Immunomodulatory metabolites i xivumbeko xa nkoka xa tumor microenvironment (TME), kambe handle ka swin’wana swintsongo, vutivi bya tona bya ha tiveka ngopfu. Laha, hi kambisisile swirhumbana na tisele ta T ku suka eka swirhumbana na ascites ya vavabyi lava nga na high-grade serous carcinoma (HGSC) ku paluxa metabolome ya swiyenge leswi swo hambana swa TME. Ascites na ti cells ta tumor swina ku hambana lokukulu ka metabolite. Loko ku pimanisiwa na ascites, ti cells ta T leti nghenaka eka tumor ti fuwile swinene hi 1-methylnicotinamide (MNA). Hambi leswi mpimo wa MNA eka tisele ta T wu tlakukeke, ku paluxiwa ka nicotinamide N-methyltransferase (enzyme leyi hlohlotelaka ku hundziseriwa ka mintlawa ya methyl ku suka eka S-adenosylmethionine ku ya eka nicotinamide) swi pimiwile eka ti fibroblasts na tisele ta tshumba. Hi ku tirha, MNA yi hlohlotela tisele ta T ku humesa cytokine tumor necrosis factor alpha leyi hlohlotelaka tshumba. Hikwalaho, MNA leyi humaka eka TME yi hoxa xandla eka vulawuri bya masocha ya miri ya tisele ta T naswona yi yimela xikongomelo xa vutshunguri bya masocha ya miri lexi nga vaka kona eka vutshunguri bya khensa ya vanhu.
Ti- metabolite leti humaka eka tshumba ti nga va ni vuyelo lebyikulu byo sivela eka masocha ya miri lama lwisanaka ni tshumba, naswona vumbhoni byo tala byi kombisa leswaku ti nga tlhela ti tirha tanihi matimba ya nkoka lama susumetelaka ku kula ka vuvabyi (1). Ku engetela eka mbuyelo wa Warburg, ntirho wa sweswinyana wu sungurile ku hlamusela xiyimo xa metabolic xa tisele ta tshumba na vuxaka bya yona na xiyimo xa masocha ya miri ya microenvironment ya tshumba (TME). Minkambisiso eka timodeli ta mbeva ni tisele ta T ta vanhu yi kombise leswaku ku cinca-cinca ka glutamine (2), ku cinca-cinca ka oxidative (3) ni ku cinca-cinca ka glucose (4) swi nga tirha hi ku tiyimela eka mintlawa leyitsongo yo hambana-hambana ya tisele ta masocha ya miri. Ti metabolites to hlaya eka tindlela leti ti sivela ntirho wa anti-tumor wa ti cells ta T. Swi kombisiwile leswaku ku pfaleriwa ka coenzyme tetrahydrobiopterin (BH4) swi nga onha ku andza ka tisele ta T, naswona ku tlakuka ka BH4 emirini swi nga ndlandlamuxa nhlamulo ya masocha ya miri ya anti-tumor leyi kongomisiwaka hi CD4 na CD8. Ku engetela kwalaho, vuyelo bya immunosuppressive bya kynurenine byi nga ponisiwa hi ku nyikiwa ka BH4 (5). Eka isocitrate dehydrogenase (IDH) mutant glioblastoma, ku humesiwa ka enantiometabolic (R)-2-hydroxyglutarate (R-2-HG) swi sivela ku tirhisiwa ka tisele ta T, ku andza na Ntirho wa cytolysis (6). Sweswinyana, swi kombisiwile leswaku methylglyoxal, xiendliwa xa le tlhelo xa glycolysis, xi endliwa hi tisele ta suppressor ta masungulo ya myeloid, naswona ku hundziseriwa ka tisele ta T ta methylglyoxal swi nga sivela ntirho wa tisele ta T ta effector. Eka vutshunguri, ku herisiwa ka methylglyoxal ku nga hlula ntirho wa tisele ta myeloid-derived suppressor (MDSC) na ku ndlandlamuxa hi ku tirhisana vutshunguri bya checkpoint blockade eka timodeli ta mbeva (7). Tidyondzo leti hi ku hlangana ti kandziyisa ntirho wa nkoka wa ti metabolites leti humaka eka TME eku lawuleni ka ntirho na ntirho wa tisele ta T.
Ku nga tirhi kahle ka tisele ta T ku vikiwile ngopfu eka khensa ya ovary (8). Leswi swi vangiwa hi ndlela yo karhi hi swihlawulekisi swa metabolic leswi nga kona eka hypoxia na misiha ya tshumba leyi nga tolovelekangiki (9), leswi endlaka leswaku ku hundzuriwa glucose na tryptophan swi va switirhisiwa swa le tlhelo swo fana na lactic acid na kynurenine. Lactate yo tlula mpimo ya le handle ka sele yi hunguta ku humesiwa ka interferon-γ (IFN-γ) naswona yi fambisa ku hambana ka mintlawa leyitsongo ya myelosuppressive ( 10 , 11 ). Ku dyiwa ka tryptophan swi sivela hi ku kongoma ku andza ka tisele ta T naswona swi sivela ku nyika swikombiso swa tisele ta T leti amukelaka ( 12-14 ). Hambi leswi ku nga na swibumabumelo leswi, ntirho wo tala lowu rhendzeleke metabolism ya masocha ya miri wu endliwile eka in vitro T cell culture hiku tirhisa optimized media, kumbe ku pimiwile eka ti homologous mouse models in vivo, laha kungariki na xin’we lexi kombisaka hiku hetiseka heterogeneity ya ti cancers ta vanhu na Physiological macro na micro environment.
Xivumbeko lexi tolovelekeke xa khensa ya ovary i ku hangalaka ka peritoneal na ku vonaka ka ascites. Ku hlengeletiwa ka mati ya tisele eka ascites swi fambisana ni vuvabyi lebyi yaka emahlweni ni ku vhumbha loku nga riki kahle ( 15 ). Hi ku ya hi swiviko, xiyenge lexi xo hlawuleka xi na hypoxic, xi na swiyimo swa le henhla swa vascular endothelial growth factor (VEGF) na indoleamine 2,3-dioxygenase (IDO), naswona xi nghenisiwa hi tisele ta T regulatory na tisele ta myeloid inhibitory ( 15-18 ). Ndhawu ya metabolic ya ascites yi nga ha hambana na ya tumor hi yoxe, hikwalaho ku phurogiremiwa nakambe ka tisele ta T eka ndhawu ya peritoneal a swi le rivaleni. Ku engetela kwalano, ku hambana ka nkoka na heterogeneity exikarhi ka ascites na ti metabolites letinga kona eka ndzhawu ya tumor swinga sivela ku nghenelela ka ti cells ta masocha ya miri na ntirho wa tona eka ti tumors, naswona vulavisisi byin’wana bya laveka.
Leswaku hi tlhantlha swiphiqo leswi, hi endlile ndlela ya sensitive cell separation na liquid chromatography tandem mass spectrometry (LC-MS/MS) ku dyondza tinxaka to hambana ta ti cells (ku katsa na ti cells ta CD4 + na CD8 + T) xikan’we na le ndzeni na le xikarhi ka ti tumors Ti metabolites ta yona ti span ti cells eka ascites yin’we na ndzhawu ya tumor ya muvabyi. Hi tirhisa ndlela leyi hi ku fambisana na high-dimensional flow cytometry na single-cell RNA sequencing (scRNA-seq) ku nyika xifaniso lexi lulamisiweke swinene xa xiyimo xa metabolic xa vaaki lava va nkoka. Endlelo leri ri paluxe ku tlakuka lokukulu eka mpimo wa 1-methylnicotinamide (MNA) eka tisele ta T ta tshumba, naswona swikambelo swa in vitro swi kombisile leswaku mbuyelo wa immunomodulatory wa MNA eka ntirho wa tisele ta T a wu nga tiviwi khale. Hi ku angarhela, endlelo leri ri paluxa ku tirhisana ka metaboliki exikarhi ka swirhumbana na tisele ta masocha ya miri, naswona ri nyika vutivi byo hlawuleka eka timetabolite ta vulawuri bya masocha ya miri, leswi nga pfunaka eka vutshunguri bya vutshunguri bya masocha ya miri lebyi simekiweke eka tisele ta T ta khensa ya ovary Minkarhi ya Vutshunguri.
Hi tirhise high-dimensional flow cytometry ku pima mpimo wa ku amukeriwa ka glucose hi nkarhi wun’we [2-(N-(7-nitrophenyl-2-oxa-1,3-diaza-4-yl)amino)-2-deoxyglucose (2-NBDG) na ntirho wa mitochondrial [MitoTracker Deep Red (MT DR)] (7, 19, 20) swi le tlhelo na tlhelo leswi tolovelekeke swimaki leswi hambanisaka tisele ta masocha ya miri na nhlayo ya tisele ta tshumba (Tafula ra S2 na Xifaniso xa S1A). Nxopaxopo lowu wu kombisile leswaku loko ku pimanyisiwa na ti cells ta T, ti ascites na ti cells ta tumor tina ti levele tale henhla ta glucose uptake, kambe tina ku hambana kutsongo eka ntirho wa mitochondrial. Xiringaniso xa ku tekiwa ka glucose eka tisele ta tshumba [CD45-EpCAM (EpCAM)+] i kanharhu ku ya eka mune ku tlula ka tisele ta T, naswona xiringaniso xa ku amukeriwa ka glucose ka tisele ta CD4 + T i 1.2 wa tiphesente ta tisele ta CD8 + T, leswi kombisaka leswaku Tumor infiltrating lymphocytes (TIL) yi na swilaveko swo hambana swa metaboliki hambi ku ri eka TME yin’we (Xifaniso 1A). Kuhambana na sweswo, ntirho wa mitochondrial eka ti cells ta tumor wufana na wa ti cells ta CD4 + T, naswona ntirho wa mitochondrial wa tinxaka letimbirhi ta ti cells wule henhla kutlula wa ti cells ta CD8 + T (Xifaniso 1B). Hiku angarhela, mbuyelo lowu wu paluxa levele ya metabolic. Ntirho wa metabolic wa ti cells ta tumor wule henhla kutlula wa ti cells ta CD4 + T, naswona ntirho wa metabolic wa ti cells ta CD4 + T wule henhla kutlula wa ti cells ta CD8 + T. Hambi leswi switandzhaku leswi eka tinxaka hinkwato ta tisele, a ku na ku hambana loku nga cincekiki eka xiyimo xa metabolic xa tisele ta CD4 + na CD8 + T kumbe mpimo wa tona lowu yelanaka eka ascites loko ku pimanisiwa na swirhumbana (Xifaniso 1C). Kuhambana na sweswo, eka xiphemu xa tisele ta CD45, xiphemu xa tisele ta EpCAM+ eka tshumba xi tlakukile loko xi pimanisiwa na ascites (Xifaniso 1D). Hi tlhele hi vona ku hambana loku nga erivaleni ka metaboliki exikarhi ka swiphemu swa tisele ta EpCAM+ na EpCAM-. Ti cells ta EpCAM+ (tumor) tina ku amukeriwa ka glucose yale henhla xikan’we na ntirho wa mitochondrial kutlula ti cells ta EpCAM-, leswinga e henhla swinene kutlula ntirho wa metabolic wa ti fibroblasts eka ti cells ta tumor eka TME (Xifaniso 1, E na F).
. (C) Nhlayo ya tisele ta CD4 + na CD8 + (ya tisele ta CD3 + T) eka ascites na tumor. (D) Xiphemu xa tisele ta tshumba ra EpCAM + eka ascites na tshumba (CD45−). (E na F) EpCAM + CD45-tumor na EpCAM-CD45-matrix glucose uptake (2-NBDG) (E) na ntirho wa mitochondrial (MitoTracker wo tshwuka wo dzwihala) (F) tigirafu leti yimelaka (eximatsini) na datha leyi nga eka tafula ( Exineneni) Ascites na tisele ta tshumba. (G) Tigirafu leti yimelaka ku paluxiwa ka CD25, CD137 na PD1 hi flow cytometry. (H na I) Ku paluxiwa ka CD25, CD137 na PD1 eka tisele ta CD4 + T (H) na tisele ta CD8 + T (I). (J na K) Tiphenotypes ta naive, central memory (Tcm), effector (Teff) na effector memory (Tem) leti simekiweke eka ku paluxiwa ka CCR7 na CD45RO. Swifaniso leswi yimelaka (exineneni) na datha ya tafula (exineneni) ya tisele ta CD4 + T (J) na tisele ta CD8 + T (K) eka ascites na swirhumbana. Mimpimo ya P leyi kumiweke hi paired t-test (*P<0.05, **P<0.01 na ***P<0.001). Layini yi yimela vavabyi lava ringanisiweke (n = 6). FMO, fluorescence ku susiwa yin’we; MFI, matimba ya le xikarhi ya fluorescence.
Nxopaxopo wun’wana wu paluxe ku hambana kun’wana lokukulu exikarhi ka xiyimo xa phenotypic xa tisele ta T lexi lulamisiweke swinene. Ku tsundzuka loku tirhisiweke (Xifaniso 1, G ku ya eka I) na effector (Xifaniso 1, J na K) eka swirhumbana swi tala ngopfu ku tlula ascites (xiphemu xa tisele ta CD3 + T). Hilaha ku fanaka, ku xopaxopa phenotype hi ku paluxiwa ka swikombiso swa ku tirha (CD25 na CD137) na swikombiso swa ku hela [programmed cell death protein 1 (PD1)] swi kombisile leswaku hambi leswi swihlawulekisi swa metaboliki swa vaaki lava swi hambaneke (Xifaniso S1, B ku ya eka E) , Kambe ku hava ku hambana lokukulu ka metaboliki loku voniweke hi ku landzelelana exikarhi ka swiyenge leswitsongo swa naive, effector kumbe memory (Xifaniso S1, F ku ya eka I). Mimbuyelo leyi yi tiyisisiwile hi ku tirhisa tindlela to dyondza hi muchini ku avela hi ku tisungulela ti-phenotype ta tisele (21), leswi yaka emahlweni swi paluxa vukona bya nhlayo leyikulu ya tisele ta marhambu ya marhambu (CD45 + / CD3- / CD4 + / CD45RO +) eka ascites ya muvabyi (Xifaniso S2A ). Exikarhi ka tinxaka hinkwato ta tisele leti tivekaka, nhlayo leyi ya tisele ta myeloid yi kombisile ku amukeriwa ka glucose yale henhla swinene na ntirho wa mitochondrial (Xifaniso S2, B kuya eka G). Mimbuyelo leyi yi kombisa ku hambana lokukulu ka metabolic exikarhi ka tinxaka to tala ta tisele leti kumekaka eka ascites na swirhumbana eka vavabyi va HGSC.
Ntlhontlho lowukulu eka ku twisisa swihlawulekisi swa metaboliko swa TIL i xilaveko xo hambanyisa tisampulu ta tisele ta T ta ku basa loku ringaneleke, khwalithi na nhlayo kusuka eka swirhumbana. Mindzavisiso ya sweswinyana yi kombisile leswaku tindlela to hlawula na ku fuwisa vuhlalu leti simekiweke eka flow cytometry tinga endla leswaku kuva na ku cinca eka ti cellular metabolite profiles ( 22-24 ). Ku hlula xiphiqo lexi, hi antswisile ndlela yo fuwisa vuhlalu ku hambanyisa na ku hambanyisa TIL eka khensa ya ovary ya munhu leyi tsemiweke hi vuhandzuri emahlweni ka nxopaxopo hi LC-MS/MS (vona Switirhisiwa na Maendlelo; Xifaniso 2A). Ku kota ku kambela mbuyelo hinkwawo wa protocol leyi eka ku cinca ka metabolite, hi pimanisile ti metabolite profiles ta ti cells ta T leti tirhisiweke hi vaphakeri lava hanyeke kahle endzhaku ka goza leri nga laha henhla ro hambanyisa vuhlalu na ti cells letinga hambanisiwanga hi vuhlalu kambe ti tshamile eka ayisi. Nxopaxopo lowu wa vulawuri bya khwalithi wu kumile leswaku kuna ku yelana lokukulu exikarhi ka swiyimo leswi swimbirhi (r = 0.77), naswona ku phindha-phindha ka xithekiniki ka ntlawa wa 86 wa ti metabolites swina ku phindha-phindha ka le henhla (Xifaniso 2B). Hikwalaho, tindlela leti ti nga endla nxopaxopo wa metabolite lowu kongomeke eka tisele leti endlaka ku fuwisiwa ka muxaka wa tisele, xisweswo ti nyika pulatifomo yo sungula ya xiboho xa le henhla xo kuma ti metabolite to karhi eka HGSC, xisweswo ti endla leswaku vanhu va kota ku kuma ku twisisa loku dzikeke ka ku kongoma ka tisele Nongonoko wa metabolism ya rimbewu.
(A) Xifaniso xa xikimi xa ku fuwa ka vuhlalu bya maginete. Loko kungase kamberiwa hi LC-MS/MS, ti cells tita hundza eka swirhendzevutani swinharhu hiku landzelelana swa magnetic bead enrichment kutani titshama eka ayisi. (B) Vuyelo bya muxaka wa ku fuwisa eka ku tala ka ti-metabolite. Avhareji ya mimpimo yinharhu eka muxaka wun’wana na wun’wana wa ku fuwisa ± SE. Layini ya grey yi yimela vuxaka bya 1:1. Ku yelana ka le ndzeni ka tlilasi (ICC) ka mimpimo leyi phindha-phindhiweke leyi kombisiweke eka lebula ya axis. NAD, ku nga nicotinamide adenine dinucleotide. (C) Xifaniso xa xikimi xa mafambelo ya ntirho ya nxopaxopo wa metabolite ya muvabyi. Ascites kumbe swirhumbana swi hlengeletiwa eka vavabyi naswona swi hlayisiwa hi cryopreserved. Xiphemu lexintsongo xa xikombiso xin’wana na xin’wana xi kamberiwile hi flow cytometry, kasi tisampulu leti saleke ti endliwile swirhendzevutani swinharhu swa ku fuwisiwa eka tisele ta CD4+, CD8+ na CD45-. Swiphemu leswi swa tisele swi kamberiwile hi ku tirhisa LC-MS/MS. (D) Mepe wo hisa wa nhlayo ya metabolite leyi ringaniseriweke. Dendrogram yiyimela ku hlengeletiwa ka Ward ka swiphemu swa Euclidean exikarhi ka swikombiso. (E) Nxopaxopo wa swiphemunkulu (PCA) wa mepe wa metabolite ya xikombiso, lowu kombisaka ku tlheriseriwa kanharhu ka xikombiso xin’wana na xin’wana, swikombiso leswi humaka eka muvabyi un’we swi hlanganisiwa hi layini. (F) PCA ya xivumbeko xa metabolite xa xikombiso lexi vekiweke eka muvabyi (i.e., ku tirhisa xiphemu xa redundancy); muxaka wa xikombiso wu pimiwile hi convex hull. PC1, xiphemu lexikulu xa 1; PC2, xiphemu lexikulu xa 2.
Endzhaku ka sweswo, hi tirhise ndlela leyi yo fuwisa ku xopaxopa 99 wa ti-metabolite eka swiphemu swa tisele ta CD4 +, CD8 + na CD45 eka ti-ascites to sungula na swirhumbana swa vavabyi va tsevu va HGSC (Xifaniso 2C, Xifaniso xa S3A na Tafula ra S3 na S4). Nhlayo ya vaaki leyi tsakeriwaka yi endla 2% kuya eka 70% wa xikombiso lexikulu xosungula xa ti cells leti hanyaka, naswona xiphemu xa ti cells xihambana swinene exikarhi ka vavabyi. Endzhaku ko hambanyisa vuhlalu, xiphemu lexi fuweke xa ntsakelo (CD4+, CD8+ kumbe CD45-) xi endla kutlula 85% wa ti cells hinkwato leti hanyaka eka xikombiso hi xiringaniso. Ndlela leyi yo fuwisa yi hi pfumelela ku kambisisa nhlayo ya tisele kusuka eka human tumor tissue metabolism, leswi swinga kotekiki ku swi endla kusuka eka tisampulu letikulu. Hi ku tirhisa protocol leyi, hi kumile leswaku l-kynurenine na adenosine, ti metabolites leti timbirhi leti tivekaka kahle ta immunosuppressive ti tlakusiwile eka tisele ta T ta tshumba kumbe tisele ta tshumba (Xifaniso S3, B na C). Hikwalaho, mimbuyelo leyi yi kombisa ku tshembeka na vuswikoti bya thekinoloji ya hina yo hambanyisa tisele na mass spectrometry ku kuma ti metabolites ta nkoka hi tlhelo ra biological eka tinyama ta muvabyi.
Nxopaxopo wa hina wu tlhele wu paluxa ku hambanisiwa lokukulu ka metaboliki ka tinxaka ta tisele endzeni na le xikarhi ka vavabyi (Xifaniso 2D na Xifaniso xa S4A). Ngopfungopfu, loko ku pimanisiwa na vavabyi van’wana, muvabyi wa 70 u kombisile swihlawulekisi swo hambana swa metaboliki (Xifaniso 2E na Xifaniso xa S4B), leswi kombisaka leswaku ku nga va na ku hambana lokukulu ka metaboliki exikarhi ka vavabyi. Swifanele ku xiya leswaku loko ku pimanyisiwa na vavabyi van’wana (1.2 kuya eka 2 wa tilitara; Tafula ra S1), nhlayo hinkwayo ya ascites leyi hlengeletiweke eka muvabyi wa 70 (80 ml) ayiri yitsongo. Ku lawuriwa ka heterogeneity exikarhi ka vavabyi hi nkarhi wa nxopaxopo wa swiphemu leswikulu (xikombiso, hi ku tirhisa nxopaxopo wa xiphemu xa redundancy) swi kombisa ku cinca loku nga cincekiki exikarhi ka tinxaka ta tisele, naswona tinxaka ta tisele na/kumbe microenvironment swi hlengeletiwile kahle ku ya hi xivumbeko xa metabolite (Xifaniso 2F ). Nxopaxopo wa ti metabolites tin’we wu kandziyisile switandzhaku leswi naswona wu paluxe ku hambana lokukulu exikarhi ka tinxaka ta tisele na microenvironment. Swi fanerile ku xiya leswaku ku hambana lokukulu swinene loku voniweke i MNA, leyi hi ntolovelo yi fuweke eka tisele ta CD45- na le ka tisele ta CD4+ na CD8+ leti nghenaka eka tshumba (Xifaniso 3A). Eka tisele ta CD4 +, mbuyelo lowu wu vonaka ngopfu, naswona MNA eka tisele ta CD8 + na yona yi vonaka yi khumbeka swinene hi mbango. Hambiswiritano, leswi a hi swa nkoka, hikuva i vavabyi vanharhu ntsena eka va tsevu lava nga kamberiwaka eka swikoweto swa CD8+ swa tshumba. Ku engetela eka MNA, eka tinxaka to hambana ta ti cells eka ascites na tumors, ti metabolites tin’wana letinga tivekiki kahle eka TIL na tona ti fuwile hindlela yohambana (Swifaniso S3 na S4). Hikokwalaho, datha leyi yi paluxa sete leyi tshembisaka ya ti immunomodulatory metabolites eka ndzavisiso lowu yaka emahlweni.
(A) Nhlayo leyi tolovelekeke ya MNA eka tisele ta CD4+, CD8+ na CD45- ku suka eka ascites na tshumba. Ploto ya bokisi yi kombisa xikarhi (layini), interquartile range (frame hinge) na data range, ku fika eka 1.5 times ya interquartile range (frame whisker). Hilaha swi hlamuseriweke hakona eka Switirhisiwa na Maendlelo ya Muvabyi, tirhisa ntikelo wa limma wa muvabyi ku kumisisa ntikelo wa P (*P<0.05 na **P<0.01). (B) Xifaniso xa xikimi xa metabolism ya MNA (60). Swilo leswi cinca-cincaka: S-adenosyl-1-methionine; SAH, S-adenosine-1-homocysteine ​​leyi nga ni swiaki swa xirhendzevutani; NA, xitsongwatsongwana xa nicotinamide; MNA, 1-methylnicotinamide leyi nga ni xitsongwatsongwana; 2-PY, 1-methyl- 2-piridone-5-khabokisamidi; 4-PY, 1-methyl-4-pyridone-5-khabokisamidi ya xirhendzevutani; NR, ribose ya nicotinamide; NMN, nicotinamide ya mononucleotide. Ti- enzyme (ya rihlaza): NNMT, nicotinamide N-methyltransferase; SIRT, ku nga ti- sirtuins; NAMPT, nicotinamide ya phosphoribosyl ya ku hundziseriwa; AOX1, aldehyde oxidase 1, ku nga xitsongwatsongwana xa 1; NRK, ku nga nicotinamide riboside kinase; NMNAT, nicotinamide mono Nyukiliyoti ya adenylate transferase; Pnp1, purine nyukiliyosayidi ya phosphorylase. (C) t-SNE ya scRNA-seq ya ascites (grey) na tumor (yo tshwuka; n = 3 wa vavabyi). (D) Ku paluxiwa ka NNMT eka vaaki vo hambana va tisele lava hlawuriweke hi ku tirhisa scRNA-seq. (E) Ku paluxiwa ka NNMT na AOX1 eka SK-OV-3, tinso ta embryonic ta munhu (HEK) 293T, tisele ta T na tisele ta T leti tshunguriweke hi MNA. Xivulavulelo lexi hlanganisiweke xi kombisiwile loko xi pimanisiwa na SK-OV-3. Xivumbeko xa ku paluxiwa na SEM xi kombisiwile (n = 6 wa vaphakeri lava hanyeke kahle). Mimpimo ya Ct leyi tlulaka 35 yi tekiwa yi nga voniwi (UD). (F) Ku paluxiwa ka SLC22A1 na SLC22A2 eka SK-OV-3, HEK293T, tisele ta T na tisele ta T leti tshunguriweke hi 8mM MNA. Xivulavulelo lexi hlanganisiweke xi kombisiwile loko xi pimanisiwa na SK-OV-3. Xivumbeko xa ku paluxiwa na SEM xi kombisiwile (n = 6 wa vaphakeri lava hanyeke kahle). Mimpimo ya Ct leyi tlulaka 35 yi tekiwa yi nga voniwi (UD). (G) Nhlayo ya MNA ya tisele eka tisele ta T ta donor leti hanyeke kahle leti tirhisiweke endzhaku ka tiawara ta 72 ta ku pfaleriwa na MNA. Xivumbeko xa ku paluxiwa na SEM xi kombisiwile (n = 4 wa vaphakeri lava hanyeke kahle).
MNA yi endliwa hiku hundzisela ntlawa wa methyl kusuka eka S-adenosyl-1-methionine (SAM) kuya eka nicotinamide (NA) hi nicotinamide N-methyltransferase (NNMT; Xifaniso 3B). NNMT yi kombisiwa ngopfu eka tikhensa to hambana ta vanhu naswona yi fambelanisiwa na ku andza, ku hlasela na ku hangalaka (25-27). Ku twisisa ku antswa xihlovo xa MNA eka tisele ta T eka TME, hi tirhise scRNA-seq ku hlamusela ku paluxiwa ka NNMT eka tinxaka hinkwato ta tisele eka ascites na swirhumbana swa vavabyi vanharhu va HGSC ( Tafula ra S5 ). Nxopaxopo wa kwalomu ka 6,500 wa tisele wu kombisile leswaku eka tindhawu ta ascites na tumor, ku paluxiwa ka NNMT a ku pimiwile eka nhlayo leyi ehleketiwaka ya fibroblast na tisele ta tshumba (Xifaniso 3, C na D). Swi fanerile ku xiya leswaku a ku na ku paluxiwa ka NNMT loku nga erivaleni eka vaaki vahi na vahi lava kombisaka PTPRC (CD45 +) (Xifaniso 3D na Xifaniso xa S5A), leswi kombisaka leswaku MNA leyi kumiweke eka spectrum ya metabolite yi nghenisiwile eka tisele ta T. Ku paluxiwa ka aldehyde oxidase 1 (AOX1) ku hundzula MNA ku va 1-methyl-2-pyridone-5-carboxamide (2-PYR) kumbe 1-methyl-4-pyridone-5-carboxamide (4- PYR); Xifaniso 3B) xi tlhela xi siveriwa eka nhlayo ya ti fibroblasts leti kombisaka COL1A1 (Xifaniso S5A), leswi loko swi hlanganisiwa swi kombisaka leswaku tisele ta T ti pfumala vuswikoti bya metabolism ya ntolovelo ya MNA. Xivumbeko xa ku paluxiwa ka tijini leti leti fambelanaka na MNA xi tiyisisiwile hi ku tirhisa sete ya vumbirhi ya datha ya tisele leti tiyimeleke ku suka eka ascites ku suka eka vavabyi va HGSC (Xifaniso S5B; n = 6) (16). Ku engetela kwalaho, nxopaxopo wa quantitative polymerase chain reaction (qPCR) wa tisele ta T ta munyiki leti hanyeke kahle leti tshunguriweke hi MNA wu kombisile leswaku loko ku pimanisiwa na tisele ta tshumba ra ovary ta vulawuri bya SK-OV-3, NNMT kumbe AOX1 a yi lava ku va yi nga kombisiwanga (Xifaniso 3E). Vuyelo lebyi byi nga languteriwangiki byi kombisa leswaku MNA yi nga ha humesiwa ku suka eka ti fibroblasts kumbe swirhumbana eka tisele ta T leti nga ekusuhi eka TME.
Hambi leswi vahlawuriwa va katsaka ndyangu wa swifambo swa organic cation 1 ku ya eka 3 (OCT1, OCT2 na OCT3) leswi khodiweke hi ndyangu wa soluble carrier 22 (SLC22) (SLC22A1, SLC22A2 na SLC22A3), swifambo leswi nga vaka kona swa MNA swa ha hlamuseriwa (28) . QPCR ya mRNA ku suka eka tisele ta T ta munyiki leti hanyeke kahle yi kombisile swiyimo swa le hansi swa ku paluxiwa ka SLC22A1 kambe swiyimo leswi nga voniwiki swa SLC22A2, leswi tiyisiseke leswaku swi vikiwile khale eka matsalwa (Xifaniso 3F) (29). Kuhambana na sweswo, layini ya tisele ta tshumba ra ovarian ya SK-OV-3 yi kombisile swiyimo swa le henhla swa swifambo leswimbirhi (Xifaniso 3F).
Leswaku ku kamberiwa ku koteka ka ti cells ta T letingana vuswikoti byo tswonga MNA ya matiko mambe, ti donor T cells leti hanyeke kahle ti byaleriwile kuringana 72 wa tiawara eka vukona bya ti concentrations to hambana ta MNA. Loko kungari na exogenous MNA, nhlayo ya ti cellular ya MNA ayi nge voniwi (Figure 3G). Hambiswiritano, tisele ta T leti tirhisiweke leti tshunguriweke hi MNA ya le handle ti kombisile ku tlakuka loku titshegeke hi mpimo eka nhlayo ya MNA eka tisele, ku fika eka 6 mM MNA (Xifaniso 3G). Mbuyelo lowu wu kombisa leswaku hambi leswi kungana xiyimo xale hansi xa transporter expression xikan’we naku pfumaleka ka enzyme leyikulu leyingana vutihlamuleri eka intracellular MNA metabolism, TIL yaha kota ku teka MNA.
Spectrum ya metabolites eka ti cells ta T ta vavabyi na swikambelo swa in vitro MNA absorption swi engetela ku koteka ka leswaku ti cancer-associated fibroblasts (CAF) ti humesa MNA naswona ti cells ta tumor tinga lawula phenotype na ntirho wa TIL. Ku kumisisa mbuyelo wa MNA eka tisele ta T, tisele ta T ta donor leti hanyeke kahle ti tirhisiwile eka vitro eka vukona kumbe ku pfumaleka ka MNA, naswona ku andza ka tona na vuhumelerisi bya cytokine swi kamberiwile. Endzhaku ka masiku ya 7 ya ku engeteriwa ka MNA eka mpimo wa le henhla, nhlayo ya ku phindha-phindha ka vaaki yi hungutiwile hi ndlela yo ringanela, kasi matimba ya hlayisiwile eka mpimo hinkwawo (Xifaniso 4A). Ku engetela kwalaho, vutshunguri bya exogenous MNA byi endle leswaku ku va na ku tlakuka ka xiphemu xa tisele ta CD4 + na CD8 + T leti kombisaka tumor necrosis factor-α (TNFα; Xifaniso 4B). Kuhambana na sweswo, vuhumelerisi bya le ndzeni ka tisele bya IFN-γ byi hungutiwile swinene eka tisele ta CD4 + T, kambe ku nga ri eka tisele ta CD8 + T, naswona a ku ri hava ku cinca lokukulu eka interleukin 2 (IL-2; Xifaniso 4, C na D). Hikokwalaho, enzyme-linked immunosorbent assay (ELISA) ya ti supernatants kusuka eka mindzhavuko leyi ya ti cell ta T leti tshunguriweke hi MNA yi kombisile ku tlakuka lokukulu eka TNFα, kuhunguteka ka IFN-γ, naswona kungari na ku cinca eka IL-2 (Xifaniso 4, E kuya eka G). . Kuhunguteka ka IFN-γ swikomba leswaku MNA yinga tlanga xiave eka ku sivela ntirho wa anti-tumor wa ti cells ta T. Ku endlela ku tekelela mbuyelo wa MNA eka cytotoxicity leyi kongomisiwaka hi tisele ta T, tisele ta chimeric antigen receptor T (FRα-CAR-T) leti kongomisaka eka tisele ta folate receptor α na CAR-T (GFP) leti lawuriwaka hi tisele ta green fluorescent protein (GFP) -CAR-T) ti endliwa hi tisele ta mononuclear ta ngati ya le matlhelo ya munyiki leti hanyeke kahle (PBMC). Ti cells ta CAR-T ti byarisiwile kuringana 24 wa tiawara eka vukona bya MNA, kutani ti byariwa swin’we na ti human SK-OV-3 ovarian tumor cells leti kombisaka folate receptor α eka effector kuya eka target ratio ya 10:1. Vutshunguri bya MNA byi endle leswaku ku va na ku hunguteka lokukulu eka ntirho wo dlaya wa tisele ta FRα-CAR-T, leswi a swi fana na tisele ta FRα-CAR-T leti tshunguriweke hi adenosine (Xifaniso 4H).
(A) Nhlayo hinkwayo ya tisele leti hanyaka na ku phindha-phindha ka vaaki (PD) hi ku kongoma ku suka eka ndhavuko hi siku ra 7. Girafamu ya tibara yi yimela xiringaniso + SEM ya vaphakeri va tsevu lava hanyeke kahle. Yi yimela datha ku suka eka kwalomu ka n = 3 wa swikambelo leswi tiyimeleke. (B ku ya eka D) CD3/CD28 na IL-2 swi tirhisiwile ku endla leswaku tisele ta T ti tirha eka nhlayo ya tona ya MNA ku ringana masiku ya 7. Loko kungase endliwa nxopaxopo, ti cells ti hlohleteriwile hi PMA/ionomycin hi GolgiStop kuringana 4 wa tiawara. Ku paluxiwa ka TNFα (B) eka tisele ta T. Xikombiso xa xifaniso (eximatsini) na datha ya tafula (exineneni) ya ku paluxiwa ka TNFα eka tisele leti hanyaka. Ku paluxiwa ka IFN-γ (C) na IL-2 (D) eka tisele ta T. Ku paluxiwa ka ti cytokines swi pimiwe hi flow cytometry. Girafamu ya tibara yi yimela xiringaniso (n = 6 wa vaphakeri lava hanyeke kahle) + SEM. Tirhisa nxopaxopo wa ndlela yin’we wa ku hambana na swipimelo leswi phindha-phindhiweke (*P<0.05 na **P<0.01) ku kumisisa ntikelo wa P. Yi yimela datha ku suka eka kwalomu ka n = 3 wa swikambelo leswi tiyimeleke. (E ku ya eka G) CD3/CD28 na IL-2 swi tirhisiwile ku endla leswaku tisele ta T ti tirha eka nhlayo ya tona ya MNA ku ringana masiku ya 7. Medium yi hlengeletiwile emahlweni na endzhaku ka 4 wa tiawara ta PMA/ionomycin stimulation. Nhlayo ya TNFα (E), IFN-γ (F) na IL-2 (G) yi pimiwe hi ELISA. Girafamu ya tibara yi yimela xiringaniso (n = 5 wa vaphakeri lava hanyeke kahle) + SEM. Nhlayo ya P yi kumiwile hi ku tirhisa nxopaxopo wa ndlela yin’we wa ku hambana na ku pima loku phindha-phindhiweke (*P<0.05). Layini ya mathonsi yi kombisa mpimo wa ku kumiwa ka ku kumiwa. (H) Xikambelo xa ku hambanisiwa ka tisele. Ti cells ta FRα-CAR-T kumbe GFP-CAR-T ti lulamisiwile hi adenosine (250μM) kumbe MNA (10 mM) kuringana 24 wa tiawara, kutani ti tshikiwa tinga tshunguriwi (Ctrl). Tiphesente ta ku dlayiwa ka tisele ta SK-OV-3 ti pimiwe. Nhlayo ya P leyi kumiweke hi Welch t test (*P<0.5 na **P<0.01).
Leswaku ku kumiwa ku twisisa ka michini ka vulawuri bya ku paluxiwa ka TNFα lebyi titshegeke hi MNA, ku cinca eka TNFα mRNA ya tisele ta T leti tshunguriweke hi MNA ku kamberiwile (Xifaniso 5A). Tisele ta T ta donor leti hanyeke kahle leti tshunguriweke hi MNA ti kombisile ku tlakuka kambirhi eka ti levele ta TNFα transcription, leswi kombisaka leswaku MNA yi titshege hi vulawuri bya TNFα transcriptional. Ku lavisisa endlelo leri ra vulawuri leri nga kotekaka, swivangelo swimbirhi leswi tivekaka swa matsalelo leswi lawulaka TNFα, ku nga activated T cell nuclear factor (NFAT) na specific protein 1 (Sp1), swi kamberiwile ku hlamula eka ku boha MNA eka proximal TNFα promoter ( 30 ). TNFα promoter yi na 6 wa tindhawu leti hlawuriweke to boha NFAT na 2 wa tindhawu to boha Sp1, leti hlanganisaka eka ndhawu yin’we [-55 base pairs (bp) ku suka eka 5'cap] (30). Chromatin immunoprecipitation (ChIP) yi kombisile leswaku loko yi tshunguriwa hi MNA, ku bohiwa ka Sp1 eka TNFα promoter swi engetelekile kanharhu. Ku nghenisiwa ka NFAT na swona swi engetelekile no tshinelela nkoka (Xifaniso 5B). Data leyi yi kombisa leswaku MNA yi lawula ku paluxiwa ka TNFα hi ku tirhisa Sp1 transcription, naswona hi mpimo wutsongo ku paluxiwa ka NFAT.
(A) Loko ku pimanisiwa na tisele ta T leti kurisiweke handle ka MNA, ku cinca ka folda ka ku paluxiwa ka TNFα eka tisele ta T leti tshunguriweke hi MNA. Xivumbeko xa ku paluxiwa na SEM xi kombisiwile (n = 5 wa vaphakeri lava hanyeke kahle). Yi yimela datha ku suka eka kwalomu ka n = 3 wa swikambelo leswi tiyimeleke. (B) TNFα promoter ya tisele ta T leti tshunguriweke hi kumbe handle ka 8 mM MNA endzhaku ka NFAT na Sp1 yi hlanganisiwile na (Ctrl) na PMA/ionomycin stimulation ku ringana 4 wa tiawara. Immunoglobulin G (IgG) na H3 switirhisiwile tani hi vulawuri byo biha na lebyinene eka immunoprecipitation, hiku landzelelana. Ku pimiwa ka nhlayo ya ChIP swikombe leswaku ku bohiwa ka Sp1 na NFAT eka TNFα promoter eka ti cells leti tshunguriweke hi MNA swi tlakukile minkarhi yo hlaya loko ku pimanyisiwa na vulawuri. Yi yimela datha ku suka eka kwalomu ka n = 3 wa swikambelo leswi tiyimeleke. Nhlayo ya P leyi kumiweke hi swikambelo swo tala swa t (*** P <0.01). (C) Loko ku pimanisiwa na ascites ya HGSC, ti cells ta T (non-cytotoxic) ti kombisile ku tlakuka ka ku paluxiwa ka TNF eka tumor. Mihlovo yi yimela vavabyi vo hambana-hambana. Ti cells leti kombisiweke ti sampled hiku landzelelana kuya eka 300 naswona ti jittered kuva ti hunguta overdrawing (** Padj = 0.0076). (D) Modele lowu ringanyetiweke wa MNA wa khensa ya ovary. MNA yi endliwa eka ti cells ta tumor na ti fibroblasts eka TME naswona yi tekiwa hi ti cells ta T. MNA yi engetela ku bohiwa ka Sp1 eka TNFα promoter, leswi yisaka eka ku tlakuka ka TNFα transcription na vuhumelerisi bya TNFα cytokine. MNA yitlhela yivanga kuhunguteka ka IFN-γ. Ku sivela ntirho wa ti cell ta T swi endla leswaku kuhunguteka vuswikoti byo dlaya xikan’we naku hatlisisa kukula ka tumor.
Hi ku ya hi swiviko, TNFα yi na switandzhaku swa le mahlweni na le ndzhaku leswi titshegeke hi anti-tumor na anti-tumor, kambe yi na xiave lexi tivekaka swinene eku hlohloteleni ka ku kula na ku hangalaka ka khensa ya ovary ( 31-33 ). Hi ku ya hi swiviko, nhlayo ya TNFα eka tinyama ta ascites na tshumba eka vavabyi lava nga na khensa ya ovary yi le henhla ku tlula eka tinyama leti nga riki na khombo ( 34-36 ). Hi mayelana na endlelo, TNFα yi nga lawula ku tirhisiwa, ntirho na ku andza ka tisele to basa ta ngati, na ku cinca phenotype ya tisele ta khensa ( 37 , 38 ). Ku fambisana na swikumiwa leswi, nxopaxopo wa ku paluxiwa ka tijini to hambana wu kombisile leswaku TNF yi lawuriwa swinene eka tisele ta T eka tinyama ta tshumba loko ku pimanisiwa na ascites (Xifaniso 5C). Ku tlakuka ka ku paluxiwa ka TNF swi vonakile ntsena eka nhlayo ya tisele ta T leti nga na phenotype leyi nga riki ya cytotoxic (Xifaniso xa S5A). Hi ku komisa, datha leyi yi seketela vonelo ra leswaku MNA yi na switandzhaku swimbirhi swa immunosuppressive na tumor promoting eka HGSC.
Ku lebula ka fluorescent loku sekeriweke eka flow cytometry ku hundzuke ndlela leyikulu yo dyondza metabolism ya TIL. Mindzavisiso leyi yi kombisile leswaku loko ku pimanisiwa na ti peripheral blood lymphocytes kumbe T cells kusuka eka swirho swa secondary lymphoid, murine na human TIL yina mboyamelo wale henhla wo amukela glucose ( 4 , 39 ) na ku lahlekeriwa hi ntirho wa mitochondrial hakatsongotsongo ( 19 , 40 ). Hambi leswi hi voneke mbuyelo lowu fanaka eka ndzavisiso lowu, nhluvukiso wa nkoka i ku pimanisa metabolism ya tisele ta tshumba na TIL ku suka eka tinyama leti fanaka ta tshumba leti tsemiweke. Ku fambisana na swin’wana swa swiviko leswi swa khale, tisele ta tshumba (CD45-EpCAM +) ku suka eka ascites na swirhumbana ti na ku amukeriwa ka glucose ya le henhla ku tlula tisele ta CD8 + na CD4 + T, leswi seketelaka leswaku ku amukeriwa ka glucose ya le henhla ya tisele ta tshumba ku nga pimanisiwa na tisele ta T. Mhaka ya mphikizano wa tisele ta T. TME. Hambiswiritano, ntirho wa mitochondrial wa tisele ta tshumba wu le henhla ku tlula wa tisele ta CD8 + T, kambe ntirho wa mitochondrial wu fana na wa tisele ta CD4 + T. Mimbuyelo leyi yi tiyisisa nhlokomhaka leyi humelelaka ya leswaku oxidative metabolism i ya nkoka eka tisele ta tshumba ( 41 , 42 ). Va tlhela va ringanyeta leswaku tisele ta CD8 + T ti nga ha va na ku tsandzeka ku tirha ka oxidative ku tlula tisele ta CD4 + T, kumbe leswaku tisele ta CD4 + T ti nga tirhisa swihlovo swa khaboni handle ka glucose ku hlayisa ntirho wa mitochondrial ( 43 , 44 ). Swi fanele ku xiyiwa leswaku hi vonile ku hambana eka ku amukeriwa ka glucose kumbe ntirho wa mitochondrial exikarhi ka CD4 + T effectors, T effector memory na T central memory cells eka ascites. Hilaha ku fanaka, xiyimo xa ku hambana ka tisele ta CD8 + T eka swirhumbana a xi fambelani na ku cinca eka ku amukeriwa ka glucose, leswi kombisaka ku hambana lokukulu exikarhi ka tisele ta T leti kurisiweke in vitro na TIL ya munhu eka vivo ( 22 ). Swibumabumelo leswi swi tlhele swi tiyisisiwa hi ku tirhisiwa ka ku averiwa ka nhlayo ya tisele ta otomatiki loku nga na xihlawuhlawu, leswi nga ya emahlweni swi paluxa leswaku tisele ta CD45 + / CD3- / CD4 + / CD45RO + leti nga na ku amukeriwa ka glucose ya le henhla na ntirho wa mitochondrial ku tlula tisele ta tshumba ti tele kambe ti na nhlayo ya tisele leti tirhaka ta Metabolic. Nhlayo leyi yi nga ha yimela subpopulation leyi ehleketiwaka ya tisele ta myeloid suppressor kumbe tisele ta plasmacytoid dendritic leti hlawuriweke eka nxopaxopo wa scRNA-seq. Hambi leswi leswi haswimbirhi swi vikiweke eka swirhumbana swa ovary ya vanhu [45], swa ha lava Ntirho wun’wana i ku hlamusela subpopulation leyi ya myeloid.
Hambi leswi tindlela leti simekiweke eka flow cytometry tinga hlamuselaka ku hambana hiku angarhela eka glucose na oxidative metabolism exikarhi ka tinxaka ta ti cell, ti metabolites leti kongomeke leti humelerisiwaka hi glucose kumbe swihlovo swin’wana swa carbon eka mitochondrial metabolism eka TME aswi se kumiwa. Ku avela vukona kumbe ku pfumaleka ka ti metabolites eka ntlawa lowu nyikiweke wa TIL swilava ku basisiwa ka nhlayo ya ti cell kusuka eka tissue leyi tsemiweke. Hikwalaho, ndlela ya hina yo fuwisa tisele leyi hlanganisiweke na mass spectrometry yi nga nyika vutivi eka ti-metabolite leti fuweke hi ku hambana eka tisele ta T na vaaki va tisele ta tshumba eka swikombiso swa vavabyi leswi fambelanaka. Hambi leswi endlelo leri ri nga na swipfuno eka ku hlawuriwa ka tisele loku tirhisiwaka hi fluorescence, tilayiburari to karhi ta metabolite ti nga khumbeka hikwalaho ka ku tshamiseka loku nga kona na/kumbe mpimo wa ku cinca hi ku hatlisa (22). Hambi swiritano, ndlela ya hina yi kotile ku kuma ti metabolites timbirhi leti tivekaka ta immunosuppressive, adenosine na kynurenine, hikuva ti hambana swinene exikarhi ka tinxaka ta xikombiso.
Nxopaxopo wa hina wa metabolite wa swirhumbana na tinxaka letitsongo ta TIL wu nyika vutivi byo tala eka ntirho wa ti-metabolite eka TME ya ovary. Xo sungula, hi ku tirhisa flow cytometry, hi kumile leswaku a ku ri hava ku hambana eka ntirho wa mitochondrial exikarhi ka swirhumbana na tisele ta CD4 + T. Hambiswiritano, nxopaxopo wa LC-MS/MS wu paluxe ku cinca lokukulu eka nhlayo ya ti metabolites exikarhi ka vaaki lava, leswi kombisaka leswaku mahetelelo mayelana na metabolism ya TIL na ntirho wa yona hinkwawo wa metabolic ya lava nhlamuselo ya vukheta. Xavumbirhi, MNA i metabolite leyingana ku hambana lokukulu exikarhi ka CD45-cells na T cells eka ascites, kungari ti tumors. Hikokwalaho, compartmentalization na ndzhawu ya tumor swinga va na switandzhaku swohambana eka TIL metabolism, leswi kombisaka heterogeneity leyi nga kotekaka eka microenvironment leyi nyikiweke. Xa vunharhu, ku paluxiwa ka enzyme leyi humesaka MNA NNMT ngopfungopfu swi pimiwile eka CAF, leyi nga tisele ta tshumba hi mpimo wutsongo, kambe swiyimo swa MNA leswi voniwaka swi voniwa eka tisele ta T leti humaka eka tshumba. Ku humesiwa ngopfu ka NNMT eka CAF ya ovarian ku na mbuyelo lowu tivekaka wo tlakusa khensa, xiphemu xin’wana hikwalaho ka ku tlakusa metabolism ya CAF, ku hlaseriwa ka tshumba na metastasis ( 27 ). Hambi leswi mpimo hinkwawo wa TIL wu nga exikarhi, ku paluxiwa ka NNMT eka CAF swi fambelana swinene na Cancer Genome Atlas (TCGA) mesenchymal subtype, leyi fambelanaka na prognosis yo biha ( 27 , 46 , 47 ). Eku heteleleni, ku paluxiwa ka enzyme AOX1 leyi nga na vutihlamuleri eka ku onhaka ka MNA na swona swi pimiwile eka vaaki va CAF, leswi kombisaka leswaku tisele ta T ti pfumala vuswikoti byo metabolize MNA. Mimbuyelo leyi yi seketela mianakanyo ya leswaku hambi leswi ntirho wun’wana wu lavekaka ku tiyisisa ku kumiwa loku, swiyimo swa le henhla swa MNA eka tisele ta T swi nga kombisa vukona bya microenvironment ya CAF leyi tshikilelaka masocha ya miri.
Hiku nyikiwa levele yale hansi ya expression ya ti MNA transporters xikan’we na ti levele letinga voniwiki ta ti protein ta nkoka leti katsekaka eka MNA metabolism, vukona bya MNA eka ti cells ta T aswi languteriwanga. NNMT kumbe AOX1 a swi nga voniwi hi scRNA-seq analysis na targeted qPCR ya ti cohorts timbirhi leti tiyimeleke. Mimbuyelo leyi yi kombisa leswaku MNA ayi endliwi hi ti cells ta T, kambe yi amukeriwa kusuka eka TME leyi rhendzeleke. Swikambelo swa in vitro swikombisa leswaku ti cells ta T titala ku hlengeleta exogenous MNA.
Tidyondzo ta hina ta in vitro ti kombisile leswaku exogenous MNA yi hlohletela ku paluxiwa ka TNFα eka tisele ta T naswona yi ndlandlamuxa ku bohiwa ka Sp1 eka TNFα promoter. Hambi leswi TNFα yi nga na mintirho yo lwisana na tshumba na ku lwisana na tshumba, eka khensa ya ovary, TNFα yi nga hlohlotela ku kula ka khensa ya ovary ( 31-33 ). Ku neutralization ka TNFα eka ovarian tumor cell culture kumbe ku herisiwa ka TNFα signal eka ti models ta mbeva swi nga antswisa TNFα-mediated inflammatory cytokine production na ku sivela ku kula ka tumor ( 32 , 35 ). Hikwalaho, eka xiyimo lexi, MNA leyi humaka eka TME yi nga tirha tanihi metabolite ya pro-inflammatory hi ku tirhisa endlelo leri titshegeke hi TNFα hi ku tirhisa autocrine loop, xisweswo yi tlakusa ku humelela na ku hangalaka ka khensa ya ovary ( 31 ). Hi ku ya hi ku koteka loku, ku pfaleriwa ka TNFα ku dyondziwa tanihi xitirhisiwa lexi nga vaka xa vutshunguri eka khensa ya ovary ( 37 , 48 , 49 ). Ku engetela kwalaho, MNA yi onha cytotoxicity ya tisele ta CAR-T eka tisele ta tshumba ra ovarian, leswi nyikaka vumbhoni byin’wana bya ku tshikileriwa ka masocha ya miri lama kongomisiwaka hi MNA. Hi ku hlangana, mimbuyelo leyi yi ringanyeta modele lowu eka wona ti tumors na ti cells ta CAF ti humesaka MNA eka extracellular TME. Hi ku tirhisa (i) ku hlohloteriwa ka ku kula ka khensa ya ovary loku hlohloteriwaka hi TNF na (ii) ku sivela ntirho wa cytotoxic wa tisele ta T loku hlohloteriwaka hi MNA, leswi swi nga va na mbuyelo wa tshumba kambirhi (Xifaniso 5D).
Hi ku gimeta, hi ku tirhisa nkatsakanyo wa ku fuwisiwa ka tisele hi xihatla, ku landzelelana ka sele yin’we na ku hambanisiwa ka metabolic, ndzavisiso lowu wu paluxe ku hambana lokukulu ka immunometabolomic exikarhi ka swirhumbana na tisele ta ascites eka vavabyi va HGSC. Nxopaxopo lowu wo angarhela wu kombisile leswaku ku na ku hambana eka ku amukeriwa ka glucose na ntirho wa mitochondrial exikarhi ka tisele ta T, naswona wu hlawurile MNA tanihi non-cell autonomous immune regulatory metabolite. Data leyi yi na nkucetelo eka ndlela leyi TME yi khumbaka metabolism ya tisele ta T eka tikhensa ta vanhu. Hambi leswi mphikizano lowu kongomeke wa swakudya exikarhi ka tisele ta T na tisele ta khensa wu vikiweke, ti metabolite ti nga tlhela ti tirha tanihi valawuri lava nga kongomangiki ku tlakusa ku kula ka tshumba naswona swi nga endleka ku tshikilela tinhlamulo ta masocha ya miri ya le ndzeni. Nhlamuselo yin’wana ya ntirho wa ntirho wa ti metabolites leti ta vulawuri yi nga pfula tindlela tin’wana to ndlandlamuxa nhlamulo ya masocha ya miri ya anti-tumor.
Swikombiso swa vavabyi na datha ya tliliniki swi kumiwile hi ku tirhisa vuhlayiselo bya tinyama bya tshumba ra khensa ya BC lebyi tiyisisiweke hi Canadian Tissue Repository Network. Hi ku ya hi phurotokholo leyi amukeriweke hi Komiti ya Mahanyelo ya Vulavisisi bya Khensa ya BC na Yunivhesiti ya British Columbia (H07-00463), swikombiso hinkwaswo swa vavabyi na datha ya tliliniki swi kumile mpfumelelo lowu tsariweke lowu nga na vutivi kumbe swi tshike mpfumelelo wa swona ximfumo. Tisampulu ti hlayisiwa eka BioBank leyi tiyisisiweke (BRC-00290). Vuxokoxoko bya swihlawulekisi swa muvabyi swi kombisiwile eka Tithebulu ta S1 na S5. Eka cryopreservation, ku tirhisiwa scalpel ku bola hi muchini xikombiso xa tshumba ra muvabyi ivi endzhaku xi susumeta hi 100-micron filter leswaku ku kumiwa ku yimisiwa ka sele yin’we. Ascites ya muvabyi yi centrifuged eka 1500 rpm kuringana 10 wa timinete eka 4°C kuva yi pellet ti cells naku susa supernatant. Ti cells leti kumiweke kusuka eka tumor na ascites ti cryopreserved eka 50% heat-inactivated human AB serum (Sigma-Aldrich), 40% RPMI-1640 (Thermo Fisher Scientific) na 10% dimethyl sulfoxide. Ti suspensions leti ta single cell leti hlayisiweke ti thawed naswona ti tirhisiwile eka metabolomics na metabolite determination leswi hlamuseriweke laha hansi.
Xikarhi lexi heleleke xi na 0.22 μm leyi sefiweke 50:50 leyi engeteriweke RPMI 1640: AimV. RPMI 1640 + 2.05 mM l-glutamine (Thermo Fisher Scientific) leyi engeteriweke hi 10% wa serum ya AB ya munhu leyi nga tirhisiwiki hi ku hisa (Sigma-Aldrich), 12.5 mM Hepes (Thermo Fisher Scientific), 2 mM l-glutamine (Thermo Fisher Scientific) Fisher Scientific), 1 x Nkatsakanyo wa penicillin Streptomycin (PenStrep) (Thermo Fisher Scientific) na 50 μMB-mercaptoethanol. AimV (Invitrogen) yi engeteriwa hi 20 mM Hepes (Thermo Fisher Scientific) na 2 mM l-glutamine (Thermo Fisher Scientific). Flow cytometer staining buffer ayi vumbiwa hi 0.22μm filtered phosphate buffered saline (PBS; Invitrogen) leyi engeteriweke hi 3% hi heat-inactivated AB human serum (Sigma). Buffer yo fuwisa ti cell yi vumbiwa hi 0.22μm ya PBS leyi sefiweke naswona yi engeteriwile hi 0.5% heat-inactivated human AB serum (Sigma-Aldrich).
Eka 37°C complete medium, ti cells ti cheriwe 10 nM MT DR na 100 μM 2-NBDG kuringana 30 wa timinete. Endzhaku ka sweswo, ti cells ti cheriwe viability dye eF506 eka 4°C kuringana 15 wa timinete. Pfuxeta ti cells eka FC Block (eBioscience) na Brilliant Stain Buffer (BD Biosciences), chela eka flow cytometer staining buffer (kuya hi swiletelo swa muendli), kutani u pfalela kuringana 10 wa timinete eka mahiselo ya kamara. Hlawula ti cells hi sete ya ti antibodies (Table S2) eka flow cytometry staining buffer eka 4°C kuringana 20 wa timinete. Yimisa ti cells eka flow cytometry staining buffer (Cytek Aurora; 3L-16V-14B-8R configuration) loko kungase kamberiwa. Tirhisa SpectroFlo na FlowJo V10 ku xopaxopa datha ya nhlayo ya tisele, naswona tirhisa GraphPad Prism 8 ku tumbuluxa datha. Median fluorescence intensity (MFI) ya 2-NBDG na MT DR yi log-normalized, kutani ku tirhisiwile xikambelo xa paired t eka nxopaxopo wa tinhlayo ku hlamusela vavabyi lava ringanisiweke. Susa vaaki hinkwavo lava nga na swiendlakalo swa le hansi ka 40 eka nxopaxopo; nghenisa ntikelo wa MFI wa 1 eka mimpimo yihi kumbe yihi yo biha u nga si endla nxopaxopo wa tinhlayo na ku vonakala ka datha.
Leswaku hi engetela maqhinga ya manual gating ya phanele ya phurosese leyi nga laha henhla, hi tirhise nhlamuselo leyi heleleke hi murhi wa xipimelo xa xivumbeko (FAUST) (21) ku avela hi ku tisungulela tisele eka vaaki endzhaku ko herisa tisele leti feke eka FlowJo. Hi lawula vuhumelerisi hi voko ku hlanganisa vaaki lava vonakaka va averiwa hi ndlela yo biha (ku hlanganisa PD1+ na tisele ta PD1-tumor) na vaaki lava hlayisiweke. Xikombiso xin’wana na xin’wana xina xiringaniso xa kutlula 2% wa ti cells, eka ntsengo wa 11 wa vaaki.
Ficoll gradient density centrifugation yitirhisiwile ku hambanyisa PBMC kusuka eka switirhisiwa swo hambanyisa ti leukocyte (STEMCELL Technologies). Ti cells ta CD8 + T ti hambanisiwile kusuka eka PBMC hiku tirhisa CD8 MicroBeads (Miltenyi) naswona ti andlariwile eka medium leyi heleleke hiku tirhisa TransAct (Miltenyi) kuringana 2 wa mavhiki kuya hi swiletelo swa muendli. Ti cells ti pfumeleriwile ku yima kuringana 5 wa masiku eka medium leyi heleleke leyingana IL-7 (10 ng/ml; PeproTech), kutani titlhela ti hlohleteriwa hi TransAct. Hi siku ravu 7, kuya hi swiletelo swa muendli, human CD45 MicroBeads (Miltenyi) yitirhisiwile ku fuwisa ti cells eka swirhendzevutani swinharhu hiku landzelelana. Ti cells ti hambanisiwile eka nxopaxopo wa flow cytometry (hilaha swi hlamuseriweke hakona laha henhla), naswona miliyoni ya ti cells ti hambanisiwile kanharhu eka nxopaxopo wa LC-MS/MS. Tisampulu ti lulamisiwile hi LC-MS/MS tani hileswi swi hlamuseriweke laha hansi. Hi ringanyete nhlayo ya metabolite leyi kayivelaka hi nhlayo ya ion ya 1,000. Xikombiso xin’wana na xin’wana xi normaliseriwa hi total ion number (TIC), xi hundzuriwa hi logarithmically naswona xi normaliseriwa hiku tisungulela eka MetaboAnalystR loko kungase endliwa nxopaxopo.
Ku yimisiwa ka sele yin’we ya muvabyi un’wana na un’wana swi thawiwile kutani swi sefiwile hi 40 μm filter kuya eka medium leyi heleleke (hilaha swi hlamuseriweke hakona laha henhla). Kuya hi protocol ya muendli, swirhendzevutani swinharhu leswi landzelelanaka swa ku hlawula lokunene hiku hambanyisa vuhlalu bya magnetic hiku tirhisa MicroBeads (Miltenyi) switirhisiwile ku fuwisa tisampulu ta ti cells ta CD8+, CD4+ na CD45- (eka ayisi). Hiku komisa, ti cells titlhela ti suspended eka cell enrichment buffer (hilaha swi hlamuseriweke hakona laha henhla) kutani ti hlayiwa. Ti cells ti pfaleriwile na vuhlalu bya CD8 bya munhu, vuhlalu bya CD4 bya munhu kumbe vuhlalu bya CD45 bya munhu (Miltenyi) eka 4°C kuringana 15 wa timinete, kutani ti hlantswiwa hi cell enrichment buffer. Xikombiso xi hundziseriwa eka kholomo ya LS (Miltenyi), naswona swiphemu swa kahle na swo biha swa hlengeletiwa. Leswaku ku hungutiwa nkarhi na ku kurisa goza ro vuyisa tisele, xiphemu xa CD8 xi tlhela xi tirhisiwa eka xirhendzevutani xa vumbirhi xa ku fuwisa CD4+, naswona xiphemu xa CD4 xi tirhisiwa eka ku fuwisa ka CD45 loku landzelaka. Hlayisa solution eka ice eka maendlelo hinkwawo yo hambanyisa.
Ku lulamisela swikombiso eka nxopaxopo wa metabolite, ti cells ti hlantswiwile kan’we hi ice-cold salt solution, naswona 1 ml ya 80% methanol yi cheriwile eka xikombiso xin’wana na xin’wana, kutani ti vortexed naswona ti snap frozen eka liquid nitrogen. Tisampulu ti endliwile eka swirhendzevutani swinharhu swa freeze-thaw naswona ti centrifuged eka 14,000 rpm kuringana 15 wa timinete eka 4°C. Supernatant leyinga na ti metabolites yi pfanganisiwa kufikela yi oma. Ti metabolites titlhele ti pfanganisiwa eka 50 μl ya 0.03% formic acid, ti vortexed kuva ti hlanganisiwa, kutani ti centrifuged kuva ti susa swilo leswinga sala.
Humesa ti metabolites tani hileswi swi hlamuseriweke laha henhla. Tlhuvulela supernatant eka bodlhela ra high performance liquid chromatography eka ndzavisiso wa metabolomics. Tirhisa protocol ya vutshunguri bya random ku tshungula xikombiso xin’wana na xin’wana hi nhlayo leyi fanaka ya ti cells ku sivela switandzhaku swa batch. Hi endle nkambisiso wa khwalithi wa ti-metabolite ta misava hinkwayo leti kandziyisiweke khale eka AB SCIEX QTRAP 5500 Triple Quadrupole Mass Spectrometer (50). Nxopaxopo wa chromatographic na ku hlanganisiwa ka ndzhawu ya nhlohlorhi swi endliwile hiku tirhisa MultiQuant version 2.1 software (Applied Biosystems SCIEX).
Nhlayo ya ion ya 1000 yitirhisiwile ku ringanyeta nkoka wa metabolite leyi kayivelaka, naswona TIC ya xikombiso xin’wana na xin’wana yitirhisiwile ku hlayela ndzhawu ya nhlohlorhi leyi tolovelekeke ya metabolite yin’wana na yin’wana leyi kumiweke ku lulamisa ku cinca loku nghenisiweke hi nxopaxopo wa switirhisiwa kusuka eka ku lulamisiwa ka xikombiso. Endzhaku ka loko TIC yi normalised, MetaboAnalystR(51) (default parameter) yi tirhisiwa eka ku hundzuriwa ka logarithmic na ku ringanisa layini ya norm ya otomatiki. Hi tirhise PCA na vegan R package ku endla nxopaxopo wo lavisisa wa ku hambana ka metabolome exikarhi ka tinxaka ta xikombiso, naswona hi tirhise nxopaxopo wa xiphemu xa redundancy ku xopaxopa vavabyi. Tirhisa ndlela ya Ward ku aka dendrogram ya mepe wo hisa ku hlengeleta mpfhuka wa Euclidean exikarhi ka swikombiso. Hi tirhise limma (52) eka standardized metabolite abundance ku kuma differentially abundant metabolites eka muxaka hinkwawo wa sele na microenvironment. Leswaku hi olovisa nhlamuselo, hi tirhisa parameter ya group mean ku boxa modele, na ku tekela enhlokweni tinxaka ta ti cell eka microenvironment tani hi ntlawa wun’wana na wun’wana (n = 6 groups); eka xikambelo xa nkoka, hi endlile mimpimo yinharhu leyi phindha-phindhiweke eka metabolite yin’wana na yin’wana Leswaku hi papalata ku andzisiwa ka mavunwa, muvabyi u katsiwile tanihi xihinga eka dizayini ya limma. Leswaku hi kambela ku hambana ka ti metabolites exikarhi ka vavabyi vo hambana, hi lulamisile modele wa limma ku katsa na vavabyi hi ndlela leyi nga cinciki. Hi vika nkoka wa ku hambana loku boxiweke ka ha ri emahlweni exikarhi ka muxaka wa sele na microenvironment ya Padj <0.05 (Benjamini-Hochberg correction).
Endzhaku ka ku fuwisiwa ka matimba hiku tirhisa Miltenyi Dead Cell Removal Kit (>80% viability), single-cell transcriptome sequencing yi endliwile eka total live frozen ascites na tisampulu ta tumor hiku tirhisa 10x 5′gene expression protocol. Ntlhanu wa timhangu letingana ti tumors leti fambelanaka na ascites ti kamberiwile, hambi leswi ku hanya ka le hansi kusuka eka xikombiso xin’we xa tumor swi sivele ku katsiwa ka yona. Leswaku hi fikelela ku hlawuriwa ko tala ka vavabyi, hi hlanganisile swikombiso swa muvabyi un’wana na un’wana eka milayeni ya 10x chromium controller, naswona hi xopaxophile tindhawu ta ascites na tshumba hi ku hambana. Endzhaku ko landzelelana [Illumina HiSeq 4000 28×98 bp paired end (PE), genome ya Quebec; xiringaniso xa 73,488 na 41,378 wa ku hlaya hi sele ya tshumba na ascites hi ku landzelelana]], hi tirhise CellSNP na Vireo (53) (leswi sekeriweke eka CellSNP tanihi The common human SNP (VCF) leyi nyikiweke hi GRCh38 yi averiwe vutivi bya munyiki.Hi tirhisa SNPRelate ku ringanyeta vutivi bya le kusuhi (IBS) bya xiyimo xa genotype ya muvabyi (IBS), ku nga katsi tisele leti nga aviwangiki na tisele leti tiviweke tanihi ti-duplex na ku fambelana ka vaphakeri exikarhi ka tisampulu ta ascites na tshumba ( 54 ), hi hlayise timhangu tinharhu leti nga na vuyimeri byo tala bya tisele eka tshumba na ascites ku endlela nxopaxopo wa le hansi endzhaku ko endla goza ra mass filtration eka scater (55) na scran (56) BioConductor packaging, leswi swi humese 6975 wa tisele (2792 and 4183 cells from tumor and ascites, respectively) for analysis. We use igraph's (57) Louvain clustering of shared nearest neighbor network (SNN) based on Jaccard distance to cluster cells by expression. The clusters were manually annotated to putative cell types based on marker gene expression and visualized with t-SNE. Cytotoxic T cells are ku hlamuseriwa hi ku paluxiwa ka CD8A na GZMA, ku nga katsiwi swirhabyani leswi nga na ku paluxiwa ka tiphrotheyini ta ribosomal ta le hansi Hi fikelela datha leyi kandziyisiweke ya Izar et al (16), ku katsa na ku nghenisiwa ka vona ka t-SNE, swi nga lawula ku hlangana ka ku paluxiwa exikarhi ka swimaki swa tisele ta masocha ya miri.
PBMC yi hambanisiwile kusuka eka switirhisiwa swo hambanyisa ti leukocyte (STEMCELL Technologies) hi Ficoll gradient density centrifugation. Ti cells ta CD3 + ti hambanisiwile kusuka eka PBMC hiku tirhisa vuhlalu bya CD3 (Miltenyi). Eka vukona kumbe ku pfumaleka ka MNA, ti cells ta CD3+ ti tirhisiwile hi plate-bound CD3 (5μg/ml), soluble CD28 (3μg/ml) na IL-2 (300 U/ml; Proleukin). Hi siku rohetelela raku ndlandlamuxiwa, ku hanya (Fixable Viability Dye eFluor450, eBioscience) na ku andza (123count eBeads, Thermo Fisher Scientific) swikamberiwile hi flow cytometry. Kambisisa ntirho wa effector hiku hlohletela ti cells hi PMA (20 ng/ml) na ionomycin (1μg/ml) hi GolgiStop kuringana 4 wa tiawara, naswona u langutisisa CD8-PerCP (RPA-T8, BioLegend), CD4-AF700 (RPA-T4) , BioLegend) na TNFα-fluorescein isothiocyanate (FITC) (MAb11, BD). Hlohlotela ti cells ta qPCR na ChIP hi PMA (20 ng/ml) na ionomycin (1μg/ml) kuringana 4 wa tiawara. Supernatant ya ELISA yi hlengeletiwile emahlweni na le ndzhaku ka ku hlohloteriwa hi PMA (20 ng/ml) na ionomycin (1 μg/ml) kuringana 4 wa tiawara.
Landzelela protocol ya muendli ku hambanyisa RNA hiku tirhisa RNeasy Plus Mini Kit (QIAGEN). Tirhisa QIAshredder (QIAGEN) ku homogenize xikombiso. Tirhisa xitirhisiwa xa RNA kuya eka cDNA xa vuswikoti byale henhla (Thermo Fisher Scientific) ku hlanganisa DNA yo tatisela (cDNA). Tirhisa TaqMan Rapid Advanced Master Mix (Thermo Fisher Scientific) ku pima nhlayo ya ku paluxiwa ka tijini (hi ku ya hi protocol ya muendli) hi ti probes leti landzelaka: Hs00196287_m1 (NNMT), Hs00154079_m1 (AOX1), Hs00427552_m1 (SLC22A1), . Hs02786624_g1 [glyceraldehyde-3-phosphate ehandle ka Hayidirojeni (GAPDH)] na Hs01010726_m1 (SLC22A2). Tisampulu ti fambisiwa eka sisiteme ya PCR ya nkarhi wa xiviri ya StepOnePlus (Applied Biosystems) (Applied Biosystems) eka MicroAmp fast optical 96-well reaction plate (Applied Biosystems) na filimi ya mahlo ya MicroAmp. Nhlayo yin’wana na yin’wana ya Ct leyi tlulaka 35 yi tekiwa yi ri ehenhla ka mpimo wa ku kumiwa naswona yi funghiwile tanihi leyi nga voniwiki.
Endla ChIP tanihilaha swi hlamuseriweke hakona khale (58). Hiku komisa, ti cells ti tshunguriwile hi formaldehyde (final concentration 1.42%) naswona ti pfaleriwile eka mahiselo ya kamara kuringana 10 wa timinete. Tirhisa buffer yo pfimba leyi engeteriweke (25 mM Hepes, 1.5 mM MgCl2, 10 mM KCl na 0.1% NP-40) eka ayisi ku ringana timinete ta 10, kutani u tlhela u yi susa eka immunoprecipitation buffer tanihilaha swi hlamuseriweke hakona (58 ). Kutani xikombiso xi sonicated hi swirhendzevutani leswi landzelaka: 10 wa swirhendzevutani (20 1-second pulses) na nkarhi wa static wa 40 wa tisekoni. Hlayisa ti antibodies ta ChIP-grade immunoglobulin G (Cell Signaling Technology; 1μl), histone H3 (Cell Signaling Technology; 3μl), NFAT (Invitrogen; 3μl) na SP1 (Cell Signaling Technology; 3μl) na xikombiso eka 4°CC shake vusiku hinkwabyo. Hlayisa vuhlalu bya protein A (Thermo Fisher Scientific) na xikombiso eka 4°C hiku rhurhumela hiku olova kuringana 1 wa awara, kutani u tirhisa vuhlalu bya chelex (Bio-Rad) ku fuwisa DNA, naswona tirhisa proteinase K (Thermo Fisher) eka ku gayela ti protein. TNFα promoter yi kumiwile hi PCR: emahlweni, GGG TAT CCT TGA TGC TTG TGT; ku hambana na sweswo, GTG CCA ACA ACT GCC TTT ATA TG (xiendliwa xa 207-bp). Swifaniso leswi swi endliwile hi Image Lab (Bio-Rad) naswona swi pimiwile hi ku tirhisa software ya ImageJ.
Supernatant ya cell culture yi hlengeletiwile tani hileswi swinga hlamuseriwa laha henhla. Ku kumiwa ku endliwile kuya hi maendlelo ya muendli wa human TNFα ELISA kit (Invitrogen), human IL-2 ELISA kit (Invitrogen) na human IFN-γ ELISA kit (Abcam). Hikuya hi protocol ya muendli, supernatant yi cheriwile 1:100 kuva yi kumiwa TNFα na IL-2, na 1:3 kuva yi kumiwa IFN-γ. Tirhisa EnVision 2104 Multilabel Reader (PerkinElmer) ku pima ku amukeriwa eka 450 nm.
PBMC yi hambanisiwile kusuka eka switirhisiwa swo hambanyisa ti leukocyte (STEMCELL Technologies) hi Ficoll gradient density centrifugation. Ti cells ta CD3 + ti hambanisiwile kusuka eka PBMC hiku tirhisa vuhlalu bya CD3 (Miltenyi). Eka vukona kumbe ku pfumaleka ka MNA, ti cells ta CD3+ ti tirhisiwile hi plate-bound CD3 (5μg/ml), soluble CD28 (3μg/ml) na IL-2 (300 U/ml; Proleukin) kuringana 3 wa masiku. Endzhaku ka masiku ya 3, ti cells ti hlengeletiwile kutani ti hlantswiwa hi 0.9% saline, naswona pellet yi snap frozen. Ku hlayiwa ka tisele swi endliwile hi flow cytometry (Cytek Aurora; 3L-16V-14B-8R configuration) hiku tirhisa 123count eBeads.
Humesa ti metabolites tani hileswi swi hlamuseriweke laha henhla. Xiphemu lexi omisiweke xi vumbiwile nakambe eka nhlayo ya 4000 cell equivalents/μl. Kambisisa xikombiso hi reversed-phase chromatography (1290 Infinity II, Agilent Technologies, Santa Clara, CA) na kholomo ya CORTECS T3 (2.1×150 mm, sayizi ya swiphemuphemu 1.6-μm, sayizi ya pore 120-Å; #186008500, Waters). Polar mass spectrometer (6470, Agilent), laha electrospray ionization yi tirhaka hi ndlela ya kahle. Xiphemu xa A lexi fambafambaka i 0.1% wa formic acid (eka H2O), xiphemu xa B lexi fambafambaka i 90% wa acetonitrile, 0.1% wa formic acid. LC gradient i 0 kuya eka 2 wa timinete eka 100% A, 2 kuya eka 7.1 wa timinete eka 99% B, na 7.1 kuya eka 8 wa timinete eka 99% B. Kutani u tlhela u ringanisela kholomo na mobile phase A eka flow rate ya 0.6 ml/min kuringana 3 wa timinete. . Nhlayo ya ku khuluka i 0.4ml/min, naswona kamara ra kholomo ri hisiwa kuya eka 50°C. Tirhisa mpimanyeto wa tikhemikhali to basa wa MNA (M320995, Toronto Research Chemical Company, North York, Ontario, Canada) ku simeka nkarhi wo khoma (RT) na ku cinca (RT = 0.882 wa timinete, ku hundzuka 1 = 137→94.1, ku hundzuka 2 = 137→92 , Ku hundzuka 3 = 137→78). Loko ku cinca hinkwako kunharhu ku humelela hi nkarhi lowu faneleke wo hlayisa, ku cinca ka 1 ku tirhisiwa ku pima nhlayo ku tiyisisa ku kongoma. Standard curve ya MNA (Toronto Research Chemical Company) yi endliwile hi ti serial dilutions ta tsevu ta stock solution (1 mg/ml) kuva ku kumiwa ti standards ta 0.1, 1.0, 10 na 100 ng/ml na 1.0 na 10μg/ml hiku landzelelana ka mati. Mpimo wa ku kumiwa i 1 ng/ml, naswona nhlamulo ya linear yile xikarhi ka 10 ng/ml na 10μg/ml. Ku cheriwa kun’wana na kun’wana ka ti microliters timbirhi ta xikombiso na standard switirhisiwa eka nxopaxopo wa LC/MS, naswona xikombiso xa vulawuri bya khwalithi leyi pfanganisiweke xi fambisiwa eka ti injection tin’wana na tin’wana ta tsevu ku tiyisisa ku tshamiseka ka pulatifomo ya nxopaxopo. Tinhlamulo ta MNA ta tisampulu hinkwato ta tisele leti tshunguriweke hi MNA a ti ri endzeni ka linear range ya assay. Nxopaxopo wa datha wu endliwile hi ku tirhisa software ya nxopaxopo wa nhlayo ya MassHunter (v9.0, Agilent).
Xitukulwana xa vumbirhi xa αFR-CAR construct xi tekiwile eka Song et al. (59) . Hi ku komisa, xivumbeko lexi xi na swilo leswi landzelaka: Ntlhandlamano wa murhangeri wa CD8a, xiphemu xa αFR-specific single-chain variable fragment, CD8a hinge na transmembrane region, CD27 intracellular domain na CD3z intracellular domain. Ntlhandlamano wa CAR lowu heleleke wu endliwile hi GenScript, kutani wu cloned eka xitukulwana xa vumbirhi xa lentiviral expression vector ehenhla ka khasete ya GFP expression leyi tirhisiweke ku kambela vukorhokeri bya transduction.
Lentivirus yi endliwa hiku hundziseriwa ka ti cells ta HEK293T [American Type Culture Collection (ATCC); yi byariwile eka Dulbecco’s modified Eagle medium leyingana 10% fetal bovine serum (FBS) na 1% PenStrep, naswona yitirhisiwile CAR-GFP vector na The packaging plasmids (psPAX2 na pMD2.G, Addgene) yitirhisa lipofection amine (Sigma-Aldrich). Supernatant leyingana xitsongwatsongwana yi hlengeletiwile endzhaku ka 48 na 72 wa tiawara endzhaku ka transfection, yi sefiwile, naswona yi hlanganisiwa hi ultracentrifugation. Hlayisa concentrated viral supernatant eka -80°C kufikela loko transduction.
PBMC yi hambanisiwa kusuka eka switirhisiwa swo hambanyisa ti donor leukocyte leswi hanyeke kahle (STEMCELL Technologies) hi Ficoll gradient density centrifugation. Tirhisa ti positive selection CD8 microbeads (Miltenyi) ku hambanyisa ti cells ta CD8+ kusuka eka PBMC. Ku hlohlotela tisele ta T hi TransAct (Miltenyi) na le ka TexMACS medium [Miltenyi; yi engeteriwile hi 3% wa serum ya munhu leyi nga tirhisiwiki hi ku hisa, 1% wa PenStrep na IL-2 (300 U/ml)]. Endzhaku ka tiawara ta makume mbirhi mune endzhaku ka ku hlohloteriwa, ti cells ta T ti hundzuriwile hi lentivirus (10 μl concentrated virus supernatant eka 106 wa ti cells). 1 kuya eka 3 wa masiku endzhaku ka transduction eka Cytek Aurora (eka FSC (Forward Scatter)/SSC (Side Scatter), Singlet, GFP+), kambela ku paluxiwa ka GFP ya ti cells ku kombisa vuswikoti bya transduction bya kwalomu ka 30% .
Ti cells ta CAR-T ti byaleriwile kuringana 24 wa tiawara eka Immunocult (STEMCELL Technologies; ti engeteriwile hi 1% PenStrep) ehansi ka swiyimo leswi landzelaka: tinga tshunguriwangiki, ti tshunguriwile hi 250 μM adenosine kumbe 10 mM MNA. Endzhaku ka vutshunguri bya le mahlweni, tisele ta CAR-T ti hlantswiwile hi PBS naswona ti hlanganisiwile na 20,000 wa tisele ta SK-OV-3 [ATCC; eka McCoy 5A medium (Sigma-Aldrich) leyi engeteriweke hi 10% FBS na 1% PenStrep eka 10: Effector kuya eka target ratio ya 1 yi engeteriwile hi ku phindha-phindha kanharhu eka Immunocult medium leyi engeteriweke. Ti cells ta SK-OV-3 na ti cells ta SK-OV-3 leti lysed hi digitalis saponin (0.5mg/ml; Sigma-Aldrich) titirhisiwile tani hi vulawuri byo biha na lebyinene, hiku landzelelana. Endzhaku ka 24 wa tiawara ta ku byariwa swin’we, supernatant yi hlengeletiwile naswona lactate dehydrogenase (LDH) yi pimiwe kuya hi swiletelo swa muendli (LDH Glo Cytotoxicity Assay Kit, Promega). LDH supernatant yi cheriwile 1:50 eka LDH buffer. Tiphesente ta ku dlayiwa ti pimiwe hiku tirhisa fomula leyi landzelaka: tiphesente ta ku dlayiwa = tiphesente ta ku lulamisiwa / mpimo lowukulu wa ku dlaya x 100%, laha tiphesente ta ku lulamisiwa = co-culture-T cells ntsena, na mpimo lowukulu wa ku dlaya = positive control-negative control.
Hilaha swi hlamuseriweke hakona eka tsalwa kumbe switirhisiwa na maendlelo, tirhisa GraphPad Prism 8, Microsoft Excel kumbe R v3.6.0 eka nxopaxopo wa tinhlayo. Loko swikombiso swo tala swi hlengeletiwile eka muvabyi un’we (ku fana na ascites na tumor), hi tirhisa xikambelo xa paired t kutani hi katsa muvabyi tanihi mbuyelo wa xitshuketa eka modele wa linear kumbe generalized tanihilaha swi faneleke hakona. Eka nxopaxopo wa metabolomics, xikambelo xa nkoka xi endliwa hi ku phindha-phindha kanharhu.
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Xivutiso lexi xi tirhisiwa ku kambela loko u ri muendzi na ku sivela ku rhumeriwa ka swipamu hi ku tisungulela.
Marisa K. Kilgour (Marisa K. Kilgour), Sarah MacPherson (Sarah MacPherson), Lauren G. Zacharias (Lauren G. Zacharias), Abigail Eli Aris G. Watson (H. Watson (H. Watson), John Stagg (John Stagg), Brad H. Nelson (Brad H. Nelson), Ralph J. De Bellardini (Ralph J. DeBerardinis), Russell G. Jones (Russell G. Jones), Phineas T. Hamilton (Phineas T., Xihlamusela-marito xa Xitsonga.
MNA yi hoxa xandla eka ku tshikileriwa ka masocha ya miri ya tisele ta T naswona yi yimela xikongomelo lexi nga vaka kona xa vutshunguri bya masocha ya miri eka vutshunguri bya khensa ya vanhu.
Marisa K. Kilgour (Marisa K. Kilgour), Sarah MacPherson (Sarah MacPherson), Lauren G. Zacharias (Lauren G. Zacharias), Abigail Eli Aris G. Watson (H. Watson (H. Watson), John Stagg (John Stagg), Brad H. Nelson (Brad H. Nelson), Ralph J. De Bellardini (Ralph J. DeBerardinis), Russell G. Jones (Russell G. Jones), Phineas T. Hamilton (Phineas T., Xihlamusela-marito xa Xitsonga.
MNA yi hoxa xandla eka ku tshikileriwa ka masocha ya miri ya tisele ta T naswona yi yimela xikongomelo lexi nga vaka kona xa vutshunguri bya masocha ya miri eka vutshunguri bya khensa ya vanhu.
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