Ibyuma bihindura ubudahangarwa bw'umubiri ni ikintu cy'ingenzi mu bidukikije by'uturemangingo (TME), ariko uretse bike, imiterere yabyo ntabwo izwi cyane. Aha, twasesenguye ibibyimba n'uturemangingo twa T biva mu turemangingo n'uturemangingo tw'abarwayi bafite kanseri yo mu bwoko bwa serous (HGSC) kugira ngo tugaragaze metabolome y'ibi bice bitandukanye bya TME. Ascites n'uturemangingo twa kanseri bifite itandukaniro rikomeye muri metabolite. Ugereranije na ascites, uturemangingo twa T twinjira mu turemangingo twinshi cyane muri 1-methylnicotinamide (MNA). Nubwo urwego rwa MNA mu turemangingo twa T ruri hejuru, imikorere ya nicotinamide N-methyltransferase (enzyme itera imbaraga kwimura amatsinda ya methyl kuva kuri S-adenosylmethionine ikajya kuri nicotinamide) igarukira kuri fibroblasts n'uturemangingo twa kanseri. Mu mikorere, MNA ituma uturemangingo twa T dusohora cytokine tumor necrosis factor alpha itera kanseri. Kubwibyo, MNA ikomoka kuri TME igira uruhare mu kugenzura ubudahangarwa bw'uturemangingo twa T kandi ikaba intego ishobora kuba iyo kuvura kanseri y'abantu.
Ibinyabutabire bikomoka ku kibyimba bishobora kugira ingaruka zikomeye ku budahangarwa bw’umubiri mu kurwanya ikibyimba, kandi ibimenyetso byinshi bigaragaza ko bishobora no kuba imbaraga z’ingenzi mu gutera imbere kw’indwara (1). Uretse ingaruka za Warburg, ubushakashatsi buherutse gutangira kugaragaza imiterere y’imikorere y’uturemangingo tw’ikibyimba n’isano bifitanye n’imiterere y’ubudahangarwa bw’uturemangingo tw’ikibyimba (TME). Ubushakashatsi ku ngero z’imbeba n’uturemangingo twa T tw’abantu bwagaragaje ko imikorere ya glutamine (2), imikorere ya oxidative (3) n’imikorere ya glucose (4) bishobora gukora ku giti cyabyo ku matsinda atandukanye y’uturemangingo tw’umubiri. Ibinyabutabire byinshi muri izi nzira bibuza imikorere ya T mu kurwanya ikibyimba. Byagaragaye ko guhagarika coenzyme tetrahydrobiopterin (BH4) bishobora kwangiza ikwirakwira ry’uturemangingo twa T, kandi kwiyongera kwa BH4 mu mubiri bishobora kongera uburyo ubudahangarwa bw’umubiri buterwa na CD4 na CD8. Byongeye kandi, ingaruka zo gukumira ubudahangarwa bw’umubiri za kynurenine zishobora gukizwa no guhabwa BH4 (5). Muri isocitrate dehydrogenase (IDH) mutant glioblastoma, gusohora kwa enantiometabolic (R)-2-hydroxyglutarate (R-2-HG) bibuza gukora, kwiyongera no gukora cytolysis ya selile T (6). Vuba aha, byagaragaye ko methylglyoxal, umusaruro wa glycolysis, ikorwa n'uturemangingo dutuma selile T zikora myeloid, kandi kohereza selile T muri methylglyoxal bishobora kubuza selile T gukora imikorere. Mu kuvura, kugabanya methylglyoxal bishobora gutsinda imikorere y'uturemangingo dutuma selile T zikora myeloid (MDSC) no kunoza uburyo bwo kuvura mu buryo bw'ikoranabuhanga mu buryo bw'imbeba (7). Ubu bushakashatsi bushimangira uruhare runini rwa metabolite zikomoka kuri TME mu kugenzura imikorere n'imikorere ya selile T.
Kudakora neza kw'uturemangingo twa T byagaragaye cyane muri kanseri y'intanga ngore (8). Ibi biterwa n'imiterere y'imikorere y'umubiri igaragara mu kubura umwuka uhagije no mu mitsi idasanzwe y'uturemangingo (9), bigatuma isukari na tryptophan bihinduka ibintu nk'ibikomoka kuri aside lactic na kynurenine. Lactate nyinshi yo hanze y'umubiri igabanya ikorwa rya interferon-γ (IFN-γ) kandi igatuma amatsinda mato agabanya myelosuppressive (10, 11). Gukoresha tryptophan bibuza mu buryo butaziguye kwiyongera kw'uturemangingo twa T kandi bikabuza T cell receptor signaling (12-14). Nubwo hari ibyo byagaragaye, akazi kenshi kajyanye na metabolism y'ubudahangarwa bw'umubiri kakozwe muri vitro hakoreshejwe uburyo bwa optimized media, cyangwa kagashyirwa ku buryo bugaragara ku mbeba zimeze nk'izisanzwe, nta na kimwe muri byo kigaragaza neza ubudasa bwa kanseri z'abantu n'ibidukikije by'umubiri.
Ikintu gikunze kugaragara ku ndwara ya kanseri y’intanga ngore ni ikwirakwira ry’uturemangingo mu nda no kugaragara kwa ascites. Kwiyongera kw’uturemangingo mu nda bijyana n’indwara zikomeye ndetse n’ingaruka mbi z’indwara (15). Nk’uko raporo zibivuga, iki gice cyihariye gifite ubushyuhe bwinshi, gifite urugero rwo hejuru rw’imitsi y’amaraso (VEGF) na indoleamine 2,3-dioxygenase (IDO), kandi cyinjizwa n’uturemangingo twa T regulatory na myeloid inhibitory cells (15-18). Imiterere y’imikorere ya ascites ishobora kuba itandukanye n’iy’ikibyimba ubwacyo, bityo gusubiramo gahunda y’uturemangingo twa T mu mwanya wa peritoneal ntibirasobanuka neza. Byongeye kandi, itandukaniro ry’ingenzi n’ubusumbane hagati ya ascites na metabolites biri mu nda bishobora kubangamira kwinjira kw’uturemangingo tw’ubudahangarwa bw’umubiri n’imikorere yabyo ku tubyimba, kandi hakenewe ubushakashatsi bw’inyongera.
Kugira ngo dukemure ibi bibazo, twakoze uburyo bwo gutandukanya ingirabuzimafatizo n’uburyo bwo gupima imiterere y’uturemangingo (LC-MS/MS) kugira ngo twige ubwoko butandukanye bw’uturemangingo (harimo CD4 + na CD8 + T cells) ndetse no hagati y’uturemangingo. Ibinyabutabire byayo bihurira mu turemangingo turi mu bice bimwe bya ascites n’aho umurwayi atuye. Dukoresha ubu buryo hamwe na cytometry yo mu rugero rwo hejuru n’uburyo RNA imwe ikurikirana (scRNA-seq) kugira ngo dutange ishusho y’imiterere y’uturemangingo tw’ingenzi. Ubu buryo bwagaragaje ubwiyongere bugaragara bw’urwego rwa 1-methylnicotinamide (MNA) mu turemangingo twa T turwaye, kandi igerageza ryakozwe muri vitro ryagaragaje ko ingaruka za MNA ku mikorere y’uturemangingo twa T zitari zizwi mbere. Muri rusange, ubu buryo bwerekana imikoranire hagati y’uturemangingo n’uturemangingo tw’ubudahangarwa, kandi butanga ubumenyi bwihariye ku mikorere y’uturemangingo tw’ubudahangarwa, bishobora kuba ingirakamaro mu kuvura kanseri y’intanga ngore ishingiye ku turemangingo twa T.
Twakoresheje uburyo bwo gupima ubwinshi bw'imiyoboro y'amaraso kugira ngo dupime icyarimwe uburyo isukari itwarwa [2-(N-(7-nitrophenyl-2-oxa-1,3-diaza-4-yl)amino)-2-deoxyglucose (2-NBDG) n'imikorere ya mitochondrial [MitoTracker Deep Red (MT DR)] (7, 19, 20) ni ibimenyetso bisanzwe bitandukanya uturemangingo tw’ubudahangarwa bw’umubiri n’uturemangingo tw’uturemangingo tw’uturemangingo (Imbonerahamwe ya S2 n’Ishusho ya S1A). Iri sesengura ryagaragaje ko ugereranije n’uturemangingo twa T, ascites n’uturemangingo tw’uturemangingo dufite urugero rwo hejuru rw’isukari itwarwa, ariko dufite itandukaniro rito mu mikorere ya mitochondrial. Impuzandengo y’isukari itwarwa n’uturemangingo twa tumor [CD45-EpCAM (EpCAM)+] ikubye inshuro eshatu cyangwa enye ugereranyije n’uturemangingo twa T, kandi impuzandengo y’isukari itwarwa n’uturemangingo twa CD4 + T ikubye inshuro 1.2 ugereranyije n’uturemangingo twa CD8 + T, bigaragaza ko lymphocytes zinjira mu tumori (TIL) zifite ibisabwa bitandukanye mu mikorere ndetse no muri TME imwe (Ishusho ya 1A). Mu buryo bunyuranye, imikorere ya mitochondrial mu turemangingo twa kanseri isa n’iy’uturemangingo twa CD4 + T, kandi imikorere ya mitochondrial y’ubwoko bwombi bw’uturemangingo iri hejuru y’iy’uturemangingo twa CD8 + T (Ishusho ya 1B). Muri rusange, ibi bisubizo bigaragaza urwego rw’imikorere ya kanseri. Umurimo wa metabolike w’uturemangingo twa kanseri uri hejuru y’utwa CD4 + T, kandi imikorere ya metabolike y’uturemangingo twa CD4 + T iri hejuru y’utwa CD8 + T. Nubwo hari izi ngaruka ku bwoko bw’uturemangingo, nta tandukaniro rihoraho mu miterere ya metabolike y’uturemangingo twa CD4 + na CD8 + T cyangwa ingano yatwo muri ascites ugereranije na kanseri (Ishusho ya 1C). Mu buryo bunyuranye, mu gice cy’uturemangingo twa CD45, ingano y’uturemangingo twa EpCAM + muri kanseri yiyongereye ugereranije na ascites (Ishusho ya 1D). Twabonye kandi itandukaniro rigaragara hagati y’ibice bya EpCAM + na EpCAM- cell. Uturemangingo twa EpCAM + (tumor) dufite glucose nyinshi ifata n’imikorere ya mitochondrial kurusha utwa EpCAM- cells, ibi bikaba biri hejuru cyane y’imikorere ya metabolike ya fibroblasts mu turemangingo twa kanseri muri TME (Ishusho ya 1, E na F).
(A na B) Ingufu z’ubushyuhe bw’impinduka (MFI) mu gufata isukari (2-NBDG) (A) n’imikorere ya mitochondrial y’uturemangingo twa CD4 + T (umutuku wijimye wa MitoTracker) (B) Imbonerahamwe zigaragaza (ibumoso) n’amakuru yashyizwe mu byiciro (Iburyo), uturemangingo twa CD8 + T n’uturemangingo twa EpCAM + CD45-tumor tuva kuri ascites na tumor. (C) Igipimo cy’uturemangingo twa CD4 + na CD8 + (twa CD3 + T) muri ascites na tumor. (D) Ingano y’uturemangingo twa EpCAM + tumor muri ascites na tumor (CD45−). (E na F) EpCAM + CD45-tumor na EpCAM-CD45-matrix glucose ifatwa (2-NBDG) (E) n’imikorere ya mitochondrial (umutuku wijimye wa MitoTracker) (F) Imbonerahamwe zigaragaza (ibumoso) n’amakuru yashyizwe mu byiciro (Iburyo) Ascites na tumor cells. (G) Imbonerahamwe zigaragaza CD25, CD137 na PD1 expression hifashishijwe flow cytometry. (H na I) Igaragara rya CD25, CD137 na PD1 ku tunyangingo twa CD4 + T (H) na CD8 + T (I). (J na K) Ishusho y’ubudahangarwa, ubwibukire bwo hagati (Tcm), effector (Teff) na effector memory (Tem) ishingiye ku igaragara rya CCR7 na CD45RO. Amashusho agaragaza (ibumoso) n'amakuru ajyanye n'imbonerahamwe (iburyo) y'utunyangingo twa CD4 + T (J) na CD8 + T (K) muri ascites na tumors. Agaciro ka P kagenwa n'ikizamini cya t (*P<0.05, **P<0.01 na ***P<0.001). Umurongo ugaragaza abarwayi bahuje (n = 6). FMO, fluorescence idafite kimwe; MFI, ubukana bw'ubudahangarwa bw'ubudahangarwa bw'ubudahangarwa.
Isesengura ryinyongera ryagaragaje irindi tandukaniro rikomeye hagati y’imiterere y’uturemangingo twa T twangiritse cyane. Ishusho yakozwe (Ishusho ya 1, G kugeza kuri I) n’iy’inyibutsa (Ishusho ya 1, J na K) mu tumori ikunze kugaragara cyane kurusha ascites (urugero rw’uturemangingo twa CD3 + T). Mu buryo nk’ubwo, gusesengura imiterere y’uturemangingo hakoreshejwe ibimenyetso by’inyibutsa (CD25 na CD137) n’ibimenyetso byo kugabanuka [poroteyine y’urupfu rw’uturemangingo yateguwe 1 (PD1)] byagaragaje ko nubwo imiterere y’imikorere y’uturemangingo y’uturemangingo itandukanye (Ishusho ya S1, B kugeza kuri E), ariko nta tandukaniro rikomeye ryagaragaye hagati y’uturemangingo tw’uturemangingo, tw’inyibutsa cyangwa tw’inyibutsa (Ishusho ya S1, F kugeza kuri I). Ibi byavuye mu bushakashatsi byemejwe hakoreshejwe uburyo bwo kwiga imashini kugira ngo hashyirweho imiterere y’uturemangingo mu buryo bwikora (21), byagaragaje ko hari umubare munini w’uturemangingo tw’umusemburo w’amagufwa (CD45 + / CD3- / CD4 + / CD45RO +) mu turemangingo tw’umurwayi (Ishusho ya S2A). Mu bwoko bwose bw'uturemangingo twagaragaye, utwo turemangingo twa myeloid twagaragaje ko isukari ikwirakwira cyane kandi ko mitochondrial ikora neza (Ishusho ya S2, B kugeza kuri G). Ibi bisubizo bigaragaza itandukaniro rikomeye hagati y'ubwoko bwinshi bw'uturemangingo buboneka muri ascites na tumors ku barwayi ba HGSC.
Ikibazo gikomeye mu gusobanukirwa imiterere ya TIL ni ugushaka gutandukanya ingero za T cells zifite ubuziranenge buhagije, ubwiza n'ingano biturutse ku tumori. Ubushakashatsi buherutse bwerekanye ko uburyo bwo gutondeka no kongera uduce tw'uturemangingo bushingiye kuri cytometry y'imiyoboro y'amaraso bushobora gutera impinduka mu miterere ya metabolite y'uturemangingo (22-24). Kugira ngo dukemure iki kibazo, twanogeje uburyo bwo kongera uduce tw'uturemangingo kugira ngo dutandukanye kandi dutandukanye TIL na kanseri y'intanga ngore y'umuntu yakuwemo mu buryo bwo kubaga mbere yo gusesengura hakoreshejwe LC-MS/MS (reba Ibikoresho n'Uburyo; Ishusho ya 2A). Kugira ngo dusuzume ingaruka rusange z'iyi protocole ku mpinduka za metabolite, twagereranije imiterere ya metabolite y'uturemangingo twa T cells twakozwe n'abatanga serivisi nziza nyuma y'intambwe yo gutandukanya uduce tw'uturemangingo twavuzwe haruguru hamwe n'uturemangingo tutatandukanijwe n'uduce tw'uturemangingo ahubwo twagumye ku rubura. Iri sesengura ryo kugenzura ubuziranenge ryagaragaje ko hari isano iri hagati y'ibi bintu bibiri (r = 0.77), kandi ubushobozi bwo gusubiramo mu buryo bw'ikoranabuhanga bw'itsinda ry'uturemangingo 86 bufite ubushobozi bwo gusubiramo bwinshi (Ishusho ya 2B). Bityo rero, ubu buryo bushobora gukora isesengura ry’uburyohe bw’uturemangingo mu turemangingo turimo kwiyongera k’ubwoko bw’uturemangingo, bityo bukaba urubuga rwa mbere rwo kumenya uburyohe bw’uturemangingo muri HGSC, bityo bigatuma abantu basobanukirwa byimbitse gahunda y’imikorere y’uturemangingo mu buryo bwihariye.
(A) Ishusho y’icyitegererezo cy’ubwinshi bw’udusaro tw’amaginito. Mbere yo gusesengura hakoreshejwe LC-MS/MS, uturemangingo tuzanyura mu byiciro bitatu bikurikirana byo kongera udusaro tw’amaginito cyangwa tukaguma ku rubura. (B) Ingaruka z’ubwoko bwo kongera udusaro ku bwinshi bwa metabolites. Impuzandengo y’ibipimo bitatu kuri buri bwoko bwo kongera udusaro ± SE. Umurongo w’imvi ugaragaza isano ya 1:1. Isano y’inyuma y’ibyiciro (ICC) by’ibipimo bisubirwamo bigaragara ku kirango cy’umurongo. NAD, nicotinamide adenine dinucleotide. (C) Ishusho y’icyitegererezo cy’imikorere y’isesengura rya metabolite y’umurwayi. Ascites cyangwa ibibyimba bikusanyirizwa mu barwayi kandi bikabikwa. Igice gito cya buri cyitegererezo cyasesenguwe hakoreshejwe cytometry y’amazi, mu gihe ingero zisigaye zasesenguwe mu byiciro bitatu byo kongera udusaro tw’amaginito CD4+, CD8+ na CD45. Ibi bice by’uturemangingo byasesenguwe hakoreshejwe LC-MS/MS. (D) Ikarita y’ubushyuhe bw’ubwinshi bw’utusaro tw’amaginito. Dendrogram igaragaza intera ya Ward y’intera ya Euclidean hagati y’ibipimo. (E) Isesengura ry'ibice by'ingenzi (PCA) by'ikarita y'icyitegererezo cya metabolite, igaragaza kopi eshatu za buri cyitegererezo, ingero z'umurwayi umwe zihuzwa n'umurongo. (F) PCA y'imiterere ya metabolite y'icyitegererezo ishingiye ku murwayi (ni ukuvuga, hakoreshejwe uburyo bwo kugabanya igice); ubwoko bw'icyitegererezo bugengwa n'igice cy'umubiri. PC1, igice cy'ingenzi cya 1; PC2, igice cy'ingenzi cya 2.
Hanyuma, twakoresheje ubu buryo bwo gukusanya no gusesengura ibice 99 bya CD4+, CD8+ na CD45-cell fractions mu bice by’ibanze bya ascites na tumors by’abarwayi batandatu ba HGSC (Ishusho ya 2C, Ishusho ya S3A n’Imbonerahamwe ya S3 na S4). Umubare w’abantu bashishikajwe ugizwe na 2% kugeza kuri 70% by’icyitegererezo kinini cy’uturemangingo tuzima, kandi umubare w’uturemangingo uratandukanye cyane hagati y’abarwayi. Nyuma yo gutandukanya uduce, igice cy’ingenzi cy’uturemangingo (CD4+, CD8+ cyangwa CD45-) kigizwe na arenga 85% by’uturemangingo twose tuzima mu rugero. Ubu buryo bwo gukusanya budufasha gusesengura umubare w’uturemangingo uhereye ku mikorere y’uturemangingo tw’abantu, ibyo bikaba bidashoboka ku bipimo binini. Dukoresheje iyi protocole, twasanze l-kynurenine na adenosine, izi metabolite ebyiri zizwi neza zo gukumira ubudahangarwa bw’umubiri zari nyinshi mu turemangingo twa T cyangwa tumori (Ishusho ya S3, B na C). Kubwibyo, ibi bisubizo bigaragaza ubudahemuka n’ubushobozi bw’ikoranabuhanga ryacu ryo gutandukanya uturemangingo n’ubwinshi bwa spectrometry mu kubona metabolite z’ingenzi mu mubiri mu turemangingo tw’abarwayi.
Isesengura ryacu ryagaragaje kandi itandukaniro rikomeye ry’uburyo imiterere y’uturemangingo itandukanya ubwoko bw’uturemangingo mu barwayi no hagati yabo (Ishusho ya 2D n’Ishusho ya S4A). By’umwihariko, ugereranije n’abandi barwayi, umurwayi wa 70 yagaragaje imiterere itandukanye y’imikorere y’uturemangingo (Ishusho ya 2E n’Ishusho ya S4B), bigaragaza ko hashobora kuba hari itandukaniro rikomeye hagati y’abarwayi. Ni ngombwa kumenya ko ugereranije n’abandi barwayi (litiro 1.2 kugeza kuri 2; Imbonerahamwe ya S1), ingano yose ya ascites yakusanyijwe mu barwayi 70 (mililitiro 80) yari nto. Igenzura ry’ubusumbane hagati y’abarwayi mu gihe cyo gusesengura ibice by’ingenzi (urugero, hakoreshejwe isesengura ry’ubusumbane) rigaragaza impinduka zihoraho hagati y’ubwoko bw’uturemangingo, kandi ubwoko bw’uturemangingo n’ibidukikije bito bikusanyijwe neza hakurikijwe imiterere ya metabolite (Ishusho ya 2F). Isesengura rya metabolite imwe ryashimangiye izi ngaruka kandi ryagaragaje itandukaniro rikomeye hagati y’ubwoko bw’uturemangingo n’ibidukikije bito. Ni ngombwa kumenya ko itandukaniro rikomeye ryagaragaye ari MNA, ikunze kuba ikunzwe mu turemangingo twa CD45 no mu turemangingo twa CD4+ na CD8+ twinjira mu kibyimba (Ishusho ya 3A). Ku turemangingo twa CD4 +, iyi ngaruka iragaragara cyane, kandi MNA mu turemangingo twa CD8 + nayo isa nkaho igizweho ingaruka zikomeye n'ibidukikije. Ariko, ibi si ingenzi, kuko batatu gusa mu barwayi batandatu ni bo bashobora gupimwa amanota ya CD8 + ya kanseri. Uretse MNA, mu bwoko butandukanye bw'uturemangingo mu turemangingo twa ascites na tumors, izindi metabolites zitagaragara neza muri TIL nazo zirakungahaye ku buryo butandukanye (Ishusho S3 na S4). Kubwibyo, aya makuru agaragaza itsinda ryizewe rya metabolites zikora ubudahangarwa bw'umubiri kugira ngo hakorwe ubushakashatsi burambuye.
(A) Ingano ya MNA isanzwe mu turemangingo twa CD4+, CD8+ na CD45- biva mu turemangingo twa ascites na tumori. Agasanduku kerekana urwego rw'imibare (umurongo), urwego rwa interquartile (umugozi w'umurabyo) n'urwego rw'amakuru, kugeza ku nshuro 1.5 urwego rwa interquartile (umugozi w'umurabyo). Nkuko byasobanuwe mu bikoresho by'umurwayi n'uburyo, koresha agaciro ka limma k'umurwayi kugira ngo umenye agaciro ka P (*P<0.05 na **P<0.01). (B) Ishusho y'ishusho y'imikorere ya MNA (60). Metabolites: 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. Enzymes (icyatsi): 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 ya scRNA-seq ya ascites (imvi) na tumori (umutuku; n = abarwayi 3). (D) Igaragara rya NNMT mu turemangingo dutandukanye ryagaragaye hakoreshejwe scRNA-seq. (E) Igaragara rya NNMT na AOX1 muri SK-OV-3, impyiko z'umwana uri mu nda (HEK) 293T, uturemangingo twa T na uturemangingo twa T twavuwe na MNA. Igaragara ryapfundutse ryerekanwa ugereranije na SK-OV-3. Imiterere y'igaragara rya SEM iragaragara (n = abatanga 6 bazima). Agaciro ka Ct karenze 35 gafatwa nk'aho katagaragara (UD). (F) Igaragara rya SLC22A1 na SLC22A2 muri SK-OV-3, HEK293T, T cells na T cells bivuwe na 8mM MNA. Igaragara ry’ishusho rihinze rigaragazwa ugereranije na SK-OV-3. Ishusho y’ishusho na SEM iragaragazwa (n = 6 abatanga serivisi nziza). Agaciro ka Ct karenze 35 gafatwa nk'aho katagaragara (UD). (G) Ingano ya MNA mu turemangingo twa T dutanga serivisi nziza nyuma y'amasaha 72 yo gukubitwa na MNA. Ishusho y’ishusho na SEM iragaragazwa (n = 4 abatanga serivisi nziza).
MNA ikorwa hifashishijwe kwimura itsinda rya methyl kuva kuri S-adenosyl-1-methionine (SAM) kugera kuri nicotinamide (NA) hakoreshejwe nicotinamide N-methyltransferase (NNMT; Ishusho ya 3B). NNMT igaragara cyane mu bwoko butandukanye bwa kanseri z'abantu kandi ifitanye isano no kwiyongera, kwinjira no kwangirika (25-27). Kugira ngo dusobanukirwe neza inkomoko ya MNA mu turemangingo twa T muri TME, twakoresheje scRNA-seq kugira ngo tugaragaze uko NNMT igaragara mu bwoko bw'uturemangingo mu turemangingo twa ascites na tumors z'abarwayi batatu ba HGSC (Imbonerahamwe ya S5). Isesengura ry'uturemangingo tugera ku 6.500 ryagaragaje ko mu turemangingo twa ascites no mu duce twa kanseri, NNMT yagaragaye gusa ku turemangingo twa fibroblast n'uturemangingo twa tumors (Ishusho ya 3, C na D). Ni ngombwa kumenya ko nta NNMT igaragara mu bantu iyo ari bo bose igaragaza PTPRC (CD45 +) (Ishusho ya 3D n'Ishusho ya S5A), bigaragaza ko MNA yabonetse mu buryo bwa metabolite yinjijwe mu turemangingo twa T. Igaragara rya aldehyde oxidase 1 (AOX1) rihindura MNA muri 1-methyl-2-pyridone-5-carboxamide (2-PYR) cyangwa 1-methyl-4-pyridone-5-carboxamide (4-PYR); Ishusho ya 3B) na yo igarukira ku mubare w’udukoko twa fibroblaste tugaragaza COL1A1 (Ishusho ya S5A), ibyo byose hamwe bigaragaza ko uturemangingo twa T tudafite ubushobozi bwo gukora MNA isanzwe. Imiterere y’igaragara ry’utu turemangingo dufitanye isano na MNA yemejwe hakoreshejwe amakuru ya kabiri yigenga y’uturemangingo yavuye ku barwayi ba HGSC (Ishusho ya S5B; n = 6) (16). Byongeye kandi, isesengura rya quantitative polymerase chain reaction (qPCR) ry’uturemangingo twa T dutanga twavuwe na MNA ryagaragaje ko ugereranije n’uturemangingo twa kanseri y’intanga ya SK-OV-3, NNMT cyangwa AOX1 bitagaragaye cyane (Ishusho ya 3E). Ibi bisubizo bitunguranye bigaragaza ko MNA ishobora gukurwa muri fibroblaste cyangwa tumori mu turemangingo twa T twegeranye muri TME.
Nubwo abakandida barimo umuryango w’abatwara cation organic kuva kuri 1 kugeza kuri 3 (OCT1, OCT2 na OCT3) bashyizwe mu byiciro n’umuryango w’abatwara 22 (SLC22) (SLC22A1, SLC22A2 na SLC22A3), abatwara MNA bashobora kuba batarasobanuwe neza (28). QPCR ya mRNA iturutse mu turemangingo twa T tw’abaterankunga tuzima yagaragaje urugero ruto rwa SLC22A1 ariko urugero rwa SLC22A2 rutagaragara, byemeje ko byari byaravuzwe mbere mu nyandiko (Ishusho ya 3F) (29). Ibinyuranye n’ibyo, umurongo w’uturemangingo twa kanseri y’intanga ya SK-OV-3 wagaragaje urugero rwo hejuru rw’uturemangingo twombi tw’abatwara (Ishusho ya 3F).
Kugira ngo hamenyekane niba selile T zishobora kwinjiza MNA y’amahanga, selile T z’abaterankunga bazima zaratewe mu gihe cy’amasaha 72 mu gihe hari ingano zitandukanye za MNA. Mu gihe nta MNA yo hanze irimo, ingano ya MNA mu turemangingo ntishobora kuboneka (Ishusho ya 3G). Ariko, selile T zavuwe na MNA yo hanze zagaragaje ubwiyongere bushingiye ku gipimo cy’ingano ya MNA mu turemangingo, kugeza kuri 6 mM MNA (Ishusho ya 3G). Ibi bisubizo bigaragaza ko nubwo hari urwego ruto rw’ubwikorezi bw’ibinyabutabire ndetse no kutagira enzyme nyamukuru ishinzwe imikorere ya MNA mu turemangingo, TIL iracyashobora gufata MNA.
Imiterere ya metabolites mu turemangingo twa T tw’abarwayi ndetse n’igerageza rya MNA ryo mu buryo bwa vitro byongera amahirwe y’uko fibroblasts zifitanye isano na kanseri (CAF) zisohora MNA kandi turemangingo twa kanseri zishobora kugenzura imiterere n’imikorere ya TIL. Kugira ngo hamenyekane ingaruka za MNA ku turemangingo twa T, turemangingo twa T twatanzwe n’abaterankunga twakozwe muri vitro mu gihe MNA ihari cyangwa idahari, kandi ikwirakwira ryazo n’umusaruro wa cytokine byarasuzumwe. Nyuma y’iminsi 7 yongeweho MNA ku gipimo cyo hejuru, umubare wikubye kabiri w’abaturage waragabanutse ku rugero, mu gihe imbaraga zakomeje kugabanuka ku gipimo cyose (Ishusho ya 4A). Byongeye kandi, kuvura MNA yo hanze byatumye umubare w’uturemangingo twa CD4 + na CD8 + T bigaragaza tumor necrosis factor-α (TNFα; Ishusho ya 4B) wiyongera. Mu buryo bunyuranye, umusaruro wa IFN-γ mu turemangingo wagabanutse cyane mu turemangingo twa CD4 + T, ariko ntabwo wagabanutse mu turemangingo twa CD8 + T, kandi nta mpinduka nini yabayeho muri interleukin 2 (IL-2; Ishusho ya 4, C na D). Kubwibyo, isuzuma rya immunosorbent assay (ELISA) ry’ibintu bikomoka kuri izi selile za T zavuwe na MNA ryagaragaje ukwiyongera gukomeye kwa TNFα, igabanuka rya IFN-γ, kandi nta mpinduka muri IL-2 (Ishusho ya 4, E kugeza kuri G). . Kugabanuka kwa IFN-γ bigaragaza ko MNA ishobora kugira uruhare mu kubuza imikorere ya selile za T kurwanya kanseri. Kugira ngo bigane ingaruka za MNA ku burozi buterwa na selile za T, selile za antigen T (FRα-CAR-T) zireba folate receptor α na CAR-T (GFP) zigenzurwa na poroteyine y’icyatsi kibisi (GFP) -CAR-T) zikorwa n’selile nziza za mononuclear zo mu maraso zitangwa na donor blood mononuclear (PBMC). Selile za CAR-T zakorewe ubworozi mu gihe cy’amasaha 24 imbere ya MNA, hanyuma zigakorerwa hamwe n’selile za SK-OV-3 zo mu tumori tw’intanga z’abantu zigaragaza folate receptor α ku gipimo cya effector ku ntego cya 10:1. Ubuvuzi bwa MNA bwatumye ibikorwa byo kwica uturemangingo twa FRα-CAR-T bigabanuka cyane, byari bisa n'uturemangingo twa FRα-CAR-T twavuwe na adenosine (Ishusho ya 4H).
(A) Umubare w’uturemangingo twose dushobora kubaho n’ubwiyongere bw’abaturage (PD) bivuye mu mikurire ku munsi wa 7. Imbonerahamwe y’imirongo igaragaza impuzandengo + SEM y’abatanga batandatu bazima. Igaragaza amakuru yavuye mu bushakashatsi bwa n = 3 bwigenga. (B kugeza D) CD3/CD28 na IL-2 byakoreshejwe mu gukora uturemangingo twa T ku gipimo cya MNA cyazo mu minsi 7. Mbere yo gusesengura, uturemangingo twashishikarijwe na PMA/ionomycin hamwe na GolgiStop amasaha 4. Ishusho ya TNFα (B) mu turemangingo twa T. Urugero rw’ishusho (ibumoso) n’amakuru y’imbonerahamwe (iburyo) y’ishusho ya TNFα mu turemangingo tuzima. Ishusho ya IFN-γ (C) na IL-2 (D) mu turemangingo twa T. Ishusho ya cytokines yapimwe hakoreshejwe flow cytometry. Imbonerahamwe y’imirongo igaragaza impuzandengo (n = 6 batanga bazima) + SEM. Koresha isesengura ry’uburyo bumwe ry’impinduka n’ibipimo bisubirwamo (*P<0.05 na **P<0.01) kugira ngo umenye agaciro ka P. Igaragaza amakuru yavuye mu bushakashatsi bwa n = 3 bwigenga. (Kuva kuri E kugeza kuri G) CD3/CD28 na IL-2 byakoreshejwe mu gukora uturemangingo twa T ku gipimo cya MNA cyazo mu minsi 7. Igikoresho cyakusanyijwe mbere na nyuma y'amasaha 4 yo gushishikariza PMA/ionomycin. Igipimo cya TNFα (E), IFN-γ (F) na IL-2 (G) cyapimwe na ELISA. Imbonerahamwe y'umurongo igaragaza impuzandengo (n = 5 abatanga serivisi nziza) + SEM. Agaciro ka P kagenwe hakoreshejwe isesengura ry'uburyo bumwe bw'impinduka n'ibipimo bisubirwamo (*P<0.05). Umurongo w'utudomo ugaragaza aho ubushobozi bwo kumenya bugarukira. (H) Isuzuma rya lysis y'uturemangingo. Uturemangingo twa FRα-CAR-T cyangwa GFP-CAR-T twahinduwe hakoreshejwe adenosine (250μM) cyangwa MNA (10 mM) mu gihe cy'amasaha 24, cyangwa ntitwitezwe (Ctrl). Ijanisha ry'iyicwa ry'uturemangingo twa SK-OV-3 ryapimwe. Agaciro ka P kagenwe n'ikizamini cya Welch t (*P<0.5 na **P<0.01).
Kugira ngo hamenyekane uburyo bwo gusobanura uburyo TNFα ikoreshwa na MNA ikoreshwa, hasuzumwe impinduka muri TNFα mRNA y’uturemangingo twa T twavuwe na MNA (Ishusho ya 5A). Uturemangingo twa T tw’abatanga serivisi nziza twavuwe na MNA twerekanye ubwiyongere bubiri bw’urugero rwa TNFα, bigaragaza ko MNA ishingiye ku buryo TNFα ikoreshwa na TNFα ikoreshwa. Kugira ngo hasuzumwe ubu buryo bushoboka bwo kugenzura, ibintu bibiri bizwi byo guhindura TNFα, ari byo T cell nuclear factor (NFAT) na poroteyine 1 yihariye (Sp1), byasuzumwe bitewe n’uko MNA ikorana na TNFα promoter (30). TNFα promoter igizwe n’ahantu 6 hazwi ho guhuza NFAT n’ahantu 2 ho guhuza Sp1, bihurirana ahantu hamwe [-55 base pairs (bp) kuva kuri 5'cap] (30). Chromatin immunoprecipitation (ChIP) yagaragaje ko iyo ivuwe na MNA, guhuza Sp1 na TNFα promoter byikubye gatatu. Kwinjira muri NFAT nabyo byariyongereye kandi biregereje akamaro (Ishusho ya 5B). Aya makuru agaragaza ko MNA igenzura uburyo TNFα ikoreshwa binyuze mu kwandika Sp1, ndetse no mu rugero ruto uburyo NFAT ikoreshwa.
(A) Ugereranije n’uturemangingo twa T twakuwemo nta MNA, impinduka mu mikorere ya TNFα mu turemangingo twa T twavuwe na MNA. Imiterere y’imikorere na SEM iragaragazwa (n = abatanga 5 bazima). Igaragaza amakuru yavuye nibura ku bushakashatsi bwa n = 3 bwigenga. (B) Iterambere rya TNFα ry’uturemangingo twa T ryavuwe cyangwa ritavuwe na MNA ya 8 mM nyuma ya NFAT na Sp1 byahujwe na (Ctrl) na PMA / ionomycin stimulation mu gihe cy’amasaha 4. Immunoglobulin G (IgG) na H3 byakoreshejwe nk’igenzura ribi n’iry’ubwirinzi bw’umubiri, uko bikurikirana. Ingano ya ChIP yagaragaje ko gufatana kwa Sp1 na NFAT kuri TNFα stimuler mu turemangingo twavuwe na MNA byiyongereye inshuro nyinshi ugereranije n’igenzura. Igaragaza amakuru yavuye nibura ku bushakashatsi bwa n = 3 bwigenga. Agaciro ka P kagenwa n’ibizamini byinshi bya t (*** P <0.01). (C) Ugereranije na ascites ya HGSC, uturemangingo twa T (tutari toxic) twagaragaje kwiyongera kwa TNF muri iki kibyimba. Amabara agaragaza abarwayi batandukanye. Uturemangingo twagaragajwe twapimwe ku buryo butunguranye kugeza kuri 300 kandi twashyizwemo umwuka kugira ngo tugabanye gukurura cyane (** Padj = 0.0076). (D) Icyitegererezo cya MNA cyatanzwe ku ndwara ya kanseri y'intanga ngore. MNA ikorerwa mu turemangingo twa kanseri na fibroblast muri TME kandi ifatwa n'uturemangingo twa T. MNA yongera uburyo Sp1 ifatana na TNFα promoter, bigatuma TNFα transcription yongera umusaruro wa TNFα cytokine. MNA kandi itera igabanuka rya IFN-γ. Kubuza imikorere ya T cells gutuma ubushobozi bwo kwica bugabanuka kandi ikibyimba cyihuta.
Nk’uko raporo zibivuga, TNFα ifite ingaruka zo kurwanya ikibyimba imbere n’inyuma, ariko ifite uruhare ruzwi mu guteza imbere ikura no kwangirika kwa kanseri y’intanga ngore (31-33). Nk’uko raporo zibivuga, ubwinshi bwa TNFα mu turemangingo tw’intanga ngore n’uturemangingo tw’intanga ngore mu barwayi ba kanseri y’intanga ngore ni bwinshi kurusha ubwo mu turemangingo tw’intanga ngore (34-36). Ku bijyanye n’imikorere, TNFα ishobora kugenzura imikorere, imikorere n’ubwiyongere bw’uturemangingo tw’amaraso yera, no guhindura imiterere y’uturemangingo twa kanseri (37, 38). Dukurikije ibi byavumbuwe, isesengura ry’itandukaniro ry’imiterere y’uturemangingo ryagaragaje ko TNF yazamutse cyane mu turemangingo twa T mu turemangingo tw’intanga ngore ugereranije na ascites (Ishusho ya 5C). Ukwiyongera kw’igaragara rya TNF byagaragaye gusa mu turemangingo twa T dufite imiterere idahumanya (Ishusho ya S5A). Muri make, aya makuru ashyigikira igitekerezo cy’uko MNA ifite ingaruka ebyiri zo gukumira ubudahangarwa bw’umubiri no guteza ikibyimba muri HGSC.
Gupima imiterere y'amaraso hakoreshejwe fluorescent hashingiwe ku buryo amaraso anyuramo byahindutse uburyo bw'ingenzi bwo kwiga imikorere ya TIL. Ubu bushakashatsi bwagaragaje ko ugereranije na lymphocytes zo mu maraso cyangwa selile T zo mu ngingo z'umubiri za lymphoid, TIL y'inyama z'umura n'iy'umuntu zifite ubushake bwo gufata isukari (4, 39) no gutakaza imikorere ya mitochondrial buhoro buhoro (19, 40). Nubwo twabonye ibisubizo bisa muri ubu bushakashatsi, iterambere ry'ingenzi ni ukugereranya imikorere y'uturemangingo twa kanseri na TIL bivuye ku turemangingo twa kanseri twakuwemo. Dukurikije zimwe muri izi raporo zabanje, uturemangingo twa kanseri (CD45-EpCAM +) dukomoka kuri ascites na tumors dufite imikorere myinshi ya glucose kurusha uturemangingo twa CD8 + na CD4 + T, bishimangira ko imikorere myinshi ya glucose mu turemangingo twa kanseri ishobora kugereranywa n'uturemangingo twa T. Igitekerezo cy'irushanwa rya T cells. TME. Ariko, imikorere ya mitochondrial y'uturemangingo twa kanseri iri hejuru kuruta iy'uturemangingo twa CD8 + T, ariko imikorere ya mitochondrial isa n'iy'uturemangingo twa CD4 + T. Ibi bisubizo bishimangira insanganyamatsiko igaragara y'uko imikorere ya oxidative ari ingenzi ku turemangingo twa kanseri (41, 42). Bavuga kandi ko uturemangingo twa CD8 + T dushobora kuba twibasirwa cyane no kudakora neza kwa ogisijeni kurusha uturemangingo twa CD4 + T, cyangwa ko uturemangingo twa CD4 + T dushobora gukoresha amasoko ya karuboni atari isukari kugira ngo dukomeze gukora kwa mitochondrial (43, 44). Byagombye kumenyekana ko nta tandukaniro twabonye mu gufata isukari cyangwa ibikorwa bya mitochondrial hagati ya CD4 + T effectors, T effector memory na T central memory cells muri ascites. Mu buryo nk'ubwo, imiterere y'itandukaniro ry'uturemangingo twa CD8 + T muri tumors ntaho ihuriye n'impinduka mu gufata isukari, bigaragaza itandukaniro rikomeye hagati ya uturemangingo twa T twakuriye muri vitro na TIL y'umuntu muri vivo (22). Ibi byanashimangiwe no gukoresha uburyo bwo gutanga uturemangingo twikora ku buryo butabogamye, byagaragaje kandi ko uturemangingo twa CD45 + / CD3- / CD4 + / CD45RO + dufite isukari nyinshi kandi imikorere ya mitochondrial kurusha uturemangingo twa tumour twinshi ariko dufite uturemangingo dukora muri metabolike. Uyu mubare ushobora kuba uhagarariye uturemangingo duto twa myeloid suppressor cyangwa uturemangingo twa plasmacytoid dendritic twagaragajwe mu isesengura rya scRNA-seq. Nubwo ibi byombi byagaragaye mu tumori tw’intanga ngore ku bantu [45], biracyakenewe ko hakorwa ikindi gikorwa cyo gusobanura iyi mibare y’abantu bafite myeloid.
Nubwo uburyo bushingiye kuri flow cytometry bushobora gusobanura itandukaniro rusange mu mikorere ya glucose na oxidative hagati y’ubwoko bw’uturemangingo, metabolite nyayo ikorwa na glucose cyangwa izindi soko za karuboni zo gutunganya imikorere ya mitochondrial muri TME ntiramenyekana. Guha metabolite kuba cyangwa kubura muri TIL runaka bisaba ko ingirabuzimafatizo zisukurwa mu bice byakuwemo. Kubwibyo, uburyo bwacu bwo kongera imiterere y’uturemangingo hamwe na mass spectrometry bushobora gutanga ubumenyi ku metabolite zikungahaye mu turemangingo twa T n’uturemangingo twa kanseri mu bipimo by’abarwayi bihuye. Nubwo ubu buryo bufite inyungu kuruta gutondeka uturemangingo dukoreshwa na fluorescence, hari ibitabo bya metabolite bishobora kugira ingaruka bitewe no kudahungabana cyangwa kwihuta kw’ihindagurika ry’uturemangingo (22). Nyamara, uburyo bwacu bwashoboye kumenya metabolite ebyiri zizwiho kugabanya ubudahangarwa bw’umubiri, adenosine na kynurenine, kuko zitandukanye cyane hagati y’ubwoko bw’icyitegererezo.
Isesengura ryacu rya metabonomike ku tumori n'ubwoko bwa TIL ritanga ibisobanuro birambuye ku ruhare rwa metabolike muri TME y'intanga. Ubwa mbere, dukoresheje flow cytometry, twasanze nta tandukaniro riri mu mikorere ya mitochondrial hagati ya tumori na CD4 + T cells. Ariko, isesengura rya LC-MS / MS ryagaragaje impinduka zikomeye mu bwinshi bwa metabolike muri abo bantu, bigaragaza ko imyanzuro yerekeye metabolike ya TIL n'imikorere yayo muri rusange ya metabolike isaba gusobanurwa neza. Icya kabiri, MNA ni metabolike ifite itandukaniro rikomeye hagati ya CD45-cells na T cells muri ascites, ntabwo ari tumori. Kubwibyo, gushyira ibice hamwe n'aho tumori iherereye bishobora kugira ingaruka zitandukanye kuri metabolike ya TIL, bigaragaza itandukaniro rishoboka mu bidukikije bito. Icya gatatu, imikorere ya enzyme ikora MNA NNMT ahanini igarukira kuri CAF, ari yo tumori tugize ku rugero ruto, ariko urugero rwa MNA ruboneka mu tumori tugize T. Kugaragara cyane kwa NNMT muri CAF y'intanga bizwiho gutera kanseri, bimwe mu biterwa no guteza imbere metabolike ya CAF, kwinjira kwa tumori no kugabanuka kwa metastasis (27). Nubwo urwego rwa TIL muri rusange ruri hagati, imiterere ya NNMT muri CAF ifitanye isano ya hafi n’ubwoko bwa mesenchymal ya Cancer Genome Atlas (TCGA), ifitanye isano no kuba MNA idakwirakwira neza (27, 46, 47). Hanyuma, imiterere ya enzyme AOX1 itera kwangirika kwa MNA nayo igarukira ku bantu ba CAF, ibyo bikaba bigaragaza ko uturemangingo twa T tudafite ubushobozi bwo gukora MNA. Ibi bisubizo bishyigikira igitekerezo cy'uko nubwo hakenewe akazi kenshi kugira ngo hagenzurwe iki kimenyetso, imiterere myinshi ya MNA muri uturemangingo twa T ishobora kugaragaza ko hari ibidukikije bito bya CAF bigabanya ubudahangarwa bw'umubiri.
Bitewe n'urwego ruto rw'ibinyabutabire bya MNA hamwe n'urwego rudasobanutse rwa poroteyine z'ingenzi zigira uruhare mu mikorere ya MNA, kuba MNA iri mu turemangingo twa T ntabwo bitunguranye. Nta NNMT cyangwa AOX1 byashoboraga kumenyekana hakoreshejwe isesengura rya scRNA-seq na qPCR y'amatsinda abiri yigenga. Ibi byavuye mu bushakashatsi bigaragaza ko MNA idakorwa na T cells, ahubwo iva muri TME ikikije. Ubushakashatsi bwakozwe muri vitro bwerekana ko T cells zikunda kwegeranya MNA yo hanze.
Ubushakashatsi bwacu bwakozwe muri vitro bwagaragaje ko MNA yo hanze ituma TNFα igaragara mu turemangingo twa T kandi ikongera uburyo Sp1 ifatana na TNFα. Nubwo TNFα ifite imikorere yo kurwanya ikibyimba no kurwanya ikibyimba, muri kanseri y'intanga ngore, TNFα ishobora guteza imbere ikura rya kanseri y'intanga ngore (31-33). Guhagarika TNFα mu mikurire y'uturemangingo twa kanseri y'intanga ngore cyangwa gukuraho ikimenyetso cya TNFα mu buryo bw'imbeba bishobora kunoza ikorwa rya cytokine iterwa na TNFα no kubuza ikura rya kanseri (32, 35). Kubwibyo, muri iki gihe, MNA ikomoka kuri TME ishobora gukora nk'ikintu gitera ubukana binyuze mu buryo buterwa na TNFα binyuze mu muyoboro wa autocrine, bityo bigatera imbere kugaragara no gukwirakwira kwa kanseri y'intanga ngore (31). Hashingiwe kuri ubu buryo, TNFα blockade irimo kwigwa nk'umuti ushobora kuvura kanseri y'intanga ngore (37, 48, 49). Byongeye kandi, MNA igabanya ubukana bwa cytotoxicity bw'uturemangingo twa CAR-T ku turemangingo twa kanseri y'intanga ngore, bigatanga ibindi bimenyetso byerekana ko ubudahangarwa bw'umubiri buterwa na MNA. Muri rusange, ibi bisubizo bigaragaza icyitegererezo aho kanseri n'uturemangingo twa CAF bisohora MNA muri TME yo hanze y'umubiri. Binyuze muri (i) gushishikariza gukura kwa kanseri y'intanga ngore iterwa na TNF na (ii) gukumira ibikorwa bya T biterwa na MNA, ibi bishobora kugira ingaruka ebyiri za kanseri (Ishusho ya 5D).
Mu gusoza, binyuze mu gukoresha uburyo bwo kongera imbaraga mu turemangingo, gukurikirana uturemangingo tw’umubiri umwe no gusuzuma imikorere y’uturemangingo, ubu bushakashatsi bwagaragaje itandukaniro rikomeye hagati y’uturemangingo tw’umubiri n’uturemangingo twa ascites mu barwayi ba HGSC. Iri sesengura ryimbitse ryerekanye ko hari itandukaniro mu gufata isukari n’imikorere ya mitochondrial hagati y’uturemangingo twa T, kandi ryagaragaje ko MNA ari metabolite idakoresha uturemangingo bwite. Aya makuru agira ingaruka ku buryo TME igira ingaruka ku mikorere y’uturemangingo twa T muri kanseri z’abantu. Nubwo hagaragaye irushanwa ry’intungamubiri hagati y’uturemangingo twa T n’uturemangingo twa kanseri, metabolite zishobora no gukora nk’igenzura ritaziguye kugira ngo zitere imbere mu gutera imbere kwa kanseri ndetse zishobora no gukumira ubwirinzi bw’umubiri. Gusobanura kurushaho uruhare rw’utu turemangingo dushobora gufungura ubundi buryo bwo kongera uburyo ubwirinzi bw’umubiri burwanya kanseri buterwa n’uturemangingo.
Ingero z'abarwayi n'amakuru y'ubuvuzi byabonetse binyuze mu bubiko bw'imitsi ya kanseri ya BC bwemejwe n'Ikigo gishinzwe Ubushakashatsi ku Miti cya Kanada. Dukurikije amabwiriza yemewe na Komite Ishinzwe Ubushakashatsi ku Miti ya BC na Kaminuza ya British Columbia (H07-00463), ingero zose z'abarwayi n'amakuru y'ubuvuzi byabonye uburenganzira bwanditse cyangwa byakuyeho uburenganzira bwabo ku mugaragaro. Ingero zibikwa muri BioBank yemewe (BRC-00290). Ibiranga umurwayi birambuye bigaragara mu mbonerahamwe S1 na S5. Mu gupima ubuziranenge, icyuma gikoreshwa mu gushonga ingero y'ikibyimba cy'umurwayi hanyuma kikayisunika mu cyuma cya mikoro 100 kugira ngo haboneke uburyo bwo kuyihagarika. Ascites y'umurwayi yashyizwe muri centrifuge ku muvuduko wa 1500 rpm mu gihe cy'iminota 10 kuri 4°C kugira ngo ikuremo uturemangingo no gukuramo supernatant. Uturemangingo twakuwe muri kanseri na ascites twabitswe muri serum ya AB y’umuntu ya 50% idakoresha ubushyuhe (Sigma-Aldrich), 40% RPMI-1640 (Thermo Fisher Scientific) na 10% dimethyl sulfoxide. Utu turemangingo twabitswe twashongeshejwe kandi dukoreshwa mu gusuzuma metabolomike no gusuzuma metabolite nkuko byasobanuwe hano hepfo.
Uburyo bwose bugizwe na 0.22 μm yayunguruwe 50:50 yiyongereyeho RPMI 1640: AimV. RPMI 1640 + 2.05 mM l-glutamine (Thermo Fisher Scientific) yiyongereyeho 10% ya serum ya AB y'umuntu idakoresha ubushyuhe (Sigma-Aldrich), 12.5 mM Hepes (Thermo Fisher Scientific), 2 mM l-glutamine (Thermo Fisher Scientific) Fisher Scientific), 1 x Penicillin Streptomycin (PenStrep) solution (Thermo Fisher Scientific) na 50 μMB-mercaptoethanol. AimV (Invitrogen) yiyongereyeho 20 mM Hepes (Thermo Fisher Scientific) na 2 mM l-glutamine (Thermo Fisher Scientific). Flow cytometer staining buffer yari igizwe na 0.22μm filtered phosphate buffered saline (PBS; Invitrogen) yiyongereyeho 3% ya serum ya AB y'umuntu idakoresha ubushyuhe (Sigma). Iyi seramu yongerera uturemangingo igizwe na PBS ya 0.22μm iyunguruye kandi ikongerwamo seramu ya AB y'umuntu idafite ubushyuhe ya 0.5% (Sigma-Aldrich).
Mu bushyuhe bwose bwa 37°C, uturemangingo twasizwe ibara rya 10 nM MT DR na 100 μM 2-NBDG mu minota 30. Hanyuma, uturemangingo twasizwe ibara rya eF506 ku bushyuhe bwa 4°C mu minota 15. Ongera uturemangingo muri FC Block (eBioscience) na Brilliant Stain Buffer (BD Biosciences), ushyiremo flow cytometer staining buffer (nk'uko amabwiriza y'uwakoze), hanyuma utwikire mu bushyuhe bw'icyumba mu minota 10. Siga uturemangingo udukoko (Table S2) muri flow cytometry staining buffer kuri 4°C mu minota 20. Ongera uturemangingo muri flow cytometry staining buffer (Cytek Aurora; 3L-16V-14B-8R configuration) mbere yo gusesengura. Koresha SpectroFlo na FlowJo V10 kugira ngo usesengure amakuru y'ibarura ry'uturemangingo, kandi ukoreshe GraphPad Prism 8 kugira ngo ukore amakuru. Ingufu z’ubushyuhe bw’ikirere (MFI) za 2-NBDG na MT DR zahinduwe ku buryo busanzwe, hanyuma hakoreshwa ikizamini cya t gikozwe hamwe mu gusesengura imibare kugira ngo harebwe abarwayi bahuye. Kuraho abantu bose bafite ibibazo biri munsi ya 40 mu isesengura; andika agaciro ka MFI ka 1 ku gaciro kose kari hasi mbere yo gukora isesengura ry’ibarurishamibare no kwerekana amakuru.
Kugira ngo twuzuze ingamba zo gushyiramo icyuma mu buryo bw'intoki z'urutonde rw'ibikorwa byavuzwe haruguru, twakoresheje ibisobanuro byuzuye bya shape restriction tree (FAUST) (21) kugira ngo dushyire uturemangingo ku baturage mu buryo bwikora nyuma yo gukuraho uturemangingo twapfuye muri FlowJo. Ducunga umusaruro ku giti cyacu kugira ngo duhuze ubwoko busa n'aho bwashyizwe mu buryo butari bwo (duhuza PD1+ n'uturemangingo twa PD1-tumor) n'ubwoko bwagumanye. Buri rugero rugizwe n'impuzandengo y'uturemangingo turenga 2%, ku bantu 11 bose hamwe.
Gupima ubucucike bwa Ficoll gradient density centrifugation byakoreshejwe mu gutandukanya PBMC n'ibicuruzwa byo gutandukanya leukocyte (STEMCELL Technologies). Uturemangingo twa CD8 + T twakuwe muri PBMC hakoreshejwe CD8 MicroBeads (Miltenyi) maze twongera mu buryo bwuzuye hakoreshejwe TransAct (Miltenyi) mu gihe cy'ibyumweru 2 hakurikijwe amabwiriza y'uruganda. Uturemangingo twemerewe guhagarara iminsi 5 mu buryo bwuzuye burimo IL-7 (10 ng/ml; PeproTech), hanyuma twongera gukangurwa na TransAct. Ku munsi wa 7, hakurikijwe amabwiriza y'uruganda, Uturemangingo twa CD45 MicroBeads (Miltenyi) tw'abantu twakoreshejwe mu gukungahaza uturemangingo mu byiciro bitatu bikurikirana. Uturemangingo twakusanyijwe kugira ngo dusesengure flow cytometry (nk'uko byasobanuwe haruguru), naho uturemangingo miliyoni twakusanyijwe inshuro eshatu kugira ngo dusesengure LC-MS/MS. Ingero zatunganyijwe na LC-MS/MS nk'uko byasobanuwe hano hepfo. Twapimye agaciro ka metabolite kabuze hamwe n'umubare wa ion 1.000. Buri gipimo gipimwa hakurikijwe umubare w’iyoni yose (TIC), kigahindurwa mu buryo bwa logarithmic hanyuma kigahindurwa mu buryo bwikora muri MetaboAnalystR mbere yo gusesengura.
Uduce duto twa buri murwayi twashongeshejwe hanyuma tugasukurwa binyuze mu kayunguruzo ka μm 40 mu buryo bwuzuye (nk'uko byasobanuwe haruguru). Dukurikije amabwiriza y'uwakoze, hakoreshejwe ibyiciro bitatu bikurikiranye byo guhitamo neza hakoreshejwe uburyo bwo gutandukanya uduce duto hakoreshejwe MicroBeads (Miltenyi) kugira ngo twongere ingero za CD8+, CD4+ na CD45- cells (ku rubura). Muri make, utwo tugingo twongera gushyirwa mu buryo bwo kongera ingufu za cells (nk'uko byasobanuwe haruguru) kandi tubarwa. Utwo tugingo twashyizwemo uduce duto twa CD8 tw'abantu, uduce duto twa CD4 tw'abantu cyangwa uduce duto twa CD45 tw'abantu (Miltenyi) ku bushyuhe bwa 4°C mu gihe cy'iminota 15, hanyuma twozwa n'uburyo bwo kongera ingufu za cells. Icyitegererezo gica mu gice cya LS (Miltenyi), hanyuma ibice byiza na bibi bigakusanywa. Kugira ngo tugabanye igihe kandi twongere intambwe yo kugarura utwo tugingo, igice cya CD8 gikoreshwa mu cyiciro cya kabiri cyo kongera ingufu za CD4+, hanyuma igice cya CD4 gikoreshwe mu kongera ingufu za CD45. Bika umuti ku rubura mu gihe cyose cyo gutandukanya.
Kugira ngo hategurwe ingero zo gusesengura metabolite, uturemangingo twogejwe rimwe n'umunyu ukonje, hanyuma hongerwamo ml 1 ya methanoli ya 80% kuri buri ngero, hanyuma hagashyirwamo umuyaga uvanze hanyuma hagakonjeshwa muri azote y'amazi. Ingero zashyizwe mu buryo butatu bwo gukonjesha no gushonga hanyuma zishyirwa kuri centrifuge ku muvuduko wa 14.000 rpm mu gihe cy'iminota 15 kuri 4°C. Supernatant irimo metabolite irahinduka umwuka kugeza yumye. Mebolifu yongeye gushonga muri 50 μl ya aside formique ya 0.03%, ikavangwa kugira ngo ivangwe, hanyuma ivangwa kugira ngo ikuremo imyanda.
Kuramo metabolite nkuko byasobanuwe haruguru. Shyira supernatant mu icupa rya chromatography y'amazi rifite imikorere myiza kugira ngo ukoreshe ubushakashatsi kuri metabolite. Koresha protocole yo kuvura mu buryo butunguranye kugira ngo uvure buri ngero hamwe n'umubare ungana w'uturemangingo kugira ngo wirinde ingaruka mbi. Twakoze isuzuma ry'imiterere ya metabolite ku isi yose ryatangajwe mbere kuri AB SCIEX QTRAP 5500 Triple Quadrupole Mass Spectrometer (50). Isesengura rya chromatografiya n'ishyirwa hamwe ry'agace k'ubuso byakozwe hakoreshejwe porogaramu ya MultiQuant verisiyo 2.1 (Applied Biosystems SCIEX).
Umubare wa iyoni 1000 wakoreshejwe mu gupima agaciro ka metabolite kabuze, kandi TIC ya buri gipimo yakoreshejwe mu kubara ubuso busanzwe bwa metabolite yabonetse kugira ngo hakosorwe impinduka zazanywe n'isesengura ry'ibikoresho bivuye mu gutunganya icyitegererezo. Nyuma y'uko TIC isanzwe, MetaboAnalystR(51) (parameter isanzwe) ikoreshwa mu guhindura logarithmic no gupima umurongo wa norm automatic. Twakoresheje PCA hamwe na vegan R package kugira ngo dukora isesengura ryimbitse ry'itandukaniro rya metabolite hagati y'ubwoko bw'icyitegererezo, kandi dukoresha isesengura rya partial redundancy kugira ngo dusesengure abarwayi. Dukoresheje uburyo bwa Ward kugira ngo twubake dendrogram y'ubushyuhe kugira ngo duhurize intera ya Euclidean hagati y'icyitegererezo. Twakoresheje limma (52) ku bwinshi bwa metabolite busanzwe kugira ngo tumenye metabolite nyinshi mu bwoko bwose bw'ingirabuzima fatizo n'ibidukikije bito. Kugira ngo tworoshye ibisobanuro, dukoresha parametre y'ikigereranyo cy'itsinda kugira ngo tugaragaze icyitegererezo, kandi tukareba ubwoko bw'ingirabuzima fatizo mu bidukikije bito nk'itsinda rya buri (n = amatsinda 6); Ku isuzuma ry’uburemere, twakoze ibipimo bitatu bisubirwamo kuri buri metabolite Kugira ngo twirinde ko isubiramo ry’ibitari byo, umurwayi yashyizwemo nk’imbogamizi mu miterere ya limma. Kugira ngo tugenzure itandukaniro riri hagati ya metabolite hagati y’abarwayi batandukanye, twahinduye icyitegererezo cya limma harimo n’abarwayi mu buryo buhamye. Twatanze raporo ku kamaro k’itandukaniro ryagenwe mbere hagati y’ubwoko bw’uturemangingo n’ibidukikije bya Padj <0.05 (Benjamini-Hochberg correction).
Nyuma yo kongera imbaraga hakoreshejwe Miltenyi Dead Cell Removal Kit (>80% ubuzima), hakozwe sequencing ya transcriptome y’uturemangingo duto ku turemangingo twose tw’uturemangingo twakonjeshejwe hamwe n’ibibyimba hakoreshejwe protocole ya 10x 5′expression. Hasesenguwe imanza eshanu zifite ibibyimba na ascites bihuye, nubwo ubushobozi buke bwo kubaho bwavuye ku kibyimba kimwe bwabujije ko byinjiramo. Kugira ngo tugere ku mahitamo menshi y’abarwayi, twahuje ingero za buri murwayi mu mirongo ya 10x chromium controller, maze dusesengura ascites n’ahantu hafite ibibyimba ukwabyo. Nyuma yo gukora sequencing [Illumina HiSeq 4000 28×98 bp paired end (PE), genome ya Quebec; impuzandengo y’amasomo 73.488 na 41.378 kuri buri selile ku ndwara ya kanseri na ascites uko bikurikirana], twakoresheje CellSNP na Vireo (53) (dushingiye kuri CellSNP nk'uko SNP isanzwe y'umuntu (VCF) itangwa na GRCh38 ihabwa umwirondoro w'umutanga. Dukoresha SNPRelate kugira ngo dufate umwirondoro wa hafi (IBS) w'imiterere y'uturemangingo tw'umurwayi (IBS), tutibagiwe uturemangingo n'uturemangingo tutaratangwa twagaragaye nk'uduce duplexes hamwe n'abatanga uturemangingo duhuza ascites n'ibipimo bya kanseri (54). Dushingiye kuri uyu murimo, twagumanye imanza eshatu zifite uturemangingo twinshi muri kanseri na ascites kugira ngo dusesengure. Nyuma yo gukora intambwe yo kuyungurura mu buryo bwinshi mu ipaki ya scater (55) na scran (56) ya BioConductor, ibi byatanze uturemangingo 6975 (uturemangingo 2792 na 4183 twavuye muri kanseri na ascites, uko bikurikirana) kugira ngo dusesengure. Dukoresha igraph's (57) Louvain clustering ya shared near near near network (SNN) ishingiye kuri Jaccard. Intera igera ku turemangingo tw’amatsinda hakurikijwe uko tugaragara. Amatsinda yashyizwe mu byiciro hakurikijwe ubwoko bw’uturemangingo bushingiye ku miterere ya gene y’ibimenyetso kandi agaragazwa na t-SNE. Uturemangingo twa T dufite uburozi buterwa na CD8A na GZMA, hatarimo uduce duto dufite poroteyine nke ya ribosomal. Twabonye amakuru yatangajwe na Izar et al. (16), harimo no gushyiramo t-SNE, bishobora kugenzura uko imiterere y’uturemangingo tw’umubiri ihurirana n’imiterere ya NNMT.
PBMC yatandukanyijwe n'ibicuruzwa byo gutandukanya leukocyte (STEMCELL Technologies) hakoreshejwe Ficoll gradient density centrifugation. Uturemangingo twa CD3 + twakuwe muri PBMC hakoreshejwe uduce twa CD3 (Miltenyi). Mu gihe MNA ihari cyangwa idafite, uturemangingo twa CD3 + twakoreshejwe hakoreshejwe CD3 (5μg/ml), CD28 ishongeshwa (3μg/ml) na IL-2 (300 U/ml; Proleukin). Ku munsi wa nyuma wo kwagura, ubushobozi bwo kubaho (Fixable Viability Dye eFluor450, eBioscience) n'ubwiyongere (123count eBeads, Thermo Fisher Scientific) byapimwe hakoreshejwe flow cytometry. Suzuma imikorere y'ingufu ukoresheje uburyo bwo gukangura uturemangingo hamwe na PMA (20 ng/ml) na ionomycin (1μg/ml) hamwe na GolgiStop mu gihe cy'amasaha 4, hanyuma ukurikirane CD8-PerCP (RPA-T8, BioLegend), CD4-AF700 (RPA-T4), BioLegend) na TNFα-fluorescein isothiocyanate (FITC) (MAb11, BD). Kangura uturemangingo twa qPCR na ChIP hamwe na PMA (20 ng/ml) na ionomycin (1μg/ml) mu gihe cy'amasaha 4. ELISA supernatant yakusanyijwe mbere na nyuma yo gukangura ukoresheje PMA (20 ng/ml) na ionomycin (1 μg/ml) mu gihe cy'amasaha 4.
Kurikiza protocole y'uwakoze kugira ngo utandukanye RNA ukoresheje RNeasy Plus Mini Kit (QIAGEN). Koresha QIAshredder (QIAGEN) kugira ngo uhuze icyitegererezo. Koresha RNA ifite ubushobozi bwinshi kuri cDNA kit (Thermo Fisher Scientific) kugira ngo uhuze ADN yuzuzanya (cDNA). Koresha TaqMan Rapid Advanced Master Mix (Thermo Fisher Scientific) kugira ngo upime imiterere ya gene (ukurikije protocole y'uwakoze) ukoresheje ibi bikurikira: Hs00196287_m1 (NNMT), Hs00154079_m1 (AOX1), Hs00427552_m1 (SLC22A1), Hs02786624_g1 [glyceraldehyde-3-phosphate off Hydrogen (GAPDH)] na Hs01010726_m1 (SLC22A2). Ingero zakoreshejwe kuri sisitemu ya StepOnePlus ya PCR y’igihe nyacyo (Applied Biosystems) (Applied Biosystems) muri MicroAmp fast optical 96-well reaction plate (Applied Biosystems) hamwe na MicroAmp optical film. Agaciro kose ka Ct karenze 35 gafatwa nk'akarenze urugero rwo kuvumbura kandi kakaba karashyizweho ikimenyetso cy'uko katagaragara.
Kora ChIP nkuko byasobanuwe mbere (58). Muri make, uturemangingo twavuwe formaldehyde (icyiciro cya nyuma cya 1.42%) hanyuma dushyirwa ku bushyuhe bw'icyumba mu gihe cy'iminota 10. Koresha inyongera y'ubushyuhe bwo kubyimba (25 mM Hepes, 1.5 mM MgCl2, 10 mM KCl na 0.1% NP-40) ku rubura mu gihe cy'iminota 10, hanyuma wongere ushyire mu buffer yo kuvura indwara nkuko byasobanuwe (58). Hanyuma icyitegererezo cyashyizwemo sonic hifashishijwe imikorere ikurikira: imikorere 10 (imitsi 20 y'isegonda 1) n'igihe kitari cyo cy'amasegonda 40. Shyiramo ChIP-grade immunoglobulin G (Seli Signaling Technology; 1μl), histone H3 (Seli Signaling Technology; 3μl), NFAT (Invitrogen; 3μl) na SP1 (Seli Signaling Technology; 3μl) antibodies hamwe n'icyitegererezo kuri 4°CC shake mu ijoro ryose. Shyira udusaro twa poroteyine A mu icupa (Thermo Fisher Scientific) hamwe n'icyitegererezo kuri 4°C hanyuma uzunguze buhoro isaha 1, hanyuma ukoreshe utusaro twa chelex (Bio-Rad) kugira ngo wongere ADN, kandi ukoreshe poroteyine K (Thermo Fisher) mu igogora rya poroteyine. Promoter ya TNFα yabonetse hakoreshejwe PCR: imbere, GGG TAT CCT TGA TGC TTG TGT; ahubwo, GTG CCA ACA ACT GCC TTT ATA TG (ibicuruzwa 207-bp). Amashusho yakozwe na Image Lab (Bio-Rad) kandi yapimwe hakoreshejwe porogaramu ya ImageJ.
Ingufu zo mu bwoko bwa selile zakusanyijwe nkuko byasobanuwe haruguru. Igenwa ryakozwe hakurikijwe uburyo bw'uruganda rwa TNFα ELISA kit (Invitrogen) y'umuntu, IL-2 ELISA kit (Invitrogen) y'umuntu na IFN-γ ELISA kit (Abcam). Dukurikije protocole y'uruganda, supernatant yagabanijwemo 1:100 kugira ngo hamenyekane TNFα na IL-2, na 1:3 kugira ngo hamenyekane IFN-γ. Koresha EnVision 2104 Multilabel Reader (PerkinElmer) kugira ngo upime uburyo bwo kwinjiza kuri 450 nm.
PBMC yatandukanyijwe n'ibicuruzwa byo gutandukanya leukocyte (STEMCELL Technologies) hakoreshejwe Ficoll gradient density centrifugation. Uturemangingo twa CD3 + twakuwe muri PBMC hakoreshejwe uduce twa CD3 (Miltenyi). Mu gihe MNA ihari cyangwa idafite, uturemangingo twa CD3 + twakoreshejwe hakoreshejwe CD3 (5μg/ml), CD28 ishongeshwa (3μg/ml) na IL-2 (300 U/ml; Proleukin) mu gihe cy'iminsi 3. Nyuma y'iminsi 3, uturemangingo twakusanyijwe twogezwa na saline ya 0.9%, maze agace gato karakonjeshwa. Umubare w'uturemangingo wakozwe hakoreshejwe flow cytometry (Cytek Aurora; 3L-16V-14B-8R configuration) hakoreshejwe 123count eBeads.
Ibinyabutabire by’ibinyabutabire nk’uko byasobanuwe haruguru. Ibinyabutabire byumye byongeye guhindurwa ku gipimo cya 4000 cell equivalent cells / μl. Sesengura icyitegererezo ukoresheje reversed-phase chromatography (1290 Infinity II, Agilent Technologies, Santa Clara, CA) na CORTECS T3 column (2.1×150 mm, particle size 1.6-μm, pore size 120-Å; #186008500, Waters). Polar mass spectrometer (6470, Agilent), aho electrospray ionization ikora mu buryo bwiza. Mobile phase A ni 0.1% formic acid (muri H2O), mobile phase B ni 90% acetonitrile, 0.1% formic acid. Ubushyuhe bwa LC ni iminota 0 kugeza kuri 2 kuri 100% A, iminota 2 kugeza kuri 7.1 kuri 99% B, na iminota 7.1 kugeza kuri 8 kuri 99% B. Hanyuma ongera uhuze inkingi na mobile phase A ku muvuduko wa 0.6 ml/minota mu gihe cy'iminota 3. . Umuvuduko w'amazi ni 0.4ml/minota, kandi icyumba cy'inkingi gishyushye kugeza kuri 50°C. Koresha standard ya MNA ya chemical pure (M320995, Toronto Research Chemical Company, North York, Ontario, Kanada) kugira ngo ushyireho igihe cyo kubika (RT) n'impinduka (RT = iminota 0.882, impinduka 1 = 137→94.1, impinduka 2 = 137→92, Impinduka 3 = 137→78). Iyo impinduka zose uko ari eshatu zibaye mu gihe gikwiye cyo kubika, impinduka 1 ikoreshwa mu gupima kugira ngo hamenyekane neza ko ari umwihariko. Ingano isanzwe ya MNA (Toronto Research Chemical Company) yakozwe hakoreshejwe uburyo butandatu bwo gupima umuti (1 mg/ml) kugira ngo haboneke ibipimo bya 0.1, 1.0, 10 na 100 ng/ml na 1.0 na 10μg/ml uko bikurikirana. Umupaka wo kumenya ni 1 ng/ml, kandi igisubizo kiri hagati ya 10 ng/ml na 10μg/ml. Buri ngero ya mikorolitiro ebyiri z'icyitegererezo n'ibisanzwe ikoreshwa mu gusesengura LC/MS, kandi icyitegererezo cy'ubuziranenge gikoreshwa buri ngero umunani kugira ngo urubuga rwo gusesengura ruhamye. Ibisubizo bya MNA by'ingero zose za selile zavuwe na MNA byari mu rugero rwa linear rw'isuzuma. Isesengura ry'amakuru ryakozwe hakoreshejwe porogaramu ya MassHunter quantitative analysis (v9.0, Agilent).
Imiterere ya αFR-CAR yo mu gisekuru cya kabiri yakuwe kuri Song et al. (59). Muri make, iyi miterere ikubiyemo ibi bikurikira: CD8a leader sequence, human αFR-specific single-chain variable fragment, CD8a hinge and transmembrane region, CD27 intracellular domain na CD3z intracellular domain. Imiterere yuzuye ya CAR yakozwe na GenScript, hanyuma ishyirwa muri lentiviral expression vector yo mu gisekuru cya kabiri iri hejuru ya kaseti ya GFP expression ikoreshwa mu gusuzuma imikorere ya transduction.
Lentivirus ikorwa binyuze mu guhindura uturemangingo twa HEK293T [American Type Culture Collection (ATCC); yatewe mu buryo bwa Dulbecco's modified Eagle medium irimo 10% by'amaraso y'inka yo mu nda (FBS) na 1% ya PenStrep, kandi hakoreshejwe CAR-GFP vector na The packaging plasmids (psPAX2 na pMD2.G, Addgene) bakoresha lipofection amine (Sigma-Aldrich). Supernatant irimo virusi yakusanyijwe nyuma y'amasaha 48 na 72 nyuma yo kuyihindura, iyungururwa, kandi ishyirwamo imbaraga nyinshi hakoreshejwe ultracentrifugation. Bika supernatant ya virusi ikomeye kuri -80°C kugeza ikoreshejwe.
PBMC itandukanyijwe n'ibicuruzwa bizima byo gutandukanya leukocyte (STEMCELL Technologies) hakoreshejwe Ficoll gradient density centrifugation. Koresha uduce duto twa CD8 (Miltenyi) kugira ngo utandukanye uturemangingo twa CD8+ na PBMC. Koroshya uturemangingo twa T ukoresheje TransAct (Miltenyi) no muri TexMACS medium [Miltenyi; yongerewemo 3% by'amaraso y'umuntu adakoresha ubushyuhe, 1% PenStrep na IL-2 (300 U/ml)]. Amasaha makumyabiri n'ane nyuma yo gukangurwa, uturemangingo twa T twashyizwemo lentivirus (10 μl ya virusi ikungahaye kuri buri turemangingo 106). Nyuma y'iminsi 1 kugeza kuri 3 nyuma yo gukangurwa kuri Cytek Aurora (kuri FSC (Forward Scatter)/SSC (Side Scatter), Singlet, GFP+), suzuma uburyo GFP igaragara mu turemangingo kugira ngo ugaragaze ko ubushobozi bwo gukangurwa bwa nibura 30%.
Uturemangingo twa CAR-T twatejwe mu gihe cy'amasaha 24 muri Immunocult (STEMCELL Technologies; twongereweho 1% PenStrep) muri ibi bikurikira: tutavuwe, twavuwe na 250 μM adenosine cyangwa 10 mM MNA. Nyuma yo kuvurwa mbere, uturemangingo twa CAR-T twogejwe na PBS maze duhuzwa na 20.000 SK-OV-3 selile [ATCC; muri McCoy 5A medium (Sigma-Aldrich) twongereweho 10% FBS na 1% PenStrep kuri 10: Igipimo cya effector to target cya 1 cyongereweho inshuro eshatu muri Immunocult medium yongereweho. Uturemangingo twa SK-OV-3 na SK-OV-3 selile zasizwe digitalis saponin (0.5mg/ml; Sigma-Aldrich) zakoreshejwe nk'uburyo bwo kugenzura bubi n'ubwiza, uko bikurikirana. Nyuma y'amasaha 24 yo guhinga hamwe, supernatant yakusanyijwe maze lactate dehydrogenase (LDH) ipimwa hakurikijwe amabwiriza y'uwakoze (LDH Glo Cytotoxicity Assay Kit, Promega). Supernatant ya LDH yagabanijwemo 1:50 muri LDH buffer. Ijanisha ry'ubwicanyi ryapimwe hakoreshejwe formula ikurikira: ijanisha ry'ubwicanyi = ijanisha ry'ikosora / igipimo ntarengwa cy'ubwicanyi x 100%, aho ijanisha ry'ikosora = selile za co-culture-T gusa, naho igipimo ntarengwa cy'ubwicanyi = igenzura ryiza ryo kugenzura cyangwa kurwanya.
Nkuko byasobanuwe mu nyandiko cyangwa ibikoresho n'uburyo, koresha GraphPad Prism 8, Microsoft Excel cyangwa R v3.6.0 mu gusesengura imibare. Iyo hakusanyijwe ingero nyinshi ku murwayi umwe (nk'uturemangingo n'ikibyimba), dukoresha ikizamini cya t gikozwe kuri paired cyangwa tugashyiramo umurwayi nk'ingaruka zidasanzwe mu buryo busanzwe cyangwa rusange uko bikwiye. Ku isesengura rya metabolomike, ikizamini cy'ingenzi gikorwa mu byiciro bitatu.
Ku bindi bikoresho by'inyongera kuri iyi nkuru, reba http://advances.sciencemag.org/cgi/content/full/7/4/eabe1174/DC1
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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 Bellardini (Ralph J. DeBerardinis).
MNA igira uruhare mu kugabanya ubudahangarwa bw'umubiri bw'uturemangingo twa T kandi ikaba ari yo ntego ishobora kuba iy'ubuvuzi bw'ubudahangarwa bw'umubiri mu kuvura kanseri y'abantu.
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 Bellardini (Ralph J. DeBerardinis).
MNA igira uruhare mu kugabanya ubudahangarwa bw'umubiri bw'uturemangingo twa T kandi ikaba ari yo ntego ishobora kuba iy'ubuvuzi bw'ubudahangarwa bw'umubiri mu kuvura kanseri y'abantu.
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