Metaboliteeji immunomodulatooruuji ko sifaaji teeŋtuɗi e nder mikroo-environnement tumor (TME), kono so wonaa seeɗa, ko ɓuri heewde e sifaaji mum en ina keddii tawa anndaaka. Ɗoo, min ƴeewtindiima ɓuuɓri e ƴiye T ummoriiɗe e ɓuuɓri e ascites ñawɓe kanseer serous toowɗo (HGSC) ngam feeñninde metabolom ɗeen ɗoon geɗe TME ceertuɗe. Ascites e ƴiiƴam ina njogii ceertugol metabolituuji keewɗi. So en tuugniima e ascites, ƴiye T gonɗe e nder ɓalndu ina keewi no feewi e 1-metilnikotinamiid (MNA). Hay so tawii noon tolno MNA e nder geese T ina toowi, baylagol nikotinamiid N-metiltransferase (enzim baawɗo saabaade mbayliigu dente metil ummoraade e S-adenosilmetionin feewde e nikotinamiid) ina famɗi e fibroblasteeji e geese ɓuuɓɗe. E nder golle, MNA ina addana ƴiye T yaltinde faktor alfa nekrosis tumor sitokine baɗoowo ɓuuɓri. Ko ɗum waɗi, MNA keɓaaɗo e TME ina wallita e reentaade cellal ɓalli T, ina jeyaa kadi e ko waawi wonde fof, ngam safrude kanseer aadee.
Metaboliteeji keɓtinaaɗi e ɓuuɓri ina mbaawi jogaade batte keewɗe e reentaade ɓuuɓri, tee ina ɓeydoo heewde seedeeji kollitooji wonde ina mbaawi kadi wonde doole guurɗe ngam yahrude yeeso ñawu nguu (1). Gaa gaa batte Warburg, golle baɗɗe faayiida puɗɗiima sifaade ngonka mbaylaandi (métabolisme) e nder geese tuumooje e jokkondiral mum e ngonka immuno-environnement tumour (TME). Wiɗtooji baɗaaɗi e mbaydiiji peƴooji e ƴiye T aadee kollitii wonde mbayliigu glutamine (2), mbayliigu oksidaa (3) e mbayliigu glukos (4) ina mbaawi golloraade koye mum en e denndaangal ƴiye immune ceertuɗe. Metaboliteeji keewɗi e nder ɗeen laabi ina kaɗa golle ƴiiƴam T ngam haɓaade mboros. Kolliraama wonde uddugol koenzim tetrahidrobiopterin (BH4) ina waawi bonnude ɓeydagol ƴiiƴam T, kadi ɓeydagol BH4 e nder ɓanndu ina waawi ɓeydude jaabawol reentaade mborosaaji ɗi CD4 e CD8 njiylotoo. Yanti heen, batte kynurenine ina mbaawi hisneede e njuɓɓudi BH4 (5). E nder glioblastoma mo isositrat dehidrogenaas (IDH) wayli, ɓuuɓri enantiometabolik (R)-2-hidroksiglutaraat (R-2-HG) ina haɗa ɓuuɓri T, ɓeydagol e golle sitolisis (6). Ko ɓooyaani koo, hollitaama wonde metilgliyoksaal, ko huunde nde glikolisis ina yalta, ina feewnee e ƴiye suppresoruuji iwdi mieloid, kadi mbayliigu ƴiye T metilgliyoksaal ina waawi haɗde golle ƴiye T efektor. E nder safaara, neutralisaasiyoŋ metilgliyoksal ina waawi foolde golle ƴiiƴam ɓuuɓɗam (MDSC) e ɓeydude sinerji e nder safaara ɓuuɓɗo e nder mbaydiiji peƴooji (7). Ɗeeɗoo wiɗtooji ina kawri e teeŋtinde darnde teeŋtunde e nder mbayliigu nguu, e nder yuɓɓinde golle e golle ƴiiƴam T.
Ko heewi e kanseer oo ina haalee ŋakkeende golle T cellal (8). Ɗumɗoo ina jeyaa e ko addani sifaaji mbaylaandi (métabolisme) gonɗi e nder hipoksiya e vaskulaaji tuumooji ɗi ngonaa no haanirta nii (9), ko ɗum addanta waylude glukos e tiriptofan e geɗe baɗɗe faayiida ko wayi no asid laktik e kynurenin. Laktat ekstraselulaar ɓurɗo heewde ina usta peewnugol interferon-γ (IFN-γ) ina addana kadi feeñninde mbaydiiji gonɗi e nder ɓalndu (10, 11). Ñaamde tiriptofan ina haɗa no feewi ɓeydagol ƴiiƴam T, ina haɗa kadi mbayliigu jaɓɓotooɗo ƴiiƴam T (12-14). Ko wonaa ɗeeɗoo teskaaɗe, golle keewɗe jowitiiɗe e mbayliigu cellal ɓalli mbaɗaama e nder pinal cellal T in vitro kuutortoongal mediaaji ɓurɗi moƴƴude, walla tawa ko e mbaydiiji peƴooji homologous e nder nguurndam, tawi heen gootal fof hollirtaa no feewi heterogeneity kanseeruuji aadee e nokkuuji makro e mikro Fisiyoloji.
Ko ɓuri heewde e kanseer ovarien ko saraade peritoneal e feeñde ascites. Moftude ndiyam cellal e nder ascites ina jokkondiri e ñawu nguu yahrude yeeso e ŋakkeende ƴeewndo (15). E wiyde ciimtol, ndeeɗoo feccere keeriinde ko hipoksiik, ina jogii tolnooji toowɗi e faktoruuji mawnugol endoteliyal vaskulaar (VEGF) e indoleamin 2,3-dioksijenaas (IDO), ina naatna heen kadi ƴiye T jogorɗe reentaade e ƴiye kaɗooje mieloid (15-18). Wertallo mbayliigu ascites ina waawi seertude e nokku ɗo ɓuuɓri ndii woni ɗoo, ɗum noon reprogramming celule T e nder nokku peritoneal laaɓaani. Yanti heen, ceertugol teeŋtungol e heterojenetik hakkunde ascites e metaboliiteeji tawaaɗi e nokkuuji ɓuuɓɗi ina mbaawi haɗde naatgol ƴiye immuno e golle mum en e ɓuuɓɗe, kadi ina haani wiɗtooji goɗɗi mbaɗee.
Ngam safrude ɗeen caɗeele, min mbaɗii feere seerndude ƴiye sensible e feere tandem mass spectrometry (LC-MS/MS) ngam janngude sifaaji ƴiye ceertuɗe (ina heen ƴiye CD4 + e CD8 + T) kam e nder e hakkunde ƴiye Metaboliteeji mum ina njahda e ƴiye e nder nokku gooto ascites e nokku ñawɗo ƴiye. En kuutorto ngool laawol e jokkondiral e sitometri ɓuuɓɗo toowɗo e ƴeewndo ARN gooto (scRNA-seq) ngam rokkude natal laaɓtungal no feewi e ngonka mbaylaandi ɗeen yimɓe teeŋtuɗe. Ndee feere hollitii ɓeydagol mawngol e tolno 1-metilnikotinamiid (MNA) e nder geese T tummbuɗe, e jarribooji baɗaaɗi e nder geese (in vitro) kollitii wonde batte immunomodulatoor MNA e golle geese T anndaaka tawo. E nder haala hee, ngool laawol ina hollita jokkondire hakkunde mborosaaji e ƴiye reenngo, ina rokka kadi humpitooji keertiiɗi e mbaydiiji reenngo reenngo, baawɗi naftoreede e safrude kanseer ovarien baɗaaɗo e ƴiye T Fartaŋŋeeji safrude.
En kuutoriima sitometri ɓuuɓɗo toowɗo ngam ƴeewde e sahaa gooto ƴettugol suukara [2-(N-(7-nitrofenil-2-oksa-1,3-diaza-4-il)amino)-2-deoksiglukoos (2-NBDG) e golle mitokondriyee [MitoTracker Deep Red, DR01) si9 maandeeji tippudi ceertuɗi e ƴiye immuno e keewal ƴiye tuumooje (Tablo S2 e Natal S1A). Ndeeɗoo wiɗto hollitii wonde so en ƴeewtindiima e ƴiye T, ƴiye ascites e ƴiye tumor ina njogii tolno ɓurɗo toowde e ƴettugol glukos, kono ina njogii ceertugol tokosol e golle mitokondriyaal. Hakindo ƴettugol suukara e nder geese ɓuuɓɗe [CD45-EpCAM (EpCAM)+] ina sowoo laabi tati haa nay ƴettugol suukara e nder geese T, hakindo ƴettugol suukara e nder geese CD4 + T ko laabi 1,2 e nder geese CD8 + T, ko ɗum hollirta ko limfositteeji gonɗi e nder geese gonɗe e nder geese (TIL) ina njogii saFig e metabolic ceertuɗi). E ko feewti heen, golle mitokondriyeeji e nder geese tuumooje ina nanndi e golle geese CD4 + T, tee golle mitokondriyeeje e nder geese ɗiɗi ɗee kala ina ɓura geese CD8 + T (Natal 1B). E nder keewgol, ɗeeɗoo geɗe ina kollita tolno mbayliigu. Kuutoragol ƴiiƴam ɓuuɓɗam ɓuri ƴiiƴam CD4 + T, kadi golle mbayliigu ƴiiƴam CD4 + T ɓuri ƴiiƴam CD8 + T. Ɗee batte fof e nder sifaaji ƴiiƴam, alaa fof ko seerndi ngonka mbayliigu ƴiiƴam CD4 + e CD8 + T walla e nder njuuteendi mum en e nder ascites so renndinaama e ɓuuɓri (Natal 1C). E ko feewti heen, e nder feccere CD45-cell, keewal ƴiiƴam EpCAM+ e nder ƴiiƴam ina ɓeydoo so renndinaama e ascites (Natal 1D). En teskiima kadi feere laaɓtunde e nder mbaydiiji mbaylaandi hakkunde EpCAM+ e EpCAM- cellal. Piɓaaji EpCAM+ (tumor) ina ɓuri piɓaaji EpCAM- ƴettude suukara e golle mitokondriyaal, ko ɗum ɓuri heewde e golle mbayliigu fibroblasteeji e nder piɓaaji tuumooji e nder TME (Natal 1, E e F).
(A e B) Tiiɗnaare ɓuuɓnde hakkundeere (MFI) e ƴettugol suukara (2-NBDG) (A) e golle mitokondriyeeji CD4 + T (MitoTracker boɗeejo ɓaleejo) (B) Nate wakiliije (naamo) e dokke taƴɗe (Nyaamo), CD8 + T e EpCAM + CD45-. (C) Njaajeendi CD4 + e CD8 + (e CD3 + T) e nder ascites e ɓuuɓri. (D) Njaajeendi ƴiiƴam EpCAM + e nder ascites e ƴiiƴam (CD45−). (E e F) EpCAM + CD45-tuumre e EpCAM-CD45-matriks ƴettugol suukara (2-NBDG) (E) e golle mitokondriyeeji (MitoTracker boɗeejo ɓaleejo) (F) nate wakilaare (naamo) e dokke taƴɗe ( Nyaamo) Ascites e ƴiye tuumre. (G) Nate wakiliije baylagol CD25, CD137 e PD1 e mbaydi sitometri ɓuuɓɗo. (H e I) CD25, CD137 e PD1 peeñgol e dow CD4 + T (H) e CD8 + T (I). (J e K) Fenotipeeji naafigaagal, ciimtol hakkundeewol (Tcm), ciimtol baɗte (Teff) e ciimtol baɗte (Tem) tuugiingol e baylagol CCR7 e CD45RO. Nate wakiliije (nano) e dokke tablo (nyaamo) CD4 + T (J) e CD8 + T (K) e nder ascites e ɓuuɓri. Nafooje P peeñninaama e t-test ɗiɗo (*P<0,05, **P<0,01 e ***P<0,001). Limre ndee ina hollita ñawɓe nannduɓe e mum en (n = 6). FMO, ko ɓuuɓol ngol ustata gooto; MFI, tiiɗndam ɓuuɓɗam hakkundeejam.
Wiɗtooji goɗɗi peeñninii ceertugol goɗngol maantinngol hakkunde ngonka fenotipik cellal T ɓuuɓngal no feewi. Sifaa kuutorteeɗo (Natal 1, G haa I) e siftorde (Natal 1, J e K) e nder ɓuuɓri ɓuri heewde e ascites (keewgol CD3 + T). Ko noon kadi, ƴeewtaade fenotiip oo e baylagol maandeeji kuutoragol (CD25 e CD137) e maandeeji ŋakkeende [poroteyiin maayɗo cellal porogaraam 1 (PD1)] hollirii wonde hay so tawii sifaaji mbaylaandi ɗiin yimɓe ina ceerti (Natal S1, B haa E) , Kono alaa ko seerndi mbaylaandi mawndi hakkunde memory walla S1. F haa I). Ɗee geɗe tabitinaama e huutoraade laabi janngugol masiŋaaji ngam rokkude otooji fenotipeeji cellal (21), ɗum noon ina ɓeyda feeñninde woodde celule keewɗe (CD45 + / CD3- / CD4 + / CD45RO +) e nder ascites ñawɗo oo (Natal S2A ). E nder denndaangal sifaaji ƴiiƴam anndaaɗi ɗii, ngool ƴiiƴam mieloid hollirii ɓurgol heewde glukos e golle mitokondriyaal (Natal S2, B haa G). Ɗee geɗe ina kollita ceertugol tiiɗngol mbayliigu hakkunde mbaydiiji cellal keewɗi tawaaɗi e ascites e ɓuuɓri e nder ñawɓe HGSC.
Caɗeele mawɗe e faamde sifaaji metabonomik TIL ko ŋakkeende seerndude sampleeji T cellal laaɓtuɗi, moƴƴi e keewal e nder ɓuuɓri. Wiɗtooji baɗanooɗi ko ɓooyaani kollitii wonde laabi sortugol e alɗugol beɗi tuugiiɗi e flow cytometry ina mbaawi addande waylooji e nder mbaydiiji mbaylaandi cellal (22-24). Ngam foolde ndee caɗeele, min njuɓɓinii feere alɗude beɗi ngam seerndude e seerndude TIL e kanseer ovarien neɗɗo mo seppo mum seerti e mum, hade mum ƴeewteede e LC-MS/MS (ƴeew Kaɓirɗe e Laabi; Natal 2A). Ngam yuurnitaade batte kuuɓtodinɗe ndeeɗoo porotokol e waylooji mbayliigaaji mbaylaandi, min njuurnitii mbayliigaaji mbaylaandi (profils mbaylaandi) e nder geese T ɗe dokkuɓe cellal kuutortoo caggal nde ɓe mbaɗi daawal seerndude beɗi dow ngal, e geese ɗe ceerndaaka beɗi kono heddiiɗe e dow ice. Ndee zoo wizto vursugol kalite hollitii wonde ina woodi jokkondire toowze hakkunde ziin sarziiji zizo (r = 0,77), e vursugol karallaagal fedde 86 metabolit ina jogii vursugol toowngol (Natal 2B). Ko ɗum waɗi, ɗeen laabi ina mbaawi waɗde ƴeewndo laaɓtungo e nder geese gonɗe e ƴellitgol sifaa geese, ɗum noon ina addana yimɓe waawde faamde geɗe keertiiɗe e nder HGSC, tawa ina addana yimɓe heɓde humpito ɓurngo luggiɗde e keertinaango geese Sexual metabolism programme.
(A) Natal skematik ngal alɗude beɗi magnetik. Ko adii nde LC-MS/MS ƴeewtotoo, maa ƴiye ɗee mbaɗtu waɗde cirƴam tati jokkondirɗe e ɓuuɓri magnetik walla heddotoo e dow ice. (B) Battane sifaa alɗude e keewal mbayliigaaji. Hakindo peeje tati wonande kala sifaa alɗude ± SE. Laral ɓalewal ina hollita jokkondire 1:1. Korrelaasiyoŋ nder-klaas (ICC) peeje puɗɗiiɗe kolliraaɗe e nate aksiyoŋ. NAD, ko nikotinamiid adenin dinukleotiid. (C) Natal skematik golle ƴeewndo metabolit ñawɗo. Ascites walla ɓuuɓri ina mooftee e ñawɓe, ina reenee e nder laylaytol (cryopreserve). Feccere tokosere e kala sample ƴeewtaama e flow cytometry, tawi sampleeji keddiiɗi ɗii mbaɗaama cirƴam tati ngam ƴellitde CD4+, CD8+ e CD45-. Ɗeeɗoo pecce cellal ƴeewtaama e kuutoragol LC-MS/MS. (D) Kartal nguleeki keewal metabolituuji gonɗi e mbaydi. Dendrogram ina hollita mooɓondiral Ward e distances Euclidean hakkunde sampleeji. (E) Anndugol komponenteeji mawɗi (PCA) kartal metabolit sample, holliroowo limtooji tati e kala sample, sampleeji ummoriiɗi e ñawɗo gooto ina njokkondiri e doggol. (F) PCA e mbaydi metabolit e sample sarɗiyankeewo e ñawɗo oo (ɗum woni huutoraade redundansi feccere) ; sifaa sample ina famɗi e huɓeere konveks. PC1, ko huunde mawnde 1; PC2, ko huunde mawnde 2.
Ko adii ɗuum, min kuutoriima ngool laawol alɗude ngam yuurnitaade 99 metabolit e nder pecce CD4 +, CD8 + e CD45-cell e nder ascites e tumooji adanɗi ñawɓe HGSC jeegom (Natal 2C, Natal S3A e alluwal S3 e S4). Yimɓe nafoore ina tolnoo e 2% haa 70% e sample mawɗo asliijo oo, tee keewal cellal ina seerti no feewi hakkunde ñawɓe. Caggal nde beɗi ɗii ceerti, feccere nafoore alɗunde ndee (CD4+, CD8+ walla CD45-) ina sowoo ko ɓuri 85% e denndaangal ƴiye guurɗe e nder sample oo e nder ordinateer. Ndee feere alɗude ina addana en ƴeewtaade keeweendi cellal ummoraade e mbayliigu ƴiiƴam aadee, ko ɗum huunde nde waawaa waɗde e sammeeji mawɗi. E kuutoragol ngool porotokol, min teskii wonde l-kynurenine e adenosine, ɗeeɗoo geɗe ɗiɗi gonɗe e sifaaji moƴƴi, ina toowa e nder geese T walla geese ɓuuɓɗe (Natal S3, B e C). Ko ɗum waɗi, ɗeeɗoo geɗe ina kollita gooŋɗinal e mbaawka karallaagal men seerndude cellal e karallaagal mass spectrometry ngam yiytude metaboliiteeji baɗɗi faayiida to bannge nguurndam e nder ƴiye ñawɗo.
Njiylawu amen hollitii kadi seerndi mbayliigu tiiɗngu e nder mbaydiiji cellal nder e hakkunde ñawɓe (Natal 2D e Natal S4A). Haa teeŋti noon, so en ƴeewtindiima e ñawɓe woɗɓe, ñawɗo 70 oo hollitii sifaaji mbayliigu ceertuɗi (Natal 2E e Natal S4B), ko ɗum hollirta wonde ina waawi tawa ina woodi mbayliigu mbayliigu mawngu hakkunde ñawɓe ɓee. Ina teskaa wonde so en ƴeewtindiima ñawɓe woɗɓe (1,2 haa 2 liiteer; alluwal S1), keewal ascites mooftaangal e ñawɗo 70 (80 ml) ina famɗi. Konngol heterojenetik hakkunde ñawɓe e nder ƴeewndo komponenteeji mawɗi (yeru, huutoraade ƴeewndo redundansi feccere) ina hollita waylooji jokkondirɗi hakkunde sifaaji cellal, e sifaaji cellal e/walla mikroo-environnement ina laaɓti ina kawree fawaade e sifaa metabolit (Natal 2F ). Njiylawu mbayliigaaji gooti teeŋtinii ɗeen batte, hollitii ceertugol mawngol hakkunde sifaaji cellal e nokkuuji tokoosi. Ina teskaa wonde feere ɓurnde tiiɗde teskaande ndee ko MNA, nde heewi alɗude ko e CD45- e nder CD4+ e CD8+ gonɗe e nder ƴiiƴam (Natal 3A). Wonande ƴiye CD4 +, ngool batte ɓuri laaɓtude, tee MNA e nder ƴiye CD8 + ina wayi no kadi ina tiiɗi e nokku oo. Kono ɗum alaa ko nafata, sibu ko tato tan e nder ñawɓe jeegom ɓee mbaawi ƴeewteede ngam anndude tolno CD8+ tumor. Ko jiidaa e MNA, e nder sifaaji cellal ceertuɗi e nder ascites e tuumooji, metaboliteeji goɗɗi ɗi ngalaa sifaa e TIL ina ceerti kadi (Natal S3 e S4). Ko ɗum waɗi, ɗeeɗoo dokke ina peeñnina mbaydi fotndi wonde e mbaydiiji immunomodulateur ngam wiɗtooji goɗɗi.
(A) Ko woni e MNA no woorunoo e nder CD4+, CD8+ e CD45- ummoraade e ascites e ɓuuɓri. Natal kaɓirgal ngal hollirta ko hakkundeere (doggol), hakkundeere (hinge frame) e hakkundeere domen, haa laabi 1,5 hakkundeere (whisker frame). No siforaa e Kaɓirɗe e Laabi Ñawɗo nii, huutoroo limre limma ñawɗo oo ngam anndude limre P (*P<0.05 e **P<0.01). (B) Natal skematik mbayliigu MNA (60). Kosam ɗam : S-adenosil-1-metionin ; SAH, ko S-adenosin-1-homosisteen; NA, ko nikotiinamiid; MNA, ko 1-metilnikotinamiid; 2-PY, 1-metil- 2-piridon-5-karboksamiid ; 4-PY, 1-metil-4-piridon-5-karboksamiid; NR, ko riboos nikotinamiid; NMN, ko nikotinamiid gooto. Enzim (haako-ñebbe) : NNMT, ko nikotinamiid N-metiltransferaas ; SIRT, ko ƴiiƴam ; NAMPT, ko ɓuuɓri fosforibosil nikotinamiid ; AOX1, ko aldehiid oksidaas 1; NRK, woni kinaase ribosiid nikotinamiid ; NMNAT, woni nikotinamiid gooto Nukleotiid adenilat mbayliigu ; Pnp1, ko fosforilaas nukleosiid purin. (C) t-SNE mo scRNA-seq mo asite (boɗeejo) e ɓuuɓri (boɗeejo; n = 3 ñawɓe). (D) Feññinagol NNMT e nder denndaangal celule ceertuɗe anndaaɗe e kuutoragol scRNA-seq. (E) Feññinde NNMT e AOX1 e nder SK-OV-3, ƴiiƴam ɓiɗɗo neɗɗo (HEK) 293T, ƴiiƴam T e ƴiiƴam T safraaɗam MNA. Haala follaande ndee hollitaama e fawaade e SK-OV-3. Ko hollirta ko sifaa baylagol e SEM (n = 6 dokkoowo celluɗo). Nafooje Ct ɓurɗe 35 ina mbiyaa ko ɗe njiyataa (UD). (F) Feññinde SLC22A1 e SLC22A2 e nder SK-OV-3, HEK293T, ƴiye T e ƴiye T safreteeɗe e MNA 8mM. Haala follaande ndee hollitaama e fawaade e SK-OV-3. Ko hollirta ko sifaa baylagol e SEM (n = 6 dokkoowo celluɗo). Nafooje Ct ɓurɗe 35 ina mbiyaa ko ɗe njiyataa (UD). (G) Ko woni e MNA cellal e nder cellal T dokkoowo celluɗo kuutorteeɗo caggal waktuuji 72 gonɗi e ɓuuɓri e MNA. Ko hollirta ko sifaa baylagol e SEM (n = 4 dokkoowo celluɗo).
MNA ina heɓee e waylude fedde metil ndee ummoraade e S-adenosil-1-metionin (SAM) feewde e nikotinamiid (NA) e nikotinamiid N-metiltransferase (NNMT; Natal 3B). NNMT ina heewi feeñde e kanseeruuji aadee keewɗi, ina jokkondiri e ɓeydagol, e ɓuuɓri e metastaas (25-27). Ngam ɓeydaade faamde iwdi MNA e nder geese T e nder TME, min kuutoriima scRNA-seq ngam sifaade baylagol NNMT e nder mbaydiiji geese e nder ascites e ɓuuɓri ñawɓe HGSC tato (Tablo S5). Njiylawu ko ina tolnoo e 6 500 ƴiiƴam hollitii wonde e nder nokkuuji ascites e tuumooji, baylagol NNMT ina reerɗi e ƴiiƴam fibroblast e ƴiiƴam teskinɗam (Natal 3, C e D). Ina teskaa wonde alaa fof feere NNMT laaɓtunde e nder kala renndo feeñninde PTPRC (CD45 +) (Natal 3D e Natal S5A), ko ɗum hollirta wonde MNA yiytaa e nder mbaydi metabolit oo, naatii e ƴiye T. Feññinagol aldehiid oksidaas 1 (AOX1) ina wayla MNA e 1-metil-2-piridon-5-karboksamiid (2-PYR) walla 1-metil-4-piridon-5-karboksamiid (4- PYR) ; Natal 3B) ina reerɗi kadi e keewal fibroblasteeji peeñninɗi COL1A1 (Natal S5A), ɗiin fof ina kollita wonde ƴiye T ina ŋakki mbaawka waylude MNA no woorunoo. Hoolkisaama no ɗeen taƴe jokkondirɗe e MNA peeñnirta nii, tawa ko e kuutoragol doosɗe cellal keertiiɗe ɗiɗmere ummoraade e ascites ummoriiɗe e ñawɓe HGSC (Natal S5B; n = 6) (16). Yanti heen, ƴeewndo limtilimtinngo (qPCR) e ƴiye T dokkooje celluɗe safreteeɗe e MNA kollitii wonde so en ƴeewtindiima e ƴiye SK-OV-3 gonɗe e ɓuuɓri ƴiiƴam, NNMT walla AOX1 ina famɗi ɗo peeñninaaka (Natal 3E). Ɗee geɗe ɗe cikkaaka ina kollita wonde MNA ina waawi yaltude e fibroblasteeji walla e tuumooji e nder geese T ɓadiiɗe e nder TME.
Hay so tawii kanndidaaji ɗii ina njeyaa e ɓesngu jolngo katyon 1 haa 3 (OCT1, OCT2 e OCT3) ngo ɓesngu jolngo 22 (SLC22) (SLC22A1, SLC22A2 e SLC22A3) koodi, jolngo MNA potngo wonde ngoo, haa jooni anndaaka (28) . QPCR mRNA ummoriiɗo e ƴiye T dokkooje celluɗe hollitii tolnooji baylagol SLC22A1 kono tolnooji SLC22A2 ɗi nganndaaka, ko ɗum holliti wonde ɗum ko adii ɗuum ina haalee e binndanɗe (Natal 3F) (29). E ko feewti heen, mbaydi ƴiiƴam SK-OV-3 ina hollita tolnooji toowɗi e jolngooji ɗii fof (Natal 3F).
Ngam ƴeewde so tawii ƴiye T ina njogii mbaawka huutoraade MNA janano, ƴiye T dokkooje celluɗe ɗee njiytiraa ko waktuuji 72 tawa ina tawee e ɓuuɓri MNA ceertundi. So MNA eksoojen alaa, ko woni e nder cellal MNA koo waawaa yiyeede (Natal 3G). Kono tan, ƴiye T kuutorteeɗe e MNA eksoojen ina kollita ɓeydagol fawaade e doosgal MNA e nder ƴiye ɗee, haa 6 mM MNA (Natal 3G). Ndeeɗoo yiytere hollitii wonde hay so tawii tolno baylagol jolngo ngoo ina famɗi, tee ina ŋakki enzim mawɗo baawɗo waylude MNA nder geese, TIL ina waawi ƴettude MNA haa jooni.
Spektoor metaboliteeji e nder geese T ñawɓe e jarribooji ɓuuɓgol MNA e nder geese ina ɓeyda mbaawka fibroblasteeji jokkondirɗi e kanseer (CAF) ina njiyloo MNA e geese tuumooje ina mbaawi reende fenotip e golle TIL. Ngam anndude batte MNA e ƴiye T, ƴiye T dokkooje celluɗe ɗee kuutoraa ko e nder geese (in vitro) tawa MNA ina woodi walla alaa, kadi ɓeydagol mum en e peewnugol sitokineeji ɗii ƴeewtaama. Caggal balɗe 7 ɓeydugol MNA e doosgal ɓurngal toowde, limoore limtooji ɗii ustaama no feewi, tawi semmbe ina jokki e doosɗe ɗee kala (Natal 4A). Yanti heen, safrude MNA eksoojenik ina addana ɓeydagol keeweendi CD4 + e CD8 + T peeñninoowo faktor nekrosis tumor-α (TNFα; Natal 4B). E ko feewti heen, peewnugol IFN-γ nder geese ina usta no feewi e nder geese CD4 + T, kono wonaa e geese CD8 + T, tee alaa fof waylo waylo mawngo woodi e interleukin 2 (IL-2; Natal 4, C e D). Ko ɗum waɗi, ƴeewndo immunosorbent jokkondirngo e enzim (ELISA) e ƴiiƴam gonɗam e ɗeen pine ƴiiƴam T gonɗe e MNA, hollitii ɓeydagol mawngol e TNFα, ustaare IFN-γ, alaa waylo waylo e IL-2 (Natal 4, E haa G). . Ustagol IFN-γ ina hollita wonde MNA ina waawi jogaade darnde e haɗde golle gonɗe e nder geese T ngam haɓaade mboros oo. Ngam siimtude batte MNA e sitotoksisiteeji gonɗi e nder ƴiiƴam T, ƴiiƴam jaɓɓoowo antigen kimerik T (FRα-CAR-T) jowitiingal e jaɓɓotooɗo folate α e CAR-T (GFP) jogorɗo rewde e ƴiiƴam poroteyiin ɓuuɓɗam (GFP) -CAR-T) ina feewnee e ƴiiƴam periferik ƴiiƴam celluɗam ( mononuPB ƴiiƴam dokkoowo). Piɓaaji CAR-T ina njuutee fotde waktuuji 24 e nder MNA, caggal ɗuum ina njuutee e piɓaaji SK-OV-3 gonɗi e nder reedu neɗɗo, peeñninoowo jaɓɓotooɗo folate α e njuuteendi 10:1. Hoore MNA ina addana ustaare mawnde e golle warngooji ƴiiƴam FRα-CAR-T, ko nanndi e ƴiiƴam FRα-CAR-T safraaɗam e adenosin (Natal 4H).
(A) Limre cellal guurngal e ɓeydagol yimɓe (PD) to bannge pinal ñalnde 7. Graafik bar ina hollita tolno + SEM dokkuɓe jeegom celluɓe. Ina hollita dokke ummoraade e ko famɗi fof n = 3 jarribooji keertiiɗi. (B haa D) CD3/CD28 e IL-2 kuutoraa ngam ustude ƴiiƴam T e nder njuuteendi MNA mum en fotde balɗe 7. Ko adii nde ƴeewndo waɗata, ƴiiƴam ɗam ɓuuɓnaama e PMA/ionomycin e GolgiStop fotde waktuuji 4. TNFα (B) feeñninde e nder ƴiye T. Yeru natal (nano) e dokke tablo (nyaamo) baylagol TNFα e nder geese guurɗe. IFN-γ (C) e IL-2 (D) ina peeñnina e nder geese T. Feññinagol sitokineeji ɗii fotnoo ko e flow cytometry. Natal bar ina hollita tolno (n = 6 dokkoowo celluɗo) + SEM. Huutoroo ƴeewndo laawol gootol e peeje mbayliigu (*P<0.05 e **P<0.01) ngam anndude nafoore P. Ina hollita dokke ummoraade e ko famɗi fof n = 3 jarribooji keertiiɗi. (E haa G) CD3/CD28 e IL-2 kuutoraa ngam ustude ƴiiƴam T e nder njuuteendi MNA mum en fotde balɗe 7. Medium oo mooftaama ko adii e caggal waktuuji 4 stimulaasiyoŋ PMA/ionomycin. Ko ELISA fotnoo ƴeewde ko heewi e TNFα (E), IFN-γ (F) e IL-2 (G). Natal bar ina hollita tolno (n = 5 dokkoowo celluɗo) + SEM. Nafoore P anndaa ko e huutoraade ƴeewndo laawol gootol e peeje mbayliigu e peeje puɗɗiiɗe (*P<0.05). Laral toɓɓere ngal hollirta ko keerol deftere deftere ndee. (H) Ƴeewndo limto cellal. Ɓowɗi FRα-CAR-T walla GFP-CAR-T ina njuutee e adenosin (250μM) walla MNA (10 mM) fotde waktuuji 24, walla ɗi ngoppaaka (Ctrl). Teemdere warngooji SK-OV-3 ina pooɗanoo. Nafoore P hollitaama e jarribo t Welch (*P<0,5 e **P<0,01).
Ngam heɓde faamde mekanismaaji no feewi e reentaade baylagol TNFα fawaade e MNA, mbayliigaaji mRNA TNFα e nder geese T safrooɓe MNA ƴeewtaama (Natal 5A). Piɓaaji T dokkooji celluɗi safreteeɗi e MNA kollitii ɓeydagol laabi ɗiɗi e nder tolnooji mbayliigu TNFα, ko ɗum hollirta wonde MNA ina fawii e njuɓɓudi mbayliigu TNFα. Ngam wiɗtude ngool laawol baawngol rewde, geɗe ɗiɗi anndaaɗe baylotooɗe e reentaade TNFα, woni faktor nukliyeer T cell (NFAT) e poroteyiin keeriiɗo 1 (Sp1), ƴeewtaama ngam jaabaade jokkondiral MNA e ƴellitoowo TNFα ɓadiiɗo ( 30 ). Promoter TNFα ina waɗi nokkuuji 6 jokkondirɗi NFAT anndaaɗi e nokkuuji 2 jokkondirɗi Sp1, ina njillondiri e nokku gooto [-55 base pairs (bp) ummoraade e 5'cap] (30). Immunopresipitaasiyoŋ kromatin (ChIP) hollitii wonde so neɗɗo safraama e MNA, jokkondiral Sp1 e ƴellitoowo TNFα ina ɓeydoo laabi tati. Naatgol NFAT ngol kadi ɓeydiima, ɓadtiima nafoore (Natal 5B). Ɗeeɗoo dokke ina kollita wonde MNA ina reena baylagol TNFα rewrude e baylagol Sp1, e nder ko famɗi fof baylagol NFAT.
(A) So en tuugniima e ƴiye T peewnaaɗe tawa MNA alaa, waylo waylo follere TNFα e nder ƴiye T safreteeɗe e MNA. Ko hollirta ko sifaa baylagol e SEM (n = 5 dokkoowo celluɗo). Ina hollita dokke ummoraade e ko famɗi fof n = 3 jarribooji keertiiɗi. (B) Promoter TNFα mo ƴiye T safreteeɗe e MNA 8 mM walla ɗe ngalaa caggal nde NFAT e Sp1 kawraa e (Ctrl) e stimulaasiyoŋ PMA/ionomycin fotde waktuuji 4. Immunoglobulin G (IgG) e H3 kuutoraa ko ngam ƴeewde ko moƴƴi e ko moƴƴi ngam ƴellitde immunoglobulin, kam en fof. Limto ChIP hollitii wonde jokkondiral Sp1 e NFAT e ƴellitoowo TNFα e nder geese safrooɓe MNA ɓeydiima laabi keewɗi so yerondiraama e ƴeewndo. Ina hollita dokke ummoraade e ko famɗi fof n = 3 jarribooji keertiiɗi. Nafoore P ina anndaa e t-tests keewɗi (*** P <0.01). (C) So en tuugniima e ascites HGSC, ƴiye T (ɗe ngonaa sitotoksiik) ina kollita ɓeydagol feeñninde TNF e nder ɓuuɓri ndii. Nokkuuji ɗii ina kolla ñawɓe ceertuɓe. Cellal hollirteengal ngal ƴettaa ko e 300 e jitter ngam ustude ƴellitaare ɓurtunde (** Padj = 0,0076). (D) Model MNA teskinɗo ngam kanseer ovarien. MNA ina feewnee e nder geese tuumorɗe e fibroblasteeji e nder TME, ko geese T ƴettata ɗum. MNA ina ɓeyda jokkondiral Sp1 e ƴellitoowo TNFα, ɗum addanta ɓeydagol mbayliigu TNFα e peewnugol sitokineeji TNFα. MNA ina addana kadi ustaade IFN-γ. Softinde golle ƴiiƴam T ina addana ustaade mbaawka warde e yaawnaade mawnude ɓuuɓri.
E wiyde ciimtol, TNFα ina jogii batte cemmbinɗe e ɓuuɓri gonɗe e yeeso e caggal, kono ina jogii darnde anndaande e wallitde mawnugol e metastaas kanseer oo (31-33). E wiyde ciimtol, keeweendi TNFα e nder ƴiye ascites e ƴiye tuumooje e nder ñawɓe kanseer oo ina ɓuri keeweendi ƴiye moƴƴe (34-36). To bannge mekanisme, TNFα ina waawi reende ƴellitaare, golle e ɓeydagol ƴiiƴam ɓalejam, e waylude fenotipe ƴiiƴam kanseer (37, 38). E tuugnaade e ɗeeɗoo geɗe, wiɗtooji baylagol taƴe ceertuɗe kollitii wonde TNF ina yaaji no feewi e nder geese T e nder ƴiye tuumooje so renndinaama e ascites (Natal 5C). Ɓeydagol baylagol TNF ina feeñi tan e nder ƴiiƴam T jogiiɗam fenotipe mo alaa sitotoksiik (Natal S5A). E raɓɓiɗinaade, ɗeeɗoo dokke ina mballita e miijo wonde MNA ina jogii batte ɗiɗi cemmbinɗe immunosuppressive e cemmbinɗe tumor e nder HGSC.
Label fluorescent tuugiiɗo e flow cytometry wontii feere mawnde ngam janngude mbayliigu TIL. Ɗeeɗoo wiɗtooji kollitii wonde so en ƴeewtindiima limfositte ƴiiƴam periferik walla limfositte T ummoriiɗe e terɗe limfoyde ɗiɗmere, TIL murine e neɗɗo ina ɓuri waawde ƴettude suukara (4, 39) e ŋakkeende golle mitokondriyeeji seeɗa-seeɗa (19, 40). Hay so en teskiima njeñtudi nanndundi e ndii e nder ndeeɗoo wiɗto, ƴellitaare mawnde ko ƴeewde mbayliigu ƴiiƴam e TIL ummoraade e ƴiiƴam ɓuuɓɗam resected gootol. E tuugnaade e yoga e ɗeeɗoo ciimtol ɓennungol, ƴiye tuumooje (CD45-EpCAM +) ummoriiɗe e ascites e ƴiye ina njogii ɓuuɓri ɓurndi heewde suukara e ƴiye CD8 + e CD4 + T, ina wallita wonde ɓuuɓri suukara ɓurndi heewde e ƴiye ɓuuɓri ina waawi yerondireede e ƴiye T. Miijo pottital T cellal. TME. Kono tan, golle mitokondriyaal e nder ƴiye tuumooje ina ɓura ƴiye CD8 + T, kono golle mitokondriyaal ina nanndi e golle ƴiye CD4 + T. Ɗee geɗe ina semmbina tiitoonde peeñnde wonde mbayliigu oksidaaji ina waɗi nafoore wonande ƴiye tuumooje (41, 42). Ɓe kolliti kadi wonde CD8 + T ina waawi ɓurde waawde ustaade e nder CD4 + T, walla tawa CD4 + T ina waawi huutoraade ƴulɓe karbon ko wonaa glukos ngam jokkude golle mitokondriyeeji (43, 44). Tesko-ɗen wonde en teskiima alaa ko seerndi ƴettugol suukara walla golle mitokondriyaal hakkunde CD4 + T, T effector memory e T cells memory central e nder ascites. Ko noon kadi, ngonka ceertugol ƴiiƴam CD8 + T e nder ƴiiƴam alaa ko jokkondiri e waylooji ƴettugol suukara, ko ɗum teeŋtini ceertugol mawngol hakkunde ƴiiƴam T pinal e nder ƴiiƴam (in vitro) e TIL neɗɗo e nder ƴiiƴam (22). Ɗeeɗoo teskuyaaji kadi kollitaama e kuutoragol peccitagol keewal ƴiiƴam otomatik ngol aldaa e paltoor, ko ɗum ɓeydi hollirde wonde ƴiiƴam CD45 + / CD3- / CD4 + / CD45RO + ɓurɗam heewde glukos e golle mitokondriyaal ɓuri ƴiiƴam tummbuɗam ina heewi kono ina jogii keewal ƴiiƴam Metabolic active. Ndeeɗoo fedde ina waawi wonde fedde teskinnde e nder ƴiye ɓuuɓnooje mieloid walla ƴiye dendritik plasmacytoid cifaaɗe e wiɗto scRNA-seq. Hay so tawii ɗeeɗoo geɗe ɗiɗi fof kollitaama e ɓuuɓri ƴiiƴam aadee [45], ina haani haa jooni Golle goɗɗe ko siftinde ndeeɗoo subpopulation myeloid.
Hay so tawii laabi tuugiiɗi e flow cytometry ina mbaawi laaɓtinde ceertugol kuuɓtodinngol hakkunde glukos e mbayliigu oksidaaji hakkunde sifaaji cellal, mbayliigu laaɓtungu ngu glukos walla ɓuuɓri karbon goɗɗi peewnata ngam mbayliigu mitokondriyaal e nder TME, anndaaka tawo. Hokkude woodde walla waasde woodde metaboliit e nder lowre TIL dottaande ina ɗaɓɓi laɓɓinde keewal cellal e nder ƴiiƴam taƴɗam. Ko ɗum waɗi, feere men alɗude cellal hawrude e spektorometri mass ina waawi rokkude humpitooji ko faati e metaboliteeji ceertuɗi alɗude e nder cellal T e limtooji cellal tumor e nder sampleeji ñawɓe nannduɗi. Hay so tawii ngalɗoo laawol ina jogii nafoore e ƴeewndorɗe cellal kuutorteeɗe e ɓuuɓri, won defte metaboliit ina mbaawi battinde sabu darnde mum en e/walla mbayliigu jaawngu (22). Ɗum fof e wayde noon, feere men ndee waawii anndude mbaydiiji ɗiɗi anndaaɗi, hono adenosine e kynurenine, sibu ina ceerti no feewi hakkunde sifaaji sampleeji.
Njiylawu men metabonomik e ɓuuɓri e mbaydiiji TIL ina addana en ɓeydaade humpitaade darnde metaboliteeji e nder TME ovarien. Ko adii fof, e kuutoragol flow cytometry, min teskii wonde alaa ko seerndi golle mitokondriyaal hakkunde ɓuuɓri e CD4 + T cells. Kono tan, wiɗto LC-MS/MS hollitii waylooji mawɗi e keewal mbayliigaaji e nder ɗeen ɗoon geɗe, kollitooji wonde ciimtol ko faati e mbayliigaaji TIL e golle mbayliigaaji mum fof ina ɗaɓɓi firo laaɓtungo. Ɗiɗaɓere ndee, ko MNA woni metabolit ɓurɗo seertude hakkunde CD45-cellules e T-cellules e nder ascites, wonaa ɓuuɓri. Ko ɗum waɗi, kompartimentaasiyoŋ e nokku ɗo tuumo oo woni ɗoo ina mbaawi jogaade batte ceertuɗe e mbayliigu TIL, ko ɗum teeŋtinta heterojenetik baawɗo wonde e nder weeyo mikroo-environnement dottaaɗo. Tataɓol ngol, ko baylagol enzim NNMT peewnoowo MNA ɓuri waawde huutoreede ko e CAF, woni ƴiiƴam ɓuuɓɗam haa ɓurti, kono toɓɓe MNA yiyteteeɗe ina teskaa e ƴiiƴam T keɓtinaaɗam e ƴiiƴam. Ko ɓuri heewde e NNMT e nder CAF ovarien ina jogii batte anndaaɗe e ɓeydagol kanseer, tawi heen geɗal ko sabu ɓeydagol mbayliigu CAF, e ɓuuɓri ɓuuɓri e metastaas (27). Hay so tawii noon tolno TIL oo fof ina famɗi, baylagol NNMT e nder CAF ina ɓadii no feewi e sifaa mesenkimal biyeteeɗo Cancer Genome Atlas (TCGA), jokkondirɗo e ŋakkeende ƴeewndo (27, 46, 47). E joofnirde, baylagol enzim AOX1 baawɗo ustude MNA ina famɗi kadi e nder yimɓe CAF, ko ɗum hollirta wonde ƴiye T ina ŋakki mbaawka waylude MNA. Ɗee geɗe ina mballita e miijo wonde hay so tawii golle goɗɗe ina poti waɗeede ngam hoolkisaade ndeeɗoo yiytunde, tolnooji toowɗi MNA e nder geese T ina mbaawi hollude wonde ina woodi mikroo-environnement CAF immunosuppressif.
E fawaade e tolno baylagol lowre MNA e tolnooji ɗi nganndaaka e nder poroteyiinuuji teeŋtuɗi jeyaaɗi e mbayliigu MNA, tawaade MNA e nder geese T ina sikkaa. Alaa fof NNMT walla AOX1 mbaawi yiyteede e ƴeewndo scRNA-seq e qPCR teskinɗo e kohorteeji ɗiɗi keertiiɗi. Ɗee geɗe kollitii wonde MNA sinndaaka e nder geese T, kono ina soomi e TME taariindi. Jarribooji baɗaaɗi e nder geese ina kollita wonde ƴiye T ina keewi mooftude MNA eksoojen.
Wiɗtooji men e nder geese kollitii wonde MNA eksoojenik ina addana TNFα feeñde e nder geese T, ina ɓeyda kadi jokkondiral Sp1 e ƴellitoowo TNFα. Hay so tawii TNFα ina jogii golle kaɓtorɗe e kaɓtorɗe, e nder kanseer oo, TNFα ina waawi wallitde mawnugol kanseer oo (31-33). Neutralisation TNFα e nder pinal celule tumor ovarien walla ittude signaal TNFα e nder moodeluuji peƴooji ina waawi moƴƴinde peewnugol sitokineeji ɓuuɓɗi baɗɗi TNFα e haɗde mawnugol tumor (32, 35). Ko ɗum waɗi, e ooɗoo sifaa, MNA keɓaaɗo e TME ina waawi wonde mbayliigu ɓuuɓngu rewrude e feere fawaande e TNFα rewrude e loowdi otokriin, ɗum noon ina addana ɗum feeñde e sarde kanseer ovarien (31). Tuugnaade e ngool mbaawka, ina janngee wonde ɓuuɓri TNFα ina waawi safrude kanseer oo (37, 48, 49). Yanti heen, MNA ina bonna sitotoksisiteeji ƴiiƴam CAR-T e ƴiiƴam ɓuuɓɗam, ɗum noon ina addana ɗum seedeeji goɗɗi ngam haɗde cellal ɓalli rewrude e MNA. E nder renndo, ɗeeɗoo geɗe ina kollita mbaadi ɗo tuumooji e ƴiye CAF njiylotoo MNA e nder TME ekstraselulaar. Rewrude e (i) ustude mawnugol kanseer ovarien baɗaaɗo e TNF e (ii) ustude golle sitotoksiik cellal T baɗaaɗo e MNA, ɗum ina waawi jogaade batte ɓuuɓɗe ɗiɗi (Natal 5D).
E joofnirde, e huutoraade denndaangal ƴellitaare cellal jaawngal, jokkondire cellal goote e ƴeewndorɗe mbaylaandi, ndeeɗoo wiɗto hollitii ceertugol mawngol immunometabolomik hakkunde ƴiye tummbuɗe e ƴiye ascites e nder ñawɓe HGSC. Ndeeɗoo wiɗto kuuɓtodinngo hollitii wonde ina woodi ceertugol e ƴettugol glukos e golle mitokondriyaal hakkunde ƴiye T, tee ina anndi MNA ko huunde nde alaa ko woni e mum so wonaa ƴiye. Ɗeeɗoo dokke ina njogii batte e no TME waylirta mbayliigu cellal T e nder kanseeruuji aadee. Hay so tawii noon pottital toowngal ngam heɓde ñamri hakkunde ƴiye T e ƴiye kanseer ina haalee, metaboliit ina waawi kadi wonde reenoowo toowɗo ngam wallitde yahrude yeeso e nder ɓalndu, ina waawi kadi haɗde jaabawuuli immuno-endogenic. Sifaa goɗɗo darnde gollal ɗeen mbayliigaaji jokkondire ina waawi udditaade peeje goɗɗe ngam ɓeydude jaabawol immuniteer anti-tumor.
Eɓɓooje ñawɓe e dokke kilinik keɓaama e nokku ɗo ƴiiƴam BC BC woni ɗoo, mo Reso desorɗe ƴiiƴam Kanadaa seedtini. E fawaade e porotokol jaɓaaɗo e Goomu Etikaaji Wiɗtooji Kanser BC e Duɗal Jaaɓi-haaɗtirde Kolommbi (H07-00463), denndaangal ƴeewndorɗe ñawɓe e dokke kilinik keɓii yamiroore binnditagol laaɓtungol walla njaɓaani jaɓgol mum en e dow laawol. Sammbaaji ɗii ina ndesndaa e BioBank (BRC-00290) seedtinaaɗo. Sifaa ñawɗo oo laaɓtuɗo ina hollira e alluuje S1 e S5. Ngam reentaade (cryopreservation), scalpel ina huutoree ngam ustude ƴiiƴam ñawɗo oo e nder masiŋaaji, caggal ɗuum ɓuuɓna ɗum e nder filtirde 100 mikron ngam heɓde suspension cellal gootol. Ascites ñawɗo oo ina senndiree e 1500 rpm fotde hojomaaji 10 e 4°C ngam pellitde ƴiiƴam ɗam e ittude ko woni e dow mum koo. Piɓaaji keɓaaɗi e ɓuuɓri e ascites, njuppaama e nder 50% e ƴiiƴam AB neɗɗo (Sigma-Aldrich) mo nguleeki mum alaa, 40% e RPMI-1640 (Thermo Fisher Scientific) e 10% e dimetil sulfoksiid. Ɗeeɗoo suspensions cellal gootal keɓtinaaɗe, ɓuuɓnaama, kuutoraa ngam metabolomik e anndinde metaboliit sifotooɗe les ɗee.
Hakkunde timmunde ina waɗi 0,22 μm filtirde 50:50 ɓeydaande RPMI 1640: AimV. RPMI 1640 + 2,05 mM l-glutamin (Ganndal Termo Fisher) ɓeydaama e 10% e ƴiiƴam AB neɗɗo mo nguleeki mum alaa (Sigma-Aldrich), Hepes 12,5 mM (Ganndal Termo Fisher), 2 mM l-glutamin (Ganndal Termo Fisher) S1 Kosam streptomisin (PenStrep) e 50 μMB-merkaptoetanol. AimV (Invitrogen) ina ɓeydee heen Hepes 20 mM (Ganndal Termo Fisher) e l-glutamin 2 mM (Ganndal Termo Fisher). Tampon mbayliigu sitomeeter ɓuuɓɗo oo ina waɗi 0,22 μm saline tampon fosfaat filtirde (PBS; Invitrogen) ɓeydaaɗo e 3% e ƴiiƴam neɗɗo AB mo nguleeki mum alaa (Sigma). Tampon alɗugol cellal ina waɗi PBS filtirde 0,22μm, ɓeydaa heen 0,5% e serum AB neɗɗo mo nguleeki mum alaa (Sigma-Aldrich).
E nder 37°C timmuɗo, ƴiiƴam ɗam ɓuuɓnaama e 10 nM MT DR e 100 μM 2-NBDG fotde hojomaaji 30. Ko adii ɗuum, ƴiye ɗee njuppaa e mbaydi nguurndam eF506 to 4°C fotde hojomaaji 15. Sosde ƴiiƴam ɗam e nder FC Block (eBioscience) e tampon ɓuuɓɗo (BD Biosciences), ɓuuɓna ɗum e nder tampon ɓuuɓɗo (hono no peewnoowo oo yamiri nii), ɓuuɓna ɗum fotde hojomaaji 10 e nguleeki suudu. Taƴde ƴiiƴam ɗam e seto antibiotik (Tablo S2) e nder tampon taƴoowo sitometri ɓuuɓɗo e 4°C fotde hojomaaji 20. Sosde ƴiye ɗee e nder tampon mbayliigu sitometri (Cytek Aurora; mbaydi 3L-16V-14B-8R) hade mum en ƴeewteede. Huutoro SpectroFlo e FlowJo V10 ngam yuurnitaade dokke limto cellal, e huutoraade GraphPad Prism 8 ngam sosde dokke ɗee. Teddeendi ɓuuɓndi (MFI) 2-NBDG e MT DR ko log-normalized, caggal ɗuum test t paired huutoraa ngam ƴeewde limtooji ngam hiisaade ñawɓe nannduɓe. Ittude denndaangal yimɓe ɓe keɓaani 40 kewu e nder analis ; naatde e teddeendi MFI 1 wonande kala teddeendi bonndi hade maa waɗde ƴeewndo limtooji e ƴeewndo dokke.
Ngam ɓeydude peeje gating manuel e panneel golle dow oo, min kuutoriima annotaasiyoŋ timmuɗo e lekki keeriindi mbaadi (FAUST) (21) ngam rokkude otomatik cellal e yimɓe caggal nde min itti cellal maayngal e nder FlowJo. Min njuɓɓinta ko yaltinnde e junngo ngam hawrude e denndaangal yimɓe nannduɓe e ko ŋakki (hawrude e PD1+ e PD1-tumor cells) e yimɓe keɓtinaaɓe. Kala sample ina waɗi ko ina tolnoo e 2% e cellal, tawi ko 11 limre.
Sentirifugaasiyoŋ densité gradient ficoll huutoraama ngam seerndude PBMC e geɗe seerndude ƴiiƴam (STEMCELL Technologies). Piɓaaji CD8 + T ɗii ceertinaama e PBMC e kuutoragol CD8 MicroBeads (Miltenyi) e yaajnude e nder weeyo timmungo kuutortooɗo TransAct (Miltenyi) fotde jonte 2 fawaade e jamirooje peewnoowo oo. Ɓowɗi ɗii ɗacciraa ko balɗe 5 e nder weeyo timmungo, tawa ina waɗi IL-7 (10 ng/ml; PeproTech), caggal ɗuum ɗi njuumta kadi e TransAct. Ñalnde 7 ndee, e fawaade e jamirooje peewnoowo oo, CD45 MicroBeads (Miltenyi) neɗɗo huutoraama ngam alɗinde ƴiiƴam e nder cirƴam tati jokkondirɗam. Piɓaaji ɗii limtaama ngam ƴeewndaade flow cytometry (hono no siforaa dow nii), e miliyoŋ piɓaaji limtaaɗi laabi tati ngam ƴeewndaade LC-MS/MS. Sammbaaji ɗii mbaɗiraa ko LC-MS/MS no siforaa les nii. En limtii teddeendi metabolit ŋakkuɗo oo e limoore iyon 1 000. Kala sample ina nominaree e limoore iyonuuji (TIC), ina waylee e loogaari, ina nominaree otomatik e nder MetaboAnalystR hade mum ƴeewteede.
Suspension cellule gooto e kala ñawɗo ina ɓuuɓna, ina filtiree e filtirde 40 μm e nder weeyo timmungo (hono no siforaa dow nii). E fawaade e porotokol peewnoowo oo, cirƴam tati jokkondirɗam e suɓagol moƴƴol e seerndude ɓulli magnetik kuutortooɗi MicroBeads (Miltenyi) kuutoraa ngam alɗinde sampleeji CD8+, CD4+ e CD45- (e dow ice). E raɓɓiɗinaade, ƴiye ɗee ina njuutee e tampon (hono no siforaa dow nii) ina limtee. Piɓaaji ɗii njuppaa e ɓuuɓri CD8 neɗɗo, ɓuuɓri CD4 neɗɗo walla ɓuuɓri CD45 neɗɗo (Miltenyi) e nguleeki 4°C fotde hojomaaji 15, caggal ɗuum loowaa e tampon ɓeydoowo piɓaaji. Sammba oo rewata ko e koloñaal LS (Miltenyi), pecce moƴƴe e bonɗe ɗee mooftee. Ngam ustude dumunna oo e ɓeydude daawal cellal, ndeen feccere CD8 ndee huutortee ko e mbaydi ɗiɗmiri CD4+, feccere CD4 ndee huutoree ngam ƴellitde CD45 caggal ɗuum. Waɗde ɓuuɓri ndii e dow ice e nder golle seerndude ɗee kala.
Ngam feewnude seedanteeje ngam yuurnitaade metaboliit, ƴiye ɗee loowaama laawol gootol e ɓuuɓri ɓuuɓndi, 1 ml metaan 80% ɓeydaa e seedantaagal kala, caggal ɗuum vortex e snap frozen e nder azot ndiyam. Sammbaaji ɗii mbaɗaa e cirkooji tati ɓuuɓɗi-ɓuuɓɗi, ɗi njuppaa e 14 000 rpm fotde hojomaaji 15 e 4°C. Ko heddii e mum koo ina waɗi metaboliteeji ɗii, ina ɓuuɓtoo haa yoora. Metaboliteeji ɗii njuumtaama kadi e nder 50 μl asid formik 0,03%, njuumtaama ngam jillondirde, caggal ɗuum njuumtaama ngam ittude mborosaaji.
Ittugol metaboliteeji no siforaa dow nii. Waɗde, ɓuuɓnude ƴiiƴam ɗam e nder butelaaji kromatogaraafi ndiyam toowɗi ngam wiɗtooji metabolomik. Huutoroo porotokol safaara ranndo ngam safrude kala sample e limoore cellal nanndunde ngam haɗde batte batte. En mbaɗii ƴeewndo moƴƴo e mbayliigaaji winndereeji baɗaaɗi ko adii ɗuum e AB SCIEX QTRAP 5500 (50). Anndugol kromatogaraafi e jokkondire nokkuuji toowɗi mbaɗaama e kuutoragol lowre MultiQuant mbaydi 2.1 (Applied Biosystems SCIEX).
Limre iyon 1000 huutoraama ngam hiisaade nafoore metaboliit mo ŋakki, e TIC kala eɓɓoore huutoraa ngam hiisaade nokku ɓurɗo toowde e kala metaboliit yiytaa ngam feewnude waylooji naatnaaɗi e analis kuutorgal ummoraade e peewnugol eɓɓoore. Caggal nde TIC noddaa, MetaboAnalystR(51) (parametru teskinɗo) huutortee ko ngam waylude loogaari e ƴellitde laabi noddaango otomatik. En kuutoriima PCA e package vegan R ngam waɗde wiɗtooji ƴeewndorɗe ceertuɗe metabolome hakkunde sifaaji sample, e kuutoragol ƴeewndo redundansi feccere ngam ƴeewndaade ñawɓe. Huutoro feere Ward ngam mahde dendrogram kartal nguleeki ngam mooftude distance Euclidean hakkunde sampleeji. En kuutoriima limma (52) e dow keewal mbayliigaaji gonɗi e mbaydi ngam anndude mbayliigaaji keewɗi ceertuɗi e nder sifaa cellal e nder nokkuuji tokoosi ɗii fof. Ngam hoybinde firo ngoo, eɗen kuutoroo parametru moƳƳo fedde ngam hollirde mbaadi ndii, e ƴeewtaade sifaaji cellal e nder mikroo-environnement hono kala fedde (n = 6 fedde); ngam jarribo maantinngo, min mbaɗii peeje tati keewɗe ngam kala metabolit Ngam daɗde e fenaande, ñawɗo oo naatnaa e caɗeele e nder diisnondiral limma. Ngam ƴeewde feere mbayliigu hakkunde ñawɓe ceertuɓe, min njuɓɓinii mbaadi limma ina waɗi ñawɓe e mbaadi tabitndi. En kaaliino nafoore feere teskinnde ko adii hakkunde sifaa cellal e mikroo-environnement Padj <0.05 (peewnugol Benjamini-Hochberg).
Caggal nde semmbe ɓeydaa e huutoraade Miltenyi Dead Cell Removal Kit (>80% e nguurndam), ƴeewndo transkriptom cellal gootol waɗaama e dow ascites e sampleeji tuumooji ɓuuɓɗi guurɗi fof e huutoraade porotokol baylagol 10x 5′gene. Caɗeele joy jogiiɗe ɓuuɓri e ascites nannduɗi ƴeewtaama, hay so tawii noon ŋakkeende nguurndam ummoraade e sample ɓuuɓri gooto haɗi ɗum naatde heen. Ngam heɓde suɓngooji keewɗi e ñawɓe, min kawri e sampleeji kala ñawɗo e nder laabi 10x gonɗi e ƴeewndo krom, min ƴeewtindorii nokkuuji ascites e nokkuuji tumor ceertuɗi. Caggal nde njuɓɓudi [Illumina HiSeq 4000 28×98 bp jokkere enɗam (PE), genooji Kebek ; hakkundeere 73 488 e 41 378 janngeteeɗe e kala cellal ngam tumor e ascites]], min kuutoriima CellSNP e Vireo (53) (fawaade e CellSNP no SNP neɗɗo gooto (VCF) mo GRCh38 rokki oo ina rokka genotype ñawɗo (genotype ñawɗo oo. Min kuutoriima SNPRelate ngam (IBS), tawa ina woɗɗi ƴiye ɗe ndokkaaka e ƴiye ɗe nganndu-ɗaa ko duplexes e dokkooje nannduɗe hakkunde ascites e sammeeji tuumooji (54), min keɓii caɗeele tati jogiiɗe ƴiye keewɗe e nder tummbudu e asciteeji ngam ƴeewndaade dow (scCoran filtration step e)6 6975 ƴiiƴam (2792 e 4183 ƴiiƴam ummoraade e ƴiiƴam e ascites, no mbaydiiji ɗii fof) ngam ƴeewndaade e yiyteede e t-SNE. Piɓaaji T cytotoxic ina cifaa e baylagol CD8A e GZMA, tawa ina jeyaa heen ɓuuɓri gonndi e baylagol poroteyiin ribosomal seeɗa En keɓii dokke bayyinaaɗe ɗe Izar et al (16), ina heen t-SNE gonɗe e mum en, ina mbaawi jogaade baylagol gonngol hakkunde piɓaaji immune.
PBMC seerndaama e geɗe ceertuɗe ƴiiƴam (STEMCELL Technologies) e mbaydi njuɓɓudi Ficoll. CD3 + ina seerti e PBMC e kuutoragol ɓuuɓri CD3 (Miltenyi). E nder MNA walla alaa, CD3+ ina huutoree e CD3 jokkondirɗo e plaque (5 μg/ml), CD28 ɓuuɓɗo (3 μg/ml) e IL-2 (300 U/ml; Proleukin). Ñalnde cakkitiinde yaajde ndee, nguurndam (Dye de Viability Fixable eFluor450, eBioscience) e ɓeydagol (123count eBeads, Thermo Fisher Scientific) ƴeewtaama e mbaydi sitometri ɓuuɓndi. Ƴeewtaade golle efektor e ustude ƴiiƴam e PMA (20 ng/ml) e iyonomisin (1μg/ml) e GolgiStop fotde waktuuji 4, e ƴeewtaade CD8-PerCP (RPA-T8, BioLegend), CD4-AF700 (RPA-T4) , BioLegend) e TNForecytein (MAb11, BD). Ƴeewndo qPCR e ChIP e PMA (20 ng/ml) e iyonomisin (1μg/ml) fotde waktuuji 4. Ko heddii e ELISA koo mooftaama ko adii e caggal nde ɓuuɓri ndii ɓuuɓnaa e PMA (20 ng/ml) e iyonomisin (1 μg/ml) fotde waktuuji 4.
Rewrude e peeje peewnoowo ngam seerndude ARN e huutoraade RNeasy Plus Mini Kit (QIAGEN). Huutoro QIA (QIAGEN) ngam ɓuuɓnude sample oo. Huutoroo kit ARN e ADN baawɗo toowde (Thermo Fisher Scientific) ngam sinkude ADN timmuɗo (ADN). Huutoro TaqMan Rapid Advanced Master Mix (Thermo Fisher Scientific) ngam limtude baylagol taƴe (fawaade e porotokol peewnoowo oo) e ɗeeɗoo ƴulɓe : Ko ɗum woni ko wiyetee « gliseraldehiid-3-fosfaat » e dow hidrojen (GAPDH)] e « m1 » (SLC22A2). Sammbaaji ɗii ndokkaama e dow njuɓɓudi PCR StepOnePlus e sahaa goonga (Biosistemaaji kuutorteeɗi) (Biosistemaaji kuutorteeɗi) e nder laylaytol jaawngol 96-ɓulli MicroAmp (Biosistemaaji kuutorteeɗi) e filmo optik MicroAmp. Kala teddeendi Ct ɓurndi 35 ina hiisee ina woni dow tolno yiytugol, ina maantiniree wonde yiytaaka.
Waɗde ChIP no siforaa adan nii (58). E raɓɓiɗinaade, ƴiye ɗee njuurnitee ko e formaldehiid (ko heewi e mum en ko 1,42%), ɗe njuumta e nguleeki suudu fotde hojomaaji 10. Huutoro tampon ɓuuɓɗo ɓeydaaɗo (Hepes 25 mM, MgCl2 1,5 mM, KCl 10 mM e NP-40 0,1%) e dow ɓuuɓri fotde hojomaaji 10, caggal ɗuum ɓuuɓna ɗum e nder tampon immunopresipitaasiyoŋ no siforaa nii (58 ). Ndeen sample oo sonikaama e ɗeeɗoo cirkooji : cirkooji 10 (20 pulse 1-second) e waktuuji 40 static. Ƴeewee ƴiiƴam G (karallaagal ƴeewndo cellal; 1 μl), histon H3 (karallaagal ƴeewndo ƴiiƴam; 3 μl), NFAT (Invitrogen; 3 μl) e SP1 (karallaagal ƴeewndo ƴiiƴam; 3 μl) e ƴiiƴam ɗam e dow CC°4 Ƴeewee ɓuuɓri poroteyiin A (Thermo Fisher Scientific) e sample oo e nguleeki 4°C tawa ina ɓuuɓna ɗum no feewi fotde waktu gooto, caggal ɗuum huutoroo ɓuuɓri chelex (Bio-Rad) ngam alɗinde ADN, huutoroo ɓuuɓri poroteyiin K (Thermo Fisher) ngam ɓuuɓnude poroteyiin. Ko PCR yiytaa ko ƴellitoowo TNFα : yeeso, GGG TAT CCT TGA TGC TGT ; ko luutndii ɗum, GTG CCA ACA ACT GCC TTT ATA TG (geɗel 207 bp). Nate ɗee ko Image Lab (Bio-Rad) peewni ɗe, limtaaɗe ɗee ko e lowre ImageJ.
Ko heddii e pinal cellal koo mooftaa ko no siforaa dow nii. Anndinde ndee waɗiraa ko e laabi peewnoowo oo, hono kaɓirɗe ELISA TNFα neɗɗo (Invitrogen), kaɓirɗe ELISA IL-2 neɗɗo (Invitrogen) e kaɓirɗe ELISA IFN-γ neɗɗo (Abcam). E fawaade e porotokol peewnoowo oo, ƴiiƴam ɗam ɓuuɓnaama 1:100 ngam yiytude TNFα e IL-2, e 1:3 ngam yiytude IFN-γ. Huutoro janngirde 2104 (PerkinElmer) ngam fooɗtude ɓuuɓol e 450 nm.
PBMC seerndaama e geɗe ceertuɗe ƴiiƴam (STEMCELL Technologies) e mbaydi njuɓɓudi Ficoll. CD3 + ina seerti e PBMC e kuutoragol ɓuuɓri CD3 (Miltenyi). E nder MNA walla alaa, ƴiiƴam CD3+ ina huutoree e CD3 jokkondirɗo e plaque (5 μg/ml), CD28 ɓuuɓɗo (3 μg/ml) e IL-2 (300 U/ml; Proleukin) fotde balɗe 3. Caggal balɗe 3, ƴiiƴam ɗam mooftaa, loowaa e 0,9% saline, pellet oo ɓuuɓnaa. Limre cellal waɗiraa ko e mbaydi sitometri (Cytek Aurora; mbaydi 3L-16V-14B-8R) e kuutoragol eBeads limtooji 123.
Ittugol metaboliteeji no siforaa dow nii. Kosam ɗam yoorɗam ɗam rewnaama e ɓuuɓri 4000 cellal/μl. Hiisaade seedantaagal ngal e mbaydi mbaylaandi (1290 Infinity II, Agilent Technologies, Santa Klara, Kaliforni) e koloñaal CORTECS T3 (2,1×150 mm, njaajeendi ɓuuɓri 1,6-μm, njaajeendi ɓuuɓri 120-Å; #186008500). Spektromeeter masse polaire (6470, Agilent), ɗo iyon elektoro-spray gollotoo e mbaadi moƴƴiri. Faandaare A moolanaande ko 0,1% asid formik (e nder H2O), faandaare B moolanaande ko 90% asetonitril, 0,1% asid formik. LC gradient ko 0 haa 2 minit wonande 100% A, 2 haa 7,1 minit wonande 99% B, e 7,1 haa 8 minit wonande 99% B. Ndeen noon, ndartinaa koloñaal oo e faɗo A moɓtoowo e njuuteendi 0,6 ml/min wonande hojomaaji 3. . Ɗaɗi ɗii ko 0,4ml/min, suudu koloñaal nduu ina ɓuuɓna haa 50°C. Huutoroo tolno kemikal laaɓtuɗo MNA (M320995, Sosiyetee kemikal wiɗto Toronto, North York, Ontario, Kanadaa) ngam tabitinde waktuuji jokkondire (RT) e waylo-waylo (RT = 0,882 minit, waylo-waylo 1 = 137→94,1, waylo-waylo 2 = 137, waylo-waylo→373). So tawii mbayliigaaji tati ɗii fof mbaɗii e sahaa jokkondire potɗe, mbayliigaaji 1 ɗii kuutortee ko ngam limtude ngam tabitinde keertinagol. Kurkureeji gonɗi e MNA (Sosiyetee kemikal wiɗto Toronto) peeñninaama e diƴƴe jeegom (6) gonɗe e ɓuuɓri gonndi e ɓuuɓri (1 mg/ml) ngam heɓde ɓuuɓri 0,1, 1,0, 10 e 100 ng/ml e 1,0 e 10 μg/ml ndiyam. Keerol yiytugol ngol ko 1 ng/ml, jaabawol lineer ngol ko hakkunde 10 ng/ml e 10μg/ml. Kala injection mikroo-liiteeruuji ɗiɗi e sample e standard ina huutoree ngam ƴeewndaade LC/MS, e sample ƴeewndo kalite jillondirɗo ina doga kala injection jeetati ngam tabitinde darnde plateau analis. Jaabawuuli MNA denndaangal sampleeji cellal MNA-treated ina ngonnoo e nder limre lineere ƴeewndo ngoo. Njiylawu doosɗe ɗee waɗiraa ko e lowre ƴeewndo keewal MassHunter (v9.0, Agilent).
Konngol αFR-CAR ngol jibinannde ɗiɗmere ndee ƴettaa ko e Song et al. (59). E raɓɓiɗinaade, konngol ngol ina waɗi ɗeeɗoo geɗe : jokkondire ardiiɓe CD8a, feccere waylowaylo αFR neɗɗo keeriiɗo, nokku CD8a hinge e transmembrane, domen CD27 nder ƴiiƴam e domen CD3z nder ƴiiƴam. Ko GenScript sinndi jokkondire CAR timmuɗe ɗee, caggal ɗuum ɗe njuɓɓinaa e mbaydi mbaylaandi lentiviral jibinannde ɗiɗmere to dow kaset baylagol GFP kuutorteeɗo ngam ƴeewde mbaadi mbayliigu.
Lentivirus ina feewnee e mbayliigu ƴiiƴam HEK293T [Koolol pinal sifaaji Amerik (ATCC); mawni e nder mbaydi Eagle mo Dulbecco wayli, ina waɗi 10% e ƴiiƴam nagge (FBS) e 1% e PenStrep, e huutoraade mbaydi CAR-GFP e Pilasmiduuji paaspooruuji (psPAX2 e pMD2.G, Addgene) ina kuutoroo amin lipofeksiyoŋ (Sigma-Aldrich). Kosam ɗam ina waɗi mboros oo, mooftaa ko waktuuji 48 e 72 caggal nde ɗam ƴettaa, ɗam filtiraa, ɗam ɓuuɓnaa e ultracentrifugation. Siftorde ɓuuɓri viral ɓuuɓndi ndii e -80°C haa ndi ɓuuɓta.
PBMC ina seerti e geɗe seerndirɗe ƴiiƴam celluɗam (STEMCELL Technologies) e mbaydi njuɓɓudi Ficoll. Huutoroo mikroo-beɗe CD8 cuɓaaɗe moƴƴe (Miltenyi) ngam seerndude CD8+ e PBMC. Ƴeewtaade ƴiiƴam T e TransAct (Miltenyi) e nder mbaydi TexMACS [Miltenyi; ɓeydaa heen 3% e ƴiiƴam neɗɗo mo nguleeki mum alaa, 1% e PenStrep e IL-2 (300 U/ml)]. Waktuuji 24 caggal ɓuuɓri, ƴiye T ɗee mbaylaa e lentivirus (10 μl ɓuuɓri mboros ɓuuɓndi e nder ƴiye 106). Balɗe 1 haa 3 caggal nde mbayliigu nguu ƴettaa e dow Cytek Aurora (e dow FSC (Ceerno yeeso)/SSC (Ceerno bannge), Singlet, GFP+), ƴeewto-ɗee baylagol GFP e nder geese ɗee ngam hollirde mbaadi mbayliigu ko famɗi fof 30% .
Piɓaaji CAR-T ina njuutee fotde waktuuji 24 e nder Immunocult (Karallaagal STEMCELL; ɓeydaama e 1% PenStrep) e nder ɗeeɗoo sarɗiiji : ɗi njuumtaani, ɗi njiytiraaka 250 μM adenosin walla 10 mM MNA. Caggal nde ƴiiƴam ɗam ƴettaa, ƴiiƴam CAR-T loowaa e PBS, hawraa e 20 000 ƴiiƴam SK-OV-3 [ATCC; e nder mbaydi McCoy 5A (Sigma-Aldrich) ɓeydaandi e 10% FBS e 1% PenStrep e 10 : Njaajeendi 1 e nder mbaydi 1 ɓeydaama e mbaydi tati e nder mbaydi Immunocult ɓeydaandi. Piɓaaji SK-OV-3 e piɓaaji SK-OV-3 limtaaɗi e saponin dijital (0,5 mg/ml; Sigma-Aldrich) kuutoraa ko no ƴeewndorɗe moƴƴe e moƴƴe, no mbaydiiji ɗii fof. Caggal waktuuji 24 e ndema, ko heddii koo mooftaa, laktat dehidrogenaas (LDH) fotnoo fotde no peewnoowo oo yamiri nii (LDH Glo Cytotoxicity Assay Kit, Promega). Ko heddii e LDH koo, ko 1:50 e nder tampon LDH. Teemdere warngooji ɗii fotnoo ko huutoraade ngalɗoo foormula : teemdere warngooji ɗii = teemdere peewnugol / tolno warngooji ɓurɗi heewde x 100%, ɗo teemdere peewnugol = co-culture-T cells tan, e tolno warngooji ɓurɗi heewde = ƴeewndo moƴƴo-konngol bonngol.
No siforaa e binndol walla kaɓirɗe e laabi, huutoroo GraphPad Prism 8, Microsoft Excel walla R v3.6.0 ngam ƴeewndaade limlebbi. So tawii seedanteeje keewɗe mooftaama e ñawɗo gooto (hono ascites e tumor), eɗen kuutoroo t test paired walla eɗen mbaɗa ñawɗo oo e batte random e nder model linear walla generalisé no haanirta nii. Ngam ƴeewtaade metabolomik, jarribo nafoore waɗaama e mbaydiiji tati.
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Marisa K. Kilgour (Marisa K. Kilgour), Sarah MacPherson (Sarah MacPherson), Lauren G. Zakariyas (Lauren G. Zakariyas), Abigail Eli Aris G. Watson (H. Watson), John Stagg (John Stagg), Brad H. Russell (John Stagg), Brad H (Rusel G. Jones), Finis T. Hamilton (Finis T.
MNA ina wallita e ustude cellal ɓalli T, ina lomtoo kadi mbaadi immunoterapi potndi huutoreede ngam safrude kanseer aadee.
Marisa K. Kilgour (Marisa K. Kilgour), Sarah MacPherson (Sarah MacPherson), Lauren G. Zakariyas (Lauren G. Zakariyas), Abigail Eli Aris G. Watson (H. Watson), John Stagg (John Stagg), Brad H. Russell (John Stagg), Brad H (Rusel G. Jones), Finis T. Hamilton (Finis T.
MNA ina wallita e ustude cellal ɓalli T, ina lomtoo kadi mbaadi immunoterapi potndi huutoreede ngam safrude kanseer aadee.
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