Ema-metabolites e-immunomodulatory ayintfo lebalulekile yesimo lesincane sesimila (TME), kepha ngaphandle kwetintfo letimbalwa, bunjalo babo buhlala bungatiwa kakhulu. Lapha, sahlatiya timila kanye nemangcamuzane e-T kusuka etimila kanye ne-ascites yetigulane letine-serous carcinoma (HGSC) lesesigabeni lesiphakeme kute sivete i-metabolome yalamagumbi lehlukene e-TME. Ascites kanye nemangcamuzane esimila babe umehluko lomkhulu we-metabolite. Nawucatsanisa ne-ascites, emaseli e-T langena esimileni anotsiswa kakhulu nge-1-methylnicotinamide (MNA). Noma lizinga le-MNA kumaseli e-T liphakeme, kuveta i-nicotinamide N-methyltransferase (i-enzyme lekhutsata kudluliselwa kwemacembu e-methyl kusuka ku-S-adenosylmethionine kuya ku-nicotinamide) kukhawulelwe kuma-fibroblasts kanye nemangcamuzane esimila. Ngendlela lesebentako, i-MNA ikhutsata emaseli e-T kutsi akhiphe sici se-cytokine sesimila lesikhutsata sifo se-alpha. Ngako-ke, i-MNA lesuselwa ku-TME ifaka sandla ekulawuleni emasotja emtimba emaseli e-T futsi imelela lisu le-immunotherapy lelingahle libe khona lekwelapha umdlavuza webantfu.
Ema-metabolites lasuselwa esimileni angaba nemtselela lomkhulu wekuvimbela kuvikeleka kwe-anti-tumor, futsi bufakazi lobunyenti bukhombisa kutsi angaphindze asebente njengemandla labalulekile ekuchubekeni kwesifo (1). Ngetulu kwemphumela weWarburg, umsebenti wakamuva sewucale kuveta simo sekugaya kudla kwemangcamuzane esimila kanye nebudlelwane bayo nesimo semasotja emtimba se-tumor microenvironment (TME). Tifundvo letimayelana nemamodeli emagundvwane kanye nemaseli e-T ebantfu tikhombise kutsi kusebenta kwe-glutamine (2), kusebenta kwe-oxidative (3) kanye nekusebenta kwe-glucose (4) kungasebenta ngekutimela kumacembu lamancane emaseli lahlukahlukene lavikela umtimba. Ema-metabolites lamanyenti kuletindlela avimbela umsebenti wekulwa netimila temaseli e-T. Kufakazelwe kutsi kuvinjelwa kwe coenzyme tetrahydrobiopterin (BH4) kungalimata kwandza kwemaseli eT, futsi kwandza kwe BH4 emtimbeni kungenta ncono kuphendvula kwemasotja emtimba lalwa netimila lokulawulwa yi CD4 ne CD8. Ngetulu kwaloko, umphumela wekuvikela umtimba we-kynurenine ungakhululwa ngekuphatfwa kwe-BH4 (5). Ku-isocitrate dehydrogenase (IDH) glioblastoma legucukako, kukhishwa kwe-enantiometabolic (R)-2-hydroxyglutarate (R-2-HG) kuvimbela kusebenta kwemaseli e-T, kwandza kanye neMsebenti we-cytolysis (6). Muva nje, kukhonjisiwe kutsi i-methylglyoxal, umkhicito we-glycolysis, ukhicitwa ngemaseli lacindzetelako emvelaphi ye-myeloid, futsi kudluliselwa kwemaseli e-T e-methylglyoxal kungavimbela umsebenti wemaseli e-T lasebentako. Ekwelapheni, ku-neutralization kwe-methylglyoxal kungancoba umsebenti wemangcamuzane lacindzetelako lasuselwa ku-myeloid (MDSC) futsi kukhulise ngekusebentisana kwelashwa kwekuvimbela indzawo yekuhlola kumamodeli emagundvwane (7). Letifundvo ngekuhlanganyela tigcizelela indzima lebalulekile ye-TME-lesuselwa ku-metabolites ekulawuleni umsebenti we-T cell kanye nemsebenti.
T cell kabi kubikwe kakhulu umdlavuza ovarian (8). Loku kungenca yetimphawu tekugaya kudla letikhona ku hypoxia kanye nemitsambo yesimila lengakavami (9), lokuholela ekuguculweni kwe glucose ne tryptophan ibe yimikhicito lehambisanako lefana ne lactic acid kanye ne kynurenine. I-lactate lengetulu kwemaseli inciphisa kukhicitwa kwe-interferon-γ (IFN-γ) futsi icondzisa umehluko wemacembu lamancane e-myelosuppressive (10, 11). Kudliwa kwe-tryptophan kuvimbela ngco kwandza kwemaseli e-T futsi kuvimbela kukhombisa kwe-T cell receptor (12-14). Naphezu kwaloku lokubonwako, umsebenti lomnyenti lozungete kusebenta kwemasotja emtimba wentiwa ku-in vitro T cell culture kusetjentiswa imidiya lelungisiwe, noma kukhawulelwe kumamodeli emagundvwane lafananako ku-vivo, kute ngisho nayinye lekhombisa ngalokugcwele i-heterogeneity yemdlavuza webantfu kanye nemvelo ye-Physiological macro kanye ne-micro.
Sici lesivamile semdlavuza wema-ovari kusakateka kwe-peritoneal kanye nekuvela kwe-ascites. Kugcwala kwemanti emaseli ku-ascites kuhlobene nesifo lesihamba embili kanye nekubiketela lokubi (15). Ngekuya kwemibiko, leligumbi lelihlukile li-hypoxic, linemazinga laphakeme ekukhula kwemitsambo ye-endothelial (VEGF) kanye ne-indoleamine 2,3-dioxygenase (IDO), futsi lifakwa ngemangcamuzane e-T lalawulako kanye nemangcamuzane lavimbela i-myeloid (15-18). Indzawo yekugaya kudla ye-ascites ingahle ihluke kuleyo yesimila sona, ngako-ke kuhlelwa kabusha kwemangcamuzane e-T esikhaleni se-peritoneal akucaci. Ngekwengeta, umehluko lomcoka kanye nekungafani emkhatsini we ascites kanye ne metabolites lekhona endzaweni yesimila kungavimbela kungena kwemangcamuzane emasotja emtimba kanye nemsebenti wawo etimileni, futsi lucwaningo loluchubekako luyadzingeka.
Kute silungise letinkinga, saklama indlela yekuhlukaniswa kwemaseli labucayi kanye nendlela ye-chromatography tandem mass spectrometry (LC-MS/MS) yekufundza tinhlobo letehlukene temaseli (kufaka ekhatsi emaseli e-CD4 + kanye ne-CD8 + T) kanye nangaphakatsi nasemkhatsini wetimila Ema-metabolites ayo ahlanganisa emaseli esimeni se-ascites lesifanako kanye nesimo sesiguli. Sisebentisa lendlela ngekuhambisana ne-cytometry yekugeleta lesetulu kanye nekulandzelana kwe-RNA yeseli linye (scRNA-seq) kuniketa umfanekiso locatululwe kakhulu wesimo sekugaya kudla kwalebantfu lababalulekile. Lendlela yaveta kukhula lokukhulu kwelizinga le-1-methylnicotinamide (MNA) kumaseli e-T esimila, futsi kuhlolwa kwe-in vitro kwakhombisa kutsi umphumela we-immunomodulatory we-MNA emsebentini wemaseli e-T bewungakatiwa ngaphambilini. Ngalokuvamile, lendlela iveza kusebentisana kwekugaya kudla lokufanako emkhatsini wetimila kanye nemangcamuzane lavikela umtimba, futsi iniketa lwati lolwehlukile kuma-metabolites ekulawula emasotja emtimba, langaba lusito ekwelapheni i-T cell-based cancer cancer immunotherapy Ematfuba ekwelapha.
Sisebentise i-cytometry yekugeleza lesetulu kute silinganise ngaso sonkhe sikhatsi kutsatfwa kweshukela. emamaki lajwayelekile lahlukanisa emaseli lavikela umtimba kanye nebantfu bemaseli esimila (Lithebula S2 kanye neMfanekiso S1A). Lokuhlatiya kwakhombisa kutsi uma kucatsaniswa nemaseli eT, ema ascites kanye nemaseli esimila anemazinga laphakeme ekutsatsa glucose, kepha anemehluko lomncane emsebentini we mitochondrial. Kutsatfwa kweshukela lokusemkhatsini kwemangcamuzane esimila [CD45-EpCAM (EpCAM)+] kuphindvwe katsatfu kuya kane kunaloko kwemangcamuzane e-T, futsi silinganiso sekutsatfwa kweshukela kwemangcamuzane e-CD4 + T kuphindvwe ka-1.2 kunaloko kwemangcamuzane e-CD8 + T, lokukhombisa kutsi ema-lymphocytes langena esimila ku-TME adzinga i-TME lehlukene). Ngalokwehlukile, umsebenti we-mitochondrial kumaseli esimila ufana nalowo wemaseli e-CD4 + T, futsi umsebenti we-mitochondrial wato totimbili tinhlobo temaseli uphakeme kunalowo wemaseli e-CD8 + T (Umfanekiso 1B). Ngalokuvamile, lemiphumela iveza lizinga lekugaya kudla. Umsebenti wekugaya emaseli esimila uphakeme kunalowo wemangcamuzane e-CD4 + T, futsi umsebenti wekugaya emaseli e-CD4 + T uphakeme kunalowo wemangcamuzane e-CD8 + T. Naphezu kwalemiphumela kuto tonkhe tinhlobo temaseli, kute umehluko lohambisanako esimeni sekugaya se-CD4 + kanye ne-CD8 + T cells noma tilinganiso tawo letihlobene ku-ascites uma kucatsaniswa netimila (Umfanekiso 1C). Ngalokwehlukile, esigabeni se-CD45-cell, linani lemaseli e-EpCAM + kulesimila lakhula uma kucatsaniswa ne-ascites (Umfanekiso 1D). Siphindze sabona umehluko locacile wekugaya emkhatsini we-EpCAM + kanye ne-EpCAM- cell tincenye. Emaseli e-EpCAM + (simila) anekutsatsa lokusetulu kweshukela kanye nemsebenti we-mitochondrial kunemaseli e-EpCAM-, lokuyintfo lephakeme kakhulu kunemsebenti wekugaya kudla we-fibroblasts kumaseli esimila ku-TME (Umfanekiso 1, E na-F).
(A na B) Kucina kwekukhanya lokusemkhatsini (MFI) kwekutsatfwa kweshukela (2-NBDG) (A) kanye nemsebenti we-mitochondrial wemangcamuzane e-CD4 + T (MitoTracker lebovu lemnyama) (B) Emagrafu lamele (ngasencele) kanye nedatha lebhalwe phansi (Ngasekudla), emaseli e-CD8 + T kanye ne-EpCAM + T cell a kusuka ku-EpCAM + CD45-. (C) Silinganiso semaseli e-CD4 + kanye ne-CD8 + (emaseli e-CD3 + T) ku-ascites kanye nesimila. (D) Inani le-EpCAM + emaseli esimila ku-ascites kanye nesimila (CD45−). (E na F) EpCAM + CD45-simila kanye ne-EpCAM-CD45-matrix glucose kutsatfwa (2-NBDG) (E) kanye nemsebenti we-mitochondrial (MitoTracker lobovu lomnyama) (F) emagrafu lamele (ngasencele) kanye nedatha lebhalwe phansi ( Ngasekudla) Ema-ascites kanye nemangcamuzane esimila. (G) Emagrafu lamele CD25, CD137 kanye PD1 kubonakaliswa ngekugeleza cytometry. (H na-I) CD25, CD137 kanye ne-PD1 kuveta kumaseli e-CD4 + T (H) kanye ne-CD8 + T emangcamuzane (I). (J na K) Naive, inkumbulo lesemkhatsini (Tcm), inkumbulo lesebentako (Teff) kanye nenkumbulo lesebentako (Tem) lesekelwe ekuboniseni i-CCR7 ne-CD45RO. Imifanekiso lemele (ngasencele) kanye nemininingwane yethebula (ngasekudla) yemaseli e-CD4 + T (J) kanye nemaseli e-CD8 + T (K) ku-ascites kanye netimila. Emanani e-P ancunywa yi-t-test lebhanjiwe (* P <0.05, ** P <0.01 kanye *** P <0.001). Umugca umelela tigulane letihambisanako (n = 6). FMO, kukhanya lokukhanyako lokususa munye; MFI, umfutfo wekukhanya lokusemkhatsini.
Kuhlatiya lokunyenti kwaveta lomunye umehluko lomkhulu emkhatsini wesimo se-T cell phenotypic lesilungiswe kakhulu. Lokusebenta (Umfanekiso 1, G kuya ku I) kanye nememori ye-effector (Umfanekiso 1, J na K) etimileni kuvame kakhulu kune-ascites (silinganiso se-CD3 + T cells). Ngendlela lefanako, kuhlatiya i-phenotype ngekuveta timphawu tekusebenta (CD25 kanye ne-CD137) kanye netimphawu tekucitsa [iprotheyini yekufa kwemaseli 1 (PD1)] yakhombisa kutsi noma timphawu tekugaya kudla talabantfu tehlukile (Umfanekiso S1, B kuya ku-E) , Kepha kute umehluko lomkhulu wekugaya kudla umphumela naFigure S1. F kuya ku I). Lemiphumela yacinisekiswa ngekusebentisa tindlela tekufundza ngemshini kute yabelwe ngekuzenzakalela ema-phenotypes emaseli (21), lokuphindze kwaveta kuba khona kwelinani lelikhulu lemangcamuzane emgogodla wematsambo (CD45 + / CD3- / CD4 + / CD45RO +) ku-ascites yesiguli (Umfanekiso S2A ). Emkhatsini wato tonkhe tinhlobo temaseli letikhonjiwe, lelinani lemaseli e-myeloid lakhombisa kutsatfwa kwe-glucose lokusetulu kanye nemsebenti we-mitochondrial (Umfanekiso S2, B kuya ku-G). Lemiphumela igcizelela umehluko locinile wekugaya emkhatsini wetinhlobo letinyenti temaseli latfolakala ku-ascites kanye netimila etigulini te-HGSC.
Inselele lenkhulu ekuvisisekeni kwetimphawu te metabonomic te TIL sidzingo sekuhlukanisa emasampula e T cell lahlantekile lokwenele, lizinga kanye nebuningi kusuka etimila. Tifundvo takamuva tikhombise kutsi kuhlunga kanye netindlela tekucebisa buhlalu letisekelwe ku-cytometry yekugeleza kungaholela ekugucukeni kwemaphrofayli e-metabolite yemaseli (22-24). Kuze sincobe lenkinga, salungisa indlela yekucebisa buhlalu kute sihlukanise futsi sihlukanise i-TIL kusuka kumdlavuza we-ovarian webantfu lohlindvwe ngaphambi kwekuhlatiya yi-LC-MS / MS (bona Tintfo Netindlela; Umfanekiso 2A). Kute sihlole umtselela jikelele wale protocol etingucukweni te-metabolite, sacatsanisa emaphrofayli e-metabolite emangcamuzane e-T lasebenta banikeli labanemphilo ngemuva kwesinyatselo sekuhlukaniswa kwebuhlalu lesingenhla nemaseli langakahlukaniswa ngebuhlalu kepha ahlala eqhweni. Lokuhlatiya kwekulawula lizinga kutfole kutsi kunekuhambisana lokusetulu emkhatsini waletimo letimbili (r = 0.77), kanye nekuphindzaphindza kwebuchwepheshe belicembu lema metabolites langu 86 kunekuphindzaphindza lokusetulu (Umfanekiso 2B). Ngako-ke, letindlela tingakhona kwenta kuhlatiya lokunembile kwe-metabolite kumaseli labhekene nekuceceshwa kweluhlobo lwemaseli, ngaleyo ndlela kuniketa inkhundla yekucala lesetulu yekukhomba ema-metabolites latsite ku-HGSC, ngaleyo ndlela kwenta bantfu batfole kucondza lokujulile kwekukhetsekile kwemaseli Luhlelo lwe-metabolism yebulili.
(A) Umdvwebo we-Schematic wekuceceshwa kwebuhlalu kazibuthe. Ngaphambi kwekuhlatiya nge LC-MS/MS, lamaseli atawudlula emizuliswaneni lemitsatfu lelandzelanako yekuceceshwa kwebuhlalu bemagnetic noma ahlale eqhweni. (B) Umphumela weluhlobo lwekucebisa ngebuningi be-metabolites. Silinganiso setilinganiso letintsatfu teluhlobo ngalunye lwekucebisa ± SE. Umugca lompunga umelela budlelwane be-1:1. Kuhambisana kwe-intra-class (ICC) kwetilinganiso letiphindzaphindziwe letikhonjiswe kulebuli ye-eksisi. I-NAD, i-nicotinamide lesetulu. (C) Umdvwebo we-Schematic wekusebenta kwekuhlatiya kwe-metabolite yesiguli. Ema-ascites noma timila tibutfwa etigulini bese tigcinwa e-cryopreserved. Incenye lencane yesampula ngayinye yahlatiya nge flow cytometry, kantsi lamasampula lasele enta imijikeleto lemitsatfu yekucebisa emaseli eCD4+, CD8+ kanye ne CD45-. Leti tincenye temaseli tahlaziywa kusetjentiswa i-LC-MS / MS. (D) Imephu yekushisa ye-metabolite lejwayelekile inani. Le dendrogram imelela kuhlanganiswa kwa Ward kwemabanga e Euclidean emkhatsini wemasampula. (E) Kuhlatiya kwetincenye letiyinhloko (PCA) kwemephu ye-metabolite yesampula, lekhombisa kuphindzaphindza lokutsatfu kwesampula ngayinye, emasampula lavela esigulini sinye ahlanganiswa ngumugca. (F) I-PCA yephrofayli ye-metabolite yesampula lecondziswe esigulini (lokusho kutsi, kusetjentiswa kusebenta lokuyincenye); luhlobo lwesampula lulinganiselwe yi-hull le-convex. PC1, ingcenye leyinhloko 1; PC2, incenye lenkhulu 2.
Ngemuva kwaloko, sasebentisa lendlela yekucebisa kuhlatiya ema-metabolites langu-99 ku-CD4 +, CD8 + kanye ne-CD45-cell fractions ku-ascites leyinhloko kanye netimila tetigulane letisitfupha te-HGSC (Umfanekiso 2C, Umfanekiso S3A kanye neLithebula S3 na-S4). Linani lebantfu labatsandzako libalelwa ku 2% kuya ku 70% wesampula lenkhulu yekucala yemaseli laphilako, futsi linani lemaseli lihluka kakhulu emkhatsini wetigulane. Ngemuva kwekuhlukanisa lobuhlalu, lencenye lecebile yenshisekelo (CD4+, CD8+ noma CD45-) ibalelwa ngetulu kwa 85% wawo onkhe emaseli laphilako kulesampula ngesilinganiso. Lendlela yekucebisa isivumela kutsi sihlatiye emaseli labantfu kusuka ku-metabolism yeticubu tesimila sebantfu, lokuyintfo lengeke yenteke kutsi yenteke kusuka kumasampula lamakhulu. Kusebentisa le protocol, satfola kutsi i-l-kynurenine ne-adenosine, lama-metabolites lamabili labonakala kahle e-immunosuppressive aphakanyiswa kumaseli e-T esimila noma emaseli esimila (Umfanekiso S3, B na-C). Ngako-ke, lemiphumela ikhombisa kwetsembeka kanye nemandla ekuhlukaniswa kwemaseli etfu kanye nebuchwepheshe be-mass spectrometry kutfola ema-metabolites labalulekile etilwaneni eticubini tesiguli.
Kuhlatiya kwetfu kwaphindze kwaveta kwehlukaniswa lokucinile kwetinhlobo temaseli ngaphakatsi nasemkhatsini wetigulane (Umfanekiso 2D kanye neMfanekiso S4A). Ikakhulukati, uma kucatsaniswa naletinye tigulane, sigulane 70 sakhombisa timphawu letehlukene tekugaya (Umfanekiso 2E kanye neMfanekiso S4B), lokukhombisa kutsi kungaba ne-heterogeneity lenkhulu yekugaya emkhatsini wetigulane. Kuyaphawuleka kutsi uma kucatsaniswa naletinye tigulane (1.2 kuya ku 2 litres; Lithebula S1), linani leliphelele le-ascites lebutfwe esigulini 70 (80 ml) belincane. Kulawula kwe-heterogeneity ye-inter-patient ngesikhatsi sekuhlatiya kwetincenye letiyinhloko (sibonelo, kusebentisa kuhlatiya kwe-redundancy lokuyincenye) kukhombisa tingucuko letingagucuki emkhatsini wetinhlobo temaseli, futsi tinhlobo temaseli kanye / noma i-microenvironment tihlanganiswa ngalokucacile ngekuya kwephrofayli ye-metabolite (Umfanekiso 2F ). Kuhlatiya ema metabolites langawodvwa kwagcizelela lemiphumela futsi kwaveta umehluko lomkhulu emkhatsini wetinhlobo temaseli kanye nendzawo lencane. Kufanele kutsi kucashelwe kutsi umehluko lowendlulele kakhulu lobonwako yi MNA, levame kuceceshwa ngemaseli eCD45- kanye nakumaseli eCD4+ kanye ne CD8+ langena kulesimila (Umfanekiso 3A). Kumaseli eCD4 +, lomphumela ubonakala kakhulu, futsi iMNA kumaseli eCD8 + nayo ibonakala itsintseka kakhulu yimvelo. Kepha, loku akubalulekile, ngoba tigulane letintsatfu kuphela kuletisithupha letingahlolwa kutsi tine-tumor CD8+ scores. Ngekwengeta ku MNA, etinhlotjeni letehlukene temaseli ku ascites kanye netimila, lamanye ema metabolites langabonakali kahle ku TIL nawo acebile ngekwehlukana (Tibalo S3 na S4). Ngako-ke, lemininingwane iveza isethi letsembisako yema-metabolites e-immunomodulatory kute kwentiwe lucwaningo loluchubekako.
(A) Lokucuketfwe lokujwayelekile kwe-MNA ku-CD4 +, CD8 + kanye ne-CD45- emaseli kusuka ku-ascites kanye nesimila. Lelibhokisi likhombisa libanga lelisemkhatsini (umugca), libanga le interquartile (i hinge yeluhlaka) kanye nelibanga lemininingwane, kuze kube tikhatsi letingu 1.5 libanga le interquartile (luhlaka lwe whisker). Njengobe kuchaziwe ku Tintfo Tesigulane Netindlela, sebentisa linani le-limma lesiguli kute utfole linani le-P (* P <0.05 na ** P <0.01). (B) Umdvwebo we-Schematic wekusebenta kwe-MNA (60). Tintfo letiguculwako: S-adenosyl-1-imethionine; I-SAH, i-S-adenosi-1-i-homocysteine; NA, i-nikotinami; I-MNA, 1-i-methilnikotinamidi; 2-PY, 1-imethili- 2-phiridoni-5-ikhabhoksamidi; 4-PY, 1-imethili-4-phiridoni-5-ikhabhoksamidi; NR, i-ribose ye-nikotinamide; NMN, i-nicotinamide lephilako. Ema-enzyme (luhlata): i-NNMT, i-N-methyltransferase; SIRT, tintfo letiphilako; I-NAMPT, i-nicotinamide ledluliswako; i-AOX1, i-aldehayidi oksidazi 1; NRK, i- kinase ye- nicotinamide; I-NMNAT, i-nicotinamide lehamba ngetinyawo; I-Pnp1, i-purine nucleoside ye-phosphorylase. (C) t-SNE ye-scRNA-seq ye-ascites (lempunga) kanye nesimila (lesibovu; n = 3 tigulane). (D) NNMT kubonakaliswa emaseli lahlukahlukene lakhonjwe kusetjentiswa scRNA-seq. (E) Kuveta i-NNMT ne-AOX1 ku-SK-OV-3, tinso tembungu webantfu (HEK) 293T, emaseli e-T kanye nemangcamuzane e-T laphatfwe nge-MNA. Lesikhulumo lesigocekile sikhonjiswa sihlobene ne-SK-OV-3. Iphethini yekuveta nge-SEM iyakhonjiswa (n = 6 banikeli labanemphilo). Emanani e-Ct langetulu kwa-35 atsatfwa njengalangabonakali (UD). (F) Kuveta i-SLC22A1 ne-SLC22A2 ku-SK-OV-3, i-HEK293T, emangcamuzane e-T kanye nemangcamuzane e-T lalashwa nge-8mM MNA. Lesikhulumo lesigocekile sikhonjiswa sihlobene ne-SK-OV-3. Iphethini yekuveta nge-SEM iyakhonjiswa (n = 6 banikeli labanemphilo). Emanani e-Ct langetulu kwa-35 atsatfwa njengalangabonakali (UD). (G) Cell MNA lokucuketfwe kusebenta empilo umnikeli T cells ngemuva amahora 72 incubation nge MNA. Iphethini yekuveta nge-SEM iyakhonjiswa (n = 4 banikeli labanemphilo).
I-MNA ikhicitwa ngekudlulisela licembu le-methyl kusuka ku-S-adenosyl-1-methionine (SAM) kuya ku-nicotinamide (NA) nge-nicotinamide N-methyltransferase (NNMT; Umfanekiso 3B). I-NNMT ikhonjiswa kakhulu emidlavuzeni leyehlukahlukene yebantfu futsi ihlobene nekwandza, kuhlasela kanye ne-metastasis (25-27). Kute sicondze kancono umtfombo we-MNA kumaseli e-T ku-TME, sasebentisa i-scRNA-seq kuveta kuveta kwe-NNMT kuto tonkhe tinhlobo temaseli ku-ascites kanye netimila tetigulane letintsatfu te-HGSC (Lithebula S5). Kuhlatiya emaseli langaba ngu-6,500 kwakhombisa kutsi etindzaweni te-ascites kanye ne-tumor, kuveta kwe-NNMT bekuvinjelwe ku-fibroblast kanye ne-tumor cell populations (Umfanekiso 3, C na D). Kufanele kucashelwe kutsi kute kuveta lokusobala kwe-NNMT kunobe ngusiphi sibalo sebantfu lesiveta i-PTPRC (CD45 +) (Umfanekiso 3D kanye neMfanekiso S5A), lokukhombisa kutsi i-MNA letfolakala ku-metabolite spectrum yetfulwe kumaseli e-T. Kuveta kwe-aldehyde oxidase 1 (AOX1) kugucula i-MNA ibe yi-1-methyl-2-pyridone-5-carboxamide (2-PYR) noma i-1-methyl-4-pyridone-5-khabhoksamide (4-PYR); Umfanekiso 3B) uphindze uvinjelwe kubantfu be-fibroblasts labaveta i-COL1A1 (Umfanekiso S5A), lokuhlangene kukhombisa kutsi emaseli e-T aswele likhono le-MNA metabolism levamile. Iphethini yekuveta lamajini lahlobene ne-MNA yacinisekiswa kusetjentiswa idatha yesibili letimele yeseli lesethiwe kusuka ku-ascites kusuka etigulini te-HGSC (Umfanekiso S5B; n = 6) (16). Ngetulu kwaloko, kuhlatiya kwekuphendvula kwe-polymerase chain (qPCR) kwemangcamuzane e-T lanikelako laphilile laphatfwa nge-MNA kwakhombisa kutsi uma kucatsaniswa nekulawula emaseli esimila se-ovarian SK-OV-3, i-NNMT noma i-AOX1 beyicishe ingavetwa (Umfanekiso 3E). Lemiphumela lengakalindzeleki ikhombisa kutsi i-MNA ingahle ikhishwe kuma-fibroblasts noma etimila ibe ngema-T cells lasedvute ku-TME.
Noma nje labakhetsiwe bafaka ekhatsi umndeni wetitfutsi te-cation letiphilako 1 kuya ku-3 (OCT1, OCT2 na-OCT3) letikhodiwe ngumndeni wetitfutsi letincibilikako 22 (SLC22) (SLC22A1, SLC22A2 na-SLC22A3), titfutsi letingaba khona te-MNA solo atikachazwa (28) . I-QPCR ye-mRNA kusuka kumaseli e-T lanikelako laphilile yakhombisa emazinga laphansi ekuveta e-SLC22A1 kepha emazinga langabonakali e-SLC22A2, lacinisekisa kutsi bekubikwe ngaphambilini etincwadzini (Umfanekiso 3F) (29). Ngalokwehlukile, umugca wemaseli esimila se-ovarian waveta emazinga laphakeme abo bobabili labatfutsi (Umfanekiso 3F).
Kute kuhlolwe kutsi kungenteka yini kutsi emaseli eT abe nemandla ekutsatsa iMNA yangaphandle, emaseli eT lanikelako laphilile akhuliswa emahora langemashumi lasikhombisa nakubili lapho khona kunetintfo letehlukene te MNA. Ngekungabi khona kwe-MNA ye-exogenous, lokucuketfwe kwemaseli e-MNA akukhoni kutfolakala (Umfanekiso 3G). Kepha, emaseli e-T lasebentako laphatfwa nge-MNA ye-exogenous akhombisa kukhula lokuncike kumthamo wekucuketfwe kwe-MNA kumaseli, kuze kube ngu-6 mM MNA (Umfanekiso 3G). Lomphumela ukhombisa kutsi noma ngabe lizinga leliphansi lekuveta umtfutsi kanye nekungabikhona kwe-enzyme lenkhulu lebhekene nekugaya kwe-MNA lengekhatsi kwemaseli, i-TIL isakhona kutsatsa i-MNA.
I-spectrum ye-metabolites kumaseli e-T etigulane kanye nekuhlolwa kwekutsatsa kwe-MNA ku-vitro kukhulisa ematfuba ekutsi ema-fibroblasts lahlobene nemdlavuza (CAF) akhiphe i-MNA kanye nemaseli esimila angalawula i-phenotype kanye nemsebenti we-TIL. Kute kutfolakale umphumela we MNA kumaseli e T, emaseli e T lanikelako laphilile asebenta ku vitro ngekubakhona noma kungabikhona kwe MNA, futsi kwandza kwawo kanye nekukhicita kwe cytokine kwahlolwa. Ngemuva kwemalanga langu 7 ekwengeta iMNA ngemthamo lophakeme, inombolo yekuphindza kabili kwebantfu yancishiswa ngesilinganiso, kantsi nemandla agcinwa kuyo yonkhe imithamo (Umfanekiso 4A). Ngetulu kwaloko, kwelashwa kwe-MNA ye-exogenous kwaholela ekwandziseni linani lemaseli e-CD4 + kanye ne-CD8 + T laveta sici se-necrosis yesimila-α (TNFα; Umfanekiso 4B). Ngalokwehlukile, kukhicitwa kwe-IFN-γ ngaphakathi kwemaseli kwancishiswa kakhulu kumaseli e-CD4 + T, kepha hhayi kumaseli e-CD8 + T, futsi bekute lushintjo lolubalulekile ku-interleukin 2 (IL-2; Umfanekiso 4, C na D). Ngako-ke, i-enzyme-lehlanganiswe ne-immunosorbent assay (ELISA) ye-supernatants kusuka kulamasiko e-T cell laphatfwe nge-MNA yakhombisa kukhula lokukhulu kwe-TNFα, kwehla kwe-IFN-γ, futsi kute lushintjo ku-IL-2 (Umfanekiso 4, E kuya ku-G). . Kwehla kwe-IFN-γ kukhombisa kutsi i-MNA ingadlala indzima ekuvimbeleni umsebenti wekulwa netimila temaseli e-T. Kute kulingisa umphumela we-MNA ku-T cell-cytotoxicity, emaseli e-chimeric antigen receptor T (FRα-CAR-T) lahloselwe ku-folate receptor α kanye ne-CAR-T (GFP) lelawulwa ngemaprotheyini laluhlata lakhanyako (GFP) -CAR-T) emaseli akhicitwa ngemaseli laphilile e-mononuPB engati lephelele yekunikela. Emaseli e-CAR-T akhuliswa emahora langu-24 ebukhoneni be-MNA, bese akhuliswa ngekubambisana nemaseli e-SK-OV-3 esimila se-ovarian laveta i-folate receptor α ngesilinganiso se-effector kuya ku-10:1. Ukwelashwa kwe-MNA kwaholela ekuncipheni lokukhulu kwemsebenti wekubulalela emaseli e-FRα-CAR-T, lebekafana nemaseli e-FRα-CAR-T lalashwa nge-adenosine (Umfanekiso 4H).
(A) Linani selilonkhe lebantfu labangemaseli laphilako kanye nekuphindvwe kabili kwebantfu (PD) ngco kusuka emasikweni ngelilanga 7. Igrafu yebha imelela lizinga + SEM lebanikeli labasitfupha labanemphilo. Imelela idatha kusuka okungenani n = 3 ukuhlolwa lokuzimele. (B kuya ku D) CD3 / CD28 kanye ne IL-2 basetjentiswa kusebenta emaseli e T ngesikhatsi sabo se MNA emalanga langu 7. Ngaphambi kwekuhlatiya, emaseli akhutsatwa nge PMA / ionomycin nge GolgiStop emahora langu 4. TNFα (B) kubonakaliswa kumaseli e-T. Sibonelo sitfombe (ngasencele) kanye nedatha tabular (ngasekudla) TNFα kubonakaliswa emangcamuzaneni aphila. IFN-γ (C) kanye IL-2 (D) kubonakaliswa kumaseli T. Kubonakala kwema-cytokines kwakalwa ngekugeleza kwe-cytometry. Igrafu yebha imelela kusho (n = 6 banikeli labanemphilo) + SEM. Sebentisa kuhlatiya kwendlela yinye yekuhluka kanye netilinganiso letiphindzaphindziwe (* P <0.05 kanye na ** P <0.01) kutfola linani le P. Imelela idatha kusuka okungenani n = 3 ukuhlolwa lokuzimele. (E kuya ku G) CD3 / CD28 kanye ne IL-2 basetjentiswa kusebenta emaseli e T ngesikhatsi sabo se MNA emalanga langu 7. Le medium yabutfwa ngaphambi nangemuva kwemahora langu-4 ekuvuselela kwe-PMA / ionomycin. Lokucuketfwe kwe-TNFα (E), i-IFN-γ (F) kanye ne-IL-2 (G) kwakalwa nge-ELISA. Igrafu yebha imelela lizinga (n = 5 banikeli labanemphilo) + SEM. P value kuncunywa kusetjentiswa kuhlatiya kwendlela yinye yekuhluka kanye nekukala lokuphindzaphindziwe (* P <0.05). Umugca lonemachashazi ukhombisa umkhawulo wekutfola wekutfola. (H) Kuhlolwa kwe-lysis yemaseli. Emaseli e-FRα-CAR-T noma e-GFP-CAR-T alungiswa nge-adenosine (250 μM) noma i-MNA (10 mM) emahora langu-24, noma ashiywa angaphatfwa (Ctrl). Iphesenti lokubulawa kwemangcamuzane e-SK-OV-3 kwalinganiswa. Inani le-P linqunywa yi-Welch t test (* P <0.5 kanye ** P <0.01).
Kute utfole kucondza kwe-mechanistic kwe-MNA-ancike ku-TNFα umtsetfo wekuveta, tingucuko ku-TNFα mRNA yemaseli e-T laphatfwe nge-MNA tahlolwa (Umfanekiso 5A). Emaseli e-T lanikelako laphilile lalashwa nge-MNA akhombisa kukhula lokuphindvwe kabili emazingeni ekubhala e-TNFα, lokukhombisa kutsi i-MNA incike ekulawulweni kwekubhala kwe-TNFα. Kuphenya lendlela yekulawula lekhonakalako, tintfo letimbili letatiwako tekubhala letilawula i-TNFα, lokungukuthi kusebenta kwe-T cell nuclear factor (NFAT) kanye nephrotheni letsite 1 (Sp1), yahlolwa ngekuphendvula ku-MNA lebopha kumgcugcuteli we-TNFα losedvute ( 30 ). Umgcugcuteli we-TNFα ucuketse tindzawo letibophako te-NFAT letikhonjiwe letingu-6 kanye netindzawo letibophako te-2 Sp1, letihlangana endzaweni yinye [-55 base pairs (bp) kusuka ku-5'cap] (30). I-Chromatin immunoprecipitation (ChIP) yakhombisa kutsi uma ilashwa nge-MNA, kuboshwa kwe-Sp1 kumgcugcuteli we-TNFα kwakhula katsatfu. Kufakwa kwe-NFAT nako kwakhula futsi kwasondzela ekubalulekeni (Umfanekiso 5B). Lemininingwane ikhombisa kutsi i-MNA ilawula kuveta kwe-TNFα ngekubhala kwe-Sp1, futsi ngesilinganiso lesincane kuveta kwe-NFAT.
(A) Nawucatsanisa nemaseli e-T lakhuliswe ngaphandle kwe-MNA, kushintja kwekugoqa kwe-TNFα kubonakaliswa kumaseli e-T laphatfwa nge-MNA. Iphethini yekuveta nge-SEM iyakhonjiswa (n = 5 banikeli labanemphilo). Imelela idatha kusuka okungenani n = 3 ukuhlolwa lokuzimele. (B) Umgcugcuteli we-TNFα wemangcamuzane e-T laphatfwe noma ngaphandle kwe-8 mM MNA ngemuva kwe-NFAT ne-Sp1 kwahlanganiswa ne-(Ctrl) kanye ne-PMA / ionomycin kuvuselela emahora langu-4. Immunoglobulin G (IgG) kanye ne H3 kwasetjentiswa njengekulawula lokubi nalokuhle kwekuvikela umtimba, ngekulandzelana. Kulinganisa kwe ChIP kwakhombisa kutsi kuboshwa kwe Sp1 ne NFAT kumgcugcuteli we TNFα kumaseli laphatfwe nge MNA kwakhula tikhatsi letinyenti uma kucatsaniswa nekulawula. Imelela idatha kusuka okungenani n = 3 ukuhlolwa lokuzimele. Inani le-P linqunywa ngekuhlolwa kwe-t lokunyenti (*** P <0.01). (C) Uma kucatsaniswa ne-ascites ye-HGSC, emaseli e-T (langasiyo i-cytotoxic) akhombisa kuveta lokunyenti kwe-TNF esimileni. Lemibala imelela tigulane letehlukene. Emaseli lakhonjisiwe atsatfwe ngekungahleleki abe ngu 300 futsi ajittered kwentela kunciphisa kudvweba kakhulu (** Padj = 0.0076). (D) Imodeli lephakanyisiwe ye-MNA yemdlavuza we-ovarian. iMNA ikhicitwa kumaseli esimila kanye nema fibroblasts ku TME futsi itsatfwa ngemaseli eT. I-MNA ikhulisa kuboshwa kwe-Sp1 kumgcugcuteli we-TNFα, lokuholela ekukhuleni kwekubhalwa kwe-TNFα kanye nekukhicita kwe-TNFα cytokine. I-MNA iphindze ibangele kwehla kwe-IFN-γ. Kuvimbela umsebenti wemaseli eT kuholela ekunciphiseni likhono lekubulala kanye nekukhula kwesimila ngekushesha.
Ngekuya kwemibiko, i-TNFα inemiphumela yekulwa ne-tumor kanye ne-anti-tumor lencike ngaphambili nangemuva, kepha inendzima leyatiwako ekukhutsateni kukhula kanye ne-metastasis yemdlavuza we-ovarian (31-33). Ngekusho kwemibiko, lokucuketfwe kwe-TNFα ku-ascites kanye neticubu tesimila etigulini letinemdlavuza we-ovarian kuphakeme kunaleto leticubu letiyingoti (34-36). Ngekuya kwendlela, i-TNFα ingalawula kusebenta, umsebenti kanye nekwandza kwemangcamuzane engati lamhlophe, futsi ishintshe i-phenotype yemangcamuzane emdlavuza (37, 38). Kuhambisana nalokutfoliwe, kuhlatiya kwekuveta ufuto lokwehlukile kwakhombisa kutsi i-TNF beyilawulwa kakhulu kumaseli e-T eticubini tesimila uma kuqhathaniswa ne-ascites (Umfanekiso 5C). Kukhula kwekuveta kwe-TNF kwabonakala kuphela kubantfu bemaseli e-T nge-phenotype lengasiyo i-cytotoxic (Umfanekiso S5A). Ngalokufishane, lemininingwane isekela umbono wekutsi i-MNA inemiphumela lemibili yekuvikela umtimba kanye nekukhutsata isimila ku-HGSC.
Kulebula kwe fluorescent lokusekelwe ku cytometry yekugeleta sekuyindlela leyinhloko yekufundzela kusebenta kwe TIL. Letifundvo tikhombise kutsi uma kucatsaniswa nema-lymphocytes engati lephelele noma emaseli e-T lavela etincenyeni te-lymphoid tesibili, i-murine kanye ne-TIL yebantfu inendlela lephakeme yekutsatsa glucose (4, 39) kanye nekulahlekelwa kancane kancane kwemsebenti we-mitochondrial (19, 40). Noma sibone imiphumela lefanako kulolu cwaningo, intfutfuko lebalulekile kucatsanisa metabolism yemangcamuzane esimila kanye ne-TIL kusuka eticubu letifanako resected isimila. Kuhambisana naleminye yalemibiko yangaphambilini, emaseli esimila (CD45-EpCAM +) lasuka ku-ascites kanye netimila anekutsatsa glucose lokusetulu kunemaseli e-CD8 + kanye ne-CD4 + T, lokusekela kutsi kutsatfwa kwe-glucose lokusetulu kwemaseli esimila kungacatsaniswa nemaseli e-T. Umcondvo wekuncintisana kwemaseli e-T. TME. Kepha, umsebenti we-mitochondrial wemangcamuzane esimila uphakeme kunalowo wemangcamuzane e-CD8 + T, kepha umsebenti we-mitochondrial uyafana nalowo wemangcamuzane e-CD4 + T. Lemiphumela icinisa ingcikitsi levelako kutsi umzimba we-oxidative ubalulekile kumaseli esimila (41, 42). Baphindze basikisele kutsi emaseli e-CD8 + T angahle abe nekungasebenti kahle kwe-oxidative kunemaseli e-CD4 + T, noma kutsi emaseli e-CD4 + T angasebentisa imithombo ye-carbon ngaphandle kwe-glucose kugcina umsebenti we-mitochondrial (43, 44). Kufanele kucashelwe kutsi asibone umehluko ekutsatfweni kweshukela noma umsebenti we-mitochondrial emkhatsini we-CD4 + T effectors, inkumbulo ye-T effector kanye ne-T inkumbulo lesemkhatsini kumaseli e-ascites. Ngendlela lefanako, simo sekuhlukaniswa kwemangcamuzane e-CD8 + T etimileni asihlangene netingucuko ekutsatfweni kwe-glucose, kugcizelela umehluko lomkhulu emkhatsini wemangcamuzane e-T lakhuliswe ku-vitro kanye ne-TIL yebantfu ku-vivo (22). Loku lokubonwe kwaphindze kwacinisekiswa ngekusebentisa kwabiwa kwemaseli latentekelako langakhetsi, lokuphindze kwaveta kutsi emaseli e-CD45 + / CD3- / CD4 + / CD45RO + lane-glucose lephakeme kanye nemsebenti we-mitochondrial kunemaseli esimila avame kakhulu kepha anelinani lemaseli lasebentako e-Metabolic. Lelinani lingamelela linani lelincane lelicatjangwako lemangcamuzane lacindzetela i-myeloid noma emaseli e-plasmacytoid dendritic lakhonjwe ekuhlaziyweni kwe-scRNA-seq. Noma kokubili loku kubikwe etimila te-ovari yebantfu [45], basadzinga Umsebenti lochubekako kutsi uchaze le-myeloid subpopulation.
Noma tindlela letisekelwe ku-flow cytometry tingacacisa umehluko jikelele ku-glucose kanye nekugaya kwe-oxidative emkhatsini wetinhlobo temaseli, ema-metabolites lacondzile lakhicitwa yi-glucose noma leminye imithombo ye-carbon yekugaya kwe-mitochondrial ku-TME ayikacaciswa. Kunika kubakhona noma kungabikhona kwema metabolites ku TIL subset leniketiwe kudzinga kuhlanjululwa kwebantfu bemaseli kusuka eticubini letikhishiwe. Ngako-ke, indlela yetfu yekucebisa emaseli lehlanganiswe ne-mass spectrometry inganiketa lwati ngema-metabolites lacebiswe ngekwehlukana kumaseli e-T kanye nebantfu bemaseli esimila ekuhambisaneni nemasampula esiguli. Noma lendlela inetinzuzo ngetulu kwekuhlunga emaseli lasebenta nge-fluorescence, imitapo letsite ye-metabolite ingahle ithinteke ngenca yekusimama lokuyimvelo kanye / noma lizinga lekujika lelisheshako (22). Nomakunjalo, indlela yetfu yakhona kubona ema metabolites lamabili labonakalako lavikela umtimba, i-adenosine ne kynurenine, ngoba ahluka kakhulu emkhatsini wetinhlobo tesampula.
Kuhlatiya kwetfu kwe-metabonomic kwetimila kanye netinhlobo letincane te-TIL kuniketa lwati lolunyenti ngendzima yema-metabolites ku-TME ye-ovarian. Kwekucala, sisebentisa i-cytometry yekugeleza, satfola kutsi bekute umehluko emsebentini we-mitochondrial emkhatsini wetimila kanye nemangcamuzane e-CD4 + T. Kepha, kuhlatiya kwe-LC-MS/MS kwaveta tingucuko letibalulekile ebuningi bema-metabolites emkhatsini walabantfu, lokukhombisa kutsi tiphetfo mayelana nekugaya kwe-TIL kanye nemsebenti wayo jikelele wekugaya tidzinga kuhumusha ngekucophelela. Kwesibili, iMNA yi metabolite lenemehluko lomkhulu emkhatsini we CD45-cells kanye ne T cells ku ascites, hhayi timila. Ngako-ke, kuhlukaniswa kanye nendzawo yesimila kungaba nemiphumela leyehlukene ku-TIL metabolism, lokugcizelela i-heterogeneity lekhonakalako endzaweni lencane leniketiwe. Kwesitsatfu, kuveta kwe-enzyme lekhicita i-MNA i-NNMT kukhawulelwe kakhulu ku-CAF, lokungemaseli esimila ngesilinganiso lesincane, kepha emazinga e-MNA labonakalako ayabonakala kumaseli e-T lasuselwa esimileni. Kuveta kakhulu i-NNMT ku-CAF ye-ovarian kunemphumela lowatiwako wekukhutsata umdlavuza, kancane ngenca yekukhutsata kusebenta kwe-CAF, kuhlaselwa kwesimila kanye ne-metastasis (27). Noma lizinga leliphelele le-TIL lilinganiselwe, kuveta kwe-NNMT ku-CAF kuhlobene kakhulu ne-Cancer Genome Atlas (TCGA) ye-mesenchymal subtype, lehambisana nekubiketela lokubi (27, 46, 47). Ekugcineni, kuveta i-enzyme AOX1 lenesibopho sekucedza i-MNA nako kukhawulelwe kubantfu be-CAF, lokukhombisa kutsi emaseli e-T aswele likhono lekugucula i-MNA. Lemiphumela isekela umbono wekutsi noma kudzingeka umsebenti lochubekako kucinisekisa loku lokutfoliwe, emazinga laphakeme e-MNA kumaseli e-T angakhombisa kuba khona kwe-immunosuppressive CAF microenvironment.
Njengoba kuniketwe lizinga leliphansi lekuveta le-MNA transporters kanye nemazinga langabonakali emaprotheyini labalulekile latsintseka ekusebentiseni kwe-MNA, kubakhona kwe-MNA kumaseli e-T akulindzelekile. Noma i-NNMT noma i-AOX1 beyingatfolakala ngekuhlatiya kwe-scRNA-seq kanye ne-qPCR lehlosiwe yema-cohorts lamabili latimele. Lemiphumela ikhombisa kutsi i-MNA ayihlanganiswa ngemaseli e-T, kepha imuncwa kusuka ku-TME letungeletile. Kuhlolwa kwe-in vitro kukhombisa kutsi emaseli e-T avame kubutsela ndzawonye i-MNA ye-exogenous.
Tifundvo tetfu te-in vitro tikhombise kutsi i-MNA ye-exogenous ikhutsata kuveta i-TNFα kumaseli e-T futsi ikhutsata kuboshwa kwe-Sp1 kumgcugcuteli we-TNFα. Noma i-TNFα inemisebenti yekulwa ne-tumor kanye ne-anti-tumor, emdlavuzeni wema-ovari, i-TNFα ingakhutsata kukhula kwemdlavuza wema-ovari (31-33). Kutsatfwa njengalokungatsatsi luhlangotsi kwe-TNFα esikweni le-ovarian tumor noma kucedvwa kwesiginali ye-TNFα kumamodeli emagundvwane kungenta ncono kukhicitwa kwe-cytokine levuvukile le-TNFα-mediated futsi kuvimbele kukhula kwesimila (32, 35). Ngako-ke, kulesi simo, i-MNA lesuselwa ku-TME ingasebenta njenge-metabolite levutsako ngendlela lencike ku-TNFα ngendlela ye-autocrine loop, ngaleyo ndlela ikhutsate kwenteka kanye nekusakateka kwemdlavuza we-ovarian (31). Kususelwa kulesi kungenteka, TNFα blockade kuyafundvwa njenge-ejenti yekwelapha lekhonakalako yemdlavuza we-ovarian (37, 48, 49). Ngetulu kwaloko, i-MNA ikhubata i-cytotoxicity yemaseli e-CAR-T kumaseli esimila se-ovarian, kuniketa lobunye bufakazi bekucindzetelwa kwemasotja emtimba lalawulwa yi-MNA. Ngekuhlanganiswa, lemiphumela iphakamisa imodeli lapho khona timila kanye nemangcamuzane e-CAF akhipha i-MNA ibe yi-TME ye-extracellular. Nge (i) TNF-lebangelwa kukhula kwemdlavuza we-ovarian kanye (ii) ne-MNA-lebangelwa kuvimbela umsebenti we-T cell cytotoxic, loku kungaba nemphumela wesimila lesiphindvwe kabili (Umfanekiso 5D).
Ekuphetseni, ngekusebentisa inhlanganisela yekucebisa emaseli ngekushesha, kulandzelana kwemaseli munye kanye nekuphrofayela kwe-metabolic, lolucwaningo lwaveta umehluko lomkhulu we-immunometabolomic emkhatsini wetimila kanye nemaseli e-ascites etigulini te-HGSC. Lokuhlatiya lokuphelele kwakhombisa kutsi kunemehluko ekutsatseni glucose kanye nemsebenti we-mitochondrial emkhatsini wemaseli e-T, futsi kwakhomba i-MNA njenge-metabolite yekulawula emasotja emtimba lengasiyo yemaseli. Lemininingwane inemtselela wekutsi i-TME ithinta njani i-T cell metabolism kumdlavuza webantfu. Noma nje kuncintisana lokucondzile kwemanutrients emkhatsini wemaseli eT kanye nemaseli emdlavuza kubikiwe, ema metabolites angaphindze asebente njengebalawuli labangacondzi kwentela kukhutsata kuchubeka kwesimila futsi kungenteka kutsi acindzetele kuphendvula kwemasotja emtimba langekhatsi. Lenye incazelo yendzima lesebentako yalama metabolites lalawulako ingavula lamanye emasu ekutfutfukisa kuphendvula kwemasotja emtimba lalwa netimila.
Tibonelo tetigulane kanye nemininingwane yemtholampilo tatfolakala ngekusebentisa indzawo yekugcina ticubu tesimila semdlavuza we-BC lecinisekisiwe yi-Canada Tissue Repository Network. Ngekuya kwendlela yekusebenta levunywe yiKomidi Yekutiphatsa Yelucwaningo Lwemdlavuza we-BC kanye neNyuvesi yaseBritish Columbia (H07-00463), tonkhe tibonelo tetigulane kanye nemininingwane yemtholampilo titfole imvume lebhalwe phansi lenelwati noma tiyekele imvume yato ngalokusemtsetfweni. Lamasampula agcinwa ku BioBank lecinisekisiwe (BRC-00290). Timphawu tesiguli letinemininingwane tikhonjiswe kuMathebula S1 na S5. Kute kugcinwe ngekushisa, kusetjentiswa i-scalpel kubola ngemshini isampula yesimila sesigulane bese iyayiphusha ngesihlungi se-100-micron kute kutfolakale kumiswa kweseli linye. I-ascites yesigulane yahlanganiswa ku 1500 rpm imizuzu lelishumi ku 4°C kwentela kufaka ema pellet emaseli bese kususwa le supernatant. Emaseli latfolakala esimileni kanye ne-ascites agcinwa ngekubandza ku-50% we-AB serum yebantfu lengasebenti ngekushisa (Sigma-Aldrich), 40% we-RPMI-1640 (I-Thermo Fisher Scientific) kanye na-10% we-dimethyl sulfoxide. Leti tintfo letigcinwe temaseli langawodvwa tancibilikiswa futsi tasetjentiselwa metabolomics kanye nekuncuma kwe metabolite lokuchazwe lapha ngentasi.
Lendlela lephelele ihlanganisa 0.22 μm lehlungiwe 50:50 lengetiwe RPMI 1640: AimV. 1640 + 2.05 mM l-glutamine (Isayensi Yemdobi Loshisako) lengetiwe nge-10% ye-AB serum yemuntfu lengasebenti ngekushisa (Sigma-Aldrich), 12.5 mM Hepes (Isayensi Yemdobi Loshisako), 2 mM l-glutamine (Isayensi Yemdobi Loshisako) Sisombululo se-Streptomycin (i-PenStrep) (sesayensi ye-Thermo Fisher) kanye ne-50 μMB-mercaptoethanol. I-AimV (I-Invitrogen) ingetiwe nge-20 mM Hepes (Isayensi Yekudoba Leshisako) kanye ne-2 mM l-glutamine (Isayensi Yekudoba Leshisako). Le buffer yekugcoba ye-cytometer yekugeleta beyine-0.22μm ye-phosphate lehlungiwe lefakwe luswayi (PBS; Invitrogen) lengetiwe nge-3% ye-AB serum yemuntfu lengasebenti ngekushisa (Sigma). Le buffer yekucebisa emaseli yakhiwe nge 0.22μm PBS lehlungiwe futsi yengetwa nge 0.5% yekushisa-kungasebenti kwebantfu AB serum (Sigma-Aldrich).
Ku 37°C lephelele lesemkhatsini, emaseli abegcotjwa nge 10 nM MT DR kanye ne 100 μM 2-NBDG imizuzu lengu 30. Ngemuva kwaloko, lamaseli agcotjwa ngedayi yekuphila eF506 ku 4°C imizuzu lengu 15. Phindza uhlanganise lamaseli ku FC Block (eBioscience) kanye ne Brilliant Stain Buffer (i BD Biosciences), ncibilikisa ku buffer yekugcoba yekugeleta kwe cytometer (ngekuya kweticondziso temkhiciti), bese uyafukamela imizuzu lelishumi ngekushisa kweligumbi. Gcobisa emaseli ngesethi yema antibodies (Lithebula S2) ku flow cytometry yekufaka emabala ku 4°C imizuzu lengemashumi lamabili. Phindza uphindze uhlanganise emaseli ku-buffer yekugcoba yekugeleta (i-Cytek Aurora; 3L-16V-14B-8R) ngaphambi kwekuhlatiya. Sebentisa iSpectroFlo ne FlowJo V10 kuhlatiya idatha yekubalwa kwemaseli, bese usebentisa iGraphPad Prism 8 kudala idatha. I-fluorescence lesemkhatsini (MFI) ye-2-NBDG ne-MT DR yayi-log-normalized, bese kusetjentiswa luhlolo lwe-t lolubhanjiwe lwekuhlatiya kwetibalo kulandzisa tigulane letihambisanako. Susa bonkhe bantfu labanetigameko letingaphansi kwa-40 ekuhlaziyeni; faka linani le-MFI le-1 kunobe ngumaphi emanani langakalungi ngaphambi kwekutsi wente kuhlatiya kwetibalo kanye nekubonakala kwemininingwane.
Kute sihlanganise isu lekuvula ngesandla lephaneli yenchubo lengenhla, sasebentisa umbhalo lophelele ngesihlahla sekuvimbela kwakheka (FAUST) (21) kute sabele emaseli ngekuzenzakalela kubantfu ngemuva kwekucedza emaseli lafile ku-FlowJo. Siphatsa ngesandla umphumela kuhlanganisa bantfu lababonakala bangabiwa kahle (kuhlanganisa i-PD1 + nemaseli e-PD1-tumor) kanye nebantfu labagcinwe. Sampula ngayinye icuketse silinganiso semangcamuzane langetulu kwa 2%, ngesibalo sebantfu labangu 11.
Kusetjentiswa kwe-Ficoll gradient kwekucina kwasetjentiswa kwehlukanisa i-PBMC kusuka kumikhicito yekuhlukanisa ema-leukocyte (I-STEMCELL Technologies). Emaseli eCD8 + T ahlukaniswe ku PBMC asebentisa ema CD8 MicroBeads (Miltenyi) futsi anwetshwa ngendlela lephelele asebentisa iTransAct (Miltenyi) emaviki lamabili ngekuya kweticondziso temkhiciti. Lamangcamuzane avunyelwe kutsi eme emalanga langu 5 endzaweni lephelele lecuketse i-IL-7 (10 ng/ml; PeproTech), bese aphindze avuselelwa nge TransAct. Ngelilanga lesi-7, ngekuya kweticondziso temkhiciti, ema-CD45 MicroBeads ebantfu (Miltenyi) asetjentiswa kucebisa emaseli ngemizuliswano lemitsatfu lelandzelanako. Lamangcamuzane ahlukaniswa kwentela kuhlatiya kwe-flow cytometry (njengoba kuchaziwe ngenhla), futsi emaseli lasigidzi sinye ahlukaniswa katsatfu kwentela kuhlatiya i-LC-MS/MS. Lamasampula acutshungulwa yi-LC-MS/MS njengoba kuchaziwe lapha ngentasi. Silinganise inani le-metabolite elahlekile ngenombolo ye-ion ye-1,000. Sampula ngayinye ijwayelekile ngenombolo ye-ion lephelele (TIC), iguculwe nge-logarithm futsi ijwayelekile ngekuzenzakalela ku-MetaboAnalystR ngaphambi kwekuhlatiya.
Lokumiswa kweseli linye lesiguli ngasinye kwancibilikiswa futsi kwahlungwa ngesihlungi se-40 μm kube ngumkhawulo lophelele (njengobe kuchaziwe ngenhla). Ngekuya kwe protocol yemkhiciti, imijikeleto lemitsatfu lelandzelanako yekukhetsa lokuhle ngekuhlukaniswa kwebuhlalu bemagnetic kusetjentiswa iMicroBeads (Miltenyi) yasetjentiswa kucebisa emasampula eCD8+, CD4+ kanye ne CD45- cells (ku ice). Ngamafuphi, emaseli aphindze ahlanganiswe ku buffer yekucebisa emaseli (njengoba kuchaziwe ngenhla) bese ayabalwa. Lamangcamuzane abefutfunyelwa ngebuhlalu be CD8 bebantfu, buhlalu be CD4 bebantfu noma buhlalu be CD45 bebantfu (Miltenyi) ku 4°C imizuzu lelishumi nesihlanu, bese ayagezwa nge buffer yekucebisa emaseli. Lesampula idluliswa ngekholomu ye LS (Miltenyi), bese tincenye letinhle naletimbi tiyabutfwa. Kute kuncishiswe sikhatsi futsi kukhuliswe sinyatselo sekululama kwemaseli, le CD8-fraction ibese isetjentiselwa umjikeleto wesibili wekucebisa iCD4+, bese le CD4-fraction isetjentiselwa kucebisa iCD45 lelandzelako. Gcina lesisombululo eqhwa kuyo yonkhe inchubo yekuhlukana.
Kulungiselela emasampula ekuhlatiya imetabolite, lamaseli agezwa kanye ngesisombululo seluswayi lolubandza njenge ice, bese 1 ml we 80% methanol wengetwa kusampula ngayinye, bese uyavutsiwe bese uyafrizwa ngekushesha ku nitrogen lengemanti. Lamasampula abekwa ngemijikeleto lemitsatfu yekubandza-kuncibilika bese aya centrifuged ku 14,000 rpm imizuzu lelishumi nesihlanu ku 4°C. Le supernatant lecuketse ema metabolites iyahwamuka kuze kube ngulapho yomile. Letintfo letigucukako taphindze tancibilikiswa ku 50 μl we 0.03% formic acid, tavutsiwe kutsi tihlanganiswe, bese tiyahlanganiswa kwentela kususa tintfo letilahliwe.
Khipha ema metabolites njengoba kuchaziwe ngenhla. Dlulisela lendzawo lesetulu ebhodleleni le-chromatography lesetulu lesebentako kwentela lucwaningo lwe-metabolomics. Sebentisa indlela yekwelapha lengahleliwe kwelapha isampula ngayinye ngenani lelifanako lemangcamuzane kuvikela imiphumela ye batch. Sentile luhlolo lwekhwalithi lwetintfo letigucukako temhlaba wonkhe lebetishicilelwe ngaphambilini ku-AB SCIEX QTRAP 5500 I-Spectrometer Yebuningi Be-Quadruple Letsatfu (50). Kuhlatiya kwe Chromatographic kanye nekuhlanganiswa kwendzawo lesetulu kwentiwa kusetjentiswa isoftware ye MultiQuant lengu 2.1 (I-SCIEX Lesetjentisiwe Yetintfo Letiphilako).
Kubalwa kwe ion kwa 1000 kwasetjentiswa kulinganisa linani le metabolite lelahlekile, bese iTIC yesampula ngayinye yasetjentiswa kubala indzawo lejwayelekile yesicoco se metabolite ngayinye letfolakele kulungisa tingucuko letetfulwa kuhlatiya kwetintfo tekusebenta kusuka ekucubunguleni isampula. Ngemuva kwe-TIC seyijwayelekile, i-MetaboAnalystR (51) (ipharamitha lejwayelekile) isetjentiselwa kuguculwa kwe-logarithmic kanye nekukala umugca wekujwayelekile lokuzenzakalelayo. Sisebentise i-PCA ngephakheji ye-vegan R kwenta kuhlatiya kwekuhlola kwemehluko we-metabolome emkhatsini wetinhlobo tesampula, futsi sasebentisa kuhlatiya kwe-redundancy lokuyincenye kuhlatiya tigulane. Sebentisa indlela ya Ward kwakha i-dendrogram yemephu yekushisa kwentela kuhlanganisa libanga le Euclidean emkhatsini wemasampula. Sisebentise i-limma (52) ngebuningi be-metabolite lejwayelekile kute sibone ema-metabolites lamanyenti lahlukile kulo lonkhe luhlobo lwemaseli kanye nemvelo lencane. Kute kwentiwe lula inchazelo, sisebentisa ipharamitha lesho licembu kucacisa imodeli, futsi sicabangele tinhlobo temaseli ku-microenvironment njengelicembu ngalinye (n = emacembu langu-6); ngekuhlolwa kwekubaluleka, senta tilinganiso letintsatfu letiphindzaphindziwe te-metabolite ngayinye Kute kugwenywe kuphindzaphindza kwemanga, lesigulane safakwa njengesihibe ekwakhiweni kwe-limma. Kute sihlole umehluko we-metabolites emkhatsini wetigulane letehlukene, salungisa imodeli ye-limma kufaka ekhatsi tigulane ngendlela lemisiwe. Sibika kubaluleka kwekucatsanisa lokucaciswe ngaphambilini emkhatsini weluhlobo lweseli kanye nemvelo lencane yePadj <0.05 (kulungiswa kwaBenjamini-Hochberg).
Ngemuva kwekucebisa ngemandla kusetjentiswa iMiltenyi Dead Cell Removal Kit (> 80% kusebenta), kulandzelana kwe-transcriptome yeseli linye kwentiwa ku-ascites lephelele lephilako lefriziwe kanye nemasampula esimila kusetjentiswa iphrothokholi yekuveta i-10x 5′gene. Emacala lasihlanu lanetimila letihambisanako kanye ne ascites ahlaziywa, noma ngabe kusebenta lokuphansi kusuka kusampula yinye yesimila kwavimbela kufakwa kwayo. Kute sifinyelele kukhetsa lokunyenti kwetigulane, sahlanganisa emasampula esiguli ngasinye emizileni ye-10x chromium controller, futsi sahlatiya i-ascites kanye netindzawo tesimila ngekwehlukana. Ngemuva kwekulandzelana [Illumina HiSeq 4000 28 × 98 bp ekupheleni lokubhanjiwe (PE), i-genome yaseQuebec; silinganiso se-73,488 kanye ne-41,378 sifundza ngeseli ngalinye lesimila kanye ne-ascites ngekulandzelana]], sasebentisa i-CellSNP ne-Vireo (53) (lesekelwe ku-CellSNP njenge-SNP yebantfu levamile (VCF) leniketwa yi-GRCh38 yabelwe bunjalo bemunikeli. (IBS), ngaphandle kwemangcamuzane langakabelwa kanye nemangcamuzane lakhonjwe njenge-duplexes kanye nebanikeli labahambisanako emkhatsini we-ascites kanye nemasampula esimila (54 ngesisekelo salomsebenti, sagcina emacala lamatsatfu ngekumeleleka kwemaseli lamanyenti esimila kanye ne-ascites yekuhlatiya lokusezansi (BiCo-filter le). kwakhicita emaseli langu-6975 (emaseli langu-2792 na-4183 lasuka esimila kanye ne-ascites, ngekulandzelana) kute ahlaziywe Sisebentisa i-igraph (57) Louvain yekuhlanganiswa kwenethiwekhi lesedvute ye-gene leyabelwana ngayo (SNN) lesekelwe ku-Jaccard ibanga kuya kumaseli e-cluster ngekuveta ngesandla kuya e-cluster. futsi kubonwa nge-t-SNE emaseli e-Cytotoxic achazwa ngekuveta kwe-CD8A kanye ne-GZMA, ngaphandle kwema-subclusters lane-ribosomal protein lephansi Sifinyelele idatha leyashicilelwa ye-Izar et al (16), kufaka ekhatsi kufaka kwabo t-SNE, kungalawula kuveta kwe-marNNker.
PBMC bahlukaniswe leukocyte imikhicito kuhlukaniswa (STEMCELL Buchwepheshe) nge Ficoll gradient buningi centrifugation. Emaseli e-CD3 + ahlukaniswe ku-PBMC asebentisa buhlalu be-CD3 (Miltenyi). Ebukhoneni noma kungabi khona kwe-MNA, emaseli e-CD3 + asebenta nge-CD3 leboshelwe emapuletini (5 μg / ml), i-CD28 lencibilikako (3 μg / ml) kanye ne-IL-2 (300 U / ml; Proleukin). Ngelilanga lekugcina lekwandziswa, kukhonakala (Idayi Yekuphila Lelungiswako eFluor450, eBioscience) kanye nekwandza (123count eBeads, Thermo Fisher Scientific) kwahlolwa ngekugeleta kwe-cytometry. Hlola umsebenti we-effector ngekutsi ukhutsate emaseli nge-PMA (20 ng / ml) kanye ne-ionomycin (1 μg / ml) nge-GolgiStop emahora langu-4, bese ugadza i-CD8-PerCP (I-MAb11, i-BD). Kugcugcutela emaseli e-qPCR kanye ne-ChIP nge-PMA (20 ng / ml) kanye ne-ionomycin (1 μg / ml) emahora langu-4. Le ELISA lesetulu yabutfwa ngaphambi nangemuva kwekuvuselela nge PMA (20 ng / ml) kanye ne ionomycin (1 μg / ml) emahora lamane.
Landzela indlela yekukhicita yehlukanisa i-RNA usebentisa i-RNeasy Plus Mini Kit (i-QIAGEN). Sebentisa i-QIAShredder (QIAGEN) kwenta isampula ibe yintfo lefanako. Sebentisa i-RNA lenemandla lamakhulu kuya kukhithi ye-cDNA (Isayensi Ye-Thermo Fisher) kuhlanganisa i-DNA lephelele (i-cDNA). Sebentisa i-TaqMan Rapid Advanced Master Mix (i-Thermo Fisher Scientific) kute ulinganise kuveta kwemajini (ngekuya kwendlela yekusebenta yemkhiciti) ngaletinhlolo letilandzelako: Hs02786624_g1 [i-hydrogen (i-GAPDH)] kanye ne-Hs01010726_m1 (SLC22A2). Lamasampula agijima eluhlelweni lwe-PCR lwesikhatsi sangempela lwe-StepOnePlus (Tinhlelo Letisetjentisiwe) (Tinhlelo Letisetjentisiwe) epuletini lekuphendvula lelisheshako le-96-well le-MicroAmp (Tinhlelo Letisetjentisiwe) ngefilimu yekubuka ye-MicroAmp. Noma nguliphi linani le Ct lelingetulu kwa 35 litsatfwa njengalelingetulu kwemkhawulo wekutfola futsi limakwe njengalelingabonakali.
Yenta ChIP njengobe kuchaziwe ngaphambilini (58). Ngamafuphi, lamaseli alashwa nge formaldehyde (kugcila kwekugcina 1.42%) bese afutfunyelwa endzaweni leshisako imizuzu lelishumi. Sebentisa buffer yekuvuvuka lengetiwe (25 mM Hepes, 1.5 mM MgCl2, 10 mM KCl kanye na 0.1% NP-40) eqhweni imizuzu lelishumi, bese uphindze uhlanganiswe ku buffer yekuvikela umtimba njengoba kuchaziwe (58 ). Lesampula yabese i-sonicated ngemijikeleto lelandzelako: imijikeleto lengu-10 (20 1-second pulses) kanye nesikhatsi lesimile semizuzwana lengu-40. Fukamela i-immunoglobulin G yelizinga le-ChIP (Buchwepheshe Bekukhombisa Emaseli; 1μl), i-histone H3 (Buchwepheshe Bekukhombisa Emaseli; 3μl), i-NFAT (Invitrogen; 3μl) kanye ne-SP1 (Buchwepheshe Bekukhombisa Emaseli; 3μl) ema-antibodies ngesampula lesesigabeni se-CC°4 Fukamela buhlalu bemaprotein A (Thermo Fisher Scientific) kanye nesampula ku 4°C ngekunyakatisa kancane lihora linye, bese usebentisa buhlalu be chelex (Bio-Rad) kwentela kucebisa iDNA, bese usebentisa iproteinase K (iThermo Fisher) kwentela kugaya emaprotein. TNFα umgcugcuteli watfolakala yi-PCR: phambili, GGG TAT CCT TGC TGC TGT; ngalokwehlukile, GTG CCA ACA UMSEBENTI GCC TTT ATA TG (umkhicito we-207-bp). Letitfombe takhicitwa yi-Image Lab (i-Bio-Rad) futsi talinganiswa kusetjentiswa isoftware ye-ImageJ.
I supernatant isiko cell kwaqoqwa njengoba kuchaziwe ngenhla. Lokuncuma kwentiwa ngekuya kwetinchubo temkhiciti wekhithi ye-TNFα ELISA yebantfu (Invitrogen), ikhithi ye-ELISA ye-IL-2 yebantfu (Invitrogen) kanye nekhithi ye-IFN-γ ELISA yebantfu (i-Abcam). Ngekuya kwendlela yemkhiciti, le supernatant yahlanjululwa nga 1:100 kute kutfolakale iTNFα ne IL-2, kanye na 1:3 kute kutfolakale iIFN-γ. Sebentisa i-EnVision 2104 Umfundzi Wemalebuli Lamanyenti (i-PerkinElmer) kukala kumuncwa ku-450 nm.
PBMC bahlukaniswe leukocyte imikhicito kuhlukaniswa (STEMCELL Buchwepheshe) nge Ficoll gradient buningi centrifugation. Emaseli e-CD3 + ahlukaniswe ku-PBMC asebentisa buhlalu be-CD3 (Miltenyi). Ebukhoneni noma kungabi khona kwe-MNA, emaseli e-CD3 + asebenta nge-CD3 leboshelwe emapuletini (5μg / ml), i-CD28 lencibilikako (3μg / ml) kanye ne-IL-2 (300 U / ml; Proleukin) emalanga lamatsatfu. Ngemuva kwemalanga lamatsatfu, lamaseli ahlanganiswa agezwa nge 0.9% saline, bese le pellet yasheshe ifakwe efrijini. Kubalwa kwemaseli kwentiwa ngekugeleta kwe-cytometry (Cytek Aurora; 3L-16V-14B-8R kucushwa) kusetjentiswa ema-eBeads langu-123count.
Khipha ema metabolites njengoba kuchaziwe ngenhla. Lesicumbi lesomile saphindze sakhiwa kabusha ngesikhatsi sekuhlanganiswa kwemaseli langu 4000/μl. Hlatiya lesampula ngekusebentisa i-chromatography yesigaba lesibuyela emuva (1290 Infinity II, Agilent Technologies, Santa Clara, CA) kanye nekholomu ye-CORTECS T3 (2.1 × 150 mm, bukhulu betinhlayiya 1.6-μm, bukhulu be-pore 120-Å; Silinganiso sebukhulu be-polar (6470, Agilent), lapho khona ionization ye-electrospray isebenta ngendlela lekahle. Sigaba lesihambako A ngu 0.1% we esidi ye formic (ku H2O), sigaba lesihambako B ngu 90% we acetonitrile, 0.1% we esidi ye formic. Le LC gradient ngu 0 kuya ku 2 imizuzu ku 100% A, 2 kuya ku 7.1 imizuzu ku 99% B, kanye nemizuzu lengu 7.1 kuya ku 8 ku 99% B. Bese uphindze ulinganise lekholomu ngesigaba lesihambako A ngesilinganiso sekugeleza se 0.6 ml/min imizuzu lemitsatfu. . Izinga lokugeleza ngu 0.4ml/min, bese igumbi lekholomu lishiswa libe ngu 50°C. Sebentisa lizinga lemakhemikhali lahlantekile le-MNA (M320995, Inkampani Yemakhemikhali Yelucwaningo YaseToronto, eNorth York, e-Ontario, eCanada) kusungulwa sikhatsi sekugcina (RT) kanye nekugucula (RT = 0.882 imizuzu, kugucula 1 = 137→94.1, kugucula 2 = 137→1372). Uma tonkhe letingucuko letintsatfu tenteka ngesikhatsi lesifanele sekugcina, tingucuko 1 tisetjentiselwa kulinganisa kwentela kucinisekisa kucacile. Le curve lejwayelekile ye MNA (Inkampani Yemakhemikhali Yelucwaningo yase Toronto) yakhicitwa ngekuhlanjululwa lokusitfupha kwekulandzelana kwesisombululo sesitoko (1 mg/ml) kutfola emazinga la 0.1, 1.0, 10 kanye na 100 ng/ml kanye na 1.0 kanye na 10μg/ml ngekulandzelana kwemanti. Umkhawulo wekutfola ngu 1 ng/ml, futsi imphendvulo lesemgceni isemkhatsini wa 10 ng/ml na 10μg/ml. Umjovo ngamunye wema microliters lamabili esampula kanye nelizinga usetjentiselwa kuhlatiya kwe LC/MS, futsi isampula yekulawula lizinga lehlanganisiwe igijima njalo ngemuva kwemijovo lesiphohlongo kwentela kucinisekisa kusimama kwepulatifomu yekuhlatiya. Letimphendvulo te-MNA tawo onkhe emasampula emaseli laphatfwe nge-MNA abengaphakatsi kweluhlu lwemugca lwe-assay. Kuhlatiya imininingwane kwentiwa kusetjentiswa luhlelo lwekuhlatiya lwesilinganiso lwe-MassHunter (v9.0, Agilent).
Situkulwane sesibili se-αFR-CAR sakhiwo satsatfwa ku-Song et al. (59). Ngamafuphi, lesakhiwo sicuketse lokucuketfwe lokulandzelako: kulandzelana kwemholi we-CD8a, sicucu se-αFR-lesikhetsekile se-single-chain legucukako, i-CD8a hinge kanye nesifundza se-transmembrane, sizinda se-CD27 se-CD27 kanye nesizinda se-CD3z se-CD3z. Lokulandzelana kwe-CAR lokuphelele kwahlanganiswa yi-GenScript, kwase kuyakhicitwa kube yi-vector yekuveta ye-lentiviral yesitukulwane sesibili lesetulu kwekhasethi yekuveta i-GFP lesetjentiselwa kuhlola kusebenta kahle kwekuhumusha.
I-Lentivirus ikhicitwa ngekutsi kutsatfwe emaseli e-HEK293T [Kugcogcwa Kwemasiko Eluhlobo LwaseMelika (ATCC); ikhuliswe endzaweni ya Dulbecco leguculwe ye Eagle lecuketse 10% we serum yetinkhomo letingakazalwa (FBS) kanye na 1% we PenStrep, futsi isetjentiswe i-CAR-GFP vector kanye ne Le plasmids yekupakisha (psPAX2 kanye ne pMD2.G, Addgene) isebentisa i-amine yekukhicita emafutsa (Sigma-Aldrich). Le supernatant leneligciwane yabutfwa ngemuva kwemahora langu 48 kanye na 72 ngemuva kwekutsi ifakwe, yahlungwa, futsi yagcizelelwa nge ultracentrifugation. Gcina le supernatant yegciwane legcizelele ku -80°C kuze kube ngulapho sekuguculwa.
Ema-PBMC ahlukaniswe nemikhicito yekuhlukanisa ema-leukocyte lanikelako laphilile (Buchwepheshe be-STEMCELL) ngekutsi kusetjentiswe bukhulu be-Ficoll. Sebentisa ema-microbeads e-CD8 lakhetsiwe kahle (Miltenyi) kuhlukanisa emaseli e-CD8+ kusuka ku-PBMC. Kuvuselela emaseli e-T nge-TransAct (iMiltenyi) kanye nasesigabeni se-TexMACS [iMiltenyi; ingetiwe nge-3% ye-serum yebantfu lengasebenti ngekushisa, 1% ye-PenStrep kanye ne-IL-2 (300 U / ml)]. Emahora langemashumi lamabili nakune ngemuva kwekuvuselela, emaseli e-T aguculwa nge-lentivirus (10 μl legcizelele igciwane le-supernatant ngemaseli langu-106). 1 kuya ku 3 emalanga ngemuva kwekuguculwa ku Cytek Aurora (ku FSC (Kusakateka Phambili) / SSC (Kusakateka Eceleni), Singlet, GFP +), hlola kuveta kwe GFP kwemangcamuzane kukhombisa kusebenta kahle kwekuguculwa lokungenani 30% .
Emaseli eCAR-T akhuliswa emahora langemashumi lamabili nakune e-Immunocult (Buchwepheshe be-STEMCELL; langetiwe nge-1% PenStrep) ngaphansi kwaletimo letilandzelako: angaphatfwa, alashwa nge-250 μM adenosine noma 10 mM MNA. Ngemuva kwekwelashwa ngaphambilini, emaseli e-CAR-T agezwa nge-PBS futsi ahlanganiswa nemaseli e-SK-OV-3 langu-20,000 [ATCC; ku McCoy 5A lesemkhatsini (Sigma-Aldrich) lengetiwe nge 10% FBS kanye na 1% PenStrep ku 10: Silinganiso se-effector kuya ku-1 sakhuliswa katsatfu ku-Immunocult lengetiwe. Emaseli e-SK-OV-3 kanye nemaseli e-SK-OV-3 lahlanganiswe nge-digitalis saponin (0.5mg / ml; Sigma-Aldrich) asetjentiswa njengekulawula lokubi nalokuhle, ngekulandzelana. Ngemuva kwemahora lamashumi lamabili nakune ekulima ngekubambisana, le supernatant yabutfwa bese le lactate dehydrogenase (LDH) yalinganiswa ngekuya kweticondziso temkhiciti (LDH Glo Cytotoxicity Assay Kit, iPromega). I-LDH lesetulu yahlanjululwa 1:50 ku-LDH buffer. Liphesenti lekubulalana lalinganiswa ngekusebentisa lefomula lelandzelako: liphesenti lekubulalana = liphesenti lekulungisa / lizinga lelikhulu lekubulala x 100%, lapho khona liphesenti lekulungisa = co-culture-T cells kuphela, kanye nelizinga lelikhulu lekubulala = kulawula lokuhle-kulawula lokubi.
Njengobe kuchaziwe embhalweni noma tintfo netindlela, sebentisa i-GraphPad Prism 8, i-Microsoft Excel noma i-R v3.6.0 yekuhlatiya tibalo. Uma emasampula lamanyenti ahlanganiswa esigulini sinye (njenge-ascites kanye nesimila), sisebentisa luhlolo lwe-t lolubhanjiwe noma sifake sigulane njengemphumela longakahleleki kumfanekiso wemugca noma jikelele njengobe kufanele. Kuhlatiya kwe metabolomics, luhlolo lwekubaluleka lwentiwa katsatfu.
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Lombuto usetjentiselwa kuhlola kutsi usivakashi yini kanye nekuvikela kuhanjiswa kwe-spam lokuzenzakalelayo.
Marisa K. Kilgour (Marisa K. Kilgour), Sara MacPherson (Sara MacPherson), Lauren G. Zacharias (Lauren G. Zacharias), Abigail Eli Aris G. (Russell G. Jones), Phineas T.
I-MNA ifaka sandla ekucindzeteleni kwemasotja emtimba emaseli e-T futsi imelela lisu le-immunotherapy lelingahle libe khona lekwelapha umdlavuza webantfu.
Marisa K. Kilgour (Marisa K. Kilgour), Sara MacPherson (Sara MacPherson), Lauren G. Zacharias (Lauren G. Zacharias), Abigail Eli Aris G. (Russell G. Jones), Phineas T.
I-MNA ifaka sandla ekucindzeteleni kwemasotja emtimba emaseli e-T futsi imelela lisu le-immunotherapy lelingahle libe khona lekwelapha umdlavuza webantfu.
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