Li-metabolite tsa immunomodulatory ke tšobotsi ea bohlokoa ea tikoloho e nyane ea hlahala (TME), empa ntle le mekhelo e 'maloa, boitsebiso ba tsona bo ntse bo sa tsejoe haholo. Mona, re hlahlobile lihlahala le lisele tsa T tse tsoang lihlahala le li-ascites tsa bakuli ba nang le serous carcinoma ea boemo bo holimo (HGSC) ho senola metabolome ea likarolo tsena tse fapaneng tsa TME. Li-ascites le lisele tsa hlahala li na le liphapang tse kholo tsa metabolite. Ha li bapisoa le li-ascites, lisele tsa T tse kenang ka hlahala li ruile haholo ho 1-methylnicotinamide (MNA). Leha boemo ba MNA liseleng tsa T bo phahame, tlhahiso ea nicotinamide N-methyltransferase (enzyme e susumetsang phetisetso ea lihlopha tsa methyl ho tloha ho S-adenosylmethionine ho ea ho nicotinamide) e lekanyelitsoe ho li-fibroblast le lisele tsa hlahala. Ka ts'ebetso, MNA e susumetsa lisele tsa T ho ntša cytokine tumor necrosis factor alpha e khothalletsang hlahala. Ka hona, MNA e tsoang ho TME e kenya letsoho taolong ea 'mele ea lisele tsa T' me e emela sepheo se ka bang teng sa kalafo ea immunotherapy bakeng sa kalafo ea mofetše oa motho.
Li-metabolite tse nkiloeng ke hlahala li ka ba le phello e tebileng ea thibelo ho sesole sa 'mele se loantšang hlahala, 'me bopaki bo bongata bo bontša hore li ka boela tsa sebetsa e le matla a bohlokoa a ho khanna tsoelo-pele ea lefu (1). Ntle le phello ea Warburg, mosebetsi oa morao tjena o qalile ho hlalosa boemo ba metabolism ea lisele tsa hlahala le kamano ea eona le boemo ba 'mele ba ho itšireletsa mafung ba tikoloho e nyenyane ea hlahala (TME). Lithuto tse entsoeng ka mehlala ea litoeba le lisele tsa T tsa batho li bontšitse hore metabolism ea glutamine (2), metabolism ea oxidative (3) le metabolism ea glucose (4) li ka sebetsa ka boikemelo lihlopheng tse fapaneng tsa lisele tsa 'mele tse loantšang hlahala. Li-metabolite tse 'maloa litseleng tsena li thibela mosebetsi oa lisele tsa T tse loantšang hlahala. Ho pakiloe hore thibelo ea coenzyme tetrahydrobiopterin (BH4) e ka senya ho ata ha lisele tsa T, 'me keketseho ea BH4 'meleng e ka ntlafatsa karabelo ea 'mele ea ho itšireletsa mafung e tsamaisoang ke CD4 le CD8. Ho phaella moo, phello ea ho thibela sesole sa 'mele ea kynurenine e ka pholosoa ke tsamaiso ea BH4 (5). Ho isocitrate dehydrogenase (IDH) mutant glioblastoma, ho ntšoa ha enantiometabolic (R)-2-hydroxyglutarate (R-2-HG) ho thibela ts'ebetso ea sele ea T, ho ata ha eona le ts'ebetso ea cytolysis (6). Haufinyane tjena, ho bontšitsoe hore methylglyoxal, sehlahisoa se tsoang ho glycolysis, e hlahisoa ke lisele tse hatellang tse simolohileng ho myeloid, 'me phetisetso ea sele ea T ea methylglyoxal e ka thibela ts'ebetso ea sele ea T e sebetsang. Kalafong, ho se sebetse hantle ha methylglyoxal ho ka hlola ts'ebetso ea lisele tse hatellang tse tsoang ho myeloid (MDSC) le ho ntlafatsa kalafo ea thibelo ea checkpoint ka kopanelo mehlaleng ea litoeba (7). Lithuto tsena ka kakaretso li totobatsa karolo ea bohlokoa ea li-metabolite tse tsoang ho TME ho laoleng ts'ebetso le ts'ebetso ea sele ea T.
Ho se sebetse hantle ha lisele tsa T ho tlalehiloe haholo kankereng ea mae a bomme (8). Sena se bakoa ke likarolo tse ling tsa metabolism tse teng ho hypoxia le methapo e sa tloaelehang ea mali ea hlahala (9), e leng se fellang ka phetoho ea tsoekere le tryptophan hore e be lihlahisoa tse ling tse kang lactic acid le kynurenine. Lactate e feteletseng ea extracellular e fokotsa tlhahiso ea interferon-γ (IFN-γ) 'me e khanna phapang ea lihlopha tse nyenyane tsa myelosuppressive (10, 11). Tšebeliso ea tryptophan e thibela ka ho toba ho ata ha lisele tsa T 'me e thibela matšoao a receptor ea lisele tsa T (12-14). Ho sa tsotellehe lintho tsena tse hlokometsoeng, mosebetsi o mongata o potileng metabolism ea 'mele o entsoe ka har'a setso sa lisele tsa T tsa in vitro ho sebelisoa mecha e ntlafalitsoeng, kapa e lekanyelitsoe ho mehlala ea litoeba tse tšoanang ka har'a vivo, tseo ho seng le e 'ngoe ea tsona e bonts'ang ka botlalo ho se tšoane ha kankere ea batho le tikoloho ea macro le micro ea fisioloji.
Tšobotsi e tloaelehileng ea kankere ea mahe a bomme ke ho ata ha peritoneal le ponahalo ea ascites. Ho bokellana ha mokelikeli oa sele ka har'a ascites ho amahanngoa le lefu le tsoetseng pele le tebello e mpe ea lefu lena (15). Ho ea ka litlaleho, karolo ena e ikhethang ha e na oksijene e ngata, e na le maemo a holimo a vascular endothelial growth factor (VEGF) le indoleamine 2,3-dioxygenase (IDO), 'me e kenella ke lisele tse laolang T le lisele tse thibelang myeloid (15-18). Tikoloho ea metabolism ea ascites e ka fapana le ea hlahala ka boeona, kahoo ho hlophisoa bocha ha lisele tsa T sebakeng sa peritoneal ha ho hlake. Ho phaella moo, liphapang tsa bohlokoa le ho se tšoane ha li-ascites le li-metabolite tse teng tikolohong ea hlahala li ka sitisa ho kenella ha lisele tsa 'mele tsa ho itšireletsa mafung le mosebetsi oa tsona ho lihlahala, 'me lipatlisiso tse ling lia hlokahala.
E le ho rarolla mathata ana, re qapile mokhoa oa karohano ea lisele tse bonolo le liquid chromatography tandem mass spectrometry (LC-MS/MS) ho ithuta mefuta e fapaneng ea lisele (ho kenyeletsoa lisele tsa CD4 + le CD8 + T) hammoho le ka hare le pakeng tsa lihlahala. Li-metabolite tsa eona li akaretsa lisele tse tšoanang tsa ascites le tikoloho ea hlahala ea mokuli. Re sebelisa mokhoa ona hammoho le cytometry ea phallo e phahameng le tatellano ea RNA ea sele e le 'ngoe (scRNA-seq) ho fana ka setšoantšo se rarollotsoeng haholo sa boemo ba metabolism ba baahi bana ba bohlokoa. Mokhoa ona o senotse keketseho e kholo boemong ba 1-methylnicotinamide (MNA) liseleng tsa T tsa hlahala, 'me liteko tsa in vitro li bontšitse hore phello ea immunomodulatory ea MNA mosebetsing oa sele ea T e ne e sa tsejoe pele. Ka kakaretso, mokhoa ona o senola likamano tse kopanetsoeng tsa metabolism pakeng tsa lihlahala le lisele tsa 'mele tsa ho itšireletsa mafung, 'me o fana ka temohisiso e ikhethang mabapi le li-metabolite tsa taolo ea' mele ea ho itšireletsa mafung, tse ka bang molemo bakeng sa kalafo ea menyetla ea kalafo ea immunotherapy ea mofetše oa ovary e thehiloeng ho T cell.
Re sebelisitse cytometry ea phallo ea tekanyo e phahameng ho lekanya ho nkuoa ha glucose ka nako e le 'ngoe [2-(N-(7-nitrophenyl-2-oxa-1,3-diaza-4-yl)amino)-2-deoxyglucose (2-NBDG) le ts'ebetso ea mitochondrial [MitoTracker Deep Red (MT DR)] (7, 19, 20) ke matšoao a tloaelehileng a bapileng a khethollang lisele tsa 'mele le baahi ba lisele tsa hlahala (Tafole ea S2 le Setšoantšo sa S1A). Tlhahlobo ena e bontšitse hore ha ho bapisoa le lisele tsa T, ascites le lisele tsa hlahala li na le maemo a holimo a ho nkuoa ha glucose, empa li na le liphapang tse nyane ts'ebetsong ea mitochondrial. Karolelano ea ho nkuoa ha glucose liseleng tsa hlahala [CD45-EpCAM (EpCAM)+] e feta makhetlo a mararo ho isa ho a mane a lisele tsa T, 'me karolelano ea ho nkuoa ha glucose liseleng tsa CD4 + T e feta makhetlo a 1.2 a CD8 + T, e leng se bontšang hore li-lymphocyte tse kenang ka hlahala (TIL) li na le litlhoko tse fapaneng tsa metabolism esita le ho TME e tšoanang (Setšoantšo sa 1A). Ka lehlakoreng le leng, mosebetsi oa mitochondrial liseleng tsa hlahala o tšoana le oa lisele tsa CD4 + T, 'me mosebetsi oa mitochondrial oa mefuta ka bobeli ea lisele o phahame ho feta oa lisele tsa CD8 + T (Setšoantšo sa 1B). Ka kakaretso, liphetho tsena li senola boemo ba metabolism. Mosebetsi oa metabolism oa lisele tsa hlahala o phahame ho feta oa lisele tsa CD4 + T, 'me mosebetsi oa metabolism oa lisele tsa CD4 + T o phahame ho feta oa lisele tsa CD8 + T. Ho sa tsotellehe liphello tsena ho pholletsa le mefuta ea lisele, ha ho na phapang e tsitsitseng boemong ba metabolism ba lisele tsa CD4 + le CD8 + T kapa boholo ba tsona bo amanang le ascites ha li bapisoa le lihlahala (Setšoantšo sa 1C). Ka lehlakoreng le leng, karolong ea lisele tsa CD45, karolo ea lisele tsa EpCAM+ hlahaleng e eketsehile ha e bapisoa le ascites (Setšoantšo sa 1D). Re boetse re bone phapang e hlakileng ea metabolism pakeng tsa likarolo tsa EpCAM+ le EpCAM- cell. Lisele tsa EpCAM+ (tumor) li na le ho monya ha glucose e ngata le mosebetsi oa mitochondrial ho feta lisele tsa EpCAM, e leng se phahameng haholo ho feta mosebetsi oa metabolism oa li-fibroblast liseleng tsa hlahala ho TME (Setšoantšo sa 1, E le F).
(A le B) Matla a bohareng a khanya ea fluorescence (MFI) ea ho monya glucose (2-NBDG) (A) le ts'ebetso ea mitochondrial ea lisele tsa CD4 + T (MitoTracker e khubelu e lefifi) (B) Li-graph tse emelang (ka letsohong le letšehali) le data e hlophisitsoeng (Ka ho le letona), lisele tsa CD8 + T le lisele tsa EpCAM + CD45-tumor tse tsoang ho ascites le hlahala. (C) Karolelano ea lisele tsa CD4 + le CD8 + (tsa lisele tsa CD3 + T) ho ascites le hlahala. (D) Palo ea lisele tsa hlahala tsa EpCAM + ho ascites le hlahala (CD45−). (E le F) EpCAM + CD45-tumor le EpCAM-CD45-matrix glucose uptake (2-NBDG) (E) le ts'ebetso ea mitochondrial (MitoTracker e khubelu e lefifi) (F) li-graph tse emelang (ka letsohong le letšehali) le data e hlophisitsoeng (Ka ho le letona) Li-Ascites le lisele tsa hlahala. (G) Li-graph tse emelang tsa polelo ea CD25, CD137 le PD1 ka cytometry ea phallo. (H le I) Pontšo ea CD25, CD137 le PD1 liseleng tsa CD4 + T (H) le lisele tsa CD8 + T (I). (J le K) Mefuta e sa tloaelehang ea memori e bohareng (Tcm), e sebetsang (Teff) le memori ea effector (Tem) e ipapisitseng le polelo ea CCR7 le CD45RO. Litšoantšo tse emelang (ka letsohong le letšehali) le data ea tafole (ka ho le letona) ea lisele tsa CD4 + T (J) le lisele tsa CD8 + T (K) ka har'a ascites le lihlahala. Boleng ba P bo khethoa ke teko ea t e kopantsoeng (*P<0.05, **P<0.01 le ***P<0.001). Mohala o emela bakuli ba tšoanang (n = 6). FMO, fluorescence ntle le e le 'ngoe; MFI, matla a fluorescence a mahareng.
Tlhahlobo e 'ngoe e senotse liphapang tse ling tse kholo lipakeng tsa boemo ba phenotypic ba sele ea T e rarollotsoeng haholo. Memori ea effector e sebetsang (Setšoantšo sa 1, G ho isa ho I) le effector (Setšoantšo sa 1, J le K) lihlahleng li atile haholo ho feta ascites (palo ea lisele tsa CD3 + T). Ka mokhoa o ts'oanang, ho sekaseka phenotype ka polelo ea matšoao a ts'ebetso (CD25 le CD137) le matšoao a ho fokotseha [protheine ea lefu la sele e hlophisitsoeng 1 (PD1)] ho bontšitse hore le hoja litšobotsi tsa metabolism tsa baahi bana li fapane (Setšoantšo sa S1, B ho isa ho E), Empa ha ho na liphapang tse kholo tsa metabolism tse ileng tsa bonoa khafetsa lipakeng tsa naive, effector kapa memori subsets (Setšoantšo sa S1, F ho isa ho I). Liphetho tsena li tiisitsoe ka ho sebelisa mekhoa ea ho ithuta ka mochini ho abela ka bohona li-phenotype tsa sele (21), e leng se ileng sa senola boteng ba palo e kholo ea lisele tsa moko oa masapo (CD45 + / CD3- / CD4 + / CD45RO +) ho ascites ea mokuli (Setšoantšo sa S2A). Har'a mefuta eohle ea lisele tse khethiloeng, palo ena ea lisele tsa myeloid e bontšitse ho monngoa ha tsoekere e ngata le ts'ebetso ea mitochondrial (Setšoantšo sa S2, B ho isa ho G). Liphetho tsena li totobatsa liphapang tse matla tsa metabolism lipakeng tsa mefuta e mengata ea lisele tse fumanoang ho ascites le lihlahala ho bakuli ba HGSC.
Bothata bo boholo ba ho utloisisa litšobotsi tsa metabonomic tsa TIL ke tlhoko ea ho arola lisampole tsa lisele tsa T tse hloekileng ka ho lekaneng, boleng le bongata ho tsoa lihlahala. Lithuto tsa morao tjena li bontšitse hore mekhoa ea ho hlophisa le ho ntlafatsa lifaha e thehiloeng ho cytometry ea phallo e ka lebisa liphetohong liprofaeleng tsa metabolite ea sele (22-24). Ho hlōla bothata bona, re ntlafalitse mokhoa oa ho ntlafatsa lifaha ho arola le ho arola TIL ho tsoa kankere ea mae a bomme ea motho e nkiloeng ka opereishene pele ho tlhahlobo ke LC-MS/MS (sheba Lisebelisoa le Mekhoa; Setšoantšo sa 2A). E le ho hlahloba tšusumetso e akaretsang ea protocol ena liphetohong tsa metabolite, re bapisitse liprofaele tsa metabolite tsa lisele tsa T tse butsoeng ke bafani ba phetseng hantle kamora mohato o kaholimo oa karohano ea lifaha le lisele tse neng li sa aroloa lifaha empa li ile tsa lula leqhoang. Tlhahlobo ena ea taolo ea boleng e fumane hore ho na le kamano e phahameng pakeng tsa maemo ana a mabeli (r = 0.77), 'me ho pheta-pheta ha botekgeniki ha sehlopha sa li-metabolite tse 86 ho na le ho pheta-pheta ho hoholo (Setšoantšo sa 2B). Ka hona, mekhoa ena e ka etsa tlhahlobo e nepahetseng ea metabolite liseleng tse ntseng li ntlafatsoa ka mofuta oa sele, kahoo e fana ka sethala sa pele sa qeto e phahameng bakeng sa ho khetholla metabolite e itseng ho HGSC, ka hona e nolofalletsa batho ho fumana kutloisiso e tebileng ea ho ikhetha ha sele Lenaneo la metabolism ea thobalano.
(A) Setšoantšo sa morero sa ho rua lifaha tsa makenete. Pele ho tlhahlobo ke LC-MS/MS, lisele li tla feta mekhahlelong e meraro e latellanang ea ho rua lifaha tsa makenete kapa li lule leqhoeng. (B) Tšusumetso ea mofuta oa ho rua bongata ba li-metabolite. Karolelano ea litekanyo tse tharo bakeng sa mofuta o mong le o mong oa ho rua ± SE. Mohala o moputsoa o emela kamano ea 1:1. Kamano ea ka hare ho sehlopha (ICC) ea litekanyo tse phetoang tse bontšitsoeng leiboleng la axis. NAD, nicotinamide adenine dinucleotide. (C) Setšoantšo sa morero sa mosebetsi oa tlhahlobo ea metabolite ea mokuli. Li-ascites kapa lihlahala li bokelloa ho bakuli 'me li bolokoa ka cryopreserved. Karolo e nyane ea sampole ka 'ngoe e ile ea hlahlojoa ke flow cytometry, ha lisampole tse setseng li ile tsa feta mekhahlelong e meraro ea ho rua bakeng sa lisele tsa CD4+, CD8+ le CD45. Likaroloana tsena tsa lisele li ile tsa hlahlojoa ho sebelisoa LC-MS/MS. (D) 'Mapa oa mocheso oa bongata ba metabolite bo tloaelehileng. Dendrogram e emela lihlopha tsa Ward tsa libaka tsa Euclidean pakeng tsa lisampole. (E) Tlhahlobo ea likarolo tse ka sehloohong (PCA) ea 'mapa oa sampole ea metabolite, e bonts'ang likopi tse tharo tsa sampole ka 'ngoe, lisampole tse tsoang ho mokuli a le mong li hokahantsoe ka mola. (F) PCA ea boemo ba metabolite ea sampole e behiloeng ho mokuli (ke hore, ho sebelisoa ho se sebelisoe ha karolo e itseng); mofuta oa sampole o lekanyelitsoe ke khetla e kobehileng. PC1, karolo e ka sehloohong ea 1; PC2, karolo e ka sehloohong ea 2.
Ka mor'a moo, re sebelisitse mokhoa ona oa ho ntlafatsa ho sekaseka li-metabolite tse 99 ho li-CD4+, CD8+ le likaroloana tsa lisele tsa CD45 ho li-ascites tsa mantlha le lihlahala tsa bakuli ba tšeletseng ba HGSC (Setšoantšo sa 2C, Setšoantšo sa S3A le Tafole ea S3 le S4). Palo ea batho ba thahasellang e etsa 2% ho isa ho 70% ea sampole e kholo ea pele ea lisele tse phelang, 'me karolo ea lisele e fapana haholo lipakeng tsa bakuli. Kamora ho arola lifaha, karolo e ruileng ea thahasello (CD4+, CD8+ kapa CD45-) e etsa ho feta 85% ea lisele tsohle tse phelang sampole ka karolelano. Mokhoa ona oa ho ntlafatsa o re lumella ho sekaseka palo ea lisele ho tsoa metabolism ea lisele tsa hlahala tsa batho, e leng ntho e ke keng ea etsoa ho tsoa lisampoleng tse kholo. Re sebelisa protocol ena, re fumane hore l-kynurenine le adenosine, li-metabolite tsena tse peli tse tsebahalang hantle tsa ho thibela immunosuppressive li ne li phahame liseleng tsa T tsa hlahala kapa liseleng tsa hlahala (Setšoantšo sa S3, B le C). Ka hona, liphetho tsena li bontša botšepehi le bokhoni ba karohano ea lisele tsa rona le theknoloji ea mass spectrometry ho fumana li-metabolite tsa bohlokoa tsa baeloji liseleng tsa bakuli.
Tlhahlobo ea rona e boetse e senotse karohano e matla ea metabolism ea mefuta ea lisele ka hare le pakeng tsa bakuli (Setšoantšo sa 2D le Setšoantšo sa S4A). Haholo-holo, ha ho bapisoa le bakuli ba bang, mokuli oa 70 o bontšitse litšobotsi tse fapaneng tsa metabolism (Setšoantšo sa 2E le Setšoantšo sa S4B), se bontšang hore ho ka ba le ho se tšoane ho hoholo ha metabolism pakeng tsa bakuli. Hoa hlokomeleha hore ha ho bapisoa le bakuli ba bang (lithara tse 1.2 ho isa ho tse 2; Tafole ea S1), kakaretso ea ascites e bokelletsoeng ho mokuli oa 70 (80 ml) e ne e le nyane. Taolo ea ho se tšoane ha bakuli nakong ea tlhahlobo ea likarolo tse ka sehloohong (mohlala, ho sebelisoa tlhahlobo ea ho se sebelisoe ha karolo) e bontša liphetoho tse tsitsitseng lipakeng tsa mefuta ea lisele, 'me mefuta ea lisele le/kapa tikoloho e nyane li bokelletsoe ka ho hlaka ho latela boemo ba metabolite (Setšoantšo sa 2F). Tlhahlobo ea metabolite e le 'ngoe e hatelitse liphello tsena 'me ea senola liphapang tse kholo lipakeng tsa mefuta ea lisele le tikoloho e nyane. Hoa hlokomeleha hore phapang e feteletseng e bonoang ke MNA, eo hangata e ruisitsoeng liseleng tsa CD45 le liseleng tsa CD4+ le CD8+ tse kenang ka har'a hlahala (Setšoantšo sa 3A). Bakeng sa lisele tsa CD4 +, phello ena e totobetse haholo, 'me MNA liseleng tsa CD8 + le eona e bonahala e angoa haholo ke tikoloho. Leha ho le joalo, sena ha se bohlokoa, hobane ke bakuli ba bararo feela ho ba tšeletseng ba ka hlahlojoang bakeng sa lintlha tsa hlahala ea CD8+. Ntle le MNA, mefuta e fapaneng ea lisele tse ascites le lihlahala, li-metabolite tse ling tse sa hlalosoang hantle ho TIL le tsona li ruile ka tsela e fapaneng (Litšoantšo S3 le S4). Ka hona, lintlha tsena li senola sete e tšepisang ea li-metabolite tsa immunomodulatory bakeng sa lipatlisiso tse ling.
(A) Dikahare tse tlwaelehileng tsa MNA ho disele tsa CD4+, CD8+ le CD45 tse tswang ho ascites le hlahala. Pontsho ya lebokose e bontsha bohareng (mola), mefuta ya di-interquartile (sehokelo sa foreimi) le mefuta ya data, ho fihlela ho makgetlo a 1.5 ho feta mefuta ya di-interquartile (foreimi ya whisker). Jwalo ka ha ho hlalositswe ho Disebediswa le Mekgwa ya Mokuli, sebedisa boleng ba limma ba mokuli ho fumana boleng ba P (*P<0.05 le **P<0.01). (B) Setšoantšo sa sekema sa metabolism ya MNA (60). Di-metabolite: S-adenosyl-1-methionine; SAH, S-adenosine-1-homocysteine; NA, nicotinamide; MNA, 1-methylnicotinamide; 2-PY, 1-methyl- 2-pyridone-5-carboxamide; 4-PY, 1-methyl-4-pyridone-5-carboxamide; NR, nicotinamide ribose; NMN, nicotinamide mononucleotide. Li-enzyme (tse tala): NNMT, nicotinamide N-methyltransferase; SIRT, sirtuins; NAMPT, nicotinamide phosphoribosyl transferase; AOX1, aldehyde oxidase 1; NRK, nicotinamide riboside kinase; NMNAT, nicotinamide mono Nucleotide adenylate transferase; Pnp1, purine nucleoside phosphorylase. (C) t-SNE ea scRNA-seq ea ascites (e putsoa) le hlahala (e khubelu; n = bakuli ba 3). (D) Ponahalo ea NNMT ho baahi ba fapaneng ba lisele tse khetholloang ho sebelisoa scRNA-seq. (E) Ponahalo ea NNMT le AOX1 ho SK-OV-3, liphio tsa lesea la embryonic (HEK) 293T, lisele tsa T le lisele tsa T tse phekoloang ke MNA. Ponahalo e mennoeng e bontšoa mabapi le SK-OV-3. Paterone ea polelo ka SEM e bontšoa (n = bafani ba 6 ba phetseng hantle). Boleng ba Ct bo fetang 35 bo nkoa bo sa fumanehe (UD). (F) Pontšo ea SLC22A1 le SLC22A2 ho SK-OV-3, HEK293T, lisele tsa T le lisele tsa T tse phekoloang ka 8mM MNA. Pontšo e mennoeng e bontšoa ha e bapisoa le SK-OV-3. Paterone ea polelo e nang le SEM e bontšoa (n = bafani ba 6 ba phetseng hantle). Boleng ba Ct bo fetang 35 bo nkoa bo sa bonahale (UD). (G) Boteng ba MNA ea sele liseleng tsa T tsa bafani ba phetseng hantle tse sebetsang ka mor'a lihora tse 72 tsa ho kenngoa ka MNA. Paterone ea polelo e nang le SEM e bontšoa (n = bafani ba 4 ba phetseng hantle).
MNA e hlahisoa ka ho fetisetsa sehlopha sa methyl ho tloha ho S-adenosyl-1-methionine (SAM) ho ea ho nicotinamide (NA) ka nicotinamide N-methyltransferase (NNMT; Setšoantšo sa 3B). NNMT e hlahisoa haholo mefuteng e fapaneng ea kankere ea batho 'me e amahanngoa le ho ata, ho hlaseloa le ho ata ha metastasis (25-27). Ho utloisisa hamolemo mohloli oa MNA liseleng tsa T ho TME, re sebelisitse scRNA-seq ho hlalosa polelo ea NNMT ho pholletsa le mefuta ea lisele ho ascites le lihlahala tsa bakuli ba bararo ba HGSC (Tafole ea S5). Tlhahlobo ea lisele tse ka bang 6,500 e bontšitse hore libakeng tsa ascites le hlahala, polelo ea NNMT e ne e lekanyelitsoe ho baahi ba fibroblast le lisele tsa hlahala tse nahanoang (Setšoantšo sa 3, C le D). Hoa bohlokoa ho hlokomela hore ha ho na polelo e totobetseng ea NNMT ho baahi leha e le bafe e hlahisang PTPRC (CD45 +) (Setšoantšo sa 3D le Setšoantšo sa S5A), e leng se bontšang hore MNA e fumanoeng ho metabolite spectrum e kentsoe liseleng tsa T. Pontšo ea aldehyde oxidase 1 (AOX1) e fetola MNA hore e be 1-methyl-2-pyridone-5-carboxamide (2-PYR) kapa 1-methyl-4-pyridone-5-carboxamide (4-PYR); Setšoantšo sa 3B) se boetse se lekanyelitsoe ho palo ea li-fibroblast tse hlahisang COL1A1 (Setšoantšo sa S5A), tseo hammoho li bontšang hore lisele tsa T ha li na bokhoni ba metabolism ea MNA e tloaelehileng. Mokhoa oa polelo oa liphatsa tsena tse amanang le MNA o netefalitsoe ho sebelisoa sete ea bobeli ea data ea sele e ikemetseng ho tsoa ho ascites ho bakuli ba HGSC (Setšoantšo sa S5B; n = 6) (16). Ho phaella moo, tlhahlobo ea quantitative polymerase chain reaction (qPCR) ea lisele tsa T tse phetseng hantle tse fuoang bafani ba phekoloang ka MNA e bontšitse hore ha ho bapisoa le lisele tsa hlahala ea ovary tsa SK-OV-3 tse laoloang, NNMT kapa AOX1 li ne li batla li sa hlahisoa (Setšoantšo sa 3E). Liphetho tsena tse sa lebelloang li bontša hore MNA e ka 'na ea ntšoa ho tsoa ho li-fibroblast kapa lihlahala ho ea ho lisele tsa T tse haufi ho TME.
Leha bakhethoa ba kenyeletsa lelapa la li-transporter tsa cation tsa tlhaho 1 ho isa ho 3 (OCT1, OCT2 le OCT3) tse ngotsoeng ke lelapa la li-transporter tse qhibilihang tsa 22 (SLC22) (SLC22A1, SLC22A2 le SLC22A3), li-transporter tse ka bang teng tsa MNA li ntse li sa hlalosoa (28). QPCR ea mRNA e tsoang liseleng tsa T tsa mofani tse phetseng hantle e bontšitse maemo a tlase a tlhahiso ea SLC22A1 empa maemo a sa bonahaleng a SLC22A2, a tiisitseng hore e ne e tlalehiloe pele libukeng (Setšoantšo sa 3F) (29). Ka lehlakoreng le leng, mola oa sele ea hlahala ea ovary ea SK-OV-3 o bontšitse maemo a holimo a li-transporter ka bobeli (Setšoantšo sa 3F).
E le ho leka monyetla oa hore lisele tsa T li be le bokhoni ba ho monya MNA ea kantle ho naha, lisele tsa T tse fanang ka bophelo bo botle li ile tsa lengoa lihora tse 72 moo ho nang le mahloriso a fapaneng a MNA. Ha ho se na MNA ea kantle ho naha, litaba tsa sele tsa MNA li ke ke tsa bonoa (Setšoantšo sa 3G). Leha ho le joalo, lisele tsa T tse sebelisitsoeng tse phekoloang ka MNA ea kantle ho naha li bontšitse keketseho e itšetlehileng ka tekanyo ea litaba tsa MNA liseleng, ho fihlela ho 6 mM MNA (Setšoantšo sa 3G). Sephetho sena se bontša hore ho sa tsotellehe boemo bo tlase ba tlhahiso ea transporter le khaello ea enzyme e ka sehloohong e ikarabellang bakeng sa metabolism ea MNA ea ka hare ho sele, TIL e ntse e ka nka MNA.
Mefuta e fapaneng ea li-metabolite liseleng tsa T tsa bakuli le litekong tsa ho monya MNA ka har'a vitro li eketsa monyetla oa hore li-fibroblast tse amanang le kankere (CAF) li ntše MNA 'me lisele tsa hlahala li ka laola phenotype le ts'ebetso ea TIL. Ho fumana phello ea MNA liseleng tsa T, lisele tsa T tse fanang ka bophelo bo botle li ile tsa kenngoa tšebetsong ka har'a vitro boteng kapa bo le sieo ba MNA, 'me keketseho ea tsona le tlhahiso ea cytokine li ile tsa hlahlojoa. Kamora matsatsi a 7 a ho eketsa MNA ka tekanyo e phahameng ka ho fetisisa, palo ea baahi ba imena habeli e ile ea fokotseha ka mokhoa o itekanetseng, ha matla a ne a bolokoa litekanyetsong tsohle (Setšoantšo sa 4A). Ho phaella moo, kalafo ea MNA ea kantle ho naha e ile ea fella ka keketseho ea karolo ea lisele tsa CD4 + le CD8 + T tse hlahisang tumor necrosis factor-α (TNFα; Setšoantšo sa 4B). Ka lehlakoreng le leng, tlhahiso ea IFN-γ ka hare ho sele e ile ea fokotseha haholo liseleng tsa CD4 + T, empa eseng liseleng tsa CD8 + T, 'me ho ne ho se na phetoho e kholo ho interleukin 2 (IL-2; Setšoantšo sa 4, C le D). Ka hona, tlhahlobo ea immunosorbent e kopantsoeng le enzyme (ELISA) ea li-supernatants tse tsoang litsong tsena tsa lisele tsa T tse phekoloang ke MNA e bontšitse keketseho e kholo ho TNFα, phokotso ea IFN-γ, le phetoho ho IL-2 (Setšoantšo sa 4, E ho isa ho G). . Phokotso ea IFN-γ e bontša hore MNA e ka bapala karolo ho thibeleng ts'ebetso ea lisele tsa T khahlanong le hlahala. E le ho etsisa phello ea MNA ho cytotoxicity e bakoang ke lisele tsa T, lisele tsa chimeric antigen receptor T (FRα-CAR-T) tse shebaneng le folate receptor α le CAR-T (GFP) tse laoloang ke lisele tsa protheine e tala ea fluorescent (GFP) -CAR-T li hlahisoa ke lisele tse phetseng hantle tsa mononuclear tsa mali tse fanang ka mali (PBMC). Lisele tsa CAR-T li ile tsa lengoa lihora tse 24 boteng ba MNA, ebe li lengoa hammoho le lisele tsa hlahala ea ovary tsa SK-OV-3 tsa batho tse hlahisang folate receptor α ka karolelano ea effector ho ea ho sepheo sa 10:1. Kalafo ea MNA e bakile phokotso e kholo ts'ebetsong ea ho bolaea ea lisele tsa FRα-CAR-T, e neng e tšoana le lisele tsa FRα-CAR-T tse phekoloang ka adenosine (Setšoantšo sa 4H).
(A) Palo yohle ya disele tse sebetsang le ho imena habeli ha palo ya batho (PD) ka kotloloho ho tswa ho setso letsatsing la 7. Grafo ya bare e emela karolelano + SEM ya bafani ba tsheletseng ba phetseng hantle. E emela data ho tswa bonyane ditekong tse ikemetseng tsa n = 3. (B ho isa ho D) CD3/CD28 le IL-2 di sebediseditswe ho kenya tshebetsong disele tsa T maemong a tsona a MNA ka matsatsi a 7. Pele ho tlhahlobo, disele di ile tsa hlasimollwa ka PMA/ionomycin ka GolgiStop ka dihora tse 4. Ponahalo ya TNFα (B) diseleng tsa T. Setšoantšo sa mohlala (ka letsohong le letshehadi) le data ya tafole (ka letsohong le letona) ya ponahalo ya TNFα diseleng tse phelang. Ponahalo ya IFN-γ (C) le IL-2 (D) diseleng tsa T. Ponahalo ya di-cytokine e lekantswe ka cytometry ya phallo. Grafo ya bare e emela karolelano (n = bafani ba phetseng hantle ba 6) + SEM. Sebedisa tlhahlobo ya tsela e le nngwe ya phapang le mehato e phetoang (*P<0.05 le **P<0.01) ho fumana boleng ba P. E emela data ho tswa bonyane ditekong tse ikemetseng tsa n = 3. (E ho isa ho G) CD3/CD28 le IL-2 li sebelisitsoe ho kenya tšebetsong lisele tsa T maemong a tsona a MNA ka matsatsi a 7. Sejana se ile sa bokelloa pele le ka mor'a lihora tse 4 tsa ho hlohlelletsa PMA/ionomycin. Maemo a TNFα (E), IFN-γ (F) le IL-2 (G) a lekantsoe ke ELISA. Grafo ea bare e emela karolelano (n = bafani ba 5 ba phetseng hantle) + SEM. Boleng ba P bo fumanoe ka ho sebelisa tlhahlobo ea tsela e le 'ngoe ea phapang le litekanyo tse phetoang (*P<0.05). Mola o nang le matheba o bontša moeli oa ho lemoha oa ho lemoha. (H) Teko ea lysis ea sele. Lisele tsa FRα-CAR-T kapa GFP-CAR-T li ile tsa fetoloa ka adenosine (250μM) kapa MNA (10 mM) ka lihora tse 24, kapa tsa tloheloa li sa phekoloa (Ctrl). Ho bolaoa ha peresente ea lisele tsa SK-OV-3 ho ile ha lekanngoa. Boleng ba P bo khethiloe ke teko ea Welch t (*P<0.5 le **P<0.01).
E le ho fumana kutlwisiso ya botekgeniki ya taolo ya tlhahiso ya TNFα e itshetlehileng ho MNA, diphetoho ho TNFα mRNA ya disele tsa T tse phekolwang ke MNA di ile tsa lekolwa (Setshwantsho sa 5A). Disele tsa T tse phetseng hantle tse phekolwang ka MNA di bontshitse keketseho e habeli maemong a phetolelo ya TNFα, e leng se bontshang hore MNA e itshetlehile hodima taolo ya phetolelo ya TNFα. Ho fuputsa mokgwa ona o ka bang teng wa taolo, dintlha tse pedi tse tsejwang tsa phetolelo tse laolang TNFα, e leng T cell nuclear factor (NFAT) e kentsweng tshebetsong le protheine e ikgethang ya 1 (Sp1), di ile tsa lekolwa ho arabela ho tlamahaneng ha MNA le promoter ya TNFα e haufi (30). Promoter ya TNFα e na le dibaka tse 6 tse hlwailweng tsa ho tlamahane ha NFAT le dibaka tse 2 tsa ho tlamahane ha Sp1, tse tshwanang sebakeng se le seng [-55 base pairs (bp) ho tloha ho 5'cap] (30). Chromatin immunoprecipitation (ChIP) e bontshitse hore ha e phekolwa ka MNA, ho tlamahane ha Sp1 le promoter ya TNFα ho ile ha eketseha hararo. Ho kenngwa ha NFAT le hona ho ile ha eketseha mme ha atamela bohlokwa (Setshwantsho sa 5B). Lintlha tsena li bontša hore MNA e laola tlhahiso ea TNFα ka phetolelo ea Sp1, 'me ka tekanyo e nyane tlhahiso ea NFAT.
(A) Ha ho bapisoa le lisele tsa T tse lengoang ntle le MNA, phetoho ea ho mena ha tlhahiso ea TNFα liseleng tsa T tse phekoloang ka MNA. Paterone ea tlhahiso ka SEM e bontšoa (n = bafani ba 5 ba phetseng hantle). E emela lintlha tse tsoang bonyane litekong tse ikemetseng tsa n = 3. (B) Mothusi oa TNFα oa lisele tsa T tse phekoloang ka kapa ntle le tsona tsa 8 mM MNA kamora NFAT le Sp1 li kopantsoe le (Ctrl) le PMA/ionomycin stimulation ka lihora tse 4. Immunoglobulin G (IgG) le H3 li sebelisitsoe e le taolo e mpe le e ntle bakeng sa immunoprecipitation, ka ho latellana. Tekanyo ea ChIP e bontšitse hore ho tlama ha Sp1 le NFAT ho mothusi oa TNFα liseleng tse phekoloang ka MNA ho eketsehile ka makhetlo a 'maloa ha ho bapisoa le taolo. E emela lintlha tse tsoang bonyane litekong tse ikemetseng tsa n = 3. Boleng ba P bo khethoa ke liteko tse ngata tsa t (*** P <0.01). (C) Ha ho bapisoa le ascites ea HGSC, lisele tsa T (tse seng tsa cytotoxic) li bontšitse keketseho ea tlhahiso ea TNF hlahaleng. Mebala e emela bakuli ba fapaneng. Lisele tse bontšitsoeng li se li hlahlobiloe ka mokhoa o sa reroang ho 300 'me li thothometsoa ho fokotsa ho hula ho feteletseng (** Padj = 0.0076). (D) Mohlala o reriloeng oa MNA bakeng sa kankere ea mahe a bomme. MNA e hlahisoa liseleng tsa hlahala le li-fibroblast ho TME 'me e nkuoa ke lisele tsa T. MNA e eketsa tlamo ea Sp1 ho promoter ea TNFα, e leng se lebisang keketsehong ea tlhahiso ea TNFα le tlhahiso ea cytokine ea TNFα. MNA e boetse e baka phokotso ea IFN-γ. Thibelo ea ts'ebetso ea sele ea T e lebisa ho fokotseng bokhoni ba ho bolaea le kholo e potlakileng ea hlahala.
Ho ea ka litlaleho, TNFα e na le litlamorao tse khahlanong le hlahala le tse khahlanong le hlahala tse itšetlehileng ka pele le ka morao, empa e na le karolo e tsebahalang haholo ho ntšetseng pele kholo le metastasis ea kankere ea mae a bomme (31-33). Ho ea ka litlaleho, mahloriso a TNFα ho ascites le lisele tsa hlahala ho bakuli ba nang le kankere ea mae a bomme a phahame ho feta a liseleng tse se nang kotsi (34-36). Mabapi le mokhoa oa ho sebetsa, TNFα e ka laola ts'ebetso, ts'ebetso le ho ata ha lisele tse tšoeu tsa mali, 'me ea fetola mofuta oa lisele tsa kankere (37, 38). Ho latela liphuputso tsena, tlhahlobo ea polelo ea liphatsa tsa lefutso e fapaneng e bontšitse hore TNF e ne e ntlafalitsoe haholo liseleng tsa T liseleng tsa hlahala ha e bapisoa le ascites (Setšoantšo sa 5C). Keketseho ea polelo ea TNF e ne e bonahala feela ho baahi ba lisele tsa T ba nang le phenotype e seng ea cytotoxic (Setšoantšo sa S5A). Ka bokhutšoanyane, lintlha tsena li tšehetsa pono ea hore MNA e na le litlamorao tse peli tsa ho thibela sesole sa 'mele le tse khothalletsang hlahala ho HGSC.
Ho ngola mabitso a fluorescent ho latela cytometry ea phallo e se e le mokhoa o ka sehloohong oa ho ithuta metabolism ea TIL. Lithuto tsena li bontšitse hore ha li bapisoa le li-lymphocyte tsa mali tse tsoang lithong tsa lymphoid tsa bobeli, li-murine le TIL tsa batho li na le tšekamelo e phahameng ea ho monya tsoekere (4, 39) le tahlehelo e fokolang ea ts'ebetso ea mitochondrial (19, 40). Leha re bone liphetho tse tšoanang thutong ena, nts'etsopele ea bohlokoa ke ho bapisa metabolism ea lisele tsa hlahala le TIL ho tsoa liseleng tse tšoanang tsa hlahala tse khaotsoeng. Ho latela tse ling tsa litlaleho tsena tse fetileng, lisele tsa hlahala (CD45-EpCAM +) tse tsoang ho ascites le lihlahala li na le ho monya tsoekere e ngata ho feta lisele tsa CD8 + le CD4 + T, tse tšehetsang hore ho monya tsoekere e ngata ha lisele tsa hlahala ho ka bapisoa le lisele tsa T. Khopolo ea tlholisano ea lisele tsa T. TME. Leha ho le joalo, ts'ebetso ea mitochondrial ea lisele tsa hlahala e phahame ho feta ea lisele tsa CD8 + T, empa ts'ebetso ea mitochondrial e tšoana le ea lisele tsa CD4 + T. Liphetho tsena li tiisa sehlooho se hlahang sa hore metabolism ea oxidative e bohlokoa bakeng sa lisele tsa hlahala (41, 42). Hape ba fana ka maikutlo a hore lisele tsa CD8 + T li ka 'na tsa e-ba kotsing e kholo ea ho se sebetse hantle ha oxidative ho feta lisele tsa CD4 + T, kapa hore lisele tsa CD4 + T li ka sebelisa mehloli ea khabone ntle le glucose ho boloka ts'ebetso ea mitochondrial (43, 44). Ho lokela ho hlokomeloa hore ha rea ​​​​bona phapang ho nkiloeng ha glucose kapa ts'ebetso ea mitochondrial pakeng tsa li-effector tsa CD4 + T, memori ea T effector le lisele tsa memori tsa T tse bohareng ho ascites. Ka mokhoa o ts'oanang, boemo ba phapang ba lisele tsa CD8 + T ho lihlahala ha bo amane le liphetoho ho nkiloeng ha glucose, bo totobatsang phapang e kholo lipakeng tsa lisele tsa T tse lengoang ka har'a vitro le TIL ea motho ka har'a vivo (22). Lintho tsena tse hlokometsoeng li boetse li tiisitsoe ke ts'ebeliso ea kabo ea palo ea lisele tse ikemetseng e sa nke lehlakore, e ileng ea boela ea senola hore lisele tsa CD45 + / CD3- / CD4 + / CD45RO + tse nang le ho nkuoa ha glucose e ngata le ts'ebetso ea mitochondrial ho feta lisele tsa hlahala li atile empa li na le palo ea lisele tse sebetsang tsa Metabolic. Palo ena ea batho e ka emela palo e nyane ea lisele tsa myeloid suppressor kapa lisele tsa plasmacytoid dendritic tse khethiloeng tlhahlobong ea scRNA-seq. Leha tsena ka bobeli li tlalehiloe lihlahleng tsa mahe a batho [45], li ntse li hloka mosebetsi o mong hape ke ho hlalosa palo ena e nyane ea myeloid.
Leha mekhoa e thehiloeng ho flow cytometry e ka hlakisa liphapang tse akaretsang tsa glucose le metabolism ea oxidative lipakeng tsa mefuta ea lisele, li-metabolite tse nepahetseng tse hlahisoang ke glucose kapa mehloli e meng ea carbon bakeng sa metabolism ea mitochondrial ho TME ha li so fumanoe. Ho abela boteng kapa ho ba sieo ha li-metabolite ho sehlopha se fanoeng sa TIL ho hloka tlhoekiso ea palo ea lisele ho tsoa liseleng tse ntšitsoeng. Ka hona, mokhoa oa rona oa ho ntlafatsa lisele o kopantsoeng le mass spectrometry o ka fana ka temohisiso mabapi le li-metabolite tse ruisitsoeng ka tsela e fapaneng liseleng tsa T le palo ea lisele tsa hlahala lisampoleng tse tsamaellanang tsa bakuli. Le hoja mokhoa ona o na le melemo ho feta ho hlopha lisele tse sebetsang ka fluorescence, lilaebrari tse ling tsa metabolite li ka angoa ke botsitso ba tlhaho le/kapa sekhahla se potlakileng sa phetoho (22). Leha ho le joalo, mokhoa oa rona o ile oa khona ho khetholla li-metabolite tse peli tse tsebahalang tsa ho thibela immunosuppressive, adenosine le kynurenine, hobane li fapana haholo lipakeng tsa mefuta ea sampole.
Tlhahlobo ea rona ea metabonomic ea lihlahala le mefuta e fapaneng ea TIL e fana ka temohisiso e batsi ka karolo ea li-metabolite ho TME ea ovarian. Taba ea pele, re sebelisa flow cytometry, re fumane hore ho ne ho se na phapang mosebetsing oa mitochondrial pakeng tsa lihlahala le lisele tsa CD4 + T. Leha ho le joalo, tlhahlobo ea LC-MS/MS e senotse liphetoho tse kholo bongata ba li-metabolite har'a baahi bana, e leng se bontšang hore liqeto mabapi le metabolism ea TIL le ts'ebetso ea eona ea metabolism ka kakaretso li hloka tlhaloso e hlokolosi. Ea bobeli, MNA ke metabolite e nang le phapang e kholo ka ho fetisisa pakeng tsa lisele tsa CD45 le lisele tsa T ho ascites, eseng lihlahala. Ka hona, karohano le sebaka sa hlahala li ka ba le litlamorao tse fapaneng ho metabolism ea TIL, e leng se totobatsang ho se tšoane ho ka bang teng tikolohong e itseng e nyenyane. Ea boraro, polelo ea enzyme e hlahisang MNA NNMT e lekanyelitsoe haholo ho CAF, e leng lisele tsa hlahala ka tekanyo e nyane, empa maemo a MNA a ka bonoang a bonoa liseleng tsa T tse nkiloeng ke hlahala. Ho pepeseha ho feteletseng ha NNMT ho CAF ea ovarian ho na le phello e tsebahalang ea ho khothalletsa kankere, karolo e 'ngoe e bakoa ke ho khothalletsa metabolism ea CAF, tlhaselo ea hlahala le metastasis (27). Leha boemo bohle ba TIL bo le boemong bo itekanetseng, tlhahiso ea NNMT ho CAF e amana haufi-ufi le mofuta oa mesenchymal oa Cancer Genome Atlas (TCGA), o amanang le tebello e mpe ea lefu la tsoekere (27, 46, 47). Qetellong, tlhahiso ea enzyme AOX1 e ikarabellang bakeng sa ho senyeha ha MNA le eona e lekanyelitsoe ho baahi ba CAF, e leng se bontšang hore lisele tsa T ha li na bokhoni ba ho fetola MNA. Liphetho tsena li tšehetsa khopolo ea hore leha ho hlokahala mosebetsi o mong ho netefatsa phumano ena, maemo a phahameng a MNA liseleng tsa T a ka bontša boteng ba tikoloho e nyenyane ea CAF e thibelang sesole sa 'mele.
Ka lebaka la boemo bo tlase ba tlhahiso ea li-transporter tsa MNA le maemo a sa bonahaleng a liprotheine tsa bohlokoa tse amehang metabolism ea MNA, boteng ba MNA liseleng tsa T ha boa lebelloa. NNMT kapa AOX1 ha lia ka tsa fumanoa ka tlhahlobo ea scRNA-seq le qPCR e reretsoeng lihlopha tse peli tse ikemetseng. Liphetho tsena li bontša hore MNA ha e bōptjoe ke lisele tsa T, empa e monngoa ho tsoa ho TME e potolohileng. Liteko tsa in vitro li bontša hore lisele tsa T li atisa ho bokella MNA ea kantle.
Lithuto tsa rona tsa in vitro li bontšitse hore MNA ea kantle e susumetsa tlhahiso ea TNFα liseleng tsa T mme e ntlafatsa ho tlamahana ha Sp1 ho promoter ea TNFα. Le hoja TNFα e na le mesebetsi ea ho loantša hlahala le ho loantša hlahala, kankereng ea mahe a bomme, TNFα e ka khothalletsa kholo ea kankere ea mahe a bomme (31-33). Ho fokotsa TNFα setsong sa lisele tsa hlahala ea mahe a bomme kapa ho felisoa ha lets'oao la TNFα mehlaleng ea litoeba ho ka ntlafatsa tlhahiso ea cytokine e ruruhang e tsamaisoang ke TNFα le ho thibela kholo ea hlahala (32, 35). Ka hona, tabeng ena, MNA e tsoang ho TME e ka sebetsa e le metabolite e khothalletsang ho ruruha ka mokhoa o itšetlehileng ka TNFα ka loop ea autocrine, ka hona e khothalletsa ho hlaha le ho ata ha kankere ea mahe a bomme (31). Ho latela monyetla ona, thibelo ea TNFα e ntse e ithutoa e le moemeli ea ka phekolang kankere ea mahe a bomme (37, 48, 49). Ho phaella moo, MNA e senya chefo ea lisele tsa CAR-T ho lisele tsa hlahala ea mahe a bomme, e fana ka bopaki bo eketsehileng ba ho hatelloa ha 'mele ke MNA. Ka kakaretso, liphetho tsena li fana ka maikutlo a mohlala oo lihlahala le lisele tsa CAF li ntšang MNA ho TME e ka ntle ho lisele. Ka (i) tlhohlelletso ea kholo ea kankere ea mae a bomme e bakoang ke TNF le (ii) thibelo ea ts'ebetso ea cytotoxic ea lisele tsa T tse bakoang ke MNA, sena se ka ba le phello e habeli ea hlahala (Setšoantšo sa 5D).
Qetellong, ka ho sebelisa motsoako oa ntlafatso ea lisele tse potlakileng, tatellano ea sele e le 'ngoe le tlhahlobo ea metabolism, phuputso ena e senotse liphapang tse kholo tsa immunometabolomic lipakeng tsa lihlahala le lisele tsa ascites ho bakuli ba HGSC. Tlhahlobo ena e felletseng e bontšitse hore ho na le liphapang tsa ho monngoa ha tsoekere le ts'ebetso ea mitochondrial pakeng tsa lisele tsa T, 'me ea khetholla MNA e le metabolite e ikemetseng ea taolo ea' mele e seng ea sele. Lintlha tsena li na le tšusumetso ea hore na TME e ama metabolism ea sele ea T joang mofetšeng oa batho. Leha tlholisano e tobileng ea limatlafatsi lipakeng tsa lisele tsa T le lisele tsa mofetše e tlalehiloe, metabolites e ka boela ea sebetsa e le balaoli ba sa tobang ho khothaletsa tsoelo-pele ea hlahala le mohlomong ho thibela likarabo tsa 'mele tsa ho itšireletsa mafung. Tlhaloso e eketsehileng ea karolo ea ts'ebetso ea metabolites tsena tsa taolo e ka bula maano a mang a ho ntlafatsa karabelo ea 'mele ea ho itšireletsa mafung khahlanong le hlahala.
Disampole tsa bakuli le tlhahisoleseding ya bongaka di fumanwe ka polokelo ya dinama tsa kankere ya BC e netefaditsweng ke Canadian Tissue Repository Network. Ho ya ka protocol e amohetsweng ke Komiti ya Boitshwaro ya Dipatlisiso tsa Kankere ya BC le Univesithi ya British Columbia (H07-00463), disampole tsohle tsa bakudi le tlhahisoleseding ya bongaka di fumane tumello e ngotsweng e nang le tsebo kapa di ile tsa tlohela tumello ya tsona semmuso. Disampole di bolokilwe ho BioBank e netefaditsweng (BRC-00290). Dibopeho tse qaqileng tsa bakudi di bontshitswe ho Ditafole S1 le S5. Bakeng sa ho boloka ka metsi, scalpel e sebediswa ho ntsha sampole ya hlahala ya mokuli ka mokgwa wa mechine ebe e e sutumelletsa ka hara sefe ya 100-micron ho fumana sele e le nngwe e emisitsweng. Ascites ya mokuli e ile ya kenngwa ka centrifuge ka 1500 rpm metsotso e 10 ho 4°C ho kenya disele le ho tlosa supernatant. Lisele tse fumanoeng ho tsoa ho hlahala le ascites li ile tsa bolokoa ka har'a serum ea AB ea motho e sa sebetseng ka mocheso ea 50% (Sigma-Aldrich), 40% RPMI-1640 (Thermo Fisher Scientific) le 10% dimethyl sulfoxide. Li-suspension tsena tsa sele e le 'ngoe tse bolokiloeng li ile tsa qhibilihisoa 'me tsa sebelisoa bakeng sa metabolomics le ho fumana metabolite tse hlalositsoeng ka tlase.
Sesebediswa se felletseng se na le 0.22 μm e sefilweng ka 50:50 e tlatselletsoang RPMI 1640: AimV. RPMI 1640 + 2.05 mM l-glutamine (Thermo Fisher Scientific) e tlatselletsoang ka 10% serum ea AB ea motho e sa sebetseng ka mocheso (Sigma-Aldrich), 12.5 mM Hepes (Thermo Fisher Scientific), 2 mM l-glutamine (Thermo Fisher Scientific) Fisher Scientific), 1 x Penicillin Streptomycin (PenStrep) tharollo (Thermo Fisher Scientific) le 50 μMB-mercaptoethanol. AimV (Invitrogen) e tlatselletsoa ka 20 mM Hepes (Thermo Fisher Scientific) le 2 mM l-glutamine (Thermo Fisher Scientific). Flow cytometer staining buffer e ne e entsoe ka 0.22μm filtered phosphate buffered saline (PBS; Invitrogen) e tlatselletsoang ka 3% heat-inactivated AB human serum (Sigma). Sesebediswa sa ho ntlafatsa disele se entswe ka PBS e sefilweng ya 0.22μm mme se tlatselletswa ka serum ya AB ya motho e sa kentsweng mochesong ya 0.5% (Sigma-Aldrich).
Ka mocheso o felletseng oa 37°C, lisele li ile tsa dauoa ka 10 nM MT DR le 100 μM 2-NBDG metsotso e 30. Ka mor'a moo, lisele li ile tsa dauoa ka dye eF506 ea ho phela ho 4°C metsotso e 15. Hlakola lisele hape ho FC Block (eBioscience) le Brilliant Stain Buffer (BD Biosciences), li qhibilihe ka har'a flow cytometer staining buffer (ho ea ka litaelo tsa moetsi), 'me u li futhumale metsotso e 10 mochesong oa kamore. Hlakola lisele ka sete ea li-antibodies (Tafole S2) ho flow cytometry staining buffer ho 4°C metsotso e 20. Hlakola lisele hape ho flow cytometry staining buffer (Cytek Aurora; 3L-16V-14B-8R configuration) pele ho tlhahlobo. Sebelisa SpectroFlo le FlowJo V10 ho sekaseka data ea palo ea lisele, 'me u sebelise GraphPad Prism 8 ho theha data. Matla a bohareng ba khanya ea leseli (MFI) ea 2-NBDG le MT DR a ile a fetoloa hore e be a tloaelehileng, 'me teko ea t e kopantsoeng e ile ea sebelisoa bakeng sa tlhahlobo ea lipalo-palo ho ikarabella bakeng sa bakuli ba tšoanang. Tlosa baahi bohle ba nang le liketsahalo tse ka tlase ho 40 tlhahlobong; kenya boleng ba MFI ba 1 bakeng sa boleng bofe kapa bofe bo fosahetseng pele u etsa tlhahlobo ea lipalo-palo le pono ea data.
E le ho tlatsetsa leano la ho kenya ka letsoho la phanele ea ts'ebetso e kaholimo, re sebelisitse tlhaloso e felletseng ea sefate sa thibelo ea sebopeho (FAUST) (21) ho abela lisele ho baahi ka bohona kamora ho felisa lisele tse shoeleng ho FlowJo. Re laola tlhahiso ka letsoho ho kopanya baahi ba bonahalang ba sa aroloa hantle (ho kopanya PD1+ le lisele tsa hlahala tsa PD1) le baahi ba bolokiloeng. Sampole ka 'ngoe e na le karolelano ea lisele tse fetang 2%, bakeng sa baahi ba 11 kaofela.
Ho ile ha sebelisoa Ficoll gradient density centrifugation ho arola PBMC ho lihlahisoa tsa karohano ea leukocyte (STEMCELL Technologies). Lisele tsa CD8 + T li ile tsa aroloa ho PBMC ho sebelisoa CD8 MicroBeads (Miltenyi) 'me tsa atolosoa ka mokhoa o felletseng ho sebelisoa TransAct (Miltenyi) libeke tse 2 ho latela litaelo tsa moetsi. Lisele li ile tsa lumelloa ho ema matsatsi a 5 ka mokhoa o felletseng o nang le IL-7 (10 ng/ml; PeproTech), ebe li tsosoa hape ka TransAct. Ka letsatsi la 7, ho latela litaelo tsa moetsi, li-CD45 MicroBeads tsa batho (Miltenyi) li ile tsa sebelisoa ho ntlafatsa lisele ka mekhahlelo e meraro e latellanang. Lisele li ile tsa aroloa bakeng sa tlhahlobo ea cytometry ea phallo (joalokaha ho hlalositsoe kaholimo), 'me lisele tse milione li ile tsa aroloa ka makhetlo a mararo bakeng sa tlhahlobo ea LC-MS/MS. Lisampole li ile tsa sebetsoa ke LC-MS/MS joalo ka ha ho hlalositsoe ka tlase. Re hakantse boleng ba metabolite bo sieo ka palo ea ion ea 1,000. Sampole ka 'ngoe e etsoa hore e be ea maemo a tloaelehileng ka palo ea li-ion kaofela (TIC), e fetoloa ka logarithmic le ho etsoa hore e be ea maemo a tloaelehileng ka bo eona ho MetaboAnalystR pele ho tlhahlobo.
Sele e le 'ngoe e emisitsoeng ea mokuli ka mong e ile ea qhibilihisoa 'me ea sefshoa ka sefe ea 40 μm ho ea seaplane se felletseng (joalokaha ho hlalositsoe kaholimo). Ho ea ka protocol ea moetsi, mekhahlelo e meraro e latellanang ea khetho e ntle ka karohano ea lifaha tsa makenete ho sebelisoa MicroBeads (Miltenyi) e sebelisitsoe ho ntlafatsa lisampole bakeng sa lisele tsa CD8+, CD4+ le CD45 (leqhoeng). Ka bokhutšoanyane, lisele li emisoa hape ka har'a buffer ea ntlafatso ea lisele (joalokaha ho hlalositsoe kaholimo) 'me li baloa. Lisele li ile tsa koaheloa ka lifaha tsa CD8 tsa batho, lifaha tsa CD4 tsa batho kapa lifaha tsa CD45 tsa batho (Miltenyi) mochesong oa 4°C metsotso e 15, ebe li hlatsuoa ka buffer ea ntlafatso ea lisele. Sampole e fetisoa ka har'a kholomo ea LS (Miltenyi), 'me likaroloana tse ntle le tse mpe lia bokelloa. E le ho fokotsa nako le ho eketsa mohato oa ho hlaphoheloa ha sele, karoloana ea CD8 e sebelisoa bakeng sa potoloho ea bobeli ea ntlafatso ea CD4+, 'me karoloana ea CD4 e sebelisoa bakeng sa ntlafatso ea CD45 e latelang. Boloka tharollo leqhoeng nakong eohle ea ts'ebetso ea karohano.
Ho lokisa disampole bakeng sa tlhahlobo ya metabolite, disele di ile tsa hlatsuwa hang ka tharollo ya letswai le batang, mme 1 ml ya 80% ya methanol e ile ya eketswa sampoleng ka nngwe, yaba e ntshwa ka mouoane mme ya hoamiswa ka naetrojene e metsi. Disampole di ile tsa kenngwa ka dipotoloho tse tharo tsa ho qhibilihisa le ho qhibilihisa mme tsa ntshwa ka sekontiri ka lebelo la 14,000 rpm metsotso e 15 ho 4°C. Supernatant e nang le metabolite e ntshwa mouoane ho fihlela e omme. Di-metabolite di ile tsa qhibilihiswa hape ka 50 μl ya 0.03% formic acid, tsa ntshwa ka sekontiri ho kopanya, mme tsa ntshwa ka sekontiri ho tlosa ditshila.
Ntša metabolites joalo ka ha ho hlalositsoe kaholimo. Fetisetsa supernatant botlolong ea chromatography ea metsi e sebetsang hantle bakeng sa lipatlisiso tsa metabolomics. Sebelisa protocol ea kalafo e sa reroang ho phekola sampole ka 'ngoe ka palo e tšoanang ea lisele ho thibela litlamorao tsa sehlopha. Re entse tlhahlobo ea boleng ba metabolites ea lefats'e e phatlalalitsoeng pele ho AB SCIEX QTRAP 5500 Triple Quadrupole Mass Spectrometer (50). Tlhahlobo ea chromatographic le kopanyo ea sebaka se phahameng li entsoe ho sebelisoa software ea MultiQuant version 2.1 (Applied Biosystems SCIEX).
Palo ea li-ion tse 1000 e sebelisitsoe ho hakanya boleng ba metabolite bo sieo, 'me TIC ea sampole ka 'ngoe e sebelisitsoe ho bala sebaka se tloaelehileng sa tlhōrō ea metabolite ka 'ngoe e fumanoeng ho lokisa liphetoho tse hlahisitsoeng ke tlhahlobo ea lisebelisoa ho tsoa ts'ebetsong ea sampole. Kamora hore TIC e fetoloe, MetaboAnalystR(51) (parameter ea kamehla) e sebelisoa bakeng sa phetoho ea logarithmic le ho lekanya mola oa norm o iketsang. Re sebelisitse PCA ka sephutheloana sa vegan R ho etsa tlhahlobo ea lipatlisiso tsa liphapang tsa metabolome lipakeng tsa mefuta ea sampole, 'me ra sebelisa tlhahlobo ea karolo ea redundancy ho sekaseka bakuli. Sebelisa mokhoa oa Ward ho haha ​​​​dendrogram ea 'mapa oa mocheso ho kopanya sebaka sa Euclidean pakeng tsa lisampole. Re sebelisitse limma (52) holim'a bongata ba metabolite bo tloaelehileng ho khetholla metabolites tse ngata ka mokhoa o fapaneng ho pholletsa le mofuta oohle oa sele le tikoloho e nyane. E le ho nolofatsa tlhaloso, re sebelisa paramethara ea karolelano ea sehlopha ho hlalosa mohlala, 'me re nahane ka mefuta ea sele tikolohong e nyane joalo ka sehlopha ka seng (n = lihlopha tse 6); Bakeng sa teko ea bohlokoa, re entse litekanyo tse tharo tse phetoang bakeng sa metabolite ka 'ngoe E le ho qoba ho ikatisa ha bohata, mokuli o ile a kenyelletsoa e le tšitiso moralong oa limma. E le ho hlahloba liphapang tsa metabolite pakeng tsa bakuli ba fapaneng, re ile ra fetola mohlala oa limma ho kenyeletsoa bakuli ka tsela e tsitsitseng. Re tlaleha bohlokoa ba phapang e boletsoeng esale pele pakeng tsa mofuta oa sele le tikoloho e nyane ea Padj <0.05 (tokiso ea Benjamini-Hochberg).
Kamora ho ntlafatsa matla ka ho sebedisa Miltenyi Dead Cell Removal Kit (>80% ya ho phela), ho etswa tatellano ya transcriptome ya sele e le nngwe hodima ascites e hoammeng e phelang le disampole tsa hlahala ho sebediswa protocol ya 10x 5′expression expression. Dinyewe tse hlano tse nang le dihlahala tse tshwanang le ascites di ile tsa sekasekwa, le hoja ho phela ho tlase ho tswa sampole e le nngwe ya hlahala ho ile ha thibela ho kenngwa ha yona. E le ho fihlella dikgetho tse ngata tsa bakudi, re kopantse disampole tsa mokuli e mong le e mong ditseleng tsa molaoli wa 10x chromium, mme ra sekaseka dibaka tsa ascites le hlahala ka thoko. Kamora ho latellana [Illumina HiSeq 4000 28×98 bp paired end (PE), genome ya Quebec; Ka karolelano ea lipalo tse 73,488 le 41,378 ka sele bakeng sa hlahala le ascites ka ho latellana]], re sebelisitse CellSNP le Vireo (53) (ho latela CellSNP kaha SNP ea motho e tloaelehileng (VCF) e fanoeng ke GRCh38 e abeloa boitsebiso ba mofani. Re sebelisa SNPRelate ho fumana boitsebiso bo haufi-ufi (IBS) ba boemo ba genotype ea mokuli (IBS), ho sa kenyeletsoe lisele le lisele tse sa abeloang tse tsejoang e le li-duplex le bafani ba tšoanang pakeng tsa ascites le lisampole tsa hlahala (54). Motheong oa mosebetsi ona, re bolokile linyeoe tse tharo tse nang le boemeli bo bongata ba lisele hlahala le ascites bakeng sa tlhahlobo e tlase. Kamora ho etsa mohato oa ho sefa bongata ka har'a sephutheloana sa scater (55) le scran (56) BioConductor, sena se hlahisitse lisele tse 6975 (lisele tse 2792 le 4183 ho tsoa hlahala le ascites, ka ho latellana) bakeng sa tlhahlobo. Re sebelisa sehlopha sa igraph (57) Louvain sa netweke e arolelanoang ea moahelani ea haufi-ufi (SNN) ho latela Jaccard sebaka ho ea ho lisele tsa sehlopha ka polelo. Lihlopha li ile tsa ngoloa ka letsoho ho ea ka mefuta ea lisele tse ipapisitseng le polelo ea liphatsa tsa lefutso tsa letšoao 'me tsa bonoa ka t-SNE. Lisele tsa T tse nang le chefo li hlalosoa ka polelo ea CD8A le GZMA, ntle le lihlopha tse nyenyane tse nang le polelo e tlase ea protheine ea ribosomal. Re fihletse lintlha tse phatlalalitsoeng tsa Izar et al. (16), ho kenyeletsoa le ho kenngoa ha tsona ha t-SNE, li ka laola ho kopana ha polelo pakeng tsa matšoao a lisele tsa 'mele le polelo ea NNMT.
PBMC e arotswe ho dihlahiswa tsa karohano ya leukocyte (STEMCELL Technologies) ka Ficoll gradient density centrifugation. Disele tsa CD3 + di arotswe ho PBMC ho sebediswa dibead tsa CD3 (Miltenyi). Ha MNA e le teng kapa e le siyo, disele tsa CD3+ di ile tsa kenngwa tshebetsong ka CD3 e tlamilweng ka poleiti (5μg/ml), CD28 e qhibilihang (3μg/ml) le IL-2 (300 U/ml; Proleukin). Letsatsing la ho qetela la katoloso, ho phela hantle (Fixable Viability Dye eFluor450, eBioscience) le ho ata ha tsona (123count eBeads, Thermo Fisher Scientific) di ile tsa lekolwa ka cytometry ya phallo. Lekola mosebetsi oa effector ka ho hlasimolla lisele tse nang le PMA (20 ng/ml) le ionomycin (1μg/ml) ka GolgiStop lihora tse 4, 'me u shebe CD8-PerCP (RPA-T8, BioLegend), CD4-AF700 (RPA-T4), BioLegend) le TNFα-fluorescein isothiocyanate (FITC) (MAb11, BD). Hlohlelletsa lisele tsa qPCR le ChIP ka PMA (20 ng/ml) le ionomycin (1μg/ml) lihora tse 4. ELISA supernatant e ile ea bokelloa pele le ka mor'a ho hlasimolla ka PMA (20 ng/ml) le ionomycin (1 μg/ml) lihora tse 4.
Latela protocol ea moetsi ho arola RNA u sebelisa RNeasy Plus Mini Kit (QIAGEN). Sebelisa QIAshredder (QIAGEN) ho etsa sampole e tšoanang. Sebelisa RNA e nang le bokhoni bo phahameng ho isa ho cDNA kit (Thermo Fisher Scientific) ho kopanya DNA e tlatsanang (cDNA). Sebelisa TaqMan Rapid Advanced Master Mix (Thermo Fisher Scientific) ho lekanya tlhahiso ea liphatsa tsa lefutso (ho latela protocol ea moetsi) ka li-probe tse latelang: Hs00196287_m1 (NNMT), Hs00154079_m1 (AOX1), Hs00427552_m1 (SLC22A1), Hs02786624_g1 [glyceraldehyde-3-phosphate off Hydrogen (GAPDH)] le Hs01010726_m1 (SLC22A2). Disampole di ile tsa sebediswa tsamaisong ya StepOnePlus ya nako ya sebele ya PCR (Applied Biosystems) (Applied Biosystems) ka hara poleiti ya karabelo ya MicroAmp e potlakileng ya 96-well (Applied Biosystems) e nang le filimi ya optical ya MicroAmp. Boleng bofe kapa bofe ba Ct bo fetang 35 bo nkuwa bo le ka hodima moedi wa ho lemoha mme bo tshwailwe e le bo sa bonahaleng.
Etsa ChIP joalo ka ha ho hlalositsoe pele (58). Ka bokhutšoanyane, lisele li ile tsa phekoloa ka formaldehyde (mahloriso a ho qetela a 1.42%) 'me tsa beoa mochesong oa kamore metsotso e 10. Sebelisa buffer e tlatselitsoeng ea ho ruruha (25 mM Hepes, 1.5 mM MgCl2, 10 mM KCl le 0.1% NP-40) leqhoeng metsotso e 10, ebe u e khutlisetsa hape ka har'a buffer ea immunoprecipitation joalo ka ha ho hlalositsoe (58). Sampole e ile ea ntan'o kenngoa ka sonic ka lipotoloho tse latelang: lipotoloho tse 10 (li-pulses tse 20 tsa metsotsoana e 1) le nako e sa fetoheng ea metsotsoana e 40. Kenya ChIP-grade immunoglobulin G (Theknoloji ea ho Bontša Sele; 1μl), histone H3 (Theknoloji ea ho Bontša Sele; 3μl), NFAT (Invitrogen; 3μl) le SP1 (Theknoloji ea ho Bontša Sele; 3μl) li-antibodies ka sampole ho 4°CC shake bosiu bohle. Kenya dibead tsa protheine A (Thermo Fisher Scientific) ka sampole ho 4°C ka ho di sisinya hanyane ka hora e le 1, ebe o sebedisa dibead tsa chelex (Bio-Rad) ho ntlafatsa DNA, mme o sebedise proteinase K (Thermo Fisher) bakeng sa tshilo ya protheine. Mohlohlelletsi wa TNFα o ile wa lemohuwa ke PCR: forward, GGG TAT CCT TGA TGC TTG TGT; ka lehlakoreng le leng, GTG CCA ACA ACT GCC TTT ATA TG (sehlahiswa sa 207-bp). Ditshwantsho di hlahisitswe ke Image Lab (Bio-Rad) mme tsa lekanngwa ho sebediswa software ya ImageJ.
Sele e nang le matla a ho hlahisa sele e ile ea bokelloa joalo ka ha ho hlalositsoe kaholimo. Ho fumanoa ha eona ho entsoe ho latela mekhoa ea moetsi ea sete ea TNFα ELISA ea motho (Invitrogen), sete ea IL-2 ELISA ea motho (Invitrogen) le sete ea IFN-γ ELISA ea motho (Abcam). Ho latela protocol ea moetsi, sete e nang le matla a ho hlahisa sele e ile ea hlapolloa ka 1:100 ho fumana TNFα le IL-2, le 1:3 ho fumana IFN-γ. Sebelisa EnVision 2104 Multilabel Reader (PerkinElmer) ho lekanya ho monya ha 450 nm.
PBMC e arotswe ho dihlahiswa tsa karohano ya leukocyte (STEMCELL Technologies) ka Ficoll gradient density centrifugation. Disele tsa CD3 + di arotswe ho PBMC ho sebediswa dibead tsa CD3 (Miltenyi). Ha MNA e le teng kapa e le siyo, disele tsa CD3+ di ile tsa kenngwa tshebetsong ka CD3 e tlamilweng ka poleiti (5μg/ml), CD28 e qhibilihang (3μg/ml) le IL-2 (300 U/ml; Proleukin) matsatsi a 3. Kamora matsatsi a 3, disele di ile tsa bokellwa mme tsa hlatsuwa ka saline ya 0.9%, mme pellet e ile ya hoamiswa ka snap. Palo ya disele e entswe ka cytometry ya phallo (Cytek Aurora; 3L-16V-14B-8R configuration) ho sebediswa 123count eBeads.
Di-metabolite tse ntšitsoeng joalokaha ho hlalositsoe kaholimo. Setlolo se omisitsoeng se ile sa tsosolosoa ka bongata ba lisele tse lekanang le 4000/μl. Hlahloba sampole ka chromatography ea mohato o khutlisitsoeng morao (1290 Infinity II, Agilent Technologies, Santa Clara, CA) le kholomo ea CORTECS T3 (2.1×150 mm, boholo ba likaroloana 1.6-μm, boholo ba pore 120-Å; #186008500, Metsi). Polar mass spectrometer (6470, Agilent), moo electrospray ionization e sebetsang ka mokhoa o motle. Mokhahlelo oa sele A ke 0.1% formic acid (ho H2O), mokhahlelo oa sele B ke 90% acetonitrile, 0.1% formic acid. Gradient ea LC ke metsotso e 0 ho isa ho e 2 bakeng sa 100% A, metsotso e 2 ho isa ho e 7.1 bakeng sa 99% B, le metsotso e 7.1 ho isa ho e 8 bakeng sa 99% B. Ebe u leka-lekanya kholomo le mokgahlelo o tsamaeang oa A ka sekhahla sa phallo sa 0.6 ml/min metsotso e 3. . Sekhahla sa phallo ke 0.4ml/min, 'me kamore ea kholomo e futhumatsoa ho fihlela ho 50°C. Sebelisa maemo a hloekileng a lik'hemik'hale a MNA (M320995, Toronto Research Chemical Company, North York, Ontario, Canada) ho theha nako ea ho boloka (RT) le phetoho (RT = metsotso e 0.882, phetoho 1 = 137→94.1, phetoho 2 = 137→92, Phetoho 3 = 137→78). Ha liphetoho tsohle tse tharo li etsahala ka nako e nepahetseng ea ho boloka, phetoho 1 e sebelisoa bakeng sa ho lekanya ho netefatsa ho hlaka. Sekhahla se tloaelehileng sa MNA (Toronto Research Chemical Company) se hlahisitsoe ka dilutions tse tsheletseng tsa tharollo ya setoko (1 mg/ml) ho fumana maemo a 0.1, 1.0, 10 le 100 ng/ml le 1.0 le 10μg/ml ka ho latellana ha metsi. Moeli wa ho lemoha ke 1 ng/ml, mme karabelo e otlolohileng e pakeng tsa 10 ng/ml le 10μg/ml. Ente e nngwe le e nngwe ya di-microliter tse pedi tsa sampole le maemo a tlwaelehileng e sebediswa bakeng sa tlhahlobo ya LC/MS, mme sampole e tswakilweng ya taolo ya boleng e tsamaiswa dienteng tse ding le tse ding tse robedi ho netefatsa botsitso ba sethala sa tlhahlobo. Dikarabo tsa MNA tsa disampole tsohle tsa sele tse phekotsweng ke MNA di ne di le ka hara sebaka se otlolohileng sa teko. Tlhahlobo ya data e entswe ho sebediswa software ya tlhahlobo ya bongata ya MassHunter (v9.0, Agilent).
Moaho oa moloko oa bobeli oa αFR-CAR o nkiloe ho Song et al. (59). Ka bokhutšoanyane, moaho o na le litaba tse latelang: tatellano ea moetapele oa CD8a, karoloana e fapaneng ea ketane e le 'ngoe ea motho ea αFR, sebaka sa hinge le transmembrane ea CD8a, sebaka sa kahare ho selefounu sa CD27 le sebaka sa kahare ho selefounu sa CD3z. Tatellano e felletseng ea CAR e entsoe ke GenScript, 'me ea kopanngoa ho vector ea polelo ea lentiviral ea moloko oa bobeli holimo ho cassette ea polelo ea GFP e sebelisoang ho lekola katleho ea phetisetso.
Lentivirus e hlahisoa ka ho fetisetsa lisele tsa HEK293T [American Type Culture Collection (ATCC); e holisitsoe ka har'a modified Eagle medium ea Dulbecco e nang le 10% fetal bovine serum (FBS) le 1% PenStrep, 'me e sebelisitsoe CAR-GFP vector le The packaging plasmids (psPAX2 le pMD2.G, Addgene) li sebelisa lipofection amine (Sigma-Aldrich). Supernatant e nang le vaerase e ile ea bokelloa lihora tse 48 le 72 kamora ho fetisetsa, ea sefshoa, 'me ea bokelloa ka ultracentrifugation. Boloka supernatant e bokelletsoeng ea vaerase ho -80°C ho fihlela e fetisetsoa.
PBMC e aroloa ho lihlahisoa tse phetseng hantle tsa karohano ea leukocyte ea bafani (STEMCELL Technologies) ka Ficoll gradient density centrifugation. Sebelisa li-microbead tsa CD8 tse khethiloeng hantle (Miltenyi) ho arola lisele tsa CD8+ ho PBMC. Hlohlelletsa lisele tsa T ka TransAct (Miltenyi) le ka har'a TexMACS medium [Miltenyi; e tlatselitsoeng ka 3% heat-inactivated human serum, 1% PenStrep le IL-2 (300 U/ml)]. Lihora tse mashome a mabeli a metso e mene kamora ho hlasimolloa, lisele tsa T li ile tsa fetisetsoa ka lentivirus (10 μl concentrated virus supernatant ka lisele tse 106). Matsatsi a 1 ho isa ho a 3 kamora ho hlakisoa ho Cytek Aurora (ho FSC (Forward Scatter)/SSC (Side Scatter), Singlet, GFP+), hlahloba polelo ea GFP ea lisele ho bontša bokhoni ba phetisetso ea bonyane 30%.
Lisele tsa CAR-T li ile tsa lengoa lihora tse 24 ho Immunocult (STEMCELL Technologies; e tlatselitsoeng ka 1% PenStrep) tlas'a maemo a latelang: li sa phekoloa, li phekoloa ka 250 μM adenosine kapa 10 mM MNA. Kamora kalafo ea pele, lisele tsa CAR-T li ile tsa hlatsuoa ka PBS 'me tsa kopanngoa le lisele tse 20,000 tsa SK-OV-3 [ATCC; ho McCoy 5A medium (Sigma-Aldrich) e tlatselitsoeng ka 10% FBS le 1% PenStrep ho 10: Karolelano ea effector ho target ea 1 e ile ea eketsoa ka makhetlo a mararo ho e tlatselitsoeng ea Immunocult medium. Lisele tsa SK-OV-3 le lisele tsa SK-OV-3 tse tlatselitsoeng ka digitalis saponin (0.5mg/ml; Sigma-Aldrich) li ile tsa sebelisoa e le taolo e mpe le e ntle, ka ho latellana. Kamora dihora tse 24 tsa ho lema hammoho, supernatant e ile ya bokellwa mme lactate dehydrogenase (LDH) ya lekanngwa ho ya ka ditaelo tsa moetsi (LDH Glo Cytotoxicity Assay Kit, Promega). Supernatant ya LDH e ile ya hlapollwa ka 1:50 ho LDH buffer. Peresente ya polao e ile ya lekanngwa ho sebediswa foromo e latelang: peresente ya polao = peresente ya tokiso / sekgahla se seholo sa polao x 100%, moo peresente ya tokiso = disele tsa co-culture-T feela, mme sekgahla se seholo sa polao = taolo e ntle ya taolo-negative.
Jwalo ka ha ho hlalositswe mongolong kapa disebedisweng le mekgweng, sebedisa GraphPad Prism 8, Microsoft Excel kapa R v3.6.0 bakeng sa tlhahlobo ya dipalopalo. Haeba disampole tse ngata di bokellwa ho mokuli a le mong (jwalo ka ascites le hlahala), re sebedisa teko ya t e kopaneng kapa re kenyeletsa mokuli e le phello e sa reroang mohlaleng o otlolohileng kapa o akaretsang kamoo ho hlokahalang. Bakeng sa tlhahlobo ya metabolomics, teko ya bohlokwa e etswa ka hararo.
Bakeng sa lisebelisoa tse ling bakeng sa sengoloa sena, ka kopo sheba http://advances.sciencemag.org/cgi/content/full/7/4/eabe1174/DC1
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Tlhokomeliso: Re kopa feela hore o fane ka aterese ea hau ea lengolo-tsoibila e le hore motho eo u mo khothaletsang leqepheng a tsebe hore u batla hore a bone lengolo-tsoibila le hore ha se spam. Re ke ke ra hapa liaterese life kapa life tsa lengolo-tsoibila.
Potso ena e sebelisetsoa ho leka hore na u moeti le ho thibela ho romelloa ha spam ka bohona.
Marisa K. Kilgour (Marisa K. Kilgour), Sarah MacPherson (Sarah MacPherson), Lauren G. Zacharias (Lauren G. Zacharias), Abigail Eli Aris G. Watson (H. Watson), John Stagg (John Stagg), Brad H. Nelson (Brad H. Nelson), Ralph J. De Bellar Jones (Russell J. G. Jones), Phineas T. Hamilton (Phineas T.
MNA e thusa ho thibela lisele tsa T 'meleng oa motho 'me e emela sepheo se ka bang teng sa phekolo ea immunotherapy bakeng sa kalafo ea mofetše oa batho.
Marisa K. Kilgour (Marisa K. Kilgour), Sarah MacPherson (Sarah MacPherson), Lauren G. Zacharias (Lauren G. Zacharias), Abigail Eli Aris G. Watson (H. Watson), John Stagg (John Stagg), Brad H. Nelson (Brad H. Nelson), Ralph J. De Bellar Jones (Russell J. G. Jones), Phineas T. Hamilton (Phineas T.
MNA e thusa ho thibela lisele tsa T 'meleng oa motho 'me e emela sepheo se ka bang teng sa phekolo ea immunotherapy bakeng sa kalafo ea mofetše oa batho.
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