imyunmodulatory mεtabolayt dεm na wan kכl fכm fכ di tכmכro maykro envayroment (TME), bכt wit fכ εksepshכn dεm, dεn aydentiti dεm stil nכ no bכku pan dεm. na ya, wi analכz di tכmכro dεm εn T sεl dεm frכm di tכmכro dεm εn ascites fכ pasεnshכn dεm we gεt hεy-grεd sεrous kכrεsכn (HGSC) fכ sho di mεtabolכm fכ dεn difrεnt TME kכmpartmεnt dεm ya. di asayt dεm εn di tכmכro sεl dεm gεt bכku mεtabolit difrεns dεm. we yu kכmpεr wit ascites, di tכmכro-infiltrating T sεl dεm de sכmtεm εnrich in 1-methylnicotinamide (MNA). pan tap we di lεvεl fכ MNA insay T sεl dεm de εlevεt, di εksprεshכn fכ nikotinamid N-mεtiltransfεrayz (wan εnzym we de katalayz di tכnfכs fכ mεtil grup dεm frכm S-adenosylmethionine to nikotinamid) de limited to fכblast dεm εn tכmכro sεl dεm. fכ wok, MNA de indyuz T sεl dεm fכ sekret di tכmכro-prכmot saytokכn tכmכro nεkrכsis fכktכ alfa. כl dis mek, TME-dεriv MNA de kכntribyut to di imyun rεguleshכn fכ T sεl dεm εn i de riprizent wan pכtεnshal imyun tεrapi tכgεt fכ di tritmεnt fכ hכman kεnsar.
di mεtabolayt dεm we kכmכt frכm di tכmכro kin gεt dip inhibitory ifekt pan di anti-tכmכr imuniti, εn mכr εn mכr pruf de sho se dεn kin sכv bak as di ki drεvin fכs fכ di sik prכgreshכn (1). apat frכm di Warburg ifekt, di wok we dεn jכs du dεn bigin fכ kכl di mεtabolik stet fכ di tכmכro sεl dεm εn in rilayshכn wit di imyun stet fכ di tכmכro maykro envayroment (TME). stכdi dεm pan maws mכdel dεm εn hכman T sεl dεm dεn sho se glutamin mεtabolism (2), כksidεtiv mεtabolism (3) εn glukכs mεtabolism (4) kin akt indipεndεnt wan pan difrεn imyun sεl sכbgrup dεm. sεvεra mεtabolit dεm na dεn path dεm ya de inhεbit di anti-tכmכr fכnshכn fכ di T sεl dεm. i pruv se di blכk we di kכεnzym tεtrahaydrobayopterin (BH4) de blok kin damej di proliferashכn fכ di T sεl dεm, εn di inkrεs fכ BH4 na di bכdi kin εnhans di anti-tכmכr imyun rεspכns we CD4 εn CD8 de mεdiet. apat frכm dat, di imyun supεsiv ifekt fכ kynurenine kin rεsכk bay di administreshכn fכ BH4 ( 5 ). insay isocitrate dehydrogenase (IDH) mutant glioblastoma, di sekreshכn fכ enantiometabolic (R)-2-hydroxyglutarate (R-2-HG) de inhεbit T sεl aktibכshכn, proliferashכn εn saytolaysis Aktiviti (6). rεsεntli, dεn sho se mεtylgliyoksal, we na bay-prכdukt fכ glycolysis, na supεr sεl dεm we kכmכt na mayloid de prodyuz, εn T sεl tכnfכs fכ mεtilglioksal kin inhεbit εfεktכ T sεl fכnshכn. insay tritmεnt, di nyutralayzeshכn fכ mεtilgliyoksal kin כvakom di aktvכti fכ myeloid-dεrivεd suprεsכr sεl dεm (MDSC) εn sinagεstikli εnhans chεkpכynt blכkεd tεrapi in maws mכdel dεm (7). dis stכdi dεm kכlektivli εnfaz di ki rol we TME-dεriv mεtabolit dεm de ple fכ rεgεl T sεl fכnshכn εn aktvכti.
di t sεl dεm we nכ de wok fayn dεn bin ripot bכku bכku wan pan ovarian kεnsar (8). dis na pat pan di mεtabolik kכntribyushכn dεm we de insay haypoksia εn abnכmal tכmכro vaskulεt (9), we de rizulta in di kכnvכshכn fכ glukכs εn triptofan to bay-prכdukt lεk laktik asid εn kynurenine. εksεsiv εkstrasεlyulכr laktat de ridyus di prodyushכn fכ intafεron-γ (IFN-γ) εn de drεb di difrεns fכ maylosuprεsiv sכbgrכp dεm ( 10 , 11 ). di kכnsכmshכn fכ triptofan dεn de inhεbit di T sεl proliferashכn dεm dεn wan εn i de inhεbit T sεl rεsεpכta signal (12-14). pan tap dεn obsכbishכn ya, plεnti wok we de arawnd imyun mεtabolism bin kכriכnt in in vitro T sεl kכlt we dεn yuz optimizεd mεdia, כ limited to homologous maus mכdel in vivo, nכn pan dεm nכ fulכp rεflek di hεtεrojεniti fכ hυman kεnsar εn Fisiolojikal makro εn maykro envayroment.
wan kכmכn tin fכ ovarian kεnsar na di pεritoneal spred εn di apinεns fכ ascites. di akyumyuleshכn fכ sεl fכluid insay ascites de sho se i gεt advans sik εn pwεl prכgnosis (15). akɔdin to ripɔt, dis yכnik kכmpartimεnt na haypoksik, i gεt hεy lεvεl dεm fכ vaskulεr εndoteyl gכt fכktכ (VEGF) εn indoleamine 2,3-dioxygenase (IDO), εn i infiltrate bay T rεgεlεtכri sεl dεm εn myeloid inhibitory sεl dεm (15-18). di mεtabolik envayroment fכ ascites kin difrεn frכm di tכmכro insεf, so di riprogramin fכ di T sεl dεm na di pεritoneal spεs nכ klia. apat frכm dat, di kכl difrεns εn hεterojεniti bitwin asayt εn mεtabolit dεm we de insay di tכmכro invayroment kin hεnd di infiltrεshכn fכ di imyun sεl dεm εn dεn fכnshכn pan tכmכro dεm, εn mכr risεch nid fכ.
fכ sכlv dεn prכblεm ya, wi disayn wan sεnsitiv sεl separeshכn εn likwid kromatografi tandem mas spεktrכmetri (LC-MS/MS) mεtכd fכ stכdi difrεn sεl tכp dεm (inklud CD4 + εn CD8 + T sεl dεm) εn bak insay εn bitwin tכmכro I mεtabolayt dεm span sεl dεm na di sem asayt εn tכmכro envayroment fכ di sikman. wi de yuz dis mεtכd in kכnjכkshכn wit hεy-dimεnshכnal fכlכ saytכmetri εn singl-sεl RNA sikεns (scRNA-seq) fכ gi wan hεli rεsכlv pכtrεt fכ di mεtabolik stetכs fכ dεn ki populeshכn dεm ya. dis mεtכd rivεl wan signifyant inkrεs pan di lεvεl fכ 1-methylnicotinamide (MNA) insay tכmכro T sεl dεm, εn in vitro εkspεriεns sho se di immunomodulatory ifekt fכ MNA pan T sεl fכnshכn bin nכ bin no bifo. in jεnarכl, dis mεtכd de sho di mכtalman mεtabolik intarakshכn bitwin di tכmכro dεm εn di imyun sεl dεm, εn i de gi yunik insayt dεm fכ di imyun rεguleshכn mεtabolayt dεm, we kin yusful fכ di tritmεnt fכ T sεl-bεys ovarian kεnsar imyun tεrapi Tritmεnt opportunities.
We yuz ay-dimɛnshɔnal flɔ saytometri fɔ simultaneɔs kwantifay glukɔs ɔptek [2-(N-(7-naytrophenyl-2-ɔksa-1,3-diaza-4-yl)amino)-2-diɔksiglukɔs (2-NBDG) ɛn maytokɔndrial aktiviti [MitoTracker Deep Red (MT DR)] (7, 19, 20) na sayd bay sayd tipik mak dεm we de difrεnt di imyun sεl dεm εn di tכmכro sεl populeshכn dεm (Tεbul S2 εn Fig S1A). dis analisis sho se we yu kכmpεr wit T sεl dεm, ascites εn tכmכro sεl dεm gεt hכy glukכs כptek lεvεl, bכt dεn gεt sכm difrεns insay maytochכndrial aktiviti. di avrej glukכs we di tכmכro sεl dεm de tek [CD45-EpCAM (EpCAM)+] na tri to fכ tεm di wan we di T sεl dεm de tek, εn di avεrej glukכs we di CD4 + T sεl dεm de tek na 1.2 tεm di wan we di CD8 + T sεl dεm de tek, we de sho se di Tumor infiltrating lymphocytes (TIL) gεt difrεn mεtabolik rikwaymεnt dεm ivin insay di sem TME (Figure 1A). difrεnt frכm dat, di maytochכndrial aktvכti na di tכmכro sεl dεm sεm lεk di wan fכ CD4 + T sεl dεm, εn di maytochכndrial aktvכti fכ di tu tכp sεl dεm hכy pas di wan fכ CD8 + T sεl dεm (Figure 1B). in jεnarכl, dεn risכlt ya de sho di mεtabolik lεvεl. di mεtabolik aktiviti fכ di tכmכro sεl dεm hכy pas di wan fכ CD4 + T sεl dεm, εn di mεtabolik aktiviti fכ CD4 + T sεl dεm hכy pas di wan fכ CD8 + T sεl dεm. pan tap dεn ifekt dεm ya akraos di tכp sεl dεm, nכ kכnsistεnt difrεns de insay di mεtabolik stetכs fכ CD4 + εn CD8 + T sεl dεm כ dεn rilitiv prכpכshכn insay ascites kכmpεr wit tכmכro dεm (Figure 1C). difrεnt frכm dat, insay di CD45-sεl frakshכn, di prכpכshכn fכ EpCAM+ sεl dεm na di tכmכro inkrεs we yu kכmpεr wit ascites (Figure 1D). Wi bin obsכv bak wan klia mεtabolik difrεns bitwin EpCAM+ εn EpCAM- sεl kכmכpכnt dεm. EpCAM+ (tכmכr) sεl dεm gεt hכy glukכs כptek εn maytochכndrial aktvכti pas EpCAM- sεl dεm, we bכku hכy pas di mεtabolik aktiviti fכ fibroblast dεm na tכmכro sεl dεm na TME (Figure 1, E εn F).
. (C) di rεshכn fכ CD4 + εn CD8 + sεl dεm (fכ CD3 + T sεl dεm) insay ascites εn tכmכro. (D) Prכpכshכn כf EpCAM + tכmכro sεl dεm na ascites εn tכmכro (CD45−). . (G) Riprεzεntεtiv grafik dεm fכ CD25, CD137 εn PD1 εksprεshכn bay fכ fכlכ saytכmetri. (H εn I) CD25, CD137 εn PD1 εksprεshכn pan CD4 + T sεl dεm (H) εn CD8 + T sεl dεm (I). (J εn K) Naiv, sεntri mεmכri (Tcm), εfεktכr (Teff) εn εfεktכ mεmכri (Tem) fεnotayp dεm we bεys pan di εksprεshכn fכ CCR7 εn CD45RO. rεprεsεntεtiv imej dεm (lεft) εn tεbul dεta (rayt) fכ CD4 + T sεl dεm (J) εn CD8 + T sεl dεm (K) insay ascites εn tכmכro dεm. P valyu dɛn we dɛn disayd bay pe t-tɛst (*P<0.05, **P<0.01 ɛn ***P<0.001). Di layn riprizent mach pasɛnt dɛn (n = 6). FMO, fluorescence mayns wan; MFI, midyan fluorescence intensiti.
fכda analisis rεvεl כda signifyant difrεns bitwin di hεli rεsכlv T sεl fεnotaypik stetכs. di aktibכt (Figure 1, G to I) εn ifektכr mεmכri (Figure 1, J εn K) insay tכmכro dεm de bכku mכr pas ascites (proporshכn fכ CD3 + T sεl dεm). semweso, we dεn analכz di fεnotayp bay di εksprεshכn fכ aktibכshכn mak dεm (CD25 εn CD137) εn diplεshכn mak dεm [programmed sεl dεd protin 1 (PD1)] sho se pan כl we di mεtabolik kכntribyushכn dεm fכ dεn populeshכn dεm ya difrεnt (Figure S1, B to E) , Bɔt nכ signifyant mεtabolik difrεns dεm bin kכnsistεntli obsεv bitwin naiv, εfεktכr כ mεmכri sכbsεt dεm (Figure S1, F to I). dis rizulεt dεm bin kכnfכm bay we dεn yuz mashin lanin mεtכd dεm fכ כtomatכk asaynd sεl fεnotayp dεm ( 21 ), we fכs rεvεl di prεsεns fכ wan big nכmba כf bon mכro sεl dεm (CD45 + / CD3- / CD4 + / CD45RO +) insay di pasεnt in ascites (Figure S2A ). pan כl di tכp sεl dεm we dεn aydentify, dis mayloid sεl populeshכn sho di hεyest glukכs כptek εn maytochכndrial aktvכti (Figure S2, B to G). dis rizulεt dεm de hεlayt di strכng mεtabolik difrεns bitwin di mכltipכl sεl tכp dεm we dεn fכnshכn insay ascites εn tכmכro dεm na HGSC pasεnshכn dεm.
di men chalenj fכ כndastand di mεtabonomik kכntribyushכn dεm fכ TIL na di nid fכ aylכt T sεl sεmpl dεm we gεt inof klin, kwaliti εn kwantiti frכm tכmכro dεm. stכdi dεm we dεn jכs du sho se di sכt εn bidz εnrichmεnt mεtכd dεm we de bays pan fכ fכlכ saytכmetri kin mek chenj dεm na di sεlyul mεtabolayt profayl dεm ( 22-24 ). Fכ כvakom dis prכblεm, wi optimiz di bidz εnrichmεnt mεtכd fכ ayzכlεt εn ayzכlεt TIL frכm sכja rεsεkt hυman ovarian kεnsar bifo analisis bay LC-MS/MS (luk Matirial εn Mεtכd; Fig 2A). fכ ases di כvala impak we dis protכkכl gεt pan mεtabolayt chenj dεm, wi kכmpεr di mεtabolayt profayl dεm fכ T sεl dεm we hεlty dכna dεm aktibכt afta di oba bidz sεparashכn stεp wit sεl dεm we nכ bin bid separet bכt we bin de pan ays. Dis kwaliti kכntrכl analisis fכn se hכy kכrεleshכn de bitwin dεn tu kכndishכn ya (r = 0.77), εn di tεknikal rεpεtabiliti fכ di grup fכ 86 mεtabolit dεm gεt hכy rεpεtabiliti (Figure 2B). fכ dat, dεn mεtכd dεm ya kin du akכrd mεtabolayt analisis insay sεl dεm we de כnda sεl tכp εnrichmεnt, so dat de gi di fכs hεy rεsכlushכn pletfכm fכ no spεsifi k mεtabolit dεm na HGSC, we de mek pipul dεn gεt dip כndastandin fכ sεl spεsifisiti Sεkswal mεtabolism program.
(A) Skematik dayagram fɔ magnɛtik bidz ɛnrichmɛnt. Bifo dεn analisis bay LC-MS/MS, di sεl dεm go כnda tri kכnsekyutiv raund dεm fכ magnεtik bidz εnrichmεnt כ rεst pan ays. (B) Di ifekt we di enrichment tayp gɛt pan di bɔku bɔku mɛtabolayt dɛn. Di avrej fɔ tri mɛzhɔmɛnt fɔ ɛni ɛnrichmɛnt tayp ± SE. Di grey layn de sho 1: 1 rilayshɔn. Di intra-klas kɔrɛleshɔn (ICC) fɔ ripit mɛzhɔmɛnt dɛn we dɛn sho na di aks lɛbl. NAD, nikotinamid adenin dinyukliɔtayd. (C) Skematik dayagram fɔ di wokflɔ fɔ di pasɛnt mɛtabolayt analisis. Dɛn kin gɛda ascites ɔ tumor frɔm di sik pipul dɛn ɛn dɛn kin kip dɛn na krayo. wan sכm pat pan εvri sεmpl dεn bin analכz bay fכlכ saytכmetri, we di rεmεnεnt sεmpl dεm bin כnda tri raund כf εnrichmεnt fכ CD4+, CD8+ εn CD45- sεl dεm. Dis sel frakshɔn dɛn bin analayz yuz LC-MS/MS. (D) Hεt map fכ standad mεtabolayt abundans. Di dendrogram riprizent Ward in klasta fɔ Yuklidian distans bitwin sampul dɛn. (E) Prinsipal kכmכpכnt analisis (PCA) fכ sεmpl mεtabolayt map, we de sho tri rεplikεt fכ εvri sεmpl, sεmpl frכm di sem pasεnt dεn kכnekt bay wan layn. (F) Di PCA fכ di mεtabolayt profayl fכ di sεmpl kכndishכn pan di pasεnt (dat na, yuz patchכl ridandans); di sampul tayp de limited bay di konveks hul. PC1, men komponent 1; PC2, men kɔmpɔnɛnt 2.
Neks, wi aplay dis enrichment mεtכd fכ analכz 99 mεtabolayt dεm na di CD4 +, CD8 + εn CD45-sεl frakshכn dεm na di praymar ascites εn tכmכro dεm fכ siks HGSC pasεnshכn dεm (Figure 2C, Figure S3A εn Table S3 εn S4). di populeshכn we intres de akכnt fכ 2% to 70% fכ di כrijinal big sεmpl fכ liv sεl dεm, εn di prכpכshכn fכ sεl dεm de difrεnt bכku bitwin di sik pipul dεm. afta dεn sεparayt di bidz dεm, di εnrich frakshכn fכ intres (CD4+, CD8+ כ CD45-) de akכnt fכ mכr dan 85% fכ כl di liv sεl dεm na di sεmpl pan avεj. dis enrichmεnt mεtכd de alaw wi fכ analכz sεl populeshכn frכm hכman tכmכro tisu mεtabolism, we nכ posεbul fכ du frכm big sεmpl dεm. Yuz dis protכkכl, wi disayd se l-kynurenine εn adenosine, dεn tu we dεn kכl imyunosuprεsiv mεtabolayt dεm we dεn kכl fayn fayn wan bin εlevεt insay tכmכro T sεl dεm כ tכmכro sεl dεm (Figure S3, B εn C). fכ dat, dεn risכlt ya de sho di fideliti εn abiliti fכ wi sεl separeshכn εn mas spεktrכmetri tεknכlכji fכ fכn bayolojikal imכtant mεtabolayt dεm na di pasεnt tisu dεm.
di analisis we wi du sho bak se wan strכng mεtabolik separeshכn fכ di tכp sεl dεm insay εn bitwin di pasεnshכn dεm (Figure 2D εn Figure S4A). In patikyula, we yu kɔmpia am wit ɔda pasɛnt dɛn, pasɛnt 70 sho difrɛn mɛtabolik kwaliti dɛn (Figure 2E ɛn Figure S4B), we sho se i kin bi se sɔbstanshal mɛtabolik itɛrojɛniti de bitwin pasɛnt dɛn. i fayn fכ no se we yu kכmpεr wit כda pasεnshכn dεm (1.2 to 2 lita; Tεbul S1), di tכtal כmכnt כf ascites we dεn kכlekt na pasεnshכn 70 (80 ml) bin sכm. di kכntrכl fכ inta-peshεnt hεtεrojεniti di tεm we prinsipal kכmכpכnt analisis (fכ egzampl, yuz patchכl ridandans analisis) sho kכnsistεnt chenj bitwin sεl tכp dεm, εn di sεl tכp dεm εn/כ maykro envayroment dεn klia wan agregεt akɔdin to di mεtabolayt profayl (Figure 2F ). di analisis fכ singl mεtabolit dεm εnfaz dεn ifekt dεm ya εn sho sכm difrεns bitwin sεl tכp dεm εn maykro envayroment. i fayn fכ no se di mכst ekstrim difrεns we dεn si na MNA, we kin εnrich insay CD45- sεl dεm εn insay CD4+ εn CD8+ sεl dεm we de infiltrate di tכmכro (Figure 3A). fכ CD4 + sεl dεm, dis ifekt de sho mכst, εn i lεk se di MNA insay CD8 + sεl dεm tu de afekt strכng wan pan di envayroment. Bɔt dis nɔ impɔtant, bikɔs na tri pan di siks pasɛnt dɛn nɔmɔ dɛn kin ebul fɔ evaluate fɔ di tumor CD8+ skɔ. apat frכm MNA, insay difrεn tכp sεl dεm na ascites εn tכmכro dεm, כda mεtabolit dεm we dεn nכ de kכl dεm fayn fayn wan na TIL dεn tu difrεnt rich (Figure S3 εn S4). fכ dat, dεn data ya de sho wan prכmis sεt fכ immunomodulatory mεtabolayt fכ fכda risεch.
(A) nכmal kכntεnt fכ MNA insay CD4+, CD8+ εn CD45- sεl dεm frכm ascites εn tכmכro. Di bɔks plot sho di midyan (layn), intakwatayl rɛnj (frɛm hinj) ɛn di data rɛnj, we go rich 1.5 tɛm di intakwatayl rɛnj (frɛm wisk). As dεn dεskrεb insay Patient Materials and Methods, yuz di pasεnt in limma valyu fכ no di P valyu (*P<0.05 εn **P<0.01). (B) Skematik dayagram fɔ MNA mɛtabolism (60). Mεtabolayt dεm: S-adenosyl-1-mεtiכnin; SAH, S-adenosin-1-homosistayn; NA, we na nikotinamid; MNA, 1-mɛtilnikɔtinamid; 2-PY, 1-methyl- 2-piridon-5-karboksamid; 4-PY, 1-methyl-4-piridon-5-karboksamid; NR, nikotinamid raybɔs; NMN, nikotinamid mononyukliɔtayd. εnzym dεm (grin): NNMT, nikotinamid N-mεtiltransfεrayz; SIRT, we na sirtuin dɛn; NAMPT, nikotinamid fosforibosil transfεrεz; AOX1, aldehayd ɔksidayz 1; NRK, nikotinamid ribosayd kinaz; NMNAT, nikotinamid mono Nyukliotayd adenylet transfεrεz; Pnp1, purin nyukliɔsayd fɔsfɔrayz. (C) t-SNE di scRNA-seq di ascites (grey) ɛn tumor (rɛd; n = 3 pasɛnt dɛn). (D) NNMT εksprεshכn in difrεnt sεl populeshכn dεm we dεn aydentify yuz scRNA-seq. (E) Eksprεshכn כf NNMT εn AOX1 in SK-OV-3, hυman εmbrayonik kidni (HEK) 293T, T sεl dεm εn MNA-trit T sεl dεm. di fold εksprεshכn de sho rilitiv to SK-OV-3. Di εksprεshכn patεn wit SEM de sho (n = 6 hεlty dכna). Ct valyu dεm we pas 35 dεn kin kכnsidr dεm as dεn nכ detektabl (UD). (F) Eksprεshכn כf SLC22A1 εn SLC22A2 in SK-OV-3, HEK293T, T sεl dεm εn T sεl dεm we dεn trit wit 8mM MNA. di fold εksprεshכn de sho rilitiv to SK-OV-3. Di εksprεshכn patεn wit SEM de sho (n = 6 hεlty dכna). Ct valyu dεm we pas 35 dεn kin kכnsidr dεm as dεn nכ detektabl (UD). (G) Sεl MNA kכntεnt in aktibכt hεlty dכna T sεl dεm afta 72 awa inkכbeshכn wit MNA. Di εksprεshכn patεn wit SEM de sho (n = 4 hεlty dכna).
MNA de prodyuz bay we dεn transfכm di mεtil grup frכm S-adenosyl-1-methionine (SAM) to nikotinamid (NA) bay nikotinamid N-methyltransferase (NNMT; Figure 3B). NNMT de ova εksprεs insay difrεn hכman kεnsar dεm εn i de sho se i de prolifera, invayshכn εn mεtastas (25-27). Fכ bεtε כndastand di sכs fכ MNA insay T sεl dεm na TME, wi yuz scRNA-seq fכ kכl di εksprεshכn fכ NNMT akrays sεl tכp dεm na di ascites εn tכmכro dεm fכ tri HGSC pasεnshכn dεm (Tεbul S5). analisis fכ aprכksimatli 6,500 sεl dεm sho se insay ascites εn tכmכro envayroment, NNMT εksprεshכn bin rεstrikt to di prεsum fכblast εn tכmכro sεl populeshכn (Figure 3, C εn D). i fayn fכ no se nכ klia NNMT εksprεshכn de insay eni populeshכn we de εksprεs PTPRC (CD45 +) (Figure 3D εn Figure S5A), we sho se di MNA we dεn dεtekt insay di mεtabolit spεktrum dεn introdכks insay T sεl dεm. di εksprεshכn fכ aldayd כksidayz 1 (AOX1) de kכnvכlt MNA to 1-mεtil-2-piridon-5-kכbכksamid (2-PYR) כ 1-mεtil-4-piridon-5-kכbכksamid (4- PYR); Fig 3B) de rεstrikt bak to di populeshכn fכ di fכblas dεm we de εksprεs COL1A1 (Figure S5A), we tכgeda de sho se T sεl dεm nכ gεt di abiliti fכ kכvεshכnal MNA mεtabolism. di εksprεshכn patεn כf dεn MNA-rεlatεd jin dεm bin vεrifik yuz wan sεkכn indipεndεnt sεl data sεt frכm ascites frכm HGSC pasεnshכn (Figure S5B; n = 6) (16). apat frכm dat, kwantiti polimεrayz chen riakshכn (qPCR) analisis fכ hεlty dכna T sεl dεm we dεn trit wit MNA sho se we dεn kכmpεr wit kכntrol SK-OV-3 ovarian tכmכr sεl dεm, NNMT כ AOX1 bin kכlכsכl nכ εksprεs (Figure 3E). dis rizulεt dεm we dεn nכ bin εkspεkt de sho se MNA kin kכmכt frכm fibroblast כ tכmכro dεm insay adjasent T sεl dεm na TME.
Although kandidet inklud di famili fכ כrganik kεshכn transpכrta 1 to 3 (OCT1, OCT2 εn OCT3) we di sכlubul kεria 22 (SLC22) famili (SLC22A1, SLC22A2 εn SLC22A3) kכd, di pכtεnshal transpכrta dεm fכ MNA stil כndafayn (28) . QPCR fכ mRNA frכm hεlty dכna T sεl dεm sho lכw εksprεshכn lεvεl fכ SLC22A1 bכt כndatektabl lεvεl fכ SLC22A2, we kכnfכm se dεn bin ripot am bifo insay di litεrachכ (Figure 3F) (29). difrεnt frכm dat, di SK-OV-3 ovarian tכmכro sεl layn εksprεs hεy lεvεl dεm fכ di tu transpכrta dεm (Figure 3F).
fכ tεst di posisibul fכ T sεl dεm fכ gεt di abiliti fכ absכp fכrin MNA, dεn kכlt hεlty dכna T sεl dεm fכ 72 awa insay di prεsεns fכ difrεn kכnsantreshכn fכ MNA. if εksojen MNA nכ de, dεn nכ kin no di sεlyul kכntεnt fכ MNA (Figure 3G). כltu, aktibכt T sεl dεm we dεn trit wit εksojen MNA sho wan dכz-dipεndεnt inkrεs pan MNA kכntεnt insay di sεl dεm, te to 6 mM MNA (Figure 3G). dis rizulεt sho se pan כl we di lכw lεvεl fכ transpכrta εksprεshכn εn di mεn εnzym we rispansabl fכ intasεlulyar MNA mεtabolism, TIL kin stil tek MNA.
di spεktrum fכ mεtabolit dεm na di pasεnshכn in T sεl dεm εn in vitro MNA absכpshכn εkspεriεns dεm de inkrεs di posisibul fכ kεnsar-asכsiet fכblas (CAF) sekret MNA εn tכmכro sεl dεm kin rεgεl di fεnotayp εn fכnshכn fכ TIL. Fכ no di ifekt we MNA gεt pan T sεl dεm, dεn bin aktibכt hεlty dכna T sεl dεm in vitro in di prεsεns כ absεns כf MNA, εn dεn proliferashכn εn saytokεn prodakshכn bin evaluate. afta 7 dez we dεn ad MNA pan di hεyest dכz, di populeshכn dכbl nכmba bin mכdarεt rεdכks, we di vigor bin mεnten pan כl di doz dεm (Figure 4A). apat frכm dat, tritmεnt fכ εksojen MNA rεsult in inkrεs pan di prכpכshכn fכ CD4 + εn CD8 + T sεl dεm we de εksprεs tכmכro nεkrכsis fכktכ-α (TNFα; Figure 4B). difrεnt frכm dat, di intasεlulyar prodakshכn fכ IFN-γ bin sכmtεm rεdכks insay CD4 + T sεl dεm, bכt nכto insay CD8 + T sεl dεm, εn nכ sכm chenj bin de insay intalyukin 2 (IL-2; Figure 4, C εn D). כl dis mek, εnzym-link immunosorbent assay (ELISA) fכ supεrnatant frכm dεn MNA-trit T sεl kכlt dεm sho sכm inkrεs pan TNFα, dεkrεshכn pan IFN-γ, εn nכ chenj in IL-2 (Figure 4, E to G). . di dכn fכ IFN-γ de sho se MNA kin ple rol fכ inhεbit di anti-tכmכr aktiviti fכ T sεl dεm. fכ simul di ifekt we MNA gεt pan T sεl-mεdiet saytכtoksisiti, chimeric antijen rεsεptכr T (FRα-CAR-T) sεl dεm we de tכk bכt fכlet rεsεptכr α εn CAR-T (GFP) we grεn fכrosεnt protin (GFP) -CAR-T) sεl dεm de rεgεl na hεlty dכna pεrifεral blכd mכnonyukliar sεl dεm (PBMC) de prodyuz. CAR-T sel dεm bin kכlt fכ 24 awa insay di prεsεns כf MNA, εn afta dat dεn kכ-kulchכr wit hυman SK-OV-3 ovarian tכmכr sεl dεm we de εksprεs fכlet rεsεptכr α pan εfεktכ to tכgεt rεshכn we na 10:1. MNA tritmεnt rεsult in wan signifyant dεkrεshכn in di kil aktvכti fכ FRα-CAR-T sεl dεm, we bin sεm lεk FRα-CAR-T sεl dεm we dεn trit wit adenosine (Figure 4H).
(A) Tכtal viable sεl kכnt εn populeshכn dכblεn (PD) dεrekt frכm kכlt pan de 7. Di bar grafik de riprizent di min + SEM fכ siks hεlty dכna dεm. Ripresent data frɔm at least n = 3 indipɛndɛnt ɛkspiriɛns. (B to D) CD3/CD28 εn IL-2 bin yuz fכ aktibכt T sεl dεm na dεn rispεktiv MNA kכnsantreshכn fכ 7 dez. Bifo analisis, sel dεm bin stimulat wit PMA/ionomycin wit GolgiStop fכ 4 awa. TNFα (B) εksprεshכn in T sεl dεm. εgzampl imej (lεft) εn tεbul data (rayt) fכ TNFα εksprεshכn insay liv sεl dεm. IFN-γ (C) ɛn IL-2 (D) ɛksprɛshɔn insay T sɛl dɛn. Di εksprεshכn כf saytokεn dεm bin mεzj bay fכ fכlכ saytכmεtri. Di bar grafik riprizent di min (n = 6 hεlty dכna) + SEM. Yuz wan-way analisis fɔ varyans ɛn ripit mɛzhɔ (*P<0.05 ɛn **P<0.01) fɔ no di P valyu. Riprɛzɛnt data frɔm at least n = 3 indipɛndɛnt ɛkspiriɛns. (E to G) CD3/CD28 εn IL-2 bin yuz fכ aktibכt T sεl dεm na dεn rispεktiv MNA kכnsantreshכn fכ 7 dez. Di mεdiכm bin kכlekt bifo εn afta 4 awa PMA/ionomycin stimulashכn. Di kɔnsɛntreshɔn dɛn fɔ TNFα (E), IFN-γ (F) ɛn IL-2 (G) bin mɛzhɔ bay ELISA. Di bar grafik de sho di min (n = 5 wɛlbɔdi donɔ dɛn) + SEM. P valyu we dɛn disayd yuz wan-way analisis fɔ varyans ɛn ripit mɛzhɔmɛnt (*P<0.05). Di dot layn de sho di ditekshɔn limit fɔ di ditekshɔn. (H) Sεl laysis asεy. FRα-CAR-T ɔ GFP-CAR-T sɛl dɛn bin ajɔst wit adenosin (250μM) ɔ MNA (10 mM) fɔ 24 awa, ɔ lɛf dɛn we dɛn nɔ trit (Ctrl). Di pasεnshכn kil fכ SK-OV-3 sεl dεm bin mεzj. P valyu we dɛn disayd bay Welch t tɛst (*P<0.5 ɛn **P<0.01).
fכ mek dεn gεt mεkanik כndastandin fכ MNA-dipεndεnt TNFα εksprεshכn rεguleshכn, dεn bin evaluate di chenj dεm na TNFα mRNA fכ MNA-trit T sεl dεm (Figure 5A). hεlty dכna T sεl dεm we dεn trit wit MNA sho tu fכl inkrεs pan TNFα transkripshכn lεvεl, we sho se MNA de dipεnd pan TNFα transkripshכnal rεguleshכn. Fכ invεstigat dis posεbul rεgεdyushכn mεkanism, tu transkripshכn fכktכ dεm we dεn no we de rεgεl TNFα, we na aktibכt T sεl nyuklia fכktכ (NFAT) εn spεsifi k protin 1 (Sp1), bin evaluate in rispכns to MNA binding to di proksimal TNFα prכmotεr ( 30 ). di TNFα promota kכntεn 6 idεntify NFAT binding sayt dεm εn 2 Sp1 binding sayt dεm, we de כvalap na wan sayt [-55 bays pe (bp) frכm di 5'cap] (30). kromatin immunoprecipitation (ChIP) sho se we dεn trit am wit MNA, di binding fכ Sp1 to di TNFα prכmotεr inkrεs tri-fol. di inkכrporεshכn כf NFAT כlso inkrεs εn aprכch imכpכtants (Figure 5B). dis data sho se MNA de rεgεl di εksprεshכn fכ TNFα tru Sp1 transkripshכn, εn to sכm sכm di εksprεshכn fכ NFAT.
(A) Kכmpεr wit T sεl dεm we dεn kכlt witout MNA, di fold chenj fכ TNFα εksprεshכn in T sεl dεm we dεn trit wit MNA. Di εksprεshכn patεn wit SEM de sho (n = 5 hεlty dכna). Riprɛzɛnt data frɔm at least n = 3 indipɛndɛnt ɛkspiriɛns. (B) Di TNFα promota fכ T sel dεm we dεn trit wit כ witout 8 mM MNA afta NFAT εn Sp1 bin kכmbayn wit (Ctrl) εn PMA/ionomycin stimulashכn fכ 4 awa. Immunoglobulin G (IgG) ɛn H3 bin yuz as nɛgitiv ɛn pɔsitiv kɔntrol fɔ immunoprecipitation, rispɛktvɔli. di kwantifikeshכn fכ ChIP sho se di binding fכ Sp1 εn NFAT to di TNFα prכmotεr in MNA-trit sεl dεm inkrεs sεvεra tεm kכmpεr wit di kכntrכl. Ripresent data frɔm at least n = 3 indipɛndɛnt ɛkspiriɛns. P valyu we dɛn disayd bay bɔku t-tɛst dɛn (*** P <0.01). (C) we yu kכmpεr wit di ascites fכ HGSC, T sεl dεm (nכn-saytotoksik) sho inkrεs εksprεshכn fכ TNF insay di tכmכro. Di kɔlɔ dɛn tinap fɔ difrɛn pasɛnt dɛn. di sεl dεm we dεn displei dεn bin rεndכm sεmpl to 300 εn jittered fכ limited כvadraw (** Padj = 0.0076). (D) Proposed model of MNA fכ ovarian kεnsar. MNA de prodyuz insay di tכmכro sεl dεm εn di fibroblast dεm na TME εn di T sεl dεm de tek am. MNA de inkrεs di binding fכ Sp1 to di TNFα prכmotεr, we de lid to inkrεs pan TNFα transkripshכn εn TNFα saytokεn prodakshכn. MNA de mek bak di IFN-γ dכn. inhibishכn fכ T sεl fכnshכn de mek i ridyus di kil abiliti εn aksεlεrat tכmכro gro.
akɔdin to ripɔt, TNFα gɛt frɔnt ɛn bak-dipɛndent anti-tכmכr εn anti-tכmכr ifekt, bכt i gεt wan rol we dεn no gud gud wan fכ protεkt di gכt εn mεtastas fכ ovarian kεnsar (31-33). akɔdin to ripɔt, di kɔnsɛntreshɔn fɔ TNFα insay ascites ɛn tumor tisu dɛn na pasɛnt dɛn we gɛt ovarian kansa pas di wan we de insay benign tisu dɛn ( 34-36 ). insay di mεkanism, TNFα kin rεgεl di aktibכshכn, fכnshכn εn proliferashכn fכ di wayt bכdi sεl dεm, εn chenj di fεnotayp fכ di kεnsar sεl dεm (37, 38). kכnsistεnt wit dεn fכnd dεm ya, difrεnt jin εksprεshכn analisis sho se TNF bin sכmtεm כp-rεgulεt in T sεl dεm insay tכmכro tisu dεm kכmpεr wit ascites (Figure 5C). di inkrεs pan TNF εksprεshכn bin כnli evidεnt in T sεl populeshכn wit wan nכn-saytotoksik fεnotayp (Figure S5A). in sכmari, dεn data ya sכpכt di vεyushכn se MNA gεt dual immunosuppressiv εn tכmכro prכmot ifekt dεm na HGSC.
Fluorescent labeling bays pan flow cytometry dεn bi di men mεtכd fכ stכdi TIL mεtabolism. dis stכdi dεn sho se we dεn kכmpεr wit pεrifεral blכd limfosayt dεm כ T sεl dεm frכm sεkכnd limfoyd כgan dεm, mכrin εn hכman TIL gεt hכy tεndens fכ tek glukכs (4, 39) εn di sכmtεm lכs fכ di maytochכndrial fכnshכn (19, 40). pan ɔl we wi dɔn si di sem kayn rizɔlt insay dis stכdi, di ki divεlכpmεnt na fכ kכmpεr di mεtabolism fכ di tכmכro sεl dεm εn TIL frכm di sem rεsεkt tכmכro tisu. kכnsist wit sכm pan dεn ripot ya we dεn bin dכn bifo, di tכmכro (CD45-EpCAM +) sεl dεm frכm ascites εn tכmכro dεm gεt hכy glukכs כptek pas CD8 + εn CD4 + T sεl dεm, we sכpכt se di hכy glukכs כptek fכ tכmכro sεl dεm kin kכmpεr wit T sεl dεm. Di kɔnsɛpt fɔ T sɛl kɔmpitishɔn. TME. כltu, di maytochכndrial aktvכti fכ di tכmכro sεl dεm hכy pas di wan fכ CD8 + T sεl dεm, bכt di maytochכndrial aktiviti fכ di CD4 + T sεl dεm. dis rizulεt dεm de rεinfכs di tεm we de kכmכt se כksidεtiv mεtabolism imכtant fכ tכmכro sεl dεm ( 41 , 42 ). dεn sho bak se CD8 + T sεl dεm kin bi mכr susεptibl fכ כksidεtiv disfכnkshכn pas CD4 + T sεl dεm, כ se CD4 + T sεl dεm kin yuz kכbכn sכs dεm כda pas glukכs fכ mεnten di maytochכndrial aktiviti ( 43 , 44 ). i fכ no se wi nכ si nכ difrεns pan glukכs כptek כ maytochכndrial aktiviti bitwin CD4 + T εfεktכr, T εfεktכ mεmכri εn T sεntri mεmכri sεl dεm na ascites. semweso, di difrεns stet fכ CD8 + T sεl dεm na di tכmכro dεm nכ gεt natin fכ du wit chenj dεm na glukכs כptek, we de hεlayt di sכm difrεns bitwin T sεl dεm we dεn kכlt in vitro εn hכman TIL in vivo ( 22 ). dis obsכbishכn dεm bin kכnfכm bak bay di yus כf כnbias כtomatכk sεl populeshכn alכkeshכn, we fכs rivεl se CD45 + / CD3- / CD4 + / CD45RO + sεl dεm wit hכy glukכs כptek εn maytochכndrial aktiviti pas tכmכro sεl dεm de prεvalεnt bכt gεt Mεtabolik aktv sεl populeshכn. dis populeshכn kin riprizent di putativ sכbpopulεshכn fכ mayloid suprεsכr sεl dεm כ plasmacytoid dεndritik sεl dεm we dεn aydentify insay di scRNA-seq analisis. pan tap dεn ripot dεn tu dεm ya na hכman ovarian tכmכro dεm [45], dεn stil nid כda wok na fכ dεskrεb dis mayloid sכbpopulεshכn.
pan tap we fכlכ saytכmetri-bεys mεtכd dεm kin klarify di jεnarכl difrεns dεm na glukכs εn כksidεtiv mεtabolism bitwin sεl tכp dεm, dεn nכ dכn disayd yet di prεsis mεtabolayt dεm we glukכs כ כda kכbכn sכs dεm de prodyuz fכ maytochכndrial mεtabolism insay TME. fכ asaynd di prεsεns כ absεns fכ mεtabolit dεm to wan givεn TIL sכbsεt nid fכ purify di sεl populeshכn frכm di tisu we dεn εksayz. כl dis mek wi sεl εnrichmεnt mεtכd we dεn kכmbayn wit mas spεktrכmetri kin gi insayt fכ di mεtabolit dεm we difrεnt wan εnrich insay T sεl dεm εn tכmכro sεl populeshכn dεm insay mεch pasεnshכn sεmpl dεm. pan ɔl we dis mεtכd gεt advantej pas fכrosεns-aktivεt sεl sכt, sכm mεtabolit laybri dεm kin afekt biכs fכ inhεrent stεbiliti εn/כ rεpid tכnכv rεt (22). pan tap dat, wi mεtכd bin ebul fכ no tu imyun supεsiv mεtabolayt dεm we dεn no, adenosin εn kynurenine, biכs dεn de difrεnt bכku bitwin di tכp dεm fכ sεmpl.
wi mεtabonomik analisis fכ di tכmכro dεm εn TIL sכbtayp dεm de gi mכr insayt fכ di rol we mεtabolit dεm de ple na ovarian TME. fכs, yuz fכ fכlכ saytכmetri, wi disayd se nכ difrεns de pan maytochכndrial aktiviti bitwin tכmכro dεm εn CD4 + T sεl dεm. כltu, LC-MS/MS analisis rivεl signifyant chenj dεm na di bכku mεtabolit dεm bitwin dεn populeshכn ya, we sho se kכnklushכn bכt TIL mεtabolism εn in כvala mεtabolik aktiviti nid fכ tek tεm intapritεshכn. sεkכn, MNA na di mεtabolayt wit di big difrεns bitwin CD45-sεl dεm εn T sεl dεm na ascites, nכto di tכmכro dεm. כl dis mek, kכmpartmεntεlayzeshn εn di tכmכro lכkεshכn kin gεt difrεn ifekt pan TIL mεtabolism, we de hεlayt di posεbul hεtεrojεniti insay wan givεn maykro envayroment. tכd, di εksprεshכn fכ di MNA-prכdyus εnzym NNMT de mεnli limited to CAF, we na tכmכro sεl dεm to sכm sεl, bכt dεtektabl MNA lεvεl dεm dεn de si insay tכmכro-dεriv T sεl dεm. di ova εksprεshכn fכ NNMT insay ovarian CAF gεt wan kεnsar-prכmot ifekt we dεn no, pat pan am biכs fכ di prכmoshכn fכ CAF mεtabolism, tכmכro invashכn εn mεtastas ( 27 ). pan ɔl we di ɔvala lɛvɛl fɔ TIL na mɔdaret, di ɛksprɛshɔn fɔ NNMT na CAF gɛt fɔ du wit di Kansa Jɛnom Atlas (TCGA) mɛsenchymal sɔbtayp, we gɛt fɔ du wit pwɛl prɔgnosis ( 27 , 46 , 47 ). fכ finish, di εksprεshכn fכ di εnzym AOX1 we rispansabl fכ MNA dεgradashכn de limited bak to di CAF populeshכn, we de sho se T sεl dεm nכ gεt di abiliti fכ mεtabolayz MNA. dis rizulεt dεm de sכpכt di aidia se pan כl we fכs wok nid fכ vεrifik dis fכnd, hεy lεvεl dεm fכ MNA insay T sεl dεm kin sho se imyunosuprεsiv CAF maykro envayroment de.
giv di lכw εksprεshכn lεvεl fכ MNA transpכrta dεm εn di כndatektabl lεvεl dεm fכ di ki protin dεm we involv insay MNA mεtabolism, di prεsεns fכ MNA insay T sεl dεm nכ de εkspεkt. Nid NNMT nכr AOX1 bin kכn dεtekt bay scRNA-seq analisis εn tכgεt qPCR כf tu indipεndεnt kכhכt. dis rizulεt dεm sho se MNA nכ de sכntez bay T sεl dεm, bכt i de abzכp frכm di TME we de rawnd am. in vitro εkspεriεns sho se T sεl dεm kin tεnd fכ akumulet εksojen MNA.
wi in vitro stכdi dεn sho se εksojen MNA de indyuz di εksprεshכn fכ TNFα insay T sεl dεm εn de εnhans di binding fכ Sp1 to di TNFα prכmotεr. pan tap we TNFα gεt כl tu di anti-tכmכr εn anti-tכmכr fכnshכn dεm, insay ovarian kεnsar, TNFα kin mek di ovarian kεnsar gro ( 31-33 ). di nyutralayzeshn fכ TNFα in ovarian tכmכro sεl kכltכr כ di εliminεshכn fכ TNFα signal in maws mכdel kin impruv TNFα-mεdiet inflammatory saytokεn prodakshכn εn inhεbit tכmכro gכd ( 32 , 35 ). כl dis mek, insay dis kes, TME-dεriv MNA kin akt lεk pro-inflammatory mεtabolayt tru wan TNFα-dipεndεnt mεkanism tru di כtokrin lכp, we de protεkt di כkכrεshכn εn spred fכ ovarian kεnsar ( 31 ). bays pan dis posisibul, dεn de stכdi TNFα blכkεd as pכtεnshal tεrapi ejen fכ ovarian kεnsar ( 37 , 48 , 49 ). apat frכm dat, MNA de impεr di saytotoksisiti fכ CAR-T sεl dεm to ovarian tכmכro sεl dεm, we de gi fכda pruf fכ MNA-mεdiet imyun sכpreshכn. kכlektif, dεn risכlt ya de sho wan mכdel we di tכmכro dεm εn CAF sεl dεm de sekret MNA insay εkstrasεlula TME. tru (i) TNF-indyus ovarian kεnsar growth stimulashכn εn (ii) MNA-indyus T sεl saytכtoksik aktvכti inhibishכn, dis kin gεt dual tכmכro ifekt (Figure 5D).
fכ kכnklud, bay we dεn aplay wan kכmbaynshכn fכ rεpid sεl εnrichmεnt, singl-sεl sikεns εn mεtabolik prכfayl, dis stכdi rivεl di big imyun mεtabolכmik difrεns bitwin tכmכro εn asayt sεl dεm insay HGSC pasεnshכn dεm. dis kכmprεhεnsiv analisis sho se difrεns de insay glukכs כptek εn maytochכndrial aktiviti bitwin T sεl dεm, εn i idεntify MNA as nכn-sεl כtכnom imyun rεgεlεtכri mεtabolayt. dis data dεm gεt impak pan aw TME de afekt T sεl mεtabolism insay hכman kεnsar. pan tap we dεn bin ripot di dayrekt kכmpεtishכn fכ nyutriεnt bitwin T sεl dεm εn kεnsar sεl dεm, mεtabolit dεm kin akt bak lεk indaykt rεgεlεt dεm fכ protεkt tכmכro prכgreshכn εn i kin sכpres di εndojεnik imyun rεspכns dεm. di fכda diskripshכn fכ di fכnshכnal rol fכ dεn rεgεdyushכn mεtabolayt dεm kin opin כda stratεji fכ εnhans di anti-tכmכr imyun rεspכns.
Dɛn bin gɛt di pasɛnt spɛshal ɛn klinik data tru di BC kansa tumor tisu ripɔsitɔri we di Kanada Tisu Ripɔsitɔri Nɛtwɔk dɔn sɛtify. Akɔdin to di protɔkɔl we di BC Kansa Risach Ɛtiks Kɔmiti ɛn di Yunivasiti ɔf British Kɔlumbia (H07-00463) dɔn gri fɔ, ɔl di pasɛnt dɛn spɛshal ɛn klinik data we dɛn gɛt we dɛn dɔn no bɔt rayt kɔnsɛnt ɔ fɔmal wan lɛf dɛn kɔnsɛnt. Di samplε dεm de stכr insay di sεtifiket BioBank (BRC-00290). Ditiɛl pasɛnt kwaliti dɛn de sho na Tebul S1 ɛn S5. fכ krayoprezεvεshכn, dεn de yuz skelpεl fכ mεkanikal dεkכmpכz di sikman in tכmכro sεmpl εn afta dat dεn de push am tru 100-maykron filta fכ gεt wan singl sεl sכspεns. di pasεnt in ascites bin sεntrifyuj na 1500 rpm fכ 10 minit na 4°C fכ pεlεt di sεl dεm εn pul di supεrnatant. Sεl dεm we dεn gεt frכm tכmכro εn ascites bin krayoprεsεv insay 50% hεt-inaktivεt hυman AB sεrum (Sigma-Aldrich), 40% RPMI-1640 (Thermo Fisher Scientific) εn 10% dimεtyl sכlfכksayd. Dis prεsεv singl sεl sכspεns dεm bin thaw εn yuz fכ mεtabolכmiks εn mεtabolayt dεtarminεshכn we dεn dεskrεb dכn.
di komplit mεdiכm kכnsis fכ 0.22 μm filta 50:50 supliment RPMI 1640: AimV. RPMI 1640 + 2.05 mM l-glutamine (Thermo Fisher Scientific) supplemented with 10% heat-inactivated human AB serum (Sigma-Aldrich), 12.5 mM Hepes (Thermo Fisher Scientific), 2 mM l-glutamine (Thermo Fisher Scientific) Fisher Scientific), 1 x Pεnisilin Strεptomaysin (PenStrep) sכlushכn (Tεrmo Fisher Scientific) εn 50 μMB-mεkaptoεtanכl. AimV (Invitrogen) de supliment wit 20 mM Hepes (Tεrmo Fisher Scientific) εn 2 mM l-glutamin (Tεrmo Fisher Scientific). di fכlכ saytכmita stεyning bכfa bin kכnsis fכ 0.22μm filta fכsfεt bכfa salin (PBS; Invitrogen) we dεn supliment wit 3% hεt-inaktivεt AB hυman sεrum (Sigma). di sεl εnrichmεnt bכfa na 0.22μm filta PBS εn i supliment wit 0.5% hεt-inaktivεt hυman AB sεrum (Sigma-Aldrich).
In 37°C komplit mεdiכm, sεl dεm bin stεyn wit 10 nM MT DR εn 100 μM 2-NBDG fכ 30 minit. Neks, di sel dεm bin stεyn wit viabiliti day eF506 na 4°C fכ 15 minit. Risuspεnd di sεl dεm insay FC Block (eBioscience) εn Brilliant Stain Buffer (BD Biosciences), dil insay fכl saytכmita stεyning bכfa (akɔdin to di manifakta in instrכkshכn), εn inkכba fכ 10 minit na rum tεmprachכ. stεn di sεl dεm wit wan sεt כf antibodi dεm (Tεbul S2) insay fכlכ saytכmεtri stεyn bכfa na 4°C fכ 20 minit. rεsuspεnd di sεl dεm insay fכlכ saytכmεtri stεyning bכfa (Cytek Aurora; 3L-16V-14B-8R kכnfigureshכn) bifo analisis. Yuz SpectroFlo ɛn FlowJo V10 fɔ analayz di sɛl kɔnt data, ɛn yuz GraphPad Prism 8 fɔ mek di data. Di midyan fluorescence intensiti (MFI) fɔ 2-NBDG ɛn MT DR bin log-nɔmal, ɛn afta dat dɛn yuz wan pe t tɛst fɔ statystik analisis fɔ akɔn fɔ mach pasɛnt dɛn. Rimov ɔl di pipul dɛm we nɔ rich 40 ivin frɔm di analisis; ɛnta wan MFI valyu we na 1 fɔ ɛni nɛgitiv valyu bifo yu du statystik analisis ɛn data vishɔnalizeshɔn.
In orda fכ supliment di manual gating strateji fכ di oba prכsεs panεl, wi yuz di ful anoteshכn bay di shep rεstrikshכn tri (FAUST) (21) fכ כtomatכk asaynd sεl dεm to di populeshכn afta wi εliminet dεd sεl dεm na FlowJo. wi manually manej di autput fכ mεrj populeshכn dεm we lεk se dεn misallocated (kכmbayn PD1+ wit PD1-tכmכr sεl dεm) εn rεtain populeshכn dεm. εvri sεmpl gεt avεj mכr dan 2% sεl dεm, fכ wan totכl 11 populeshכn dεm.
Ficoll gradient density centrifugation bin yuz fɔ separet PBMC frɔm lukosayt separeshɔn prodakt (STEMCELL Technologies). CD8 + T sel dεm bin ayzכlεt frכm PBMC yuz CD8 MicroBeads (Miltenyi) εn εkspεnd insay komplit mεdiכm yuz TransAct (Miltenyi) fכ 2 wiks akɔdin to di manifakta in instrכkshכn. Di sel dεm bin alaw fכ tinap fכ 5 dez insay komplit mεdiכm we kכntεn IL-7 (10 ng/ml; PeproTech), εn afta dat dεn ri-stimulεt wit TransAct. pan de 7, akɔdin to di manifakta in instrɔkshɔn, dɛn yuz mɔtalman CD45 MicroBeads (Miltenyi) fɔ ɛnrich sɛl dɛn insay tri kɔnsɛkutiv rawnd. Di sel dεm bin alikכt fכ fכ fכlכ saytכmetri analisis (as dεn dεskrεb am oba), εn wan miliכn sεl dεm bin alikכt tri tεm fכ LC-MS/MS analisis. Di samplε dεm bin prכsεs bay LC-MS/MS lεk aw dεn dεskrεb dεn dכn. Wi εstimat di mis mεtabolayt valyu wit wan ayכn nכmba we na 1,000. εvri sεmpl de nכmal bay di tכtal ayכn nכmba (TIC), lכgaritmik kכnvכlt εn כtomatik nכmal insay MetaboAnalystR bifo analisis.
di singl sel suspεns fכ εvri pasεnt bin thaw εn filt tru 40 μm filta insay komplit mεdiכm (as dεn dεskrεb am oba). Akɔdin to di manifakta in protɔkɔl, dɛn yuz tri kɔnsɛkutiv rawnd fɔ pɔsitiv sɛlɛkshɔn bay magnɛtik bidz separeshɔn we yuz MicroBeads (Miltenyi) fɔ ɛnrich di sampul dɛn fɔ CD8+, CD4+ ɛn CD45- sɛl dɛn (pan ays). in sכt, dεn de rεsuspεnd di sεl dεm insay sεl εnrichmεnt bכfa (as dεn dεskrεb am oba) εn kכnt dεm. di sεl dεm bin inkכba wit hυman CD8 bidz, hυman CD4 bidz כ hυman CD45 bidz (Miltenyi) na 4°C fכ 15 minit, εn afta dat dεn was dεm wit sεl εnrichmεnt bכfa. Dɛn pas di sampul tru di LS kɔlɔm (Miltenyi), ɛn dɛn kin gɛda di pɔsitiv ɛn nɛgitiv frakshɔn dɛn. fכ ridyus di tεm we di sεl de tek εn maksimayz di sεl rεkכvri stεp, dεn de yuz di CD8-frakshכn fכ di sεkכn raun fכ CD4+ εnrichmεnt, εn dεn de yuz di CD4-frakshכn fכ di CD45-εnrichmεnt we de afta dat. Kip di sɔlvushɔn pan ays ɔlsay na di separeshɔn prɔses.
Fכ pripia sεmpl fכ mεtabolayt analisis, dεn was di sεl dεm wan tεm wit ays-kol sכlt sכlushכn, εn 1 ml 80% mεtanכl bin ad to εvri sεmpl, dεn vorteks εn snap fכz insay likwid naytrojen. Di samplε dεm bin sכbjεkt to tri friz-thaw saykl εn sεntrifyuj na 14,000 rpm fכ 15 minit na 4°C. di supεrnatant we gεt di mεtabolit dεm de εvapכret te i dray. Di mεtabolayt dεm bin ri-disכlv insay 50 μl 0.03% fכmik asid, vכrtεks fכ miks, εn afta dat dεn sεntrifug fכ pul di dεbris.
Ekstrakt mεtabolayt dεm lεk aw dεn dεskrεb am oba. Transfεr di supεrnatant to wan ay pεrformεns likwid kromatografi bכtul fכ mεtabolכmiks risεch. Yuz wan random tritmεnt protכkכl fכ trit εvri sεmpl wit di sem nכmba כf sεl dεm fכ prεvεnt batch ifekt. Wi bin du wan kwalitatif asɛsmɛnt fɔ di glob ɔl mɛtabolayt dɛn we dɛn bin dɔn pablish bifo tɛm pan di AB SCIEX QTRAP 5500 Tripul Kwadrupɔl Mas Spɛktromita ( 50 ). Kromatografik analisis ɛn pik eria intagreshɔn bin de yuz MultiQuant vɛshɔn 2.1 softwe (Applied Biosystems SCIEX).
Wan ayɔn kɔnt we na 1000 bin yuz fɔ ɛstimat di mis mɛtabolayt valyu, ɛn di TIC fɔ ɛni sampul bin yuz fɔ kɔl di nɔmalayz pik eria fɔ ɛni mɛtabolayt we dɛn dɔn detekt fɔ kɔrɛkt fɔ chenj dɛn we di instrɔmɛnt analisis introduks frɔm sampul prɔsesin. Afta TIC dɔn nɔmal, dɛn yuz MetaboAnalystR(51) (difɔlt paramita) fɔ logaritmik kɔnvɔshɔn ɛn ɔtomatik norm layn skel. Wi yuz PCA wit vegan R pakej fɔ du ɛksplɔrɔshɔn analisis fɔ mɛtabolɔm difrɛns bitwin sampul tayp dɛn, ɛn yuz patɛl ridandans analisis fɔ analayz pasɛnt dɛn. Yuz Wod mεtכd fכ kכnstrכkt wan hεt map dεndrכgram fכ kכlכs di Yuklidian distans bitwin sεmpl dεm. We yuz limma (52) pan standad mεtabolayt abundans fכ aydentify difrεntli abundant mεtabolayt akrays di כl sεl tכp εn maykro envayroment. fכ mek di εksplen simpul, wi de yuz di grup min paramita fכ spεsifa di mכdel, εn kכnsidr di sεl tכp dεm na di maykro envayroment as εvri grup (n = 6 grup dεm); fכ di signifyans tεst, wi du tri ripit mεzjכmεnt fכ εvri mεtabolayt Fכ avכyd fכs rεplikshכn, dεn bin inklud di pasεnt as obstakl insay di limma disayn. fכ chεk di difrεns insay mεtabolayt bitwin difrεn pasεnshכn dεm, wi adjכst di limma mכdel inklud pasεnshכn dεm insay wan fiks we. Wi ripɔt di siginifikɛns fɔ di prɛ-spɛsifikɛd kɔntrast bitwin di sɛl tayp ɛn di maykroɛnvayrɔmɛnt fɔ Padj <0.05 (Benjamini-Hochberg kɔrɛkshɔn).
Afta vigor enrichment yuz Miltenyi Dead Cell Removal Kit (>80% viability), singl-sεl transkriptכm sikεnsεshכn bin pεrform pan di tכtal layv frכzεn ascites εn tכmכr sεmpl yuz wan 10x 5′jin εksprεshכn protכkכl. Fayv kes dɛm wit maching tumor ɛn ascites bin analayz, pan ɔl we di lɔw viabiliti frɔm wan tumor sampul bin mek dɛn nɔ put am insay. Fכ mek wi ebul fכ achy mכltipכl sεlεkshכn fכ di pasεnshכn dεm, wi kכmbayn di sεmpl dεm fכ εvri pasεnt na di len dεm fכ di 10x kromiכm kכntrola, εn analכz di ascites εn tכmכro sayt dεm sεparat wan. Afta sikwins [Illumina HiSeq 4000 28×98 bp pe εnd (PE), Kwibɛk jεnom; wan avrej 73,488 ɛn 41,378 rid pan wan sɛl fɔ tumor ɛn ascites rispɛktvɔli]], wi yuz CellSNP ɛn Vireo (53) (bays pan CellSNP as Di kɔmɔn mɔtalman SNP (VCF) we GRCh38 gi, dɛn gi am wan dona aydentiti.Wi yuz SNPRelate fɔ infɛr di klos aydentiti (IBS) fɔ di pɔsin in jɛnɛtayp stetɔs (IBS), excluding unassigned sel dεm εn sεl dεm we dεn aydentify as duplexes εn matching donors bitwin ascites εn tumor sεmpl dεm (54) Pan di bεsis fכ dis task, wi rεtεn tri kes dεm wit abundant sεl rεprεsεntεshכn in di tכmכro εn ascites fכ dכwnstrכm analisis Afta dεn du wan mas filtrεshכn stεp in di skata (55) εn scran (56) BioConductor packaging, dis giv 697 sεl dεm (2792 and 4183 cells from tumor and ascites, respectively) for analysis. We use igraph's (57) Louvain clustering of shared nearest neighbor network (SNN) based on Jaccard distance to cluster cells by expression. The clusters were manually annotated to putative cell types based on marker gene expression and visualized with t-SNE. Cytotoxic T cells are difayn bay di εksprεshכn fכ CD8A εn GZMA, we nכ de kכl sabklכsta wit lכw ribosכmal protin εksprεshכn Wi akses di pכblished data fכ Izar et al (16), inklud dεn t-SNE εmbedin, kin kכntrכl di εksprεshכn כvlεp bitwin imyun sεl makεr εn NNMT εksprεshכn.
PBMC bin separet frɔm lukosayt separeshɔn prodakt (STEMCELL Technologies) bay Ficoll gradient density sɛntrifugayshɔn. CD3 + sel dεm bin ayzכlεt frכm PBMC yuz CD3 bidz (Miltenyi). In di prεsεns כ absεns כf MNA, CD3+ sεl dεm bin aktibכt wit plet-bכnd CD3 (5μg/ml), sכlubul CD28 (3μg/ml) εn IL-2 (300 U/ml; Proleukin). Na di las de we dɛn ɛkspɛnshɔn, dɛn bin evalyu di viabiliti (Fixable Viability Dye eFluor450, eBioscience) ɛn proliferashɔn (123count eBeads, Thermo Fisher Scientific) bay flɔ saytometri. Evaluate ifektכr fכnshכn bay we yu stimulate sεl dεm wit PMA (20 ng/ml) εn ionomycin (1μg/ml) wit GolgiStop fכ 4 awa, εn mכnitor CD8-PerCP (RPA-T8, BioLegend), CD4-AF700 (RPA-T4) , BioLegend) εn TNFα-fluorescein isothiocyanate (FITC) (MAb11, BD). Stimulεt qPCR εn ChIP sεl dεm wit PMA (20 ng/ml) εn ionomycin (1μg/ml) fכ 4 awa. Di ELISA supεrnatant bin kכlekt bifo εn afta stimulashכn wit PMA (20 ng/ml) εn ionomycin (1 μg/ml) fכ 4 awa.
Fɔ fala di manifakta in protɔkɔl fɔ aylɔlɔ RNA yuz RNeasy Plus Mini Kit (QIAGEN). Yuz QIAshredder (QIAGEN) fɔ homogenize di sampul. Yuz ay-kapasiti RNA to cDNA kit (Thermo Fisher Scientific) fɔ sεntez kɔmplimɛnt DNA (cDNA). Yuz TaqMan Rapid Advans Masta Miks (Thermo Fisher Scientific) fɔ kwantayz jin ɛksprɛshɔn (akɔdin to di manifakta in protɔkɔl) wit dɛn prob ya: Hs00196287_m1 (NNMT), Hs00154079_m1 (AOX1), Hs00427552_m1 (SLC22A1), Hs02786624_g1 [glisεraldehayd-3-fכsfεt כf Haydrojεn (GAPDH)] εn Hs01010726_m1 (SLC22A2). Di samplε dεm bin rכn pan di StepOnePlus rial-taym PCR sistεm (Applied Biosystems) (Applied Biosystems) insay di MicroAmp fast optik 96-wεl riakshɔn plet (Applied Biosystems) wit MicroAmp optik film. eni Ct valyu we pas 35 dεn kin tek am se i de oba di dεtekshכn thrεshold εn dεn mak am se i nכ detekt.
Pεrform ChIP lεk aw dεn bin dεskrεb am bifo (58). In shot, di sel dεm bin trit wit fכmaldehayd (faynal kכnsantreshכn 1.42%) εn inkכba na rum tεmprachכ fכ 10 minit. Yuz supliment swεlin bכfa (25 mM Hepes, 1.5 mM MgCl2, 10 mM KCl εn 0.1% NP-40) pan ays fכ 10 minit, dεn rεsuspεnd insay imyunoprεsipitεshכn bכfa lεk aw dεn dεskrεb am ( 58 ). Di sampul bin den sonicated wit di fכlכw saykl dεm: 10 saykl (20 1-sεkכnd puls) εn wan statik tεm 40 sεkכnd. Inkubayt ChIP-grεd imyunoglobulin G (Sεl Sayn Tεknכlכji; 1μl), histכn H3 (Sεl Sayn Tεknכlכji; 3μl), NFAT (Invitrogen; 3μl) εn SP1 (Sεl Sayn Tεknכlכji; 3μl) antibodi dεm wit di sεmpl na 4°CC shek כvnayt. Inkubayt protin A bidz (Thermo Fisher Scientific) wit di sampul na 4°C wit shek jisnɔ fɔ 1 awa, dɔn yuz chɛlɛks bidz (Bio-Rad) fɔ ɛnrich di DNA, ɛn yuz proteinase K (Thermo Fisher) fɔ protin digɛst. TNFα prכmotεr bin dεtekt bay PCR: fכwכd, GGG TAT CCT TGA TGC TTG TGT; di kכntribyushכn, GTG CCA ACA ACT GCC TTT ATA TG (207-bp prodakt). Di imej dɛn na Imej Lab (Bio-Rad) bin mek dɛn ɛn dɛn bin kwantifay dɛn yuz ImageJ softwe.
Di sel kכlt supεrnatant bin kכlekt lεk aw dεn dεskrεb am oba. Di ditarminashɔn bin kɔmɔt akɔdin to di manifakta in prosidyuz fɔ mɔtalman TNFα ELISA kit (Invitrogen), mɔtalman IL-2 ELISA kit (Invitrogen) ɛn mɔtalman IFN-γ ELISA kit (Abcam). akɔdin to di manifakta in protɔkɔl, dɛn bin dil di supamatant 1:100 fɔ no TNFα ɛn IL-2, ɛn 1:3 fɔ no IFN-γ. Yuz EnVision 2104 Multilabel Reader (PerkinElmer) fɔ mɛzhɔ absɔpshɔn na 450 nm.
PBMC bin separet frɔm lukosayt separeshɔn prodakt (STEMCELL Technologies) bay Ficoll gradient density sɛntrifugayshɔn. CD3 + sel dεm bin ayzכlεt frכm PBMC yuz CD3 bidz (Miltenyi). In di prεsεns כ absεns כf MNA, CD3+ sεl dεm bin aktyuv wit plet-bכnd CD3 (5μg/ml), sכlubul CD28 (3μg/ml) εn IL-2 (300 U/ml; Proleukin) fכ 3 dez. Afta 3 dez, dεn kכlekt di sεl dεm εn was wit 0.9% salin, εn di pεlεt bin snap fכz. Sεl kכnt bin pεrform bay fכ fכlכ saytכmetri (Cytek Aurora; 3L-16V-14B-8R kכnfigureshכn) yuz 123count eBeads.
Ekstrakt mεtabolayt dεm lεk aw dεn dεskrεb am oba. Di dray ekstrakt bin rikonstituyt pan wan kɔnsɛntreshɔn we na 4000 sɛl ikwal/μl. Analayz di sampul bay rivεs-fεz kromatografi (1290 Infinity II, Agilent Technologies, Santa Klara, CA) εn CORTECS T3 kכlכm (2.1×150 mm, patikyula saiz 1.6-μm, pore saiz 120-Å; #186008500, Waters). Polar mas spɛktromita (6470, Agilent), we ilɛktrɔsprɛy ayɔnizashɔn de wok insay pɔzitiv mɔd. Mobayl fεz A na 0.1% fכmik asid (insay H2O), mobayl fεz B na 90% asetonitril, 0.1% fכmik asid. di LC gradient na 0 to 2 minit fכ 100% A, 2 to 7.1 minit fכ 99% B, εn 7.1 to 8 minit fכ 99% B. Dɔn ri-equilibrate di kɔlɔm wit mobayl fεz A pan flɔ rεt we na 0.6 ml/min fכ 3 minit. . di fכlכ rεt na 0.4ml/min, εn di kכlכm chεmba de hεt to 50°C. Yuz MNA in pure kemikal standad (M320995, Toronto Research Chemical Company, North York, Ontario, Canada) fɔ establish ritɛnshɔn tɛm (RT) ɛn transfɔmeshɔn (RT = 0.882 minit, transfɔmeshɔn 1 = 137→94.1, transfɔmeshɔn 2 = 137→92 , Kɔnvɔshɔn 3 = 137→78). We ɔl di tri transishɔn dɛn apin na di kɔrɛkt ritɛnshɔn tɛm, dɛn de yuz transishɔn 1 fɔ kwantifayeshɔn fɔ mek shɔ se spɛsifisiti. Di standad kεrv כf MNA (Toronto Risach Kεmikכl Kכmni) bin jεnarεt bay siks siriכs dilushכn כf di stכk sכlushכn (1 mg/ml) fכ gεt standad כf 0.1, 1.0, 10 εn 100 ng/ml εn 1.0 εn 10μg/ml rispεktivli likwid. di dεtekshכn limit na 1 ng/ml, εn di lεnya rεspכns de bitwin 10 ng/ml εn 10μg/ml. Ɛni injɛkshɔn fɔ tu maykrolit sampul ɛn standad dɛn de yuz fɔ LC/MS analisis, ɛn dɛn de rɔn wan miks kwaliti kɔntrol sampul ɛvri et injɛkshɔn fɔ mek shɔ se di analisis pletfɔm stebul. di MNA rispכns dεm fכ כl di MNA-trit sεl sεmpl dεm bin de insay di linya rεnj fכ di asεy. Data analisis bin du yuz MassHunter kwantiti analisis softwe (v9.0, Agilent).
Di sεkכn jεnereshכn αFR-CAR kכnstrכkt bin tek frכm Song et al. (59) we dɛn kɔl. in sכt, di kכnstrכkt gεt di fכlכw kכntεnt dεm: CD8a lida sikεns, hכman αFR-spεsifi k singl-chεn vεryuεbl fragmεnt, CD8a hinj εn transmεmbran rijyכn, CD27 intasεlulyar domεn εn CD3z intasεlulyar domεn. di komplit CAR sikεns bin sεntez bay GenScript, εn afta dat dεn klon am insay di sεkכn-jεnereshכn lentiviral εksprεshכn vεktכr apstrim כf di GFP εksprεshכn kεsεt we dεn yuz fכ evaluate di transdyushכn efyushכn.
lεntivayrus de prodyuz bay transfεkshכn fכ HEK293T sεl dεm [American Type Culture Collection (ATCC); we dεn gro insay Dulbecco in modifyed Eagle mεdiכm we gεt 10% fetal bovin sεrum (FBS) εn 1% PenStrep, εn yuz CAR-GFP vεktכr εn Di pak plasmid dεm (psPAX2 εn pMD2.G, Addgene) de yuz lipofεkshכn amin (Sigma-Aldrich). Di vayrɔs-kɔntinɛnt supamantant bin kɔlɛkt 48 ɛn 72 awa afta transfɛkshɔn, filta, ɛn kɔnsɛntret bay ultracentrifugation. stכr di kכnsantreyt vayral supεrnatant na -80°C te transdyushכn.
PBMC dεn separet frכm hεlty dכna lukosayt separeshכn prodakt dεm (STEMCELL Technologies) bay Ficoll gradient density centrifugation. yuz positifu selekshכn CD8 maykrobid (Miltenyi) fכ aylכt CD8+ sεl dεm frכm PBMC. Stimulεt T sεl dεm wit TransAct (Miltenyi) εn insay TexMACS mεdiכm [Miltenyi; supliment wit 3% hεt-inaktivεt hυman sεrum, 1% PenStrep εn IL-2 (300 U/ml)]. Twεnti fכ awa afta stimulashכn, T sεl dεm bin transdyuz wit lεntivayrus (10 μl kכnsantreyt vayrus supεrnatant pan 106 sεl dεm). 1 to 3 de afta transdyushכn pan Cytek Aurora (pan FSC (Forward Scatter)/SSC (Side Scatter), Singlet, GFP+), evaluate di GFP εksprεshכn fכ di sεl dεm fכ sho wan transdyushכn efyushכn we at le 30% .
CAR-T sel dεm bin kכlt fכ 24 awa insay Immunocult (STEMCELL Technologies; supliment wit 1% PenStrep) כnda di fכlכw kכndishכn dεm: dεn nכ trit, trit wit 250 μM adenosine כ 10 mM MNA. Afta prεtritmεnt, dεn was CAR-T sεl dεm wit PBS εn kכmbayn wit 20,000 SK-OV-3 sεl dεm [ATCC; in McCoy 5A mεdiכm (Sigma-Aldrich) supliment wit 10% FBS εn 1% PenStrep na 10: Di εfεktכ to tכgεt rεshכ fכ 1 bin amplify in triplikεt in supliment Immunocult mεdiכm. SK-OV-3 sel dεm εn SK-OV-3 sεl dεm we layz wit digitalis saponin (0.5mg/ml; Sigma-Aldrich) bin yuz as nεgεtiv εn positifu kכntrכl, rispεktivli. Afta 24 awa we dɛn kɔ-kultiv, dɛn gɛda di supamatant ɛn dɛn mɛzhɔ di laktat dihaydrojɛnɛz (LDH) akɔdin to di manifakta in instrɔkshɔn (LDH Glo Cytotoxicity Assay Kit, Promega). Di LDH supamantant bin diluted 1:50 in LDH bafa. Dɛn bin mɛzhɔ di pasɛnt we dɛn kil bay we dɛn yuz dis fɔmula: pasɛnt we dɛn kil = pasɛnt we dɛn kɔrɛkshɔn / maksimal kil rit x 100%, usay pasɛnt we dɛn kɔrɛkshɔn = kɔ-kɔlchɔ-T sɛl dɛn nɔmɔ, ɛn maksimal rit we dɛn kil = pɔsitiv kɔntrol-nɛgitiv kɔntrol.
As dɛn tɔk bɔt am na di tɛks ɔ di tin dɛn ɛn di we aw dɛn de du am, yuz GraphPad Prism 8, Microsoft Excel ɔ R v3.6.0 fɔ statystik analisis. If dεn kכlekt mכltipכl sεmpl frכm di sem pasεnt (lεk ascites εn tumor), wi de yuz paired t tεst כ inklud di pasεnt as random ifekt in wan linya כ jεnaralised mכdel as i aprכpriet. fכ mεtabolכmiks analisis, dεn de du di imכpכtants tεst insay triplikεt.
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Marisa K. Kilgour (Marisa K. Kilgour), Sera Makfasɔn (Sara Makfasɔn), Lauren G. Zacharias (Lauren G. Zacharias), Abigɛl Ɛli Aris G. Watson (H. Watson), Jɔn Stag (Jɔn Stag), Brad H. Nɛlson (Brad H. Nɛlson), Rɔlf J. Di Bɛlardini (Ralf J. Diberadinis), Rɔsɛl G. Jɔns (Rɔsɛl G. Jɔns), Fɛnas T. Hamilton (Finɛs T.
MNA de kכntribyut to di imyun sכpreshכn fכ di T sεl dεm εn i de riprizent wan pכtεnshal imyun tεrapi tכgεt fכ di tritmεnt fכ hכman kεnsar.
Marisa K. Kilgour (Marisa K. Kilgour), Sera Makfasɔn (Sara Makfasɔn), Lauren G. Zacharias (Lauren G. Zacharias), Abigɛl Ɛli Aris G. Watson (H. Watson), Jɔn Stag (Jɔn Stag), Brad H. Nɛlson (Brad H. Nɛlson), Rɔlf J. Di Bɛlardini (Ralf J. Diberadinis), Rɔsɛl G. Jɔns (Rɔsɛl G. Jɔns), Fɛnas T. Hamilton (Finɛs T.
MNA de kכntribyut to di imyun sכpreshכn fכ di T sεl dεm εn i de riprizent wan pכtεnshal imyun tεrapi tכgεt fכ di tritmεnt fכ hכman kεnsar.
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