezulu kunoweeseli ze-CD8 + T, kodwa umsebenzi we-mitochondrial ufana noweeseli ze-CD4 + T. Ezi ziphumo ziqinisa umxholo ovelayo wokuba imetabolism ye-oxidative ibalulekile kwiiseli ze-tumor (41, 42). Bakwacebisa ukuba iiseli ze-CD8 + T zisenokuba sesichengeni sokungasebenzi kakuhle kwe-oxidative kuneeseli ze-CD4 + T, okanye ukuba iiseli ze-CD4 + T zingasebenzisa imithombo yekhabhoni ngaphandle kweglucose ukugcina umsebenzi we-mitochondrial (43, 44). Kufuneka kuqatshelwe ukuba asibonanga mahluko ekuthathweni kweglucose okanye umsebenzi we-mitochondrial phakathi kwee-effectors ze-CD4 + T, i-T effector memory kunye neeseli zememori ze-T central kwi-ascites. Ngokufanayo, imeko yokwahluka kweeseli ze-CD8 + T kwi-tumor ayinanto yakwenza notshintsho ekuthathweni kweglucose, nto leyo egxininisa umahluko obalulekileyo phakathi kweeseli ze-T ezikhuliswe kwi-vitro kunye ne-human TIL in vivo (22). Ezi ngqwalasela zikwaqinisekiswe kukusetyenziswa kokwabiwa kwe-automatic cell population allocation, nto leyo ekwatyhila ukuba iiseli ze-CD45 + / CD3- / CD4 + / CD45RO + ezine-glucose uptake ephezulu kunye nomsebenzi we-mitochondrial kuneeseli ze-tumor zixhaphakile kodwa zine-Metabolic active cell population. Olu luntu lunokumela inani elincinci leeseli ze-myeloid suppressor okanye iiseli ze-plasmacytoid dendritic ezichongiweyo kuhlalutyo lwe-scRNA-seq. Nangona zombini ezi zixeliwe kwiithumba zesibeleko zabantu [45], zisafuneka Umsebenzi ongaphezulu kukuchaza olu hlobo lwe-myeloid.
Nangona iindlela ezisekelwe kwi-flow cytometry zinokucacisa umahluko ngokubanzi kwi-glucose kunye ne-oxidative metabolism phakathi kweentlobo zeeseli, ii-metabolites ezichanekileyo eziveliswa yi-glucose okanye eminye imithombo yekhabhoni ye-mitochondrial metabolism kwi-TME azikamiselwa okwangoku. Ukwabela ubukho okanye ukungabikho kwee-metabolites kwi-TIL subset ethile kufuna ukucocwa koluntu lweeseli kwizicubu ezikhutshiweyo. Ke ngoko, indlela yethu yokufumisa iiseli kunye ne-mass spectrometry inokubonelela ngengqiqo kwi-metabolites ezityebiswe ngokwahlukileyo kwiiseli ze-T kunye ne-tumor cell populations kwiisampuli zesigulana ezifanayo. Nangona le ndlela ineenzuzo ngaphezu kokuhlelwa kweeseli ezisebenza nge-fluorescence, iilayibrari ezithile ze-metabolite zinokuchaphazeleka ngenxa yokuzinza okungokwemvelo kunye/okanye izinga lokujika ngokukhawuleza (22). Nangona kunjalo, indlela yethu ikwazile ukuchonga ii-metabolites ezimbini eziqatshelweyo ze-immunosuppressive, i-adenosine kunye ne-kynurenine, kuba zahlukile kakhulu phakathi kweentlobo zeesampuli.
Uhlalutyo lwethu lwe-metabonomic lwee-tumor kunye ne-TIL subtypes lubonelela ngolwazi oluthe kratya ngendima yee-metabolites kwi-ovarian TME. Okokuqala, sisebenzisa i-flow cytometry, sifumanise ukuba akukho mahluko kwimisebenzi ye-mitochondrial phakathi kwee-tumor kunye nee-CD4 + T cells. Nangona kunjalo, uhlalutyo lwe-LC-MS/MS lutyhile utshintsho olukhulu kubuninzi bee-metabolites phakathi kwezi qela, nto leyo ebonisa ukuba izigqibo malunga ne-TIL metabolism kunye nomsebenzi wayo we-metabolism iyonke zifuna ukutolikwa ngononophelo. Okwesibini, i-MNA yi-metabolite enomahluko omkhulu phakathi kwee-CD45-cells kunye nee-T cells kwi-ascites, kungekhona ii-tumor. Ke ngoko, ukwahlulwahlulwa kunye nendawo ye-tumor kunokuba nemiphumo eyahlukileyo kwi-TIL metabolism, nto leyo egxininisa ukungafani okunokwenzeka kwindawo ethile encinci. Okwesithathu, ukubonakaliswa kwe-enzyme evelisa i-MNA i-NNMT ikakhulu kunqunyelwe kwi-CAF, eyi-tumor cells kancinci, kodwa amanqanaba e-MNA abonwayo abonwa kwii-T cells ezivela kwi-tumor. Ukubonakaliswa kakhulu kwe-NNMT kwi-ovarian CAF kunesiphumo esaziwayo sokukhuthaza umhlaza, ngokuyinxenye ngenxa yokukhuthaza i-CAF metabolism, ukuhlasela kwe-tumor kunye ne-metastasis (27). Nangona inqanaba lilonke le-TIL liphakathi, ukubonakaliswa kwe-NNMT kwi-CAF kunxulumene ngokusondeleyo ne-Cancer Genome Atlas (TCGA) mesenchymal subtype, enxulunyaniswa nokubikezela okubi (27, 46, 47). Okokugqibela, ukubonakaliswa kwe-enzyme AOX1 enoxanduva lokuwohloka kwe-MNA kukwakhawulelwe kubemi be-CAF, nto leyo ebonisa ukuba ii-T cells azinawo amandla okugaya i-MNA. Ezi ziphumo zixhasa ingcamango yokuba nangona kufuneka umsebenzi ongakumbi ukuqinisekisa oku kufunyanisiweyo, amanqanaba aphezulu e-MNA kwii-T cells anokubonisa ukuba kukho indawo encinci ye-CAF ethintela ukhuseleko lwe-immunosuppressive.
Ngenxa yenqanaba eliphantsi lokubonakaliswa kwe-MNA transporters kunye namanqanaba angabonakaliyo eeproteni eziphambili ezibandakanyeka kwi-metabolism ye-MNA, ubukho be-MNA kwiiseli ze-T abulindelekanga. Akukho NNMT okanye i-AOX1 enokubonwa ngohlalutyo lwe-scRNA-seq kunye ne-qPCR ekujoliswe kuyo yamaqela amabini azimeleyo. Ezi ziphumo zibonisa ukuba i-MNA ayidalwanga ziiseli ze-T, kodwa ifunxwa kwi-TME ejikelezileyo. Uvavanyo lwe-in vitro lubonisa ukuba iiseli ze-T zihlala ziqokelela i-MNA yangaphandle.
Izifundo zethu ze-in vitro zibonise ukuba i-MNA yangaphandle ibangela ukubonakaliswa kwe-TNFα kwiiseli ze-T kwaye iphucula ukubopha kwe-Sp1 kwi-promoter ye-TNFα. Nangona i-TNFα inemisebenzi yokulwa ne-tumor kunye ne-anti-tumor, kumhlaza we-ovarian, i-TNFα inokukhuthaza ukukhula komhlaza we-ovarian (31-33). Ukungathinteli i-TNFα kwinkcubeko yeseli ye-ovarian tumor okanye ukususwa kwesignali ye-TNFα kwiimodeli zegundane kunokuphucula ukuveliswa kwe-cytokine ebangelwa yi-TNFα kunye nokuthintela ukukhula kwe-tumor (32, 35). Ke ngoko, kule meko, i-MNA evela kwi-TME inokusebenza njenge-metabolite ekhuthaza ukuvuvukala ngokusebenzisa indlela exhomekeke kwi-TNFα nge-autocrine loop, ngaloo ndlela ikhuthaza ukwenzeka kunye nokusasazeka komhlaza we-ovarian (31). Ngokusekelwe kolu nokwenzeka, i-TNFα blockade ifundwa njengearhente enokubakho yonyango lomhlaza we-ovarian (37, 48, 49). Ukongeza, i-MNA iphazamisa i-cytotoxicity yeeseli ze-CAR-T kwiiseli ze-ovarian tumor, inika ubungqina obongezelelweyo bokucinezelwa komzimba okubangelwa yi-MNA. Ngokudibeneyo, ezi ziphumo zibonisa imodeli apho iithumba kunye neeseli zeCAF zikhupha i-MNA kwi-extracellular TME. Ngokusebenzisa (i) ukuvuselela ukukhula komhlaza wesibeleko obangelwa yi-TNF kunye (ii) nokuthintela umsebenzi we-cytotoxic cell T obangelwa yi-MNA, oku kunokuba nefuthe le-tumor eliphindwe kabini (Umfanekiso 5D).
Ukuqukumbela, ngokusebenzisa indibaniselwano yokuqiniswa kweeseli ngokukhawuleza, ukulandelelana kweeseli enye kunye nokuchonga i-metabolic, olu phononongo lutyhile umahluko omkhulu we-immunometabolomic phakathi kwee-tumor kunye nee-ascites cells kwizigulane ze-HGSC. Olu hlalutyo lubanzi lubonise ukuba kukho umahluko ekuthathweni kweglucose kunye nomsebenzi we-mitochondrial phakathi kweeseli ze-T, kwaye luchonge i-MNA njenge-metabolite elawulayo yomzimba engeyiyo iseli. Le datha inefuthe kwindlela i-TME echaphazela ngayo imetabolism yeeseli ze-T kumhlaza wabantu. Nangona ukhuphiswano oluthe ngqo lwezondlo phakathi kweeseli ze-T kunye neeseli zomhlaza luye lwabikwa, ii-metabolites zinokusebenza njengabalawuli abangathanga ngqo ukukhuthaza ukuqhubela phambili kwe-tumor kwaye mhlawumbi zicinezele iimpendulo zomzimba ezingapheliyo. Inkcazo engaphezulu yendima yokusebenza kwezi metabolites ezilawulayo inokuvula amaqhinga ahlukeneyo okuphucula impendulo yomzimba yokulwa ne-tumor.
Iisampulu zezigulane kunye nedatha yeklinikhi zifunyenwe nge-BC cancer tumor tissue repository eqinisekisiwe yiCanadian Tissue Repository Network. Ngokweprotokholi evunyiweyo yi-BC Cancer Research Ethics Committee kunye neYunivesithi yaseBritish Columbia (H07-00463), zonke iisampulu zezigulane kunye nedatha yeklinikhi zifumene imvume ebhaliweyo okanye zarhoxisa ngokusesikweni imvume yazo. Iisampulu zigcinwa kwi-BioBank eqinisekisiweyo (BRC-00290). Iimpawu zesigulane ezineenkcukacha ziboniswe kwiTheyibhile S1 kunye ne-S5. Kwi-cryopreservation, i-scalpel isetyenziselwa ukubola isampuli yesifo sesigulane ngomatshini kwaye emva koko iyityhale nge-100-micron filter ukuze kufunyanwe iseli enye. I-ascites yesigulane yafakwa kwi-centrifuge kwi-1500 rpm imizuzu eli-10 kwi-4°C ukuze kufakwe iiseli kwaye kususwe i-supernatant. Iiseli ezifunyenwe kwi-tumor kunye ne-ascites zigcinwe kwi-50% ye-heat-inactivated human AB serum (Sigma-Aldrich), i-40% RPMI-1640 (Thermo Fisher Scientific) kunye ne-10% ye-dimethyl sulfoxide. Ezi suspensions zeseli enye ezigciniweyo zinyibilikisiwe zaza zasetyenziselwa i-metabolomics kunye nokuqinisekiswa kwe-metabolite echazwe ngezantsi.
Isixhobo esipheleleyo siquka i-0.22 μm ehluziweyo ene-50:50 eyongeziweyo ye-RPMI 1640: AimV. RPMI 1640 + 2.05 mM l-glutamine (Thermo Fisher Scientific) eyongeziweyo nge-10% ye-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), isisombululo se-1 x Penicillin Streptomycin (PenStrep) (Thermo Fisher Scientific) kunye ne-50 μMB-mercaptoethanol. I-AimV (Invitrogen) yongezwe yi-20 mM Hepes (Thermo Fisher Scientific) kunye ne-2 mM l-glutamine (Thermo Fisher Scientific). I-flow cytometer staining buffer yayine-0.22μm filtered phosphate buffered saline (PBS; Invitrogen) eyongeziweyo yi-3% ye-heat-inactivated AB human serum (Sigma). I-cell enrichment buffer yenziwe yi-0.22μm filtered PBS kwaye yongezwa yi-0.5% heat-inactivated human AB serum (Sigma-Aldrich).
Kwi-medium epheleleyo engama-37°C, iiseli zafakwa i-10 nM MT DR kunye ne-100 μM 2-NBDG imizuzu engama-30. Okulandelayo, iiseli zafakwa i-viability dye eF506 kwi-4°C imizuzu eli-15. Phinda uxhome iiseli kwi-FC Block (eBioscience) kunye ne-Brilliant Stain Buffer (BD Biosciences), udibanise i-flow cytometer staining buffer (ngokwemiyalelo yomenzi), kwaye uzifake kwi-incubator imizuzu eli-10 kubushushu begumbi. Faka i-stain kwiiseli ngeseti yee-antibodies (Itheyibhile S2) kwi-flow cytometry staining buffer kwi-4°C imizuzu engama-20. Phinda uxhome iiseli kwi-flow cytometry staining buffer (Cytek Aurora; 3L-16V-14B-8R configuration) ngaphambi kohlalutyo. Sebenzisa i-SpectroFlo kunye ne-FlowJo V10 ukuhlalutya idatha yokubalwa kweeseli, kwaye usebenzise i-GraphPad Prism 8 ukwenza idatha. I-median fluorescence intensity (MFI) ye-2-NBDG kunye ne-MT DR yenziwe yaba yesiqhelo, emva koko kwasetyenziswa uvavanyo lwe-t oludibeneyo kuhlalutyo lwezibalo ukuze kujongwe izigulane ezifanayo. Susa zonke iindawo ezineziganeko ezingaphantsi kwama-40 kuhlalutyo; faka ixabiso le-MFI elingu-1 kuzo naziphi na ixabiso elibi ngaphambi kokuba wenze uhlalutyo lwezibalo kunye nokuboniswa kwedatha.
Ukuze songeze icebo lokugada ngesandla lephaneli yenkqubo engentla, sisebenzise isichazi-magama esipheleleyo somthi wokuthintela imilo (FAUST) (21) ukuze sinike iiseli ngokuzenzekelayo kuluntu emva kokususa iiseli ezifileyo kwiFlowJo. Silawula imveliso ngesandla ukuze sidibanise uluntu olubonakala ngathi alusasazwanga kakuhle (sidibanisa iPD1+ neeseli ze-PD1-tumor) kunye noluntu olugciniweyo. Isampulu nganye ine-avareji yeeseli ezingaphezu kwe-2%, kuluntu olupheleleyo oluyi-11.
I-Ficoll gradient density centrifugation yasetyenziswa ukwahlula i-PBMC kwiimveliso zokwahlulwa kwe-leukocyte (STEMCELL Technologies). Iiseli ze-CD8 + T zahlulwe kwi-PBMC kusetyenziswa ii-CD8 MicroBeads (Miltenyi) zaza zandiswa kwi-medium epheleleyo kusetyenziswa i-TransAct (Miltenyi) iiveki ezi-2 ngokwemiyalelo yomenzi. Iiseli zavunyelwa ukuba zime iintsuku ezi-5 kwi-medium epheleleyo equlethe i-IL-7 (10 ng/ml; PeproTech), zaze zavuselelwa kwakhona nge-TransAct. Ngomhla wesi-7, ngokwemiyalelo yomenzi, ii-CD45 MicroBeads zabantu (Miltenyi) zasetyenziswa ukutyebisa iiseli kwimijikelo emithathu elandelelanayo. Iiseli zahlulwe ukuze kuhlalutywe i-flow cytometry (njengoko kuchaziwe apha ngasentla), kwaye iiseli ezisisigidi zahlulwe kathathu ukuze kuhlalutywe i-LC-MS/MS. Iisampuli zacutshungulwa yi-LC-MS/MS njengoko kuchaziwe apha ngezantsi. Siqikelele ixabiso le-metabolite elingekhoyo ngenani le-ion eliyi-1,000. Isampulu nganye ilungiswa ngokwenani lee-ion ezipheleleyo (TIC), iguqulwe ngokwe-logarithmic kwaye ilungiswe ngokuzenzekelayo kwiMetaboAnalystR ngaphambi kohlalutyo.
Ukumiswa kweseli enye yesigulane ngasinye kunyibilikisiwe kwaye kwahluzwa ngesihluzo se-40 μm ukuya kwindawo epheleleyo (njengoko kuchaziwe apha ngasentla). Ngokwenkqubo yomenzi, imijikelo emithathu elandelelanayo yokukhethwa okulungileyo ngokwahlukana kweentsimbi zemagnethi kusetyenziswa iiMicroBeads (Miltenyi) yasetyenziswa ukutyebisa iisampulu zeeseli zeCD8+, CD4+ kunye neCD45 (kwiqhwa). Ngamafutshane, iiseli ziphinda zixhonywe kwi-buffer yokutyebisa iiseli (njengoko kuchaziwe apha ngasentla) kwaye zibalwe. Iiseli zifakwe iintsimbi zeCD8 zabantu, iintsimbi zeCD4 zabantu okanye iintsimbi zeCD45 zabantu (Miltenyi) kwi-4°C imizuzu eli-15, emva koko zihlanjwe nge-buffer yokutyebisa iiseli. Isampulu idluliselwa kwikholamu ye-LS (Miltenyi), kwaye kuqokelelwe iinxalenye ezilungileyo nezingalunganga. Ukuze kuncitshiswe ixesha kwaye kuphuculwe inyathelo lokubuyisela iiseli, i-CD8-fraction emva koko isetyenziselwa umjikelo wesibini wokutyebisa iiseli zeCD4+, kwaye i-CD4-fraction isetyenziselwa ukutyebisa iiseli zeCD45 ezilandelayo. Gcina isisombululo kwiqhwa kuyo yonke inkqubo yokuhlukana.
Ukulungiselela iisampulu zohlalutyo lwe-metabolite, iiseli zahlanjwa kanye ngesisombululo setyuwa ebandayo, kwaye i-1 ml ye-80% ye-methanol yongezwa kwisampulu nganye, emva koko yahluzwa yaza yaqandiswa kwi-nitrogen engamanzi. Iisampulu zafakwa kwimijikelo emithathu yokuqandisa-ukunyibilika kwaye zaxutywa kwi-centrifuge kwi-14,000 rpm imizuzu eli-15 kwi-4°C. I-supernatant equlethe i-metabolite iyasuswa ide yome. I-metabolite zaphinda zanyibilika kwi-50 μl ye-0.03% ye-formic acid, zaxutywa ukuze zixutywe, zaza zaxutywa ukuze kususwe inkunkuma.
Khipha ii-metabolites njengoko kuchaziwe apha ngasentla. Dlulisa i-supernatant kwibhotile ye-chromatography yolwelo esebenza kakuhle kakhulu ukuze kwenziwe uphando lwe-metabolomics. Sebenzisa iprotokholi yonyango olungacwangciswanga ukunyanga isampuli nganye ngenani elifanayo leeseli ukuthintela iziphumo ze-batch. Senze uvavanyo lomgangatho wee-metabolites zehlabathi ezipapashwe ngaphambili kwi-AB SCIEX QTRAP 5500 Triple Quadrupole Mass Spectrometer (50). Uhlalutyo lwe-Chromatographic kunye nokuhlanganiswa kwendawo ende kwenziwe kusetyenziswa isoftware ye-MultiQuant version 2.1 (Applied Biosystems SCIEX).
Inani lee-ion eliyi-1000 lisetyenzisiwe ukuqikelela ixabiso le-metabolite elingekhoyo, kwaye i-TIC yesampulu nganye isetyenzisiwe ukubala indawo yencochoyi eqhelekileyo ye-metabolite nganye efunyenweyo ukulungisa utshintsho oluvezwe luhlalutyo lwezixhobo ezivela ekucwangcisweni kwesampulu. Emva kokuba i-TIC iqhelekile, i-MetaboAnalystR(51) (ipharamitha emiselweyo) isetyenziselwa ukuguqulwa kwe-logarithmic kunye nokulinganisa umgca we-normal ozenzekelayo. Sisebenzise i-PCA enephakheji ye-vegan R ukwenza uhlalutyo lokuhlola umahluko we-metabolome phakathi kweentlobo zeesampulu, kwaye sisebenzise uhlalutyo lwe-redundancy oluyingxenye ukuhlalutya izigulane. Sebenzisa indlela ye-Ward ukwakha i-dendrogram yemephu yobushushu ukuhlanganisa umgama we-Euclidean phakathi kweesampulu. Sisebenzise i-limma (52) kwi-standard metabolite abuse ukuchonga ii-metabolites ezininzi ngokwahlukileyo kulo lonke uhlobo lweseli kunye ne-microenvironment. Ukuze kube lula ukuchaza, sisebenzisa i-group mean parameter ukucacisa imodeli, kwaye siqwalasele iintlobo zeeseli kwi-microenvironment njengeqela ngalinye (n = amaqela ayi-6); kuvavanyo lokubaluleka, senze imilinganiselo emithathu ephindaphindwayo kwi-metabolite nganye Ukuze siphephe ukuphindaphinda okungamanga, isigulane safakwa njengomqobo kuyilo lwe-limma. Ukuze sijonge umahluko kwi-metabolites phakathi kwezigulane ezahlukeneyo, silungise imodeli ye-limma kuquka nezigulane ngendlela emiselweyo. Sixela ukubaluleka komahluko ochazwe kwangaphambili phakathi kohlobo lweseli kunye ne-microenvironment yePadj <0.05 (ulungiso lweBenjamini-Hochberg).
Emva kokuphuculiswa kwamandla kusetyenziswa iMiltenyi Dead Cell Removal Kit (>80% viability), kwenziwe ulandelelwano lwe-transcriptome yeseli enye kwi-ascites ephilayo eqandisiweyo kunye neesampuli ze-tumor kusetyenziswa i-protocol ye-10x 5′gene expression. Amatyala amahlanu ane-tumor kunye ne-ascites ezifanayo ahlalutywe, nangona ukusinda okuphantsi kwisampulu enye ye-tumor kuthintele ukufakwa kwayo. Ukuze kufezekiswe ukhetho oluninzi lwezigulane, sidibanise iisampulu zesigulane ngasinye kwimigca ye-10x chromium controller, saza sahlalutya ii-ascites kunye neendawo ze-tumor ngokwahlukeneyo. Emva kokulandelelana [Illumina HiSeq 4000 28×98 bp paired end (PE), i-Quebec genome; umyinge we-73,488 kunye ne-41,378 yokufunda ngeseli nganye ye-tumor kunye ne-ascites ngokulandelelana], sisebenzise i-CellSNP kunye ne-Vireo (53) (ngokusekelwe kwi-CellSNP njengoko i-SNP yomntu eqhelekileyo (i-VCF) enikezelwa yi-GRCh38 inikwe isazisi somnikeli. Sisebenzisa i-SNPRelate ukugqiba isazisi esisondeleyo (i-IBS) sesimo se-genotype yesigulane (i-IBS), ngaphandle kweeseli ezingabelwanga kunye neeseli ezichongiweyo njengee-duplexes kunye nabanikeli abahambelanayo phakathi kwee-ascites kunye neesampuli ze-tumor (54). Ngokusekelwe kulo msebenzi, sigcine amatyala amathathu anokumelwa kweeseli ezininzi kwi-tumor kunye ne-ascites ukuze kuhlalutywe ngezantsi. Emva kokwenza inyathelo lokucoca ngobuninzi kwi-scater (55) kunye ne-scran (56) BioConductor packaging, oku kuvelise iiseli ezingama-6975 (iiseli ezingama-2792 kunye nama-4183 ezivela kwi-tumor kunye ne-ascites, ngokulandelelana) ukuze kuhlalutywe. Sisebenzisa i-igraph's (57) Louvain clustering yenethiwekhi yommelwane ekufutshane (i-SNN) ekwabelwana ngayo ngokusekelwe kwi-Jaccard umgama ukuya kwiiseli zeqela ngokwembonakalo. Iiqoqo zachazwa ngesandla kwiintlobo zeeseli ezibonisa ukuba zisekelwe kwimbonakalo yejini ye-marker kwaye zabonwa nge-t-SNE. Iiseli ze-T ezine-cytotoxic zichazwa ngokubonakaliswa kwe-CD8A kunye ne-GZMA, ngaphandle kwee-subclusters ezine-ribosomal protein expression ephantsi. Sifikelele kwidatha epapashwe ngu-Izar et al. (16), kubandakanya ukufakwa kwe-t-SNE yabo, inokulawula ukugqithana kwembonakalo phakathi kweempawu zeeseli zomzimba kunye nokubonakaliswa kwe-NNMT.
I-PBMC yahlulwe kwiimveliso zokwahlulwa kwe-leukocyte (STEMCELL Technologies) nge-Ficoll gradient density centrifugation. Iiseli ze-CD3 + zahlulwe kwi-PBMC kusetyenziswa ii-CD3 beads (Miltenyi). Ukuba kukho okanye kungekho MNA, iiseli ze-CD3+ zavuselelwa nge-plate-bound CD3 (5μg/ml), i-soluble CD28 (3μg/ml) kunye ne-IL-2 (300 U/ml; Proleukin). Ngomhla wokugqibela wokwandiswa, i-viability (Fixable Viability Dye eFluor450, eBioscience) kunye ne-proliferation (123count eBeads, Thermo Fisher Scientific) zavavanywa nge-flow cytometry. Vavanya umsebenzi we-effector ngokuvuselela iiseli ezine-PMA (20 ng/ml) kunye ne-ionomycin (1μg/ml) kunye ne-GolgiStop iiyure ezi-4, kwaye ujonge i-CD8-PerCP (RPA-T8, BioLegend), i-CD4-AF700 (RPA-T4), i-BioLegend) kunye ne-TNFα-fluorescein isothiocyanate (FITC) (MAb11, BD). Vuselela iiseli ze-qPCR kunye ne-ChIP nge-PMA (20 ng/ml) kunye ne-ionomycin (1μg/ml) iiyure ezi-4. I-ELISA supernatant yaqokelelwa ngaphambi nasemva kokuvuselela nge-PMA (20 ng/ml) kunye ne-ionomycin (1 μg/ml) iiyure ezi-4.
Landela iprotocol yomenzi ukuze wahlukanise i-RNA usebenzisa i-RNeasy Plus Mini Kit (QIAGEN). Sebenzisa i-QIAshredder (QIAGEN) ukuze wenze isampuli ibe yi-homogenize. Sebenzisa i-RNA enamandla aphezulu ukuya kwi-cDNA kit (Thermo Fisher Scientific) ukuze udibanise i-DNA eyongezelelekileyo (cDNA). Sebenzisa i-TaqMan Rapid Advanced Master Mix (Thermo Fisher Scientific) ukuze ulinganise ukubonakaliswa kwe-gene (ngokweprotocol yomenzi) ngezi probes zilandelayo: Hs00196287_m1 (NNMT), Hs00154079_m1 (AOX1), Hs00427552_m1 (SLC22A1), Hs02786624_g1 [glyceraldehyde-3-phosphate off Hydrogen (GAPDH)] kunye ne-Hs01010726_m1 (SLC22A2). Iisampulu ziqhutywe kwinkqubo ye-StepOnePlus ye-PCR yexesha langempela (Applied Biosystems) (Applied Biosystems) kwi-MicroAmp fast optical 96-well reaction plate (Applied Biosystems) enefilimu ye-MicroAmp optical. Naliphi na ixabiso le-Ct elidlula ama-35 lithathwa njengelingaphezulu komda wokufumanisa kwaye liphawulwe njengelingafumanekiyo.
Yenza i-ChIP njengoko kuchaziwe ngaphambili (58). Ngamafutshane, iiseli ziphathwe nge-formaldehyde (uxinzelelo lokugqibela oluyi-1.42%) zaza zafakwa kwiqondo lobushushu begumbi imizuzu eli-10. Sebenzisa i-buffer yokuvuvukala eyongezelelweyo (25 mM Hepes, 1.5 mM MgCl2, 10 mM KCl kunye ne-0.1% NP-40) kwiqhwa imizuzu eli-10, uze uphinde uxhonywe kwi-immunoprecipitation buffer njengoko kuchaziwe (58). Emva koko isampuli yafakwa kwi-sonicated ngale mijikelo ilandelayo: imijikelo eli-10 (ii-pulses ezingama-20 zemizuzwana eli-1) kunye nexesha elingashukumiyo lemizuzwana engama-40. Faka i-immunoglobulin G ye-ChIP-grade (iTekhnoloji yoKubonisa iiSeli; 1μl), i-histone H3 (iTekhnoloji yoKubonisa iiSeli; 3μl), i-NFAT (i-Invitrogen; 3μl) kunye ne-SP1 (iTekhnoloji yoKubonisa iiSeli; 3μl) kunye ne-SP1 (iTekhnoloji yoKubonisa iiSeli; 3μl) kunye nesampuli kwi-4°CC shake ubusuku bonke. Faka ii-protein A beads (Thermo Fisher Scientific) kwisampuli kwi-4°C ngokuzishukumisa kancinci iyure e-1, uze usebenzise ii-chelex beads (Bio-Rad) ukuze utyebise i-DNA, kwaye usebenzise i-proteinase K (Thermo Fisher) ekugayweni kweproteni. I-TNFα promoter ifunyenwe yi-PCR: phambili, i-GGG TAT CCT TGA TGC TTG TGT; ngokuchaseneyo, i-GTG CCA ACA ACT GCC TTT ATA TG (imveliso ye-207-bp). Imifanekiso iveliswe yi-Image Lab (Bio-Rad) kwaye yalinganiswa kusetyenziswa isoftware ye-ImageJ.
I-supernatant yenkcubeko yeseli iqokelelwe njengoko kuchaziwe apha ngasentla. Ukuchonga kwenziwe ngokweenkqubo zomenzi we-human TNFα ELISA kit (Invitrogen), i-human IL-2 ELISA kit (Invitrogen) kunye ne-human IFN-γ ELISA kit (Abcam). Ngokwemigaqo yomenzi, i-supernatant ixutywe nge-1:100 ukuze kufunyanwe i-TNFα kunye ne-IL-2, kunye ne-1:3 ukuze kufunyanwe i-IFN-γ. Sebenzisa i-EnVision 2104 Multilabel Reader (PerkinElmer) ukulinganisa ukufunxwa kwi-450 nm.
I-PBMC yahlulwe kwiimveliso zokwahlulwa kwe-leukocyte (STEMCELL Technologies) nge-Ficoll gradient density centrifugation. Iiseli ze-CD3 + zahlulwe kwi-PBMC kusetyenziswa ii-CD3 beads (Miltenyi). Ukubakho okanye ukungabikho kwe-MNA, iiseli ze-CD3+ zavuselelwa nge-plate-bound CD3 (5μg/ml), i-soluble CD28 (3μg/ml) kunye ne-IL-2 (300 U/ml; Proleukin) kangangeentsuku ezi-3. Emva kweentsuku ezi-3, iiseli zaqokelelwa zaza zahlanjwa nge-0.9% saline, kwaye i-pellet yaqandiswa. Ukubalwa kweeseli kwenziwa nge-flow cytometry (Cytek Aurora; 3L-16V-14B-8R configuration) kusetyenziswa i-123count eBeads.
Iimetabolites ezikhutshiweyo njengoko kuchaziwe apha ngasentla. I-extract eyomileyo yaphinda yahlanganiswa kwi-concentration ye-4000 cell equals/μl. Hlalutya isampuli nge-reversed-phase chromatography (1290 Infinity II, Agilent Technologies, Santa Clara, CA) kunye ne-CORTECS T3 column (2.1×150 mm, ubukhulu be-particle 1.6-μm, ubungakanani be-pore 120-Å; #186008500, Waters). I-Polar mass spectrometer (6470, Agilent), apho i-electrospray ionization isebenza kwi-positive mode. I-Mobile phase A yi-0.1% formic acid (kwi-H2O), i-mobile phase B yi-90% acetonitrile, 0.1% formic acid. I-LC gradient yimizuzu eyi-0 ukuya kweyi-2 kwi-100% A, imizuzu emi-2 ukuya kweyi-7.1 kwi-99% B, kunye nemizuzu eyi-7.1 ukuya kweyi-8 kwi-99% B. Emva koko phinda ulinganise ikholamu ne-mobile phase A kwisantya sokuhamba se-0.6 ml/min imizuzu emi-3. . Isantya sokuhamba yi-0.4ml/min, kwaye igumbi lekholamu lifudunyezwa ukuya kuma-50°C. Sebenzisa umgangatho we-MNA wekhemikhali ococekileyo (M320995, Toronto Research Chemical Company, North York, Ontario, Canada) ukuseka ixesha lokugcina (RT) kunye nokuguqulwa (RT = 0.882 imizuzu, ukuguqulwa 1 = 137→94.1, ukuguqulwa 2 = 137→92, Ukuguqulwa 3 = 137→78). Xa zonke iinguqu ezintathu zenzeka ngexesha elifanelekileyo lokugcina, utshintsho 1 lusetyenziselwa ukulinganisa ukuqinisekisa ukucaciswa. I-standard curve ye-MNA (iToronto Research Chemical Company) iveliswe ngokuxutywa okulandelelanayo okuthandathu kwesisombululo sesitokhwe (1 mg/ml) ukuze kufunyanwe imigangatho ye-0.1, 1.0, 10 kunye ne-100 ng/ml kunye ne-1.0 kunye ne-10μg/ml ngokulandelelana kolwelo. Umda wokufumanisa yi-1 ng/ml, kwaye impendulo ethe ngqo iphakathi kwe-10 ng/ml kunye ne-10μg/ml. Inaliti nganye yee-microliters ezimbini zesampuli kunye ne-standard isetyenziselwa uhlalutyo lwe-LC/MS, kwaye isampuli yokulawula umgangatho oxutyiweyo iqhutywa rhoqo emva kokufakwa kwe-injection ezisibhozo ukuqinisekisa uzinzo lweqonga lohlalutyo. Iimpendulo ze-MNA zazo zonke iisampuli zeeseli eziphathwe yi-MNA zazingaphakathi koluhlu oluthe ngqo lovavanyo. Uhlalutyo lwedatha lwenziwe kusetyenziswa isoftware yohlalutyo oluyi-MassHunter (v9.0, Agilent).
Ulwakhiwo lwesizukulwana sesibini se-αFR-CAR luthathwe kuSong et al. (59). Ngamafutshane, ulwakhiwo luqulathe oku kulandelayo: ulandelelwano lwenkokeli ye-CD8a, isiqwenga se-human αFR-specific single-chain variable, i-CD8a hinge kunye ne-transmembrane region, i-CD27 intracellular domain kunye ne-CD3z intracellular domain. Ulandelelwano olupheleleyo lwe-CAR lwenziwe yi-GenScript, lwaza lwadityaniswa kwi-vector ye-lentiviral expression yesizukulwana sesibini phezulu kwekhasethi ye-GFP expression esetyenziselwa ukuvavanya ukusebenza kakuhle kwe-transduction.
ILentivirus iveliswa ngokufakelwa kweeseli zeHEK293T [iAmerican Type Culture Collection (ATCC); ikhuliswe kwiDulbecco's modified Eagle medium equlethe i-10% ye-fetal bovine serum (FBS) kunye ne-1% yePenStrep, kwaye kusetyenziswe i-CAR-GFP vector kunye ne-The packaging plasmids (psPAX2 kunye ne-pMD2.G, i-Addgene) zisebenzisa i-lipofection amine (Sigma-Aldrich). I-supernatant equlethe intsholongwane yaqokelelwa emva kweeyure ezingama-48 kunye nama-72 emva kokufakelwa, yahluzwa, yaza yagxininiswa yi-ultracentrifugation. Gcina i-supernatant ehlanganisiweyo yentsholongwane kwi--80°C ide idluliselwe.
Ii-PBMC zahlulwe kwiimveliso zokwahlulwa kwe-leukocyte ezinempilo (STEMCELL Technologies) nge-Ficoll gradient density centrifugation. Sebenzisa ii-microbeads ze-CD8 zokukhetha okuhle (iMiltenyi) ukuze zahlukanise iiseli ze-CD8+ kwi-PBMC. Vuselela iiseli ze-T nge-TransAct (iMiltenyi) nakwi-TexMACS medium [iMiltenyi; yongezwe yi-3% ye-human serum engasebenziyo ngobushushu, i-1% yePenStrep kunye ne-IL-2 (300 U/ml)]. Iiyure ezingamashumi amabini anesine emva kokuvuselelwa, iiseli ze-T zadluliselwa nge-lentivirus (10 μl concentrated virus supernatant kwiiseli ezili-106). Kwiintsuku ezi-1 ukuya kwezi-3 emva kokudluliselwa kwi-Cytek Aurora (kwi-FSC (Forward Scatter)/SSC (Side Scatter), Singlet, GFP+), vavanya ukubonakaliswa kwe-GFP kweeseli ukubonisa ukusebenza kakuhle kokudluliselwa kwe-transduction okungenani i-30%.
Iiseli ze-CAR-T zakhuliswa iiyure ezingama-24 kwi-Immunocult (STEMCELL Technologies; zongezwe nge-1% PenStrep) phantsi kwezi meko zilandelayo: zinganyangwanga, zanyangwa nge-250 μM adenosine okanye i-10 mM MNA. Emva konyango lwangaphambi konyango, iiseli ze-CAR-T zahlanjwa nge-PBS zaza zadityaniswa neeseli ze-SK-OV-3 ezingama-20,000 [ATCC; kwi-McCoy 5A medium (Sigma-Aldrich) zongezwe nge-10% FBS kunye ne-1% PenStrep kwi-10: Umlinganiselo we-effector to target we-1 wandiswa nge-triplicate kwi-Immunocult medium eyongeziweyo. Iiseli ze-SK-OV-3 kunye neeseli ze-SK-OV-3 ezifakwe i-digitalis saponin (0.5mg/ml; Sigma-Aldrich) zasetyenziswa njengolawulo olubi nolulungileyo, ngokulandelelana. Emva kweeyure ezingama-24 zokulima kunye, i-supernatant yaqokelelwa kwaye i-lactate dehydrogenase (LDH) yalinganiswa ngokwemiyalelo yomenzi (LDH Glo Cytotoxicity Assay Kit, Promega). I-LDH supernatant yancitshiswa nge-1:50 kwi-LDH buffer. Ipesenti yokubulala yalinganiswa kusetyenziswa le fomyula ilandelayo: ipesenti yokubulala = ipesenti yokulungiswa / izinga eliphezulu lokubulala x 100%, apho ipesenti yokulungiswa = iiseli ze-co-culture-T kuphela, kwaye izinga eliphezulu lokubulala = ulawulo oluhle nolungachanekanga.
Njengoko kuchaziwe kwisicatshulwa okanye kwizixhobo nakwiindlela, sebenzisa iGraphPad Prism 8, iMicrosoft Excel okanye iR v3.6.0 kuhlalutyo lwezibalo. Ukuba iisampuli ezininzi ziqokelelwa kwisigulana esinye (ezifana ne-ascites kunye ne-tumor), sisebenzisa uvavanyo lwe-t oludibeneyo okanye siquke isigulana njengesiphumo esingacwangciswanga kwimodeli ethe tye okanye ebanzi njengoko kufanelekile. Kuhlalutyo lwe-metabolomics, uvavanyo lokubaluleka lwenziwa ngokuphindwe kathathu.
Ukuze ufumane ezinye izinto ezongezelelweyo kweli nqaku, nceda ujonge http://advances.sciencemag.org/cgi/content/full/7/4/eabe1174/DC1
Eli linqaku elivulelekileyo elisasazwa phantsi kwemigaqo yeLayisensi yeCreative Commons Attribution-Non-Commercial, evumela ukusetyenziswa, ukusasazwa kunye nokuveliswa kwakhona kuyo nayiphi na indlela, lo gama nje ukusetyenziswa kokugqibela kungekuko ukuzuza kwezorhwebo kwaye ingqikelelo kukuba umsebenzi wokuqala uchanekile. Isalathiso.
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UMarisa K. Kilgour (uMarisa K. Kilgour), uSarah MacPherson (uSarah MacPherson), uLauren G. Zacharias (uLauren G. Zacharias), u-Abigail Eli Aris G. Watson (H. Watson), uJohn Stagg (uJohn Stagg), uBrad H. Nelson (uBrad H. Nelson), uRalph J. Alph De Bellar (uRussell. G. Jones), uPhineas T. Hamilton (Phineas T.
I-MNA inegalelo ekucinezeleni amasosha omzimba eeseli ze-T kwaye imele indawo ekujoliswe kuyo kunyango lomhlaza ebantwini.
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