Ivyo bihimba bifasha umubiri guhangana n’indwara ni ikintu nyamukuru kiranga ibidukikije bitobito vy’ibivyimba (TME), ariko uretse bikeyi, akaranga kavyo karacari ahanini katazwi. Aha, twasuzumye ibivyimba n’utugingo ngengabuzima T tuva mu bivyimba n’ibivyimba vy’abarwayi bafise kanseri y’amaraso y’agaciro kanini (HGSC) kugira ngo tubone metabolome y’ivyo bice bitandukanye vya TME. Ascites n’ingingo z’ibivyimba zifise itandukaniro rikomeye ry’ibifungurwa. Ugereranije n’indwara y’ibivyimba, utugingo ngengabuzima T twinjira mu gisebe turatunze cane 1-methylnicotinamide (MNA). Naho urugero rwa MNA mu ngingo ngenderwako T ruri hejuru, ukugaragara kwa nikotinamide N-methyltransferase (intungamubiri ituma imigwi ya methyl iva muri S-adenosylmethionine ikaja muri nikotinamide) kugarukira gusa ku ngingo ngengabuzima zitwa fibroblastes be n’ingingo ngenderwako z’ibivyimba. Mu buryo bukora, MNA ituma utugingo ngengabuzima T dusohora igihimba c’umubiri gitera igihimba c’umubiri citwa cytokine alpha. Ku bw’ivyo, MNA ikomoka kuri TME irafasha mu gutunganya ubudahangarwa bw’umubiri bw’ingingo ngenderwako T kandi iserukira uburyo bwo gukingira umubiri bushobora gukoreshwa mu kuvura kanseri y’umuntu.
Ivyo bihimba biva mu bivyimba birashobora kugira ingaruka zikomeye ku bijanye n’ubudahangarwa bw’umubiri ku bivyimba, kandi ibimenyamenya vyinshi vyerekana ko bishobora kandi gukora nk’inkomezi nyamukuru zituma indwara zitera imbere (1). Uretse ingaruka za Warburg, ibikorwa biherutse gukorwa vyatanguye kugaragaza uko utugingo ngengabuzima tw’ibivyimba dukora be n’isano rifise n’ingene umubiri ugira ubudahangarwa bw’umubiri (TME). Ivyigwa vyakozwe ku bigereranyo vy’imbeba no ku turemangingo twa T tw’umuntu vyerekanye ko uguhindura glutamine (2), uguhindura oxidative (3) n’uguhindura glucose (4) bishobora gukora bidashingiye ku migwi itandukanye y’ingingo z’umubiri zigwanira umubiri. Ibintu vyinshi bihinduka muri izo nzira birabuza igikorwa c’ingingo ngenderwako T co kurwanya ibivyimba. Vyerekanwe ko guhagarika coenzyme tetrahydrobiopterin (BH4) bishobora kwonona ubwinshi bw’ingingo ngenderwako T, kandi ukwiyongera kwa BH4 mu mubiri bishobora kwongereza ubushobozi bwo kurwanya ibivyimba bufashijwe na CD4 na CD8. Ikindi, ingaruka z’ubudahangarwa bw’umubiri za kynurenine zishobora gukizwa n’ugufata BH4 (5). Mu glioblastoma ihinduka y’umubiri (IDH), ugusohoka kw’imitsi y’umubiri (R-2-HG) birabuza utugingo ngengabuzima T gukora, ukwiyongera n’ugukora kw’utugingo ngengabuzima (6). Vuba aha, vyerekanwe ko methylglyoxal, igihimba c’inyuma c’ihinduka ry’amaraso, gikomoka ku turemangingo tw’inkomoko ya myeloïde, kandi ko utugingo ngengabuzima T turungika methylglyoxal bishobora guhagarika igikorwa c’utugingo ngengabuzima T dukora. Mu kuvura, ugukuraho methylglyoxal birashobora gutsinda igikorwa c’ingingo z’umubiri zikomoka ku myeloid (MDSC) no kwongerera ubushobozi ubuvuzi bwo guhagarika ivyiyumviro mu bigereranyo vy’imbeba (7). Izo nyigisho zose hamwe zishimika ku ruhara ruhambaye rw’ibintu biva muri TME mu gutunganya ibikorwa n’ibikorwa vy’ingingo ngenderwako T.
Ugukora nabi kw’utugingo ngengabuzima T kwaravuzwe cane muri kanseri y’amabere (8). Ivyo biva ku bimenyetso vy’imibiri biva ku kubura umwuka mwiza n’imitsi y’ibivyimba idasanzwe (9), ivyo bikaba bituma glucose na tryptophane bihinduka ibintu biva mu bihimba vy’umubiri nk’aside lactique na kynurenine. Ivyo bita lactate birenze urugero bigabanya ivyimburwa vy’interferon-γ (IFN-γ) kandi bituma habaho itandukaniro ry’imigwi y’imitsi y’umubiri (10, 11). Gufata tryptophan birabuza ata guca ku ruhande utugingo ngengabuzima T kwiyongera kandi bikabuza utugingo ngengabuzima T gutanga ikimenyetso (12-14). Naho ivyo vyabonetse, igikorwa kinini kijanye n’uguhinduka kw’imitsi y’umubiri carakozwe mu mico kama y’ingingo ngenderwako T hakoreshejwe ubuhinga bwiza, canke bigarukira ku bigereranyo vy’imbeba zihuye mu buzima, nta na kimwe muri vyo kigaragaza neza ubudasa bw’imigera ya kanseri y’abantu n’ibidukikije vy’umubiri bikomeye n’ibitobito.
Ikintu gisanzwe kigaragaza kanseri y’amabere ni ugukwiragira mu gifu no kugaragara kw’ibisebe vy’amabere. Ukwirundanira kw’amazi y’ingingo mu mfuruka y’umubiri bifitaniye isano n’ingwara iteye imbere be n’ukuntu umuntu adashobora gutera neza (15). Nk’uko amakuru abivuga, ico gice kidasanzwe kirafise ubukene bw’umwuka, gifise urugero runini rw’ibitera gukura kw’imitsi (VEGF) n’indoleamine 2,3-dioxygenase (IDO), kandi kigizwe n’ingingo ngenderwako zigenzura imitsi T be n’ingingo ngenderwako zibuza imitsi (15-18). Ivyo bishobora kuba bitandukanye n’ivyo igihimba c’umubiri gikora ubwaco, ni co gituma ugusubira gukora porogarama y’ingingo ngenderwako T mu kibanza c’umubiri bidatomoye. Ikindi, itandukaniro ry’ingenzi n’ubudasa hagati y’ibihimba vy’irondoka n’ibihimba vy’umubiri biri mu bidukikije vy’ibivyimba bishobora gutuma ingingo z’umubiri zigwanira umubiri zidashobora kwinjira mu mubiri n’igikorwa cazo ku bivyimba, kandi birakenewe ko haba ubushakashatsi bundi.
Kugira ngo ivyo bibazo bitorerwe umuti, twateguye uburyo bwo gutandukanya utugingo ngengabuzima n’uburyo bwo gupima uburemere bw’utugingo ngengabuzima (LC-MS/MS) kugira ngo twige ubwoko butandukanye bw’utugingo ngengabuzima (harimwo utugingo ngengabuzima CD4 + na CD8 + T) be n’imbere mu bivyimba no hagati yavyo. Turakoresha ubu buryo hamwe n’ubuhinga bwo gupima uruja n’uruza rw’amaraso n’ugukurikiranya ARN y’ingingo imwe (scRNA-seq) kugira ngo dutange ishusho nziza cane y’ingene ivyo bihimba vy’umubiri bigenda. Ubwo buryo bwagaragaje ko urugero rwa 1-methylnicotinamide (MNA) mu turemangingo T tw’ibivyimba rwongerekanye cane, kandi ivyigwa vyakozwe mu bipimo vy’ibivyimba vyerekanye ko ingaruka za MNA ku bikorwa vy’uturemangingo T zitazwi mbere. Muri rusangi, ubu buryo burahishura imigenderanire hagati y’ibivyimba n’ingingo z’umubiri zikingira umubiri, kandi buratanga ubumenyi bwihariye ku bijanye n’ingingo z’umubiri zigenzura ubudahangarwa bw’umubiri, bishobora kuba ngirakamaro mu kuvura kanseri y’amabere ishingiye ku ntungamubiri T Amahirwe yo kuvura.
Twakoresheje ubuhinga bwo gupima uruja n’uruza rw’amaraso kugira ngo tumenye icarimwe urugero rw’isukari ifata mu mubiri [2-(N-(7-nitrophenyl-2-oxa-1,3-diaza-4-yl)amino)-2-deoxyglucose (2-NBDG) n’ibikorwa vy’imitochondrie [MitoTracker Deep Red, MT,DR01) ibimenyetso bimenyerewe bitandukanya utugingo ngengabuzima tugwanira umubiri n’utugingo ngengabuzima tw’ibivyimba (Imbonerahamwe S2 n’Igishushanyo S1A). Iryo sesangura ryerekanye ko ugereranyije n’ingingo za T, ingingo z’umubiri zitwa ascites n’ingingo z’ibivyimba zifise urugero rwo gufata isukari nyinshi, ariko zikaba zifise itandukaniro ritoyi mu bikorwa vy’imitochondrie. Igitigiri c’isukari gifatwa n’ingingo z’ibivyimba [CD45-EpCAM (EpCAM)+] ni incuro zitatu canke zine z’ingingo za T, kandi igitigiri c’isukari gifatwa n’ingingo za CD4 + T ni incuro 1,2 z’ingingo za CD8 + T, ivyo bikaba vyerekana ko utugingo ngengabuzima twinjira mu bivyimba (TIL) dufise ibisabwa bitandukanye). Mu buryo butandukanye n’ubwo, igikorwa c’imitochondrie mu ngingo z’ibivyimba gisa n’ic’ingingo za CD4 + T, kandi igikorwa c’imitochondrie c’ubwo bwoko bwompi bw’ingingo ni kinini kuruta ic’ingingo za CD8 + T (Ishusho 1B). Muri rusangi, ivyo bimenyetso birerekana urugero rw’imibiri. Ibikorwa vy’uguhindura ibiribwa vy’ingingo z’ibivyimba biraruta ivy’ingingo za CD4 + T, kandi ibikorwa vy’uguhindura ibiribwa vy’ingingo za CD4 + T biraruta ivy’ingingo za CD8 + T. Naho ivyo bitera ingaruka ku bwoko bwose bw’ingingo, nta tandukaniro ridahinduka mu bijanye n’ingene ingingo za CD4 + na CD8 + T zikora canke mu bijanye n’imiterere yazo mu bivyimba vy’umubiri ugereranyije n’ibivyimba (Ishusho 1C). Mu buryo butandukanye n’ubwo, mu gice c’ingingo za CD45, igitigiri c’ingingo za EpCAM+ mu gisebe caragwiriye ugereranyije n’igisebe c’amaraso (Ishusho 1D). Twarabonye kandi itandukaniro rigaragara hagati y’ibihimba vy’ingingo za EpCAM+ n’ivya EpCAM-. Utugingo ngengabuzima twa EpCAM+ (tumor) dufise ubushobozi bwo gufata isukari n’ibikorwa vyinshi vy’imitochondrie kurusha utugingo ngengabuzima twa EpCAM-, ivyo bikaba ari vyinshi cane kuruta ibikorwa vy’uguhindura ibiribwa vy’utugingo ngengabuzima tw’ibivyimba muri TME (Ishusho 1, E na F).
(A na B) Uburemere bw’umuco w’izuba (MFI) bw’ugufata isukari (2-NBDG) (A) n’ibikorwa vy’imitochondrie vy’ingingo za CD4 + T (MitoTracker umutuku mwiza) (B) Ivyerekana (ibubamfu) n’amakuru y’urutonde (Iburyo), ingingo za CD8 + T n’ingingo za EpCAM + T n’ingingo za EpCAM + CD45-. (C) Igitigiri c’ingingo za CD4 + na CD8 + (z’ingingo za CD3 + T) mu bihimba vy’umubiri bita ascite n’ibivyimba. (D) Igitigiri c’ingingo z’ibivyimba za EpCAM + mu bihimba vy’umubiri n’ibivyimba (CD45−). (E na F) EpCAM + CD45-igihimba c’umubiri n’igihimba c’umubiri gifata isukari (2-NBDG) (E) n’igikorwa c’imitochondrie (MitoTracker umutuku mwiza) (F) ibishushanyo vyerekana (ibubamfu) n’amakuru ari mu mbonerahamwe ( Iburyo) Ascites n’ingingo z’igihimba c’umubiri. (G) Ivyerekana CD25, CD137 na PD1 bikoresheje ubuhinga bwo gupima umuvuduko w’amaraso. (H na I) CD25, CD137 na PD1 zigaragara ku turemangingo twa CD4 + T (H) no ku turemangingo twa CD8 + T (I). (J na K) Ivyiyumviro bitari vyo, ubwonko bwo hagati (Tcm), ubwonko bukora (Teff) n’ubwonko bukora (Tem) bishingiye ku nsiguro ya CCR7 na CD45RO. Amashusho aserukira (ibubamfu) n’amakuru y’urutonde (iburyo) y’ingingo za CD4 + T (J) n’ingingo za CD8 + T (K) mu bivyimba n’ibivyimba. Ivyiyumviro vya P bigenwa n’ikigeragezo ca t (*P<0,05, **P<0,01 na ***P<0,001). Umurongo ugereranya abarwayi bahuye (n = 6). FMO, ivy’uguca ibibatsi bikuweko kimwe; MFI, uburemere bw’umuco w’izuba.
Ibindi bipimo vyerekanye ibindi bitandukanye bihambaye hagati y’imiterere y’imiterere y’ingingo ngenderwako T zitowe umuti cane. Ivyiyumviro bikora (Ishusho 1, G gushika kuri I) n’ivy’ukwibuka bikora (Ishusho 1, J na K) mu bivyimba birakunda cane kuruta ascite (igice c’ingingo za CD3 + T). Na vyo nyene, gusuzuma ivy’umubiri bikoresheje imvugo y’ibimenyetso vy’ugukora (CD25 na CD137) be n’ibimenyetso vy’ugutakaza [proteine ​​y’urupfu rw’ingingo ngenderwako 1 (PD1)] vyerekanye ko naho ibiranga imibiri y’abantu bitandukanye (Ishusho S1, B gushika kuri E) , Ariko nta tandukaniro rikomeye ry’imibiri y’abantu ryari hagati y’imibiri y’abantu canke ry’imibiri y’abantu. F gushika kuri I). Ivyo vyavuyemwo vyemejwe hakoreshejwe uburyo bwo kwiga n’imashini kugira ngo umuntu ashobore gutanga utugingo ngengabuzima (21), ivyo bikaba vyarongeye vyerekana ko hariho utugingo ngengabuzima twinshi tw’amagufa (CD45 + / CD3- / CD4 + / CD45RO +) mu mfuruka y’umurwayi (Ishusho S2A ). Mu bwoko bwose bw’ingingo zamenyekanye, iyo ngingo y’umubiri yitwa myeloïde ni yo yerekanye ko ari yo ifata cane isukari n’ibikorwa vy’imitochondrie (Ishusho S2, B gushika kuri G). Ivyo bimenyetso birerekana itandukaniro rikomeye ry’imibiri hagati y’ubwoko bwinshi bw’ingingo ziboneka mu bivyimba vy’umubiri n’ibivyimba vy’abarwayi ba HGSC.
Ingorane nyamukuru mu gutahura ibiranga TIL ni ugutandukanya ivyerekanwa vy’ingingo ngenderwako T zifise ubusuku buhagije, uburyo n’ingero bihagije ku bivyimba. Ivyigwa biherutse gukorwa vyerekanye ko uburyo bwo gutoranya no gutunganya amabuye bushingiye ku buhinga bwa flow cytometry bushobora gutuma habaho amahinduka mu mice y’ibihimba vy’umubiri (22-24). Kugira ngo tuneshe iyo ngorane, twatunganije neza uburyo bwo gutunganya amabuye kugira ngo TIL itandukanye kandi itandukanye na kanseri y’amabere y’umuntu yaciwe mu kubagwa imbere y’uko isesengurwa hakoreshejwe LC-MS/MS (raba Ibikoresho n’Uburyo; Igishushanyo 2A). Kugira ngo dusuzume ingaruka rusangi z’iyi porotokole ku mahinduka y’ibinyabuzima, twagereranije imiterere y’ibinyabuzima vy’ingingo za T zakoreshejwe n’abatanga amagara meza inyuma y’intambwe yo gutandukanya amabuye iri hejuru n’ingingo zitatandukanijwe n’amabuye ariko zagumye ku barafu. Iryo sesangura ry’uburyo bwo kugenzura uburyo ryasanze hariho isano rikomeye hagati y’ivyo bimenyetso bibiri (r = 0,77), kandi ubushobozi bwo gusubiramwo mu buryo bw’ubuhinga bw’umugwi w’ibinyabuzima 86 bufise ubushobozi bwo gusubiramwo cane (Ishusho 2B). Ku bw’ivyo, ubwo buryo burashobora gukora isesengura ry’ukuri ry’ibihimba vy’umubiri mu ngingo ziriko ziratunganirizwa ubwoko bw’ingingo, gutyo bikaba bitanga urubuga rwa mbere rufise ubushobozi bwo kumenya ibintu vy’umubiri vyihariye biri muri HGSC, gutyo bikaba bituma abantu bashobora gutahura neza cane ivy’ibihimba vy’umubiri vy’umubiri Porogarama y’ibihimba vy’umubiri vy’igitsina.
(A) Igishushanyo c’ugutunganywa kw’amabuye y’agaciro. Imbere y’uko izo nsoro zisesangurwa hakoreshejwe ubuhinga bwa LC-MS/MS, zizoca mu bice bitatu bikurikirana vy’ugutunganywa n’amabuye y’agaciro canke zigume ku barafu. (B) Ingaruka z’ubwoko bw’ubutunzi ku bwinshi bw’ibinyabuzima bihindura ibiribwa. Igitigiri c’ingero zitatu ku bwoko bwose bw’ubutunzi ± SE. Umurongo w’imvi ugereranya ubucuti bwa 1:1. Ihuriro ry’imbere mu rwego (ICC) ry’ingero zisubirwamwo ryerekanywe ku rutonde rw’umurongo. NAD, ni nikotinamide y’umubiri. (C) Igishushanyo c’ingene igikorwa co gusuzuma ivy’umubiri w’umurwayi gigenda. Ascites canke ibivyimba bikusanywa ku barwayi bikabikwa mu mazi. Igice gitoyi c’ivyo bipimo vyasuzumwe hakoreshejwe ubuhinga bwa flow cytometry, mu gihe ibindi bipimo vyasigaye vyakozwe mu bice bitatu vy’ugutunganya utugingo ngengabuzima CD4+, CD8+ na CD45-. Ivyo bice vy’ingingo vyasuzumwe hakoreshejwe ubuhinga bwa LC-MS/MS. (D) Ikarata y’ubushuhe y’ubwinshi bw’ibintu bihindura ibiribwa. Ico gishushanyo c’ibiti kigereranya ugushira hamwe kwa Ward kw’intambwe z’ivya Euclide hagati y’ibigereranyo. (E) Isesengura ry’ibice nyamukuru (PCA) ry’ikarata y’ibinyabuzima, yerekana ibisubizo bitatu vy’inyandiko imwe imwe, ivyerekanwa biva ku murwayi umwe bihuzwa n’umurongo. (F) PCA y’ivyo bihimba vy’umubiri vy’ivyo bihimba vy’umubiri bishingiye ku murwayi (ni ukuvuga gukoresha igice c’ivyo bihimba vy’umubiri); ubwoko bw’akarorero buragabanywa n’igice c’umubiri gipfutse. PC1, igice nyamukuru ca 1; PC2, igice nyamukuru ca 2.
Inyuma y’aho, twakoresheje ubu buryo bwo gutunganya kugira ngo dusuzume ibintu 99 bihinduka mu bice vy’utugingo ngengabuzima CD4 +, CD8 + na CD45 mu bihimba vy’umubiri vy’intango n’ibivyimba vy’abarwayi batandatu ba HGSC (Ishusho 2C, Igishushanyo S3A n’Imbonerahamwe S3 na S4). Igitigiri c’abantu bafise inyungu ni 2% gushika kuri 70% vy’urugero runini rw’intango rw’ingingo ngenderwako zifise ubuzima, kandi igitigiri c’ingingo ngenderwako kiratandukanye cane hagati y’abarwayi. Amaze gutandukanya amabuye, igice c’inyungu gitunganijwe (CD4+, CD8+ canke CD45-) gifise ibice birenga 85% vy’ingingo zose zifise ubuzima ziri mu citegererezo ku rugero rwa mwayeni. Ubwo buryo bwo gutunganya buratuma dushobora gusuzuma igitigiri c’ingingo ngenderwako zivuye mu guhindura ibihimba vy’umubiri vy’ibivyimba vy’umuntu, ivyo bikaba bidashoboka gukorwa biciye ku bipimo vyinshi. Dukoresheje iyo porotokole, twabonye ko l-kynurenine na adenosine, ivyo bibiri bifise ibimenyetso vyiza vy’ubudahangarwa bw’umubiri vyari vyuzuye mu turemangingo T tw’ibivyimba canke mu turemangingo tw’igituntu (Ishusho S3, B na C). Ku bw’ivyo, ivyo bimenyetso vyerekana ubudahemuka n’ubushobozi bw’ubuhinga bwacu bwo gutandukanya utugingo ngengabuzima n’ubuhinga bwo gupima uburemere bw’ibintu kugira ngo turonke ibintu bihambaye mu binyabuzima mu bihimba vy’umubiri vy’abarwayi.
Isesengura ryacu kandi ryerekanye ugutandukanya gukomeye kw’ubwoko bw’ingingo z’umubiri hagati y’abarwayi (Ishusho 2D n’Ishusho S4A). Cane cane, ugereranyije n’abandi barwayi, umurwayi 70 yerekanye ibirango bitandukanye vy’imibiri (Ishusho 2E n’Ishusho S4B), ivyo bikaba vyerekana ko hoshobora kubaho ubudasa bukomeye bw’imibiri hagati y’abarwayi. Birabereye kumenya ko ugereranije n’abandi barwayi (litiro 1,2 gushika kuri 2; Imbonerahamwe S1), umubare wose w’amaraso y’umubiri yaronkejwe ku murwayi 70 (80 ml) wari muto. Igenzura ry’ubudasa hagati y’abarwayi mu gihe c’isesengura ry’ibice nyamukuru (nk’akarorero, hakoreshejwe isesengura ry’ibice vy’inyuma) ryerekana amahinduka adahinduka hagati y’ubwoko bw’ingingo, kandi ubwoko bw’ingingo n’/canke ibidukikije bitobito birahurizwa hamwe neza hakurikijwe imiterere y’ibinyabuzima (Ishusho 2F ). Isesengurwa ry’ibintu bimwe bimwe bihinduka mu mubiri ryashimikiye kuri izo ngaruka kandi ryerekanye itandukaniro rikomeye hagati y’ubwoko bw’ingingo ngenderwako n’ibidukikije bitobito. Birabereye kumenya ko itandukaniro rikomeye cane ryabonetse ari MNA, akenshi itunganywa n’ingingo za CD45- be n’ingingo za CD4+ na CD8+ zica mu gisebe (Ishusho 3A). Ku turemangingo twa CD4 +, iyo ngaruka iragaragara cane, kandi MNA iri mu turemangingo twa CD8 + na yo nyene isa n’iyishikirwa cane n’ibidukikije. Ariko ivyo si ngombwa, kuko batatu gusa mu barwayi batandatu ni bo bashobora gusuzumwa ku bijanye n’ibiharuro vy’ibivyimba CD8+. Uretse MNA, mu bwoko butandukanye bw’ingingo z’umubiri ziri mu nzitiramibu n’ibivyimba, ibindi bihimba vy’umubiri bifise akamaro kabi muri TIL na vyo nyene birafise ubutunzi butandukanye (Ibishushanyo S3 na S4). Ku bw’ivyo, ayo makuru yerekana ko hariho ibintu vyiza bishobora gutuma umubiri ugira ubudahangarwa bw’umubiri kugira ngo hakorwe ubushakashatsi bundi.
(A) Ibiri muri MNA bisanzwe mu ngirabuzima fatizo za CD4+, CD8+ na CD45- zivuye mu bihimba vy’umubiri n’ibivyimba. Igishushanyo c’agasanduku kirerekana umurongo wo hagati (umurongo), urutonde rw’ibice bine (igihimba c’ibarabara) n’urutonde rw’amakuru, gushika ku ncuro 1,5 z’urutonde rw’ibice bine (uruzitiro rw’ibarabara). Nk’uko vyavuzwe mu Bikoresho n’Uburyo bw’Umurwayi, koresha agaciro ka limma k’umurwayi kugira ngo umenye agaciro ka P (*P<0.05 na **P<0.01). (B) Igishushanyo c’ingene MNA ihinduka (60). Ivyo bihimba bihindura ibiribwa: S-adenosil-1-metionine; SAH, S-adenosine-1-umusemburo w’umubiri; NA, nikotinami; MNA, 1-metilinikotinamide; 2-PY, 1-metili- 2-piridoni-5-karboksamide; 4-PY, 1-metili-4-piridoni-5-karboksamidi; NR, ribose y’nikotinamide; NMN, ni nikotinamide imwe gusa. Enzymes (icatsi kibisi): NNMT, nikotinamide N-methyltransferase; SIRT, ivyuma vy’umubiri; NAMPT, ni nikotinamide ihindura umubiri; AOX1, ni ukuvuga 1; NRK, ni kotinamide y’umubiri; NMNAT, ni nikotinamide imwe gusa y’umubiri; Pnp1, ni umusemburo w’ivyatsi bita purine. (C) t-SNE ya scRNA-seq y’ibisebe (imvi) n’igisebe (umutuku; n = abarwayi 3). (D) NNMT igaragara mu migwi itandukanye y’ingingo zamenyekanye hakoreshejwe scRNA-seq. (E) Ivyerekana NNMT na AOX1 muri SK-OV-3, impyiko y’urusoro rw’umuntu (HEK) 293T, utugingo ngengabuzima T n’utugingo ngengabuzima T twavuwe na MNA. Invugo ipfutse yerekanwa ijyanye na SK-OV-3. Igishushanyo c’imvugo gifise SEM kirerekanwa (n = 6 abaterankunga bafise amagara meza). Ivyiyumviro vya Ct birenze 35 bifatwa nk’ibidashobora kumenyekana (UD). (F) Ivyerekana SLC22A1 na SLC22A2 muri SK-OV-3, HEK293T, utugingo ngengabuzima T n’utugingo ngengabuzima T twavuwe na MNA 8mM. Invugo ipfutse yerekanwa ijyanye na SK-OV-3. Igishushanyo c’imvugo gifise SEM kirerekanwa (n = 6 abaterankunga bafise amagara meza). Ivyiyumviro vya Ct birenze 35 bifatwa nk’ibidashobora kumenyekana (UD). (G) Ibiri muri MNA y’ingingo ngenderwako mu ngingo ngenderwako T z’abaterankunga zifise amagara meza zikora inyuma y’amasaha 72 y’uguterwa na MNA. Igishushanyo c’imvugo gifise SEM kirerekanwa (n = 4 abaterankunga bafise amagara meza).
MNA ivugwa mu guhindura umugwi w’ivyuma bita methyl kuva muri S-adenosyl-1-methionine (SAM) ukaja mu bita nikotinamide (NA) bikoresheje nikotinamide N-methyltransferase (NNMT; Igishushanyo 3B). NNMT igaragara cane mu migera itandukanye y’abantu kandi ifatanye n’ukwiyongera, gutera n’ugukwiragira (25-27). Kugira ngo dutahure neza inkomoko ya MNA mu ngingo za T muri TME, twakoresheje scRNA-seq kugira ngo tugaragaze ukuntu NNMT igaragara mu bwoko bwose bw’ingingo mu bivyimba vy’abarwayi batatu ba HGSC (Imbonerahamwe S5). Isesengurwa ry’ingingo ngenderwako zishika 6.500 ryerekanye ko mu bidukikije vy’ibivyimba n’ibivyimba, NNMT igaragara gusa ku ngingo ngenderwako z’ibivyimba n’ingingo z’ibivyimba (Ishusho 3, C na D). Birabereye kumenya ko ata nkuru igaragara ya NNMT mu bantu bose bagaragaza PTPRC (CD45 +) (Ishusho 3D n’Ishusho S5A), ivyo bikaba vyerekana ko MNA yabonetse mu rwego rw’ibihimba vy’umubiri vyinjijwe mu ngirabuzima fatizo za T. Ivyo bimenyetso vy’uko aldehyde oxidase 1 (AOX1) ihindura MNA ikaba 1-metil-2-piridoni-5-karboksamide (2-PYR) canke 1-metil-4-piridoni-5-karboksamide (4- PYR); Igishushanyo 3B) na co nyene kigarukira ku bantu b’ibihimba vy’umubiri bita fibroblastes bigaragaza COL1A1 (Igishushanyo S5A), ivyo vyose hamwe vyerekana ko utugingo ngengabuzima T tutagira ubushobozi bwo guhindura ibintu bisanzwe bita MNA. Ivyo bimenyetso vy’ivyo bihimba vy’umubiri bifitaniye isano na MNA vyagenzuwe hakoreshejwe amakuru y’ingingo ngenderwako yigenga ya kabiri yashizwe ahabona avuye mu bihimba vy’umubiri vy’abarwayi ba HGSC (Ishusho S5B; n = 6) (16). Ikindi, isesengura ry’uruhererekane rwa polymerase (qPCR) ry’ingingo ngenderwako T z’abaterankunga zifise amagara meza zavuwe na MNA ryerekanye ko ugereranije n’ingingo ngenderwako z’ibivyimba vy’amabere SK-OV-3, NNMT canke AOX1 hafi ntivyagaragaye (Ishusho 3E). Ivyo bintu bitari bitezwe vyerekana ko MNA ishobora gusohoka mu bihimba vy’umubiri bita fibroblastes canke mu bivyimba bikaja mu bihimba vy’umubiri bita T cellules biri hafi.
Naho abashaka gutora harimwo umuryango w’ibinyabuzima bitwara ivyuma 1 gushika kuri 3 (OCT1, OCT2 na OCT3) bifise amakode n’umuryango w’ibinyabuzima bitwara 22 (SLC22) (SLC22A1, SLC22A2 na SLC22A3), ivyuma bishobora gutwara MNA ntibirasobanurwa (28) . QPCR ya mRNA ivuye mu ngirabuzimafatizo T z’abaterankunga zifise amagara meza yerekanye urugero ruto rw’imvugo ya SLC22A1 ariko urugero rudashobora kumenyekana rwa SLC22A2, ivyo bikaba vyemeza ko vyari vyaravuzwe mbere mu bitabu (Ishusho 3F) (29). Mu buryo bunyuranye, umurongo w’ingingo z’ibivyimba vy’amabere SK-OV-3 waragaragaje urugero rwo hejuru rw’ivyo bihimba vyose bibiri (Ishusho 3F).
Kugira ngo hasuzumwe ko utugingo ngengabuzima T dushobora gufata MNA y’amahanga, utugingo ngengabuzima T tw’abatanga amagara meza twatewe mu kiringo c’amasaha 72 hariho utugingo ngengabuzima dutandukanye twa MNA. Iyo ata MNA ivuye hanze, ibiri muri MNA mu turemangingo ntibishobora kumenyekana (Ishusho 3G). Ariko rero, utugingo ngengabuzima T twakoreshejwe twavuwe na MNA y’inyuma yerekanye ko hariho ukwiyongera kw’ibirimwo MNA mu ngingo ngenderwako bivanye n’urugero, gushika ku 6 mM MNA (Ishusho 3G). Ico ciyumviro kigaragaza ko naho urugero rw’uguserura kw’abatwara ibintu ruri hasi kandi ata n’intungamubiri nyamukuru ishinzwe guhindura MNA mu turemangingo, TIL irashobora gufata MNA.
Ivyiyumviro vy’ibintu bihinduka mu turemangingo twa T tw’abarwayi be n’igerageza ryo gufata MNA mu bipimo vy’amaraso, birongereza ubushobozi bw’uko utugingo ngengabuzima tujanye n’indwara ya kanseri (CAF) dushobora gusohora MNA kandi utugingo ngengabuzima tw’ibivyimba dushobora gutunganya uburyo TIL ikora be n’ingene ikora. Kugira ngo hamenyekane ingaruka MNA igira ku ngirabuzimafatizo T, ingingo ngenderwako T zifise amagara meza zakoreshejwe mu buhinga bwa vitro iyo MNA iriho canke itahari, hanyuma hasuzumwa ukuntu zigwira be n’ukuntu zikora cytokine. Inyuma y’imisi 7 yongeyeko MNA ku rugero runini, igitigiri c’abantu bakubye kabiri caragabanutse mu buryo buringaniye, mu gihe inguvu zagumyeho ku rugero rwose (Ishusho 4A). Ikindi, kuvura MNA iva hanze vyatumye habaho ukwiyongera kw’igitigiri c’ingingo ngenderwako CD4 + na CD8 + T ziserura igihimba c’umubiri gitera igisebe-α (TNFα; Igishushanyo 4B). Mu buryo bunyuranye, uguhingura IFN-γ mu turemangingo twagabanutse cane mu turemangingo twa CD4 + T, ariko ntivyagabanutse mu turemangingo twa CD8 + T, kandi nta mpinduka ikomeye yabaye mu turemangingo twa interleukin 2 (IL-2; Igishushanyo 4, C na D). Ku bw’ivyo, igerageza ry’ubudahangarwa bw’umubiri (ELISA) ry’ibihimba vy’umubiri biva muri izo nzira z’utugingo ngengabuzima T zavuwe na MNA ryerekanye ko TNFα yiyongera cane, IFN-γ igabanuka, kandi ko ata mpinduka ya IL-2 (Ishusho 4, E gushika kuri G). . Igabanuka rya IFN-γ ryerekana ko MNA ishobora kugira uruhara mu kubuza igikorwa co kurwanya ibivyimba c’ingingo ngenderwako T. Kugira ngo twigane ingaruka za MNA ku bumara bw’imitsi buterwa n’ingingo ngenderwako T, ingingo ngenderwako zifata antigène T (FRα-CAR-T) zigamije kwakira folate α na CAR-T (GFP) zigenzurwa n’ingingo ngenderwako z’ivyatsi bitoto zitwa proteine ​​(GFP) -CAR-T) zikorwa n’ingingo ngenderwako z’amaraso y’umuterankunga afise amagara meza ( mononunuPBMC). Utugingo ngengabuzima twa CAR-T twarimwe mu kiringo c'amasaha 24 hari MNA, hanyuma duterwa hamwe n'utugingo ngengabuzima tw'abantu SK-OV-3 tugaragaza folate receptor α ku rugero rw'ibikorwa n'intumbero rwa 10:1. Ubuvuzi bwa MNA bwatumye igikorwa co kwica c’ingingo za FRα-CAR-T kigabanuka cane, ivyo bikaba vyari bisa n’ingingo za FRα-CAR-T zavuwe na adenosine (Ishusho 4H).
(A) Igitigiri cose c’ingingo zishobora kubaho n’ugukubita kabiri kw’abantu (PD) biva ku mico kama ku musi wa 7. Igishushanyo c’imirongo kigereranya umubare + SEM w’abaterankunga batandatu bafise amagara meza. Iserukira amakuru ava n = 3 y’igerageza ryigenga. (B gushika kuri D) CD3/CD28 na IL-2 vyakoreshejwe mu gutuma utugingo ngengabuzima T dukora ku rugero rwavyo rwa MNA mu misi 7. Imbere y’uko hasuzumwa, utugingo ngengabuzima twavyuriwe umutima na PMA/ionomycin na GolgiStop mu kiringo c’amasaha 4. TNFα (B) igaragara mu ngingo za T. Akarorero k’ishusho (ibubamfu) n’amakuru y’urutonde (iburyo) y’imvugo ya TNFα mu ngingo z’ubuzima. IFN-γ (C) na IL-2 (D) zigaragara mu ngingo za T. Ivyo bihimba vy’umubiri vyitwa cytokines vyapimwa hakoreshejwe ubuhinga bwa flow cytometry. Igishushanyo c’imirongo kigereranya umubare (n = 6 w’abaterankunga bafise amagara meza) + SEM. Koresha isesengura ry’inzira imwe ry’uguhinduka n’ingero zisubirwamwo (*P<0.05 na **P<0.01) kugira ngo umenye agaciro ka P. Iserukira amakuru ava n = 3 y’igerageza ryigenga. (E to G) CD3/CD28 na IL-2 vyakoreshejwe mu gutuma utugingo ngengabuzima T dukora ku rugero rwavyo rwa MNA mu kiringo c’imisi 7. Ivyo bikoresho vyakoranijwe imbere n’inyuma y’amasaha 4 y’ugutera PMA/ionomycin. Ivyiyumviro vy’ivyo bihimba vy’umubiri bita TNFα (E), IFN-γ (F) na IL-2 (G) vyagereranywa n’ubuhinga bwa ELISA. Igishushanyo c’imirongo kigereranya umubare usanzwe (n = 5 abaterankunga bafise amagara meza) + SEM. Agaciro ka P kamenyekana hakoreshejwe isesengura ry’inzira imwe ry’uguhinduka n’ingero zisubirwamwo (*P<0.05). Umurongo w’utudomo werekana urugero rw’ugutahura rw’ugutahura. (H) Igerageza ry’ugusenyuka kw’utugingo ngengabuzima. Utugingo ngengabuzima twa FRα-CAR-T canke GFP-CAR-T twahinduwe hakoreshejwe adenosine (250μM) canke MNA (10 mM) mu kiringo c’amasaha 24, canke tukasigazwa tutavuwe (Ctrl). Ijanisha ry’ukwica kw’ingingo za SK-OV-3 ryagereranywa. Agaciro ka P kagenwa n’ikigeragezo ca Welch t (*P<0,5 na **P<0,01).
Kugira ngo umuntu ashobore gutahura neza ingene TNFα igaragara bishingiye kuri MNA, hasuzumwe amahinduka mu mRNA ya TNFα y’ingingo za T zavuwe na MNA (Ishusho 5A). Utugingo ngengabuzima T tw’abatanga amagara twavuwe na MNA twarerekanye ko urugero rw’inyandiko ya TNFα rwongerekanye incuro zibiri, ivyo bikaba vyerekana ko MNA iva ku ntunganyo y’inyandiko ya TNFα. Kugira ngo hakorwe itohoza kuri ubwo buryo bushoboka bwo gutunganya, ibintu bibiri bizwi vy’inyandiko bitunganya TNFα, ni ukuvuga umugera wa T cell nuclear factor (NFAT) na proteine ​​yihariye 1 (Sp1), vyasuzumwe mu kwishura ku gufatanya kwa MNA n’umurongozi wa TNFα wo hafi ( 30 ). Igitera TNFα kirimwo ibibanza 6 vyamenyekanye bifatanya NFAT n’ibibanza 2 bifatanya Sp1, bifatanya ahantu hamwe [-55 base pairs (bp) kuva ku 5’cap] (30). Ivyo bimenyetso vyerekanye ko iyo umuntu avuwe na MNA, ukuntu Sp1 ifatanya n’umugera wa TNFα vyiyongera incuro zitatu. Ukwinjizwa kwa NFAT na kwo nyene kwarongerekanye kandi kwegera akamaro (Ishusho 5B). Ivyo bimenyetso vyerekana ko MNA itunganya ukuntu TNFα igaragara biciye ku kwandika Sp1, kandi ku rugero rutoyi itunganya ukuntu NFAT igaragara.
(A) Ugereranije n’ingingo za T zavuwe ata MNA, ihinduka ry’incuro ry’imvugo ya TNFα mu ngingo za T zavuwe na MNA. Igishushanyo c’imvugo gifise SEM kirerekanwa (n = 5 abaterankunga bafise amagara meza). Iserukira amakuru ava n = 3 y’igerageza ryigenga. (B) TNFα promoter y’ingingo za T zavuwe canke zitavuwe 8 mM MNA inyuma ya NFAT na Sp1 zifatanijwe na (Ctrl) n’ugukangura PMA/ionomycin mu kiringo c’amasaha 4. Immunoglobulin G (IgG) na H3 vyakoreshejwe nk’ibipimo bibi n’ibipimo vyiza vy’ubudahangarwa bw’umubiri, uko bikurikirana. Igitigiri ca ChIP cerekanye ko ukuboha kwa Sp1 na NFAT n’umurongozi wa TNFα mu ngingo zavuwe na MNA kwariyongereye incuro nyinshi ugereranyije n’ivyo gucungera. Iserukira amakuru ava n = 3 y’igerageza ryigenga. Agaciro ka P kagenwa n’ibipimo vyinshi vya t (*** P <0.01). (C) Ugereranije n’amaraso y’umubiri (ascites) ya HGSC, utugingo ngengabuzima T (tutagira ubumara) twarerekanye ko TNF igaragara cane mu gisebe. Amabara agereranya abarwayi batandukanye. Ivyo bihimba vyerekanwa vyafashwe mu buryo bw’impfagusa gushika ku 300 kandi bihungabanywa kugira ngo bigabanye gushushanya birenze urugero (** Padj = 0.0076). (D) Ivyitegererezo vy’akarorero ka MNA ku kanseri y’amabere. MNA ivugwa mu ngingo z’ibivyimba no mu ngingo z’ibivyimba ziri muri TME kandi ifatwa n’ingingo za T. MNA yongera gufatanya Sp1 n’umurongozi wa TNFα, bikaba bituma TNFα igira ubushobozi bwo kwandika no gukora cytokine za TNFα. MNA na yo nyene ituma IFN-γ igabanuka. Guhagarika igikorwa c’utugingo ngengabuzima T bituma ubushobozi bwo kwica bugabanuka kandi n’igihimba c’umubiri kigakura ningoga.
Nk’uko amakuru abivuga, TNFα irafise ingaruka zivana n’imbere n’inyuma zirwanya ibivyimba n’ibivyimba, ariko irafise uruhara ruzwi cane mu guteza imbere ukukura no gukwiragira kwa kanseri y’amabere (31-33). Nk’uko amakuru abivuga, igitigiri ca TNFα mu bihimba vy’umubiri bita ascites n’ibivyimba vy’umubiri mu barwayi bafise kanseri y’amabere ni kinini kuruta ico mu bihimba vy’umubiri bitagira akaga (34-36). Ku bijanye n’uburyo ikora, TNFα irashobora gutunganya ugukora, ugukora n’ugukwiragira kw’utugingo ngengabuzima twera, no guhindura uburyo utugingo ngengabuzima twa kanseri dukora (37, 38). Mu buryo buhuye n’ivyo vyavumbuwe, isesengura ry’imiterere y’imitsi itandukanye ryerekanye ko TNF yari itunganijwe cane mu ngingo za T mu bihimba vy’ibivyimba ugereranyije n’indwara y’ibivyimba (Ishusho 5C). Ukwo kwiyongera kw’imvugo ya TNF kwagaragaye gusa mu migwi y’utugingo ngengabuzima T dufise umubiri udatera ubumara bw’utugingo ngengabuzima (Ishusho S5A). Mu ncamake, ayo makuru ashigikira iciyumviro c’uko MNA ifise ingaruka zibiri zo guhagarika ubudahangarwa bw’umubiri no gutera imbere ibivyimba muri HGSC.
Ivyo gushirako amazina y’izuba bishingiye ku buhinga bwa flow cytometry vyacitse uburyo nyamukuru bwo kwiga ukuntu TIL ihinduka. Izo nyigisho zerekanye ko ugereranije n’utugingo ngengabuzima tw’amaraso canke utugingo ngengabuzima T tuva mu bihimba vy’amaraso vy’inyuma, TIL y’imbwa n’iy’umuntu ifise impengamiro nyinshi yo gufata isukari (4, 39) kandi igatakaza buhoro buhoro igikorwa c’imitochondrie (19, 40). Naho twabonye ibintu bisa n’ivyo muri iki cigwa, ikintu nyamukuru co gutera imbere ni ukugereranya ukuntu utugingo ngengabuzima tw’igihimba c’umubiri dukora be n’ingene TIL ikora biva ku gihimba kimwe c’igihimba c’umubiri caciwe. Mu buryo buhuye na zimwe muri izo raporo za kera, utugingo ngengabuzima tw’ibivyimba (CD45-EpCAM +) tuva mu bivyimba n’ibivyimba dufise ubushobozi bwo gufata isukari nyinshi kuruta utugingo ngengabuzima twa CD8 + na CD4 + T, ivyo bikaba bishigikira ko utugingo ngengabuzima tw’ibivyimba dushobora gufata isukari nyinshi dushobora kugereranywa n’utugingo ngengabuzima twa T. Iciyumviro c’uguhiganwa kw’utugingo ngengabuzima T. TME. Ariko rero, igikorwa c’imitochondrie c’ingingo z’ibivyimba kiraruta ic’ingingo za CD8 + T, ariko igikorwa c’imitochondrie kirasa n’ic’ingingo za CD4 + T. Ivyo bimenyetso bikomeza insiguro iriko iraseruka y’uko uguhindura ibiribwa mu mubiri (oxidative metabolism) ari ngirakamaro ku ngirabuzima fatizo z’ibivyimba (41, 42). Bavuga kandi ko utugingo ngengabuzima twa CD8 + T dushobora gushikirwa n’ingorane z’imitsi y’umubiri kuruta utugingo ngengabuzima twa CD4 + T, canke ko utugingo ngengabuzima twa CD4 + T dushobora gukoresha ibindi bikomoka kuri carbone atari glucose kugira ngo tugumye dukora (43, 44). Ni ngombwa kumenya ko ata tandukaniro twabonye mu gufata isukari canke mu bikorwa vy’imitochondrie hagati y’ibihimba vy’umubiri bikora CD4 + T, ubwonko bw’ibihimba vy’umubiri bikora T n’ingingo z’umubiri zikora T hagati mu ndwara y’umusonga. Na vyo nyene, ukuntu utugingo ngengabuzima CD8 + T dutandukanye mu bivyimba ntaco bihuriyeko n’uguhinduka kw’ugufata isukari, ivyo bikaba bigaragaza itandukaniro rikomeye hagati y’utugingo ngengabuzima T twatewe mu bihimba vy’umubiri n’utugingo ngengabuzima twa TIL tw’umuntu mu bihimba vy’umubiri (22). Ivyo vyiyumviro vyaremejwe kandi n’ugukoresha uburyo bwo gutanga utugingo ngengabuzima tutagira aho tugarukira, ivyo bikaba vyagaragaje kandi ko utugingo ngengabuzima twa CD45 + / CD3- / CD4 + / CD45RO + dufise ubushobozi bwo gufata isukari nyinshi n’ibikorwa vy’imitochondrie kuruta utugingo ngengabuzima tw’ibivyimba ari two twinshi ariko dufise utugingo ngengabuzima dukora cane. Ivyo bishobora guserukira igitigiri c’abantu bashobora kuba ari utugingo ngengabuzima duhagarika amaraso canke utugingo ngengabuzima dutera amaraso (plasmacytoid dendritic) twabonetse mu gusesangura scRNA-seq. Naho ivyo vyose vyavuzwe mu bivyimba vy’amabere y’abantu [45], biracari bikeneye Ikindi gikorwa ni ugudondora iyo subpopulation myeloïde.
Naho uburyo bushingiye ku gupima umuvuduko w’amazi (flow cytometry) bushobora gusobanura itandukaniro rusangi riri hagati y’ubwoko bw’ingingo z’umubiri n’ivy’imibiri y’abantu, ibintu vy’ukuri biva muri glucose canke ibindi bikomoka kuri carbone kugira ngo bihindure ivy’umubiri mu mitochondrie muri TME ntibiramenyekana. Gushinga ukubaho canke ukutabaho kw’ibinyabuzima bihindura ibiribwa mu gice c’umubiri c’umubiri bisaba gusukura igitigiri c’ingingo ngenderwako zivuye mu nyama zaciwe. Ku bw’ivyo, uburyo bwacu bwo gutunganya utugingo ngengabuzima bufatanijwe n’ubuhinga bwo gupima uburemere bw’ibintu birashobora gutanga ubumenyi ku bihimba vy’umubiri bitunganywa mu buryo butandukanye mu ngingo ngenderwako T n’ingingo ngenderwako z’ibivyimba mu guhuza ivyerekanwa vy’abarwayi. Naho ubu buryo bufise akamaro kuruta gutoranya utugingo ngengabuzima dukoresheje umuco w’izuba, amasomero amwamwe y’ibinyabuzima ashobora gushikirwa n’ingorane kubera ukudahungabana kw’ibinyabuzima be n’/canke urugero rw’uguhinduka vyihuta (22). Naho ari ukwo, uburyo bwacu bwarashoboye kumenya ibintu bibiri vyemewe bifasha umubiri guhangana n’indwara, ari vyo adenosine na kynurenine, kubera ko bitandukanye cane hagati y’ubwoko bw’ivyerekanwa.
Isesengura ryacu ry’imibiri y’ibivyimba n’ubwoko butoya bwa TIL ritanga ubumenyi bwinshi ku ruhara rw’imibiri mu TME y’amabere. Ica mbere, dukoresheje ubuhinga bwa flow cytometry, twabonye ko ata tandukaniro riri hagati y’ibikorwa vy’imitochondrie hagati y’ibivyimba n’ingingo za CD4 + T. Ariko rero, isesengura rya LC-MS/MS ryerekanye ko hariho amahinduka akomeye mu bwinshi bw’ibinyabuzima bihindura ibiribwa muri abo bantu, ivyo bikaba vyerekana ko imyanzuro yerekeye uguhinduka kw’ibinyabuzima TIL n’igikorwa cayo cose c’uguhindura ibinyabuzima bisaba gusobanurwamwo neza. Ica kabiri, MNA ni co kintu gihindura ihinduka rifise itandukaniro rikomeye hagati y’utugingo ngengabuzima CD45 n’utugingo ngengabuzima T mu bivyimba, atari ibivyimba. Ku bw’ivyo, ugucapura n’aho igihimba c’umubiri kiri bishobora kugira ingaruka zitandukanye ku migenderanire ya TIL, ivyo bikaba bigaragaza ukuntu ibintu bishobora kuba bitandukanye mu bidukikije bitobito. Ica gatatu, ukugaragara kw’intungamubiri ikora MNA NNMT ahanini kugarukira kuri CAF, ari yo ngingo z’ibivyimba ku rugero rutoyi, ariko urugero rwa MNA rushobora kumenyekana ruraboneka mu ngingo za T zikomoka ku bivyimba. Uguseruka cane kwa NNMT mu CAF y’amabere birafise ingaruka zizwi zitera kanseri, bimwe biterwa n’ugutera imbere kw’imibiri ya CAF, gutera ibivyimba n’ugukwiragira (27). Naho urugero rusangi rwa TIL ruri hagati, ukugaragara kwa NNMT muri CAF kurafitaniye isano cane n’ubwoko butoya bw’imitsi y’umubiri (TCGA), bufitaniye isano n’ugutahura nabi (27, 46, 47). Ubwa nyuma, ukugaragara kw’intungamubiri AOX1 ishinzwe gusenyuka kwa MNA na kwo nyene kugarukira ku bantu bo muri CAF, ivyo bikaba vyerekana ko utugingo ngengabuzima T tutagira ubushobozi bwo guhindura MNA. Ivyo bimenyetso birashigikira iciyumviro c’uko naho hariho ibindi bikorwa bikenewe kugira ngo ivyo bimenyekane, urugero runini rwa MNA mu ngirabuzimafatizo T rushobora kwerekana ko hariho ikirere gitoyi c’umubiri gishobora gutuma umubiri udashobora kurwanya indwara.
Kubera ko urugero rw’imvugo y’ibitwara MNA ruri hasi cane be n’urugero rudashobora kumenyekana rw’amaproteine ​​y’ingenzi akora mu guhindura MNA, ukubaho kwa MNA mu ngingo za T ntivyitezwe. Nta NNMT canke AOX1 vyashobora kumenyekana biciye ku gusesangura scRNA-seq no ku qPCR y’imigwi ibiri yigenga. Ivyo bimenyetso vyerekana ko MNA idakorwa n’ingingo ngenderwako T, ahubwo ko ifatwa n’ingingo ngenderwako zikikuje TME. Ivyagezwe vyakozwe mu bipimo vy’ibikoko vyerekana ko utugingo ngengabuzima T dukunda kwirundanira MNA iva hanze.
Ivyigwa vyacu vy’in vitro vyerekanye ko MNA iva hanze ituma TNFα igaragara mu ngingo za T kandi igatuma Sp1 ifatanya n’umurongo wa TNFα. Naho TNFα ifise ubushobozi bwo kurwanya ibivyimba n’ibivyimba, muri kanseri y’amabere, TNFα irashobora gutuma kanseri y’amabere ikura (31-33). Ugukuraho TNFα mu mico kama y’ingingo z’ibivyimba vy’amabere canke gukuraho ikimenyetso ca TNFα mu bigereranyo vy’imbeba birashobora gutuma TNFα ikora cytokine zitera umururumba no kubuza gukura kw’ibivyimba (32, 35). Rero, muri iki gihe, MNA ikomoka kuri TME irashobora gukora nk’umuti utera umururumba biciye ku buryo bushingiye kuri TNFα biciye ku nzira y’umubiri (autocrine loop), gutyo igatuma kanseri y’amabere ishobora gutera no gukwiragira (31). Hashingiwe kuri ivyo bishoboka, TNFα blockade iriko irigwa nk’umuti ushobora kuvura kanseri y’amabere (37, 48, 49). Ikindi, MNA irahungabanya ubumara bw’ingingo za CAR-T ku ngingo z’ibivyimba vy’amabere, ivyo bikaba bitanga ibindi bimenyamenya vy’uko MNA ishobora guhagarika ubudahangarwa bw’umubiri. Muri rusangi, ivyo bimenyetso vyerekana akarorero aho ibivyimba n’ingingo za CAF zisohora MNA mu TME y’inyuma y’ingingo. Biciye (i) mu gutuma kanseri y’amabere ikura bitewe na TNF no (ii) mu guhagarika ibikorwa vy’ubumara bw’utugingo ngengabuzima T bitewe na MNA, ivyo bishobora kugira ingaruka zibiri ku gisebe (Ishusho 5D).
Mu gusozera, mu gukoresha ubuhinga bwo gutunganya utugingo ngengabuzima vyihuta, ugukurikiranya utugingo ngengabuzima tumwetumwe be n’ugupima ivyiyumviro vy’imibiri, iki cigwa carahishuye itandukaniro rinini ry’imibiri hagati y’ibivyimba n’ingingo z’umubiri zitwa ascite mu barwayi ba HGSC. Iryo sesangura ry’ibintu vyose ryerekanye ko hariho itandukaniro mu gufata isukari n’ibikorwa vy’imitochondrie hagati y’ingingo ngenderwako T, kandi ryerekanye ko MNA ari ikintu kitari ingingo ngenderwako zigenzura ubudahangarwa bw’umubiri. Aya makuru arafise ingaruka ku kuntu TME igira ico ikoze ku migenderanire y’ingingo ngenderwako T mu ndwara za kanseri z’abantu. Naho nyene hariho uguhiganwa gutaziguye hagati y’ingingo ngenderwako T n’ingingo ngenderwako za kanseri, ibintu bihindura ibiribwa birashobora kandi gukora nk’ibintu bitaziguye kugira ngo bitere imbere mu gutera imbere kw’ibivyimba kandi bikaba bishoboka ko bihagarika inyishu z’ubudahangarwa bw’umubiri. Iyindi ndondoro y’uruhara rw’ivyo bihimba bishobora gutuma habaho ubundi buryo bwo kwongerera ubushobozi bwo kurwanya ibivyimba.
Ivyitegererezo vy’abarwayi n’amakuru y’ubuvuzi vyaronswe biciye mu bubiko bw’imitsi y’ibivyimba vya kanseri ya BC bwemejwe n’ishirahamwe ry’ububiko bw’imitsi ryo muri Kanada. Dushingiye ku mategeko yemejwe na Komite y’Inyifato runtu y’Ubushakashatsi ku ndwara ya Kanseri yo muri BC be na Kaminuza ya British Columbia (H07-00463), ivyerekanwa vy’abarwayi bose n’amakuru y’ubuvuzi vyaronse ukwemera kwanditse canke bihevye ukwemera kwabo ku buryo butegekanijwe. Ivyo bipimo bibikwa muri BioBank yemejwe (BRC-00290). Ibirango vy’umurwayi vyerekanywe mu ndome S1 na S5. Kugira ngo umuntu ashobore kuzigama igihimba c’umubiri c’umurwayi, hakoreshwa igikoresho co gucapura kugira ngo kiboze igihimba c’umubiri c’umurwayi, hanyuma kigisunikire mu gicuncuro c’amamikro 100 kugira ngo kironke igihimba kimwe c’umubiri gihagaze. Ascite y’umurwayi yaciwe mu gitereko c’amazi (centrifuge) ku 1500 rpm mu minota 10 ku bushuhe bwa 4°C kugira ngo utugingo ngengabuzima tube nk’ibipande maze hakurwemwo ivyuma vyari hejuru. Utugingo ngengabuzima twaronswe mu bivyimba no mu bivyimba vy’umubiri twabitswe mu bice 50 kw’ijana vy’amaraso y’umuntu AB (Sigma-Aldrich), 40% vy’amaraso y’umuntu (Thermo Fisher Scientific) n’ibice 10 kw’ijana vy’amaraso y’umuntu. Ivyo bihimba vy’umubiri vyari vyazigamye vyarafutswe maze bikoreshwa mu gupima ivy’imibiri n’ivy’imibiri nk’uko bivugwa aha hepfo.
Ivyo bikoresho vyose birimwo 0,22 μm biyunguruwe 50:50 vyuzuye RPMI 1640: AimV. RPMI 1640 + 2,05 mM l-glutamine (Thermo Fisher Scientific) yongeweko 10% y’amaraso y’umuntu AB adakora kubera ubushuhe (Sigma-Aldrich), 12,5 mM Hepes (Ivy’Ubushuhe), 2 mM l-glutamine (Ivy’Ubushuhe) Igisubizo c’umuti witwa streptomycine (PenStrep) (Thermo Fisher Scientific) n’ibindi bihimba vy’umubiri bita 50 μMB-mercaptoéthanol. AimV (Invitrogen) yongeweko Hepes 20 mM (Ivy’ubuhinga bw’abarovyi) n’imiti 2 mM ya l-glutamine (Ivy’ubuhinga bw’abarovyi). Igikoresho co gutera ibara c’umubiri cari gifise umunyu w’amazi (PBS; Invitrogen) ufise uburebure bwa 0,22 μm, wongereweko 3% vy’amaraso y’umuntu (Sigma) atagira ubushuhe. Igikoresho co gutunganya utugingo ngengabuzima gifise 0,22μm ya PBS yayunguruwe kandi kigashirwamwo 0,5% y’amaraso y’umuntu AB adakora kubera ubushuhe (Sigma-Aldrich).
Mu 37°C yuzuye, utugingo ngengabuzima twashizweko ibara n’10 nM MT DR na 100 μM 2-NBDG mu minota 30. Hanyuma, izo nsoro zarashizweko irangi ry’ubuzima ryitwa eF506 ku bushuhe bwa 4°C mu minota 15. Usubire gushiramwo izo nsoro mu FC Block (eBioscience) no mu gitereko c’amabara y’agaciro (BD Biosciences), uzishire mu gitereko c’amabara c’umuyagankuba (nk’uko amabwirizwa y’uwazikoze abivuga), hanyuma uziterere iminota 10 mu bushuhe bwo mu cumba. Gutera ibara ingingo n’umugwi w’ibirwanya indwara (Imbonerahamwe S2) mu gikoresho co gutera ibara ry’umubiri (flow cytometry buffer) ku bushuhe bwa 4°C mu minota 20. Gusubira gushiramwo utugingo ngengabuzima mu gikoresho co gutera ibara (Cytek Aurora; 3L-16V-14B-8R) imbere y’uko dusuzumwa. Koresha SpectroFlo na FlowJo V10 kugira ngo usesengure amakuru y’ibara ry’ingingo, kandi ukoreshe GraphPad Prism 8 kugira ngo ureme amakuru. Igitigiri c’abafise ubushobozi bwo guca ibibatsi (MFI) ca 2-NBDG na MT DR cari gisanzwe, hanyuma hakoreshwa ikigeragezo ca t c’ababiri kugira ngo hakorwe isesengura ry’imibare kugira ngo haboneke abarwayi bahuye. Gukuraho abantu bose bafise ibintu bitari munsi ya 40 mu gusesangura; winjize agaciro ka MFI ka 1 ku gaciro kose kabi imbere yo gukora isesengura ry'imibare n'ugushushanya amakuru.
Kugira ngo twongereko ingamba zo gufunga amarembo y’amaboko y’ivyo biganiro biri hejuru, twakoresheje inyishu yuzuye n’igiti c’uburenganzira bw’imiterere (FAUST) (21) kugira ngo dushire utugingo ngengabuzima ku bantu inyuma yo gukuraho utugingo ngengabuzima twapfuye muri FlowJo. Turacungera n’amaboko igisohoka kugira ngo dufatanye abantu basa n’abatanzwe nabi (gufatanya PD1+ n’ingingo za PD1-tumor) n’abantu bagumye. Ico kigereranyo cose kirimwo ingingo zirenga 2%, ku bantu 11 bose hamwe.
Ivyo bikoresho vyakoreshejwe mu gutandukanya PBMC n’ibikoresho vyo gutandukanya utugingo ngengabuzima (STEMCELL Technologies). Utugingo ngengabuzima twa CD8 + T twatandukanijwe na PBMC hakoreshejwe CD8 MicroBeads (Miltenyi) hanyuma twagurwa mu buryo bushitse hakoreshejwe TransAct (Miltenyi) mu kiringo c’indwi 2 hakurikijwe amabwirizwa y’uwadukoze. Utugingo ngengabuzima twarekuriwe guhagarara imisi 5 mu mazi yuzuye arimwo IL-7 (10 ng/ml; PeproTech), hanyuma tugasubira guterwa n’imiti yitwa TransAct. Ku musi wa 7, nk’uko amabwirizwa y’uwayikoze abivuga, hakoreshejwe ubuhinga bwa CD45 MicroBeads (Miltenyi) bw’abantu kugira ngo butunganye utugingo ngengabuzima mu bice bitatu bikurikirana. Utugingo ngengabuzima twaciwemwo ibice kugira ngo dusuzumwe n’ubuhinga bwa flow cytometry (nk’uko vyavuzwe haruguru), kandi utugingo ngengabuzima miliyoni imwe twaciwemwo ibice incuro zitatu kugira ngo dusuzumwe hakoreshejwe ubuhinga bwa LC-MS/MS. Ivyo bipimo vyakoreshejwe ubuhinga bwa LC-MS/MS nk’uko bivugwa aha hepfo. Twagereranyije agaciro k'ibinyabuzima bibura n'umubare w'iyon 1.000. Igishushanyo cose gisanzwe n’umubare wose w’iyon (TIC), kigahindurwa mu buryo bwa logarithme kandi kigasanzwe mu MetaboAnalystR imbere y’uko gisesangurwa.
Igipande c’ingingo imwe c’umurwayi wese carafunze maze kicungururwa biciye mu gicuncuro c’uburebure bwa 40 μm kikaja mu gice c’umubiri gishitse (nk’uko vyavuzwe haruguru). Dushingiye ku mategeko y’umuhinguzi, ibice bitatu bikurikirana vy’uguhitamwo neza biciye mu gutandukanya amabuye y’agaciro hakoreshejwe MicroBeads (Miltenyi) vyakoreshejwe kugira ngo hatunganywe ivyerekanwa vy’ingingo za CD8+, CD4+ na CD45- (ku barafu). Muri make, izo nsoro zisubira gushirwa mu gitereko c’ugutunganya ingingo (nk’uko vyavuzwe haruguru) zigaharurwa. Utugingo ngengabuzima twatewe n’amabuye y’umuntu CD8, amabuye CD4 y’umuntu canke amabuye CD45 y’umuntu (Miltenyi) ku bushuhe bwa 4°C mu minota 15, hanyuma tugasukurwa n’umuti wo gutunganya utugingo ngengabuzima. Ico kigereranyo gica mu nkingi ya LS (Miltenyi), hanyuma imice myiza n’imibi igakwegeranywa. Kugira ngo igihe kigabanuke kandi intambwe yo gusubirana kw’ingingo ngenderwako igere ku rugero rwo hejuru, igice ca CD8 gica gikoreshwa mu rugendo rwa kabiri rwo gutunganya CD4+, igice ca CD4 na co kigakoreshwa mu gutunganya CD45 gikurikira. Gumana umuti ku barafu mu kiringo cose co gutandukanya.
Kugira ngo hategurwe ivyerekanwa vyo gusuzuma ibihimba vy’umubiri, izo nsoro zarasukuwe rimwe n’umunyu ukonnye nk’urubura, hanyuma militiro 1 ya methanol 80% yongerwa ku nkuru imwe imwe, hanyuma zigahindurwa zigaca zikonja mu mazi y’azote. Ivyo bipimo vyaciye bikoreshwa mu nzira zitatu zo gukonja no gukonja, hanyuma bica bishirwa mu nzira y’umuriro ku 14.000 rpm mu minota 15 ku bushuhe bwa 4°C. Ivyo bihimba vy’umubiri birimwo ivyo bihimba vy’umubiri bica bihinduka umwuka gushika vyumye. Ivyo bihimba vy’umubiri vyasubiwemwo mu 50 μl y’aside formique 0,03%, bica bivugururwa kugira ngo bivange, hanyuma bica bivugururwa kugira ngo bikureho ibisigazwa.
Gukuraho ibintu bihindura ibiribwa nk’uko vyavuzwe haruguru. Ivyo bihimba vy’umubiri bishirwa mu cupa ry’amazi ry’ubuhinga bwinshi kugira ngo hakorwe ubushakashatsi ku bijanye n’imibiri. Koresha uburyo bwo kuvura butari bwo kugira ngo uvure igipimo cose gifise umubare usa n’uw’ingingo kugira ngo wirinde ingaruka z’imigwi. Twarakoze isuzuma ry’uburyo bw’ibinyabuzima vyo kw’isi yose vyari vyasohowe mbere ku gipimo c’uburemere bw’ibinyabuzima (50) AB SCIEX QTRAP 5500. Isesengurwa ry’ibara ry’umubiri n’ugushiramwo ibice vy’isonga vyakozwe hakoreshejwe porogarama ya MultiQuant verisiyo 2.1 (Applied Biosystems SCIEX).
Igitigiri c’iyon 1000 carakoreshejwe mu kugereranya agaciro k’ibinyabuzima bibura, kandi TIC y’igipimo kimwekimwe cose yakoreshejwe mu kubara uburebure bw’isonga busanzwe bw’ibinyabuzima vyose vyabonetse kugira ngo hakosorwe amahinduka yazanywe n’isesengura ry’ibikoresho rivuye mu gutunganya igipimo. Inyuma y’aho TIC ihindukiye, MetaboAnalystR(51) (parameter mburabuzi) ikoreshwa mu guhindura logarithme no gupima umurongo w’imirongo y’imirongo. Twakoresheje PCA n’umuzigo wa vegan R kugira ngo dukore isesengura ry’itandukaniro ry’imibiri hagati y’ubwoko bw’ibigereranyo, kandi dukoresheje isesengura ry’ibice vy’ugusubira inyuma kugira ngo dusuzume abarwayi. Koresha uburyo bwa Ward kugira ngo wubake ikarita y’ubushuhe kugira ngo ushire hamwe urugendo rwa Euclide hagati y’ibigereranyo. Twakoresheje limma (52) ku bwinshi bw’ibinyabuzima kugira ngo tumenye ibinyabuzima vyinshi bitandukanye mu bwoko bwose bw’ingingo n’ibidukikije bitobito. Kugira ngo tworohereze insobanuro, dukoresha umurongo w’uburinganire bw’umugwi kugira ngo tugaragaze urugero, kandi tuzirikane ubwoko bw’ingingo mu bidukikije bitobito nk’umugwi umwumwe wose (n = 6 groups); ku bijanye n’ikigeragezo c’agaciro, twakoze ingero zitatu zisubirwamwo ku kintu cose gihindura umubiri Kugira ngo ntihagire ugusubiramwo kw’ibinyoma, umurwayi yashizwemwo nk’intambamyi mu mugambi wa limma. Kugira ngo tugenzure itandukaniro riri hagati y’ibinyabuzima bihindura ibiribwa hagati y’abarwayi batandukanye, twahinduye uburyo bwa limma harimwo n’abarwayi mu buryo butahinduka. Turatanga raporo y’akamaro k’itandukaniro ry’imbere y’igihe hagati y’ubwoko bw’ingingo n’ibidukikije bitobito vya Padj <0.05 (ugukosora kwa Benjamini-Hochberg).
Inyuma y’ugutunganywa kw’inguvu hakoreshejwe igikoresho co gukuraho utugingo ngengabuzima twapfuye (>80% viability), urutonde rw’utugingo ngengabuzima tumwe rwakozwe ku bihimba vyose vy’umubiri vy’umubiri n’ibivyimba hakoreshejwe uburyo bwo guserura 10x 5′gene. Ivyiyumviro bitanu vy’ibivyimba n’ibivyimba bihuye vyarasuzumwe, naho nyene ubushobozi buke bwo kubaho buva ku citegererezo kimwe c’igihimba c’umubiri bwabujije ko gishirwamwo. Kugira ngo dushobore guhitamwo abarwayi benshi, twahuje ivyerekanwa vy’umurwayi wese mu mirongo y’igikoresho co gucungera 10x chromium, maze dusuzuma aho ascite n’ibivyimba biri ukwavyo. Inyuma yo gukurikiranya [Illumina HiSeq 4000 28×98 bp iherezo ry’ababiri (PE), igihimba c’umubiri ca Québec; 73.488 na 41.378 zisomwa ku nsoro imwe imwe ku gisebe n’igihimba c’umubiri]], twakoresheje CellSNP na Vireo (53) (bishingiye kuri CellSNP nk’uko SNP rusangi y’umuntu (VCF) itangwa na GRCh38 ihabwa akaranga k’umuterankunga (akaranga k’umurwayi. (IBS), hatarimwo utugingo ngengabuzima tutagenewe n’utugingo ngengabuzima twamenyekanye ko ari duplexes n’abaterankunga bahuye hagati y’ibipimo vy’ibivyimba n’ivy’ibivyimba (54), twagumye dufise ibibazo bitatu bifise utugingo ngengabuzima twinshi mu bivyimba n’ibivyimba vy’ibivyimba kugira ngo dukore isesengura ry’amazi (BiCoran filtering this na)6. 6975 utugingo ngengabuzima (2792 na 4183 utugingo ngengabuzima tuva ku gisebe n’utugingo ngengabuzima tw’umubiri, uko bikurikirana) kugira ngo dusuzumwe Dukoresha igraph (57) Louvain clustering y’urusobe rw’utugingo ngengabuzima twasangiye hafi (SNN) rushingiye ku ntambwe ya Jaccard ku ntumbero z’utugingo ngengabuzima biciye ku guserura n’amaboko. kandi zigaragazwa n’ingingo z’umubiri zitwa Cytotoxic T zisobanurwa n’imvugo ya CD8A na GZMA, hatarimwo amatsinda mato mato afise imvugo y’amaproteine ​​y’imitsi y’umubiri (ribosome) n’imitsi y’umubiri, harimwo n’imvugo y’imitsi y’umubiri.
PBMC zatandukanywe n’ibikoresho vyo gutandukanya utugingo ngengabuzima (STEMCELL Technologies) hakoreshejwe ubuhinga bwa Ficoll. Utugingo ngengabuzima twa CD3 + twatandukanijwe na PBMC hakoreshejwe amabuye ya CD3 (Miltenyi). Iyo MNA iriho canke itahari, utugingo ngengabuzima twa CD3+ twakoreshwa n’imiti ifatanye n’amasahani (5μg/ml), CD28 ishobora gusenyuka (3μg/ml) na IL-2 (300 U/ml; Proleukin). Ku musi wa nyuma wo kwagura, ubushobozi bwo kubaho (Ibara ry’Ubuzima Rishobora Guhinduka eFluor450, eBioscience) n’ugukwiragira (123count eBeads, Thermo Fisher Scientific) vyasuzumwe hakoreshejwe ubuhinga bwo gupima uruja n’uruza rw’imitsi. Gusuzuma igikorwa c’abakora mu gukangura utugingo ngengabuzima dukoresheje PMA (20 ng/ml) na ionomycin (1μg/ml) dukoresheje GolgiStop mu kiringo c’amasaha 4, no kugenzura CD8-PerCP (RPA-T8, BioLegend), CD4-AF700 (RPA-T4) , BioLegend) na TNForecycytein (MAb11, BD). Gukangura utugingo ngengabuzima twa qPCR na ChIP hakoreshejwe PMA (20 ng/ml) na ionomycine (1μg/ml) mu kiringo c’amasaha 4. Ivyo bihimba vy’umubiri vya ELISA vyakwegeranijwe imbere n’inyuma y’ugukangura hakoreshejwe PMA (20 ng/ml) na ionomycin (1 μg/ml) mu kiringo c’amasaha 4.
Kurikiza amategeko y’umuhinguzi kugira ngo utandukanye ARN ukoresheje igikoresho gitoyi ca RNeasy Plus (QIAGEN). Koresha igikoresho co gucapura (QIAGEN) kugira ngo ushiremwo ivyerekanwa bimwe. Koresha ikintu gifise ubushobozi bwinshi bwo guhindura ARN ku ADN (Thermo Fisher Scientific) kugira ngo ukore ADN yuzuzanya (ADN). Koresha TaqMan Rapid Advanced Master Mix (Thermo Fisher Scientific) kugira ngo ugereranije ivyerekeye uruvyaro (bishingiye ku mategeko y’umuhinguzi) ukoresheje ibi bipimo bikurikira: 1010726_m1 (SLC22A2). Ivyo bipimo vyakoreshejwe ku buryo bwa PCR bw’igihe nyaco bwa StepOnePlus (Ivyuma Bikoreshwa) (Ivyuma Bikoreshwa) mu gipande c’amaso 96 c’amaso yihuta ca MicroAmp (Ivyuma Bikoreshwa) gifise filime y’amaso ya MicroAmp. Agaciro kose ka Ct karenze 35 gafatwa ko kari hejuru y’urugero rwo kumenya kandi gashirwako ikimenyetso c’uko katamenyekana.
Gukora ChIP nk’uko vyavuzwe mbere (58). Muri make, izo nsoro zavuwe n’umuti witwa formaldehyde (umuti w’iherezo 1,42%) hanyuma ziterwa mu bushuhe bwo mu cumba iminota 10. Koresha umuti wo gukingira ukuvyimba (25 mM Hepes, 1,5 mM MgCl2, 10 mM KCl na 0,1% NP-40) ku barafu mu minota 10, hanyuma wongere ushire mu muti wo gukingira umubiri nk’uko vyavuzwe (58 ). Ico kigereranyo caraciye gikoreshwa n’ijwi ry’umuyagankuba hakoreshejwe inzinguzingu zikurikira: inzinguzingu 10 (impumuro 20 z’isegonda 1) n’igihe kidahinduka c’amasegonda 40. Gutera ivyatsi bifise ubushobozi bwo gukingira umubiri G (Ikoranabuhanga ryo gutanga ikimenyetso c’ingingo ngenderwako; 1 μl), histone H3 (Ikoranabuhanga ryo gutanga ikimenyetso c’ingingo ngenderwako; 3 μl), NFAT (Invitrogen; 3 μl) na SP1 (Ikoranabuhanga ryo gutanga ikimenyetso c’ingingo ngenderwako; 3 μl) n’ivyo bihimba vy’umubiri bifise uburebure burenze CC. Gutera amabuye ya poroteyine A (Thermo Fisher Scientific) n’ico gipimo ku bushuhe bwa 4°C ugizunguruka buhoro buhoro mu kiringo c’isaha 1, hanyuma ukoreshe amabuye ya chelex (Bio-Rad) kugira ngo utunganye ADN, hanyuma ukoreshe poroteyine K (Thermo Fisher) kugira ngo poroteyine ifungurwe. Igitera TNFα cabonetse biciye ku nzira ya PCR: imbere, GGG TAT CCT TGA TGC TTG TGT; ahubwo, GTG CCA ACA ITEGEKO GCC TTT ATA TG (igicuruzwa c’amabara 207). Amashusho yakozwe n’ikigo Image Lab (Bio-Rad) maze agereranywa hakoreshejwe porogarama ya ImageJ.
Ivyo bihimba vy’umubiri vyari vyerekanwa nk’uko vyavuzwe haruguru. Ivyo bipimo vyakozwe hakurikijwe uburyo bw’abahinguzi b’ibikoresho vyo gupima TNFα y’umuntu (Invitrogen), ibikoresho vyo gupima IL-2 y’umuntu (Invitrogen) n’ibikoresho vyo gupima IFN-γ y’umuntu (Abcam). Dukurikije amategeko y’umuhinguzi, iyo mazi yari hejuru y’ivyo bikoresho yarahinduwe 1:100 kugira ngo hamenyekane TNFα na IL-2, na 1:3 kugira ngo hamenyekane IFN-γ. Koresha igikoresho co gusoma ibimenyetso vyinshi (PerkinElmer) kugira ngo upime ubushobozi bwo gufata amazi ku 450 nm.
PBMC zatandukanywe n’ibikoresho vyo gutandukanya utugingo ngengabuzima (STEMCELL Technologies) hakoreshejwe ubuhinga bwa Ficoll. Utugingo ngengabuzima twa CD3 + twatandukanijwe na PBMC hakoreshejwe amabuye ya CD3 (Miltenyi). Iyo MNA iriho canke itahari, utugingo ngengabuzima twa CD3+ twakoreshwa n’imiti CD3 iboshe ku masahani (5μg/ml), CD28 ishobora gusenyuka (3μg/ml) na IL-2 (300 U/ml; Proleukin) mu misi 3. Inyuma y’imisi 3, izo nsoro zarakwegeranijwe zigasukurwa n’umunyu w’ibice 0,9%, hanyuma iyo nkoko irakonja. Ibara ry’utugingo ngengabuzima ryakozwe hakoreshejwe ubuhinga bwo gupima uruja n’uruza rw’utugingo ngengabuzima (Cytek Aurora; 3L-16V-14B-8R) hakoreshejwe ubuhinga bwa eBeads 123count.
Gukuraho ibintu bihindura ibiribwa nk’uko vyavuzwe haruguru. Ivyo bihimba vyumye vyasubiwemwo ku rugero rw’ibice 4000 vy’ingingo ngenderwako/μl. 1290 Infinity II, Agilent Technologies, Santa Clara, CA) n’inkingi ya CORTECS T3 (2,1×150 mm, ubunini bw’ibice 1,6-μm, ubunini bw’ibihengeri 120-Å; #186008500). Igikoresho co gupima uburemere bw’ibintu (6470, Agilent), aho ubuhinga bwo gukoresha umuyagankuba bukora mu buryo bwiza. Iciyumviro A gigendagenda ni 0,1% c’aside formique (mu H2O), igice B gigendagenda ni 90% acetonitrile, 0,1% aside formique. Igiharuro ca LC ni iminota 0 gushika kuri 2 ku 100% A, iminota 2 gushika ku 7,1 ku 99% B, n’iminota 7,1 gushika ku 8 ku 99% B. Hanyuma wongere ugereranije inking n’inzira A igendagenda ku rugero rw’uruzi rwa 0,6 ml/min ku minota 3. . Igitigiri c’amazi atemba ni 0,4ml/min, kandi icumba c’inkingi kirashushwa gushika ku 50°C. Koresha urugero rw’imiti isukuye rwa MNA (M320995, Ishirahamwe ry’ubushakashatsi ry’imiti ryo muri Toronto, North York, Ontario, Kanada) kugira ngo ushireho igihe co kugumya (RT) n’uguhinduka (RT = iminota 0,882, uguhinduka 1 = 137→94,1, uguhinduka 2 = 137→1933). Iyo izo mpinduka zose zitatu zibaye ku gihe kibereye co kugumya, impinduka 1 ikoreshwa mu gupima igitigiri kugira ngo haboneke ukuntu ibintu vyihariye. Igiharuro c’ivya MNA (Toronto Research Chemical Company) cavuye mu gutera ivyatsi bitandatu vy’uruhererekane vy’umunyu w’ivyatsi (1 mg/ml) kugira ngo haboneke ibiharuro vya 0,1, 1,0, 10 na 100 ng/ml na 1,0 na 10 μg/ml vy’amazi. Igitigiri co kumenya ni 1 ng/ml, kandi inyishu y’umurongo iri hagati ya 10 ng/ml na 10μg/ml. Buri rushinge rwa microlitres zibiri z’urugero n’urugero rukoreshwa mu gusesangura LC/MS, kandi urushinge rwo kugenzura uburyo ruvanze rukoreshwa buri rushinge umunani kugira ngo urubuga rwo gusesangura rube rudahungabana. Inyishu za MNA z’ibipimo vyose vy’ingingo zavuwe na MNA zari mu rutonde rw’umurongo rw’igerageza. Isesengurwa ry’amakuru ryakozwe hakoreshejwe porogarama y’isesengura ry’ingero ryitwa MassHunter (v9.0, Agilent).
Ivyubatswe vy’uruvyaro rwa kabiri αFR-CAR vyakuwe kuri Song n’abandi. (59). Muri make, iyo nyubakwa irimwo ibi bikurikira: Urutonde rw’abarongozi ba CD8a, igice c’uruzitiro rumwe ruhinduka rw’umuntu αFR, akarere ka CD8a k’uruzitiro n’akarere k’uruzitiro, uruzitiro rwa CD27 n’uruzitiro rwa CD3z. Urutonde rwa CAR rwose rwakozwe na GenScript, hanyuma ruca ruhindurwa mu nzira y’imvugo y’uruvyaro rwa kabiri rw’imigera ya lentiviral iri hejuru y’uruzitiro rw’imvugo rwa GFP rwakoreshejwe mu gusuzuma ubushobozi bwo guhindura.
Lentivirus iterwa n’uguhindura utugingo ngengabuzima twa HEK293T [Ikoraniro ry’imico kama y’ubwoko bw’Abanyamerika (ATCC); 10% vy’amaraso y’inka y’uruhinja (FBS) n’1% vy’amaraso y’inka, kandi hakoreshejwe umugera wa CAR-GFP be n’umugera wa CAR-GFP be n’umugera wa CAR-GFP be n’umugera wa CAR-GFP be n’umugera wa CAR-GFP. Ivyo bihimba vy’umubiri birimwo umugera vyakwegeranijwe amasaha 48 n’amasaha 72 inyuma y’aho bihinduriwe, bicungururwa, hanyuma bishirwa hamwe hakoreshejwe ubuhinga bwa ultracentrifugation. Bika ivyuma vy’umugera vyihariye ku -80°C gushika bihinduwe.
PBMC zitandukanywa n’ibikoresho vyo gutandukanya utugingo ngengabuzima tw’amaraso (STEMCELL Technologies) hakoreshejwe ubuhinga bwa Ficoll. Koresha utugingo ngengabuzima dutoduto twa CD8 (Miltenyi) kugira ngo utandukanye utugingo ngengabuzima twa CD8+ na PBMC. Gukangura utugingo ngengabuzima T hakoreshejwe ubuhinga bwa TransAct (Miltenyi) no mu buhinga bwa TexMACS [Miltenyi; vyongeweko 3% vy’amaraso y’umuntu adakora kubera ubushuhe, 1% vya PenStrep na IL-2 (300 U/ml)]. Amasaha mirongo ibiri n’ane inyuma y’ugukangura, utugingo ngengabuzima T twahinduwe n’umugera wa lentivirus (10 μl y’umugera wihariye ku turemangingo 106). 1 gushika ku misi 3 inyuma yo guhindura kuri Cytek Aurora (ku FSC (Imbere)/SSC (Imbere), Singlet, GFP+), suzuma ukuntu GFP igaragara mu ngingo kugira ngo yerekane ubushobozi bwo guhindura nibura 30% .
Utugingo ngengabuzima twa CAR-T twatewe amasaha 24 mu gikoresho citwa Immunocult (STEMCELL Technologies; twongeweko 1% ya PenStrep) mu bihe bikurikira: tutavuwe, twavuwe n’umugera wa 250 μM w’adenosine canke 10 mM wa MNA. Inyuma y’ugutera imbere, utugingo ngengabuzima twa CAR-T twayozwe na PBS hanyuma tugafatanywa n’utugingo ngengabuzima 20.000 twa SK-OV-3 [ATCC; mu gikoresho ca McCoy 5A (Sigma-Aldrich) congereweko 10% FBS na 1% PenStrep ku 10: Igitigiri c’abakora n’ico bashaka ca 1 carakomejwe incuro zitatu mu gikoresho congereweko Immunocult. Utugingo ngengabuzima twa SK-OV-3 n’utugingo ngengabuzima twa SK-OV-3 twaciwemwo n’umuti witwa digitalis saponin (0,5mg/ml; Sigma-Aldrich) twakoreshejwe nk’ibipimo bibi n’ibipimo vyiza, uko bikurikirana. Inyuma y’amasaha 24 y’uguhinga hamwe, amazi yari hejuru y’ivyo bihimba yarakorakoranijwe, hanyuma hapimwa ivyimburwa vy’amazi (LDH) hakurikijwe amabwirizwa y’uwabikoze (LDH Glo Cytotoxicity Assay Kit, Promega). Ivyo bihimba vy’umubiri vyitwa LDH vyaciwemwo 1:50 mu buffer ya LDH. Ijanisha ry’ubwicanyi ryagereranywa hakoreshejwe iyi formile: ijana ry’ubwicanyi = ijana ry’ugukosora / igipimo c’ubwicanyi kinini x 100%, aho ijana ry’ugukosora = co-culture-T cells gusa, n’igipimo c’ubwicanyi kinini = ubugenzuzi bwiza-ubugenzuzi bubi.
Nk’uko vyavuzwe mu nyandiko canke ibikoresho n’uburyo, koresha GraphPad Prism 8, Microsoft Excel canke R v3.6.0 kugira ngo ukore isesengura ry’imibare. Iyo hafashwe ivyerekanwa vyinshi ku murwayi umwe (nk’indwara y’umusonga n’igihimba c’umubiri), dukoresha ikigeragezo ca t c’ababiri canke tugashiramwo umurwayi nk’ingaruka y’imburakimazi mu buryo bw’umurongo canke rusangi nk’uko bikwiye. Ku bijanye n’isesengura ry’imibiri, ikigeragezo c’akamaro gikorwa mu bice bitatu.
Ku bijanye n’ibikoresho vy’inyongera vy’iyi ngingo, raba kuri http://advances.sciencemag.org/cgi/ibirimwo/vyuzuye/7/4/eabe1174/DC1.
Iyi ni ingingo ishobora gukoreshwa n’abantu bose igakwiragizwa hakurikijwe amategeko y’uruhusha rwo gukoresha, gukwiragiza no gusubiramwo mu buryo ubwo ari bwo bwose, igihe cose ikoreshwa rya nyuma ritari ryo gushaka inyungu z’ubudandaji kandi ikintu nyamukuru ari uko igikorwa c’intango ari co. Urutonde.
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Iki kibazo gikoreshwa mu kugerageza nimba uri umushitsi no kubuza gutanga ubutumwa butari bwo.
Marisa K. Kilgour (Marisa K. Kilgour), Sarah MacPherson (Sarah MacPherson), Lauren G. Zakariya (Lauren G. Zakariya), Abigayili Eli Aris G. Watson (H. Watson), John Stagg (John Stagg), Brad H. (Rusel G. Jones), Fineyasi T. Hamiltoni (Fineyasi T.
MNA irafasha mu guhagarika ubudahangarwa bw’umubiri bw’ingingo ngenderwako T kandi iserukira uburyo bwo gukingira umubiri bushobora gukoreshwa mu kuvura kanseri y’umuntu.
Marisa K. Kilgour (Marisa K. Kilgour), Sarah MacPherson (Sarah MacPherson), Lauren G. Zakariya (Lauren G. Zakariya), Abigayili Eli Aris G. Watson (H. Watson), John Stagg (John Stagg), Brad H. (Rusel G. Jones), Fineyasi T. Hamiltoni (Fineyasi T.
MNA irafasha mu guhagarika ubudahangarwa bw’umubiri bw’ingingo ngenderwako T kandi iserukira uburyo bwo gukingira umubiri bushobora gukoreshwa mu kuvura kanseri y’umuntu.
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