Cov tshuaj metabolites uas ua rau lub cev tiv thaiv kab mob (immunomodulatory metabolites) yog ib qho tseem ceeb ntawm qhov chaw ib puag ncig ntawm lub qog (TME), tab sis muaj ob peb qhov tshwj xeeb, lawv cov cim tseem tsis tau paub meej. Hauv qhov no, peb tau tshuaj xyuas cov qog thiab cov qe ntshav dawb (T cell) los ntawm cov qog thiab cov qog hauv cov neeg mob uas muaj cov qog serous carcinoma (HGSC) kom pom cov metabolilome ntawm cov TME compartments sib txawv no. Cov qog hauv cov qog thiab cov qe ntshav dawb muaj ntau qhov sib txawv ntawm cov metabolite. Piv nrog cov qog hauv cov qog, cov qe ntshav dawb uas nkag mus rau hauv cov qog muaj ntau yam hauv 1-methylnicotinamide (MNA). Txawm hais tias qib ntawm MNA hauv cov qe ntshav dawb siab dua, qhov kev qhia tawm ntawm nicotinamide N-methyltransferase (ib qho enzyme uas ua rau muaj kev hloov pauv ntawm cov pab pawg methyl los ntawm S-adenosylmethionine mus rau nicotinamide) tsuas yog txwv rau cov fibroblasts thiab cov qe ntshav dawb xwb. Ua haujlwm tau zoo, MNA ua rau cov qe ntshav dawb (T cell) tso tawm cov cytokine tumor necrosis factor alpha uas ua rau cov qog ua haujlwm. Yog li ntawd, MNA uas tau los ntawm TME pab txhawb kev tswj hwm kev tiv thaiv kab mob ntawm cov qe ntshav dawb (T cell) thiab sawv cev rau lub hom phiaj kho mob rau kev kho mob qog hauv tib neeg.
Cov metabolites uas los ntawm cov qog nqaij hlav tuaj yeem muaj cov nyhuv tiv thaiv kev tiv thaiv kab mob qog nqaij hlav, thiab ntau thiab ntau qhov pov thawj qhia tau tias lawv kuj tuaj yeem ua lub zog tseem ceeb rau kev nce qib ntawm tus kab mob (1). Ntxiv rau qhov cuam tshuam Warburg, cov haujlwm tsis ntev los no tau pib piav qhia txog lub xeev metabolic ntawm cov qog nqaij hlav thiab nws txoj kev sib raug zoo nrog lub xeev tiv thaiv kab mob ntawm qhov chaw ib puag ncig ntawm cov qog nqaij hlav (TME). Kev tshawb fawb ntawm cov qauv nas thiab cov hlwb T tib neeg tau qhia tias glutamine metabolism (2), oxidative metabolism (3) thiab glucose metabolism (4) tuaj yeem ua haujlwm ywj pheej ntawm ntau pawg kab mob tiv thaiv kab mob. Ntau cov metabolites hauv cov kev no inhibit qhov kev ua haujlwm tiv thaiv kab mob qog nqaij hlav ntawm cov hlwb T. Nws tau raug pov thawj tias qhov kev thaiv ntawm coenzyme tetrahydrobiopterin (BH4) tuaj yeem ua rau puas tsuaj rau kev loj hlob ntawm cov hlwb T, thiab qhov nce ntawm BH4 hauv lub cev tuaj yeem txhim kho qhov kev tiv thaiv kab mob qog nqaij hlav uas tau txais los ntawm CD4 thiab CD8. Tsis tas li ntawd, cov nyhuv tiv thaiv kab mob ntawm kynurenine tuaj yeem cawm tau los ntawm kev tswj hwm ntawm BH4 (5). Hauv isocitrate dehydrogenase (IDH) mutant glioblastoma, qhov kev tso tawm ntawm enantiometabolic (R)-2-hydroxyglutarate (R-2-HG) inhibits T cell activation, proliferation thiab cytolysis Activity (6). Tsis ntev los no, nws tau pom tias methylglyoxal, ib qho by-product ntawm glycolysis, yog tsim los ntawm cov hlwb suppressor ntawm myeloid keeb kwm, thiab T cell hloov ntawm methylglyoxal tuaj yeem inhibit effector T cell function. Hauv kev kho mob, qhov neutralization ntawm methylglyoxal tuaj yeem kov yeej qhov kev ua haujlwm ntawm myeloid-derived suppressor cells (MDSC) thiab synergistically txhim kho checkpoint blockade therapy hauv nas qauv (7). Cov kev tshawb fawb no ua ke hais txog lub luag haujlwm tseem ceeb ntawm TME-derived metabolites hauv kev tswj hwm T cell function thiab kev ua haujlwm.
Kev ua haujlwm tsis zoo ntawm T cell tau raug tshaj tawm dav dav hauv mob qog nqaij hlav zes qe menyuam (8). Qhov no yog ib feem vim yog cov yam ntxwv ntawm kev ua haujlwm hauv lub cev uas muaj nyob hauv hypoxia thiab cov hlab ntsha tsis zoo (9), uas ua rau muaj kev hloov pauv ntawm glucose thiab tryptophan mus rau hauv cov khoom lag luam xws li lactic acid thiab kynurenine. Kev noj ntau dhau ntawm cov tshuaj lactate sab nraud txo qhov kev tsim cov interferon-γ (IFN-γ) thiab tsav qhov sib txawv ntawm cov pab pawg myelosuppressive (10, 11). Kev noj tryptophan ncaj qha txwv tsis pub T cell proliferation thiab txwv tsis pub T cell receptor signaling (12-14). Txawm hais tias muaj cov kev soj ntsuam no, ntau txoj haujlwm nyob ib puag ncig kev ua haujlwm ntawm lub cev tiv thaiv kab mob tau ua tiav hauv kev cog qoob loo T cell hauv vitro siv cov xov xwm zoo tshaj plaws, lossis txwv rau cov qauv nas homologous hauv vivo, tsis yog qhov twg qhia txog qhov sib txawv ntawm cov qog nqaij hlav tib neeg thiab Physiological macro thiab micro ib puag ncig.
Ib qho tshwj xeeb ntawm mob qog noj ntshav zes qe menyuam yog qhov kis mus rau hauv plab thiab qhov tshwm sim ntawm ascites. Kev sib sau ua ke ntawm cov kua dej hauv ascites yog txuam nrog kev mob hnyav thiab kev kwv yees tsis zoo (15). Raws li cov ntawv ceeb toom, qhov chaw tshwj xeeb no yog hypoxic, muaj cov qib siab ntawm cov vascular endothelial growth factor (VEGF) thiab indoleamine 2,3-dioxygenase (IDO), thiab raug infiltrated los ntawm T regulatory cells thiab myeloid inhibitory cells (15-18). Ib puag ncig metabolic ntawm ascites yuav txawv ntawm qhov ntawm qog nws tus kheej, yog li qhov kev rov ua dua ntawm T cell hauv qhov chaw peritoneal tsis meej. Tsis tas li ntawd, qhov sib txawv tseem ceeb thiab heterogeneity ntawm ascites thiab metabolites uas muaj nyob hauv ib puag ncig qog yuav cuam tshuam kev nkag mus ntawm cov hlwb tiv thaiv kab mob thiab lawv txoj haujlwm ntawm qog, thiab kev tshawb fawb ntxiv yog qhov xav tau.
Yuav kom daws tau cov teeb meem no, peb tau tsim ib txoj kev sib cais ntawm cov hlwb rhiab heev thiab kua chromatography tandem mass spectrometry (LC-MS/MS) los kawm txog ntau hom hlwb sib txawv (xws li CD4 + thiab CD8 + T hlwb) nrog rau sab hauv thiab ntawm cov qog nqaij hlav. Nws cov metabolites kis mus rau cov hlwb hauv tib qho ascites thiab qog nqaij hlav ib puag ncig ntawm tus neeg mob. Peb siv txoj kev no ua ke nrog high-dimensional flow cytometry thiab single-cell RNA sequencing (scRNA-seq) los muab ib daim duab zoo ntawm cov xwm txheej metabolic ntawm cov pej xeem tseem ceeb no. Txoj kev no tau qhia txog kev nce ntxiv ntawm 1-methylnicotinamide (MNA) hauv cov qog nqaij hlav T hlwb, thiab kev sim hauv vitro tau qhia tias cov nyhuv immunomodulatory ntawm MNA ntawm T cell ua haujlwm tsis tau paub ua ntej. Feem ntau, txoj kev no qhia txog kev sib cuam tshuam metabolic ntawm cov qog nqaij hlav thiab cov hlwb tiv thaiv kab mob, thiab muab kev nkag siab tshwj xeeb rau cov metabolites tswj kev tiv thaiv kab mob, uas yuav pab tau rau kev kho mob ntawm T cell-based ovarian cancer immunotherapy.
Peb siv cov txheej txheem ntws ntawm cov kab mob siab (high-dimensional flow cytometry) los ntsuas qhov kev nqus ntawm cov piam thaj (2-(N-(7-nitrophenyl-2-oxa-1,3-diaza-4-yl)amino)-2-deoxyglucose (2-NBDG) thiab kev ua haujlwm ntawm mitochondrial [MitoTracker Deep Red (MT DR)] (7, 19, 20) yog cov cim qhia ib txwm muaj uas sib txawv ntawm cov hlwb tiv thaiv kab mob thiab cov hlwb qog (Rooj S2 thiab Daim Duab S1A). Qhov kev tshuaj xyuas no qhia tau hais tias piv rau cov hlwb T, cov hlwb ascites thiab cov hlwb qog muaj cov qib nqus ntawm cov piam thaj ntau dua, tab sis muaj qhov sib txawv me dua hauv kev ua haujlwm ntawm mitochondrial. Qhov nruab nrab ntawm kev nqus ntawm cov piam thaj ntawm cov hlwb qog [CD45-EpCAM (EpCAM)+] yog peb txog plaub npaug ntawm cov hlwb T, thiab qhov nruab nrab ntawm kev nqus ntawm cov piam thaj ntawm CD4 + T yog 1.2 npaug ntawm cov hlwb CD8 + T, uas qhia tias cov qog infiltrating lymphocytes (TIL) muaj cov kev xav tau sib txawv txawm tias nyob hauv tib lub TME (Daim Duab 1A). Qhov sib piv, qhov kev ua haujlwm mitochondrial hauv cov hlwb qog nqaij hlav zoo ib yam li cov hlwb CD4 + T, thiab qhov kev ua haujlwm mitochondrial ntawm ob hom hlwb siab dua li ntawm CD8 + T (Daim Duab 1B). Feem ntau, cov txiaj ntsig no qhia txog qib metabolic. Qhov kev ua haujlwm metabolic ntawm cov hlwb qog nqaij hlav siab dua li ntawm CD4 + T, thiab qhov kev ua haujlwm metabolic ntawm CD4 + T hlwb siab dua li ntawm CD8 + T hlwb. Txawm hais tias muaj cov teebmeem no thoob plaws hom hlwb, tsis muaj qhov sib txawv ntawm cov xwm txheej metabolic ntawm CD4 + thiab CD8 + T hlwb lossis lawv cov feem sib piv hauv ascites piv rau cov qog nqaij hlav (Daim Duab 1C). Qhov sib piv, hauv CD45-cell feem, qhov feem ntawm EpCAM + hlwb hauv qog nqaij hlav nce ntxiv piv rau ascites (Daim Duab 1D). Peb kuj tau pom qhov sib txawv ntawm cov metabolism ntawm EpCAM + thiab EpCAM- hlwb. EpCAM + (qog nqaij hlav) hlwb muaj kev nqus glucose ntau dua thiab kev ua haujlwm mitochondrial ntau dua li EpCAM- hlwb, uas siab dua li qhov kev ua haujlwm metabolic ntawm fibroblasts hauv cov hlwb qog nqaij hlav hauv TME (Daim Duab 1, E thiab F).
(A thiab B) Qhov nruab nrab ntawm qhov ci ntsa iab (MFI) ntawm kev nqus cov piam thaj (2-NBDG) (A) thiab kev ua haujlwm mitochondrial ntawm CD4 + T hlwb (MitoTracker tsaus liab) (B) Cov duab sawv cev (sab laug) thiab cov ntaub ntawv tabulated (Sab xis), CD8 + T hlwb thiab EpCAM + CD45-qog hlwb los ntawm ascites thiab qog. (C) Qhov piv ntawm CD4 + thiab CD8 + hlwb (ntawm CD3 + T hlwb) hauv ascites thiab qog. (D) Feem pua ​​ntawm EpCAM + qog hlwb hauv ascites thiab qog (CD45−). (E thiab F) EpCAM + CD45-qog hlwb thiab EpCAM-CD45-matrix glucose uptake (2-NBDG) (E) thiab kev ua haujlwm mitochondrial (MitoTracker tsaus liab) (F) cov duab sawv cev (sab laug) thiab cov ntaub ntawv tabulated (Sab xis) Ascites thiab qog hlwb. (G) Cov duab sawv cev ntawm CD25, CD137 thiab PD1 kev qhia los ntawm kev ntws cytometry. (H thiab kuv) CD25, CD137 thiab PD1 kev qhia tawm ntawm CD4 + T hlwb (H) thiab CD8 + T hlwb (I). (J thiab K) Naive, lub cim xeeb hauv nruab nrab (Tcm), effector (Teff) thiab effector nco (Tem) phenotypes raws li kev qhia tawm ntawm CCR7 thiab CD45RO. Cov duab sawv cev (sab laug) thiab cov ntaub ntawv tabular (sab xis) ntawm CD4 + T hlwb (J) thiab CD8 + T hlwb (K) hauv ascites thiab qog. P tus nqi txiav txim siab los ntawm kev sib koom ua ke t-xeem (*P <0.05, **P <0.01 thiab ***P <0.001). Kab sawv cev rau cov neeg mob sib phim (n = 6). FMO, fluorescence rho tawm ib qho; MFI, nruab nrab fluorescence zog.
Kev tshuaj xyuas ntxiv tau qhia txog lwm qhov sib txawv tseem ceeb ntawm cov xwm txheej T cell phenotypic uas tau daws teeb meem siab heev. Qhov ua haujlwm (Daim Duab 1, G txog I) thiab kev nco txog effector (Daim Duab 1, J thiab K) hauv cov qog nqaij hlav muaj ntau dua li ascites (feem pua ​​ntawm CD3 + T hlwb). Ib yam li ntawd, kev tshuaj xyuas cov phenotype los ntawm kev qhia tawm ntawm cov cim qhia kev ua haujlwm (CD25 thiab CD137) thiab cov cim qhia kev poob qis [programmed cell death protein 1 (PD1)] tau qhia tias txawm hais tias cov yam ntxwv metabolic ntawm cov pej xeem no sib txawv (Daim Duab S1, B txog E), Tab sis tsis muaj qhov sib txawv tseem ceeb ntawm metabolic tau pom tseeb ntawm naive, effector lossis nco subsets (Daim Duab S1, F txog I). Cov txiaj ntsig no tau lees paub los ntawm kev siv cov txheej txheem kev kawm tshuab los txiav txim siab cov cell phenotypes (21), uas tau qhia ntxiv tias muaj ntau tus lej ntawm cov hlwb pob txha (CD45 + / CD3- / CD4 + / CD45RO +) hauv tus neeg mob ascites (Daim Duab S2A). Ntawm txhua hom cell uas tau txheeb xyuas, cov myeloid cell no tau qhia txog kev nqus cov piam thaj ntau tshaj plaws thiab kev ua haujlwm ntawm mitochondrial (Daim Duab S2, B txog G). Cov txiaj ntsig no qhia txog qhov sib txawv ntawm kev ua haujlwm ntawm ntau hom cell uas pom muaj nyob rau hauv ascites thiab qog nqaij hlav hauv cov neeg mob HGSC.
Qhov kev sib tw tseem ceeb hauv kev nkag siab txog cov yam ntxwv ntawm TIL yog qhov xav tau cais cov qauv T cell uas muaj kev huv si, zoo thiab ntau txaus los ntawm cov qog nqaij hlav. Cov kev tshawb fawb tsis ntev los no tau qhia tias kev cais thiab cov txheej txheem nplua nuj ntawm cov hlaws dai raws li cov cytometry ntws tuaj yeem ua rau muaj kev hloov pauv hauv cov qauv metabolite cellular (22-24). Txhawm rau kov yeej qhov teeb meem no, peb tau ua kom zoo dua txoj kev nplua nuj ntawm cov hlaws dai kom cais thiab cais TIL los ntawm kev phais mob qog nqaij hlav ntawm tib neeg ua ntej kev tshuaj xyuas los ntawm LC-MS / MS (saib Cov Khoom Siv thiab Cov Txheej Txheem; Daim Duab 2A). Txhawm rau txhawm rau ntsuas qhov cuam tshuam tag nrho ntawm cov txheej txheem no rau kev hloov pauv metabolite, peb tau piv cov qauv metabolite ntawm T cell uas tau ua los ntawm cov neeg pub dawb noj qab haus huv tom qab cov kauj ruam sib cais hlaws dai saum toj no nrog cov hlwb uas tsis tau sib cais hlaws dai tab sis tseem nyob ntawm dej khov. Qhov kev tshuaj xyuas kev tswj hwm zoo no pom tias muaj kev sib raug zoo ntawm ob qho xwm txheej no (r = 0.77), thiab qhov rov ua dua ntawm pawg ntawm 86 metabolites muaj qhov rov ua dua siab (Daim Duab 2B). Yog li ntawd, cov txheej txheem no tuaj yeem ua qhov kev tshuaj xyuas metabolite raug hauv cov cell uas tab tom ua kom muaj cov cell ntau ntxiv, yog li muab thawj lub platform daws teeb meem siab rau kev txheeb xyuas cov metabolites tshwj xeeb hauv HGSC, yog li ua rau tib neeg nkag siab tob dua txog kev tshwj xeeb ntawm cell.
(A) Daim duab qhia txog kev nplua nuj ntawm cov hlaws dai sib nqus. Ua ntej kev tshuaj xyuas los ntawm LC-MS/MS, cov hlwb yuav dhau peb qhov kev sib law liag ntawm cov hlaws dai sib nqus lossis nyob twj ywm ntawm dej khov. (B) Qhov cuam tshuam ntawm hom kev nplua nuj ntawm qhov muaj ntau ntawm cov metabolites. Qhov nruab nrab ntawm peb qhov kev ntsuas rau txhua hom kev nplua nuj ± SE. Kab grey sawv cev rau kev sib raug zoo 1: 1. Kev sib raug zoo hauv chav kawm (ICC) ntawm kev ntsuas rov ua dua qhia hauv daim ntawv lo axis. NAD, nicotinamide adenine dinucleotide. (C) Daim duab qhia txog kev ua haujlwm ntawm kev tshuaj xyuas cov metabolite ntawm tus neeg mob. Ascites lossis qog nqaij hlav raug sau los ntawm cov neeg mob thiab cryopreserved. Ib feem me me ntawm txhua tus qauv tau tshuaj xyuas los ntawm kev ntws cytometry, thaum cov qauv seem tau dhau peb qhov kev nplua nuj rau CD4 +, CD8 + thiab CD45- hlwb. Cov feem ntawm cov hlwb no tau tshuaj xyuas siv LC-MS/MS. (D) Daim ntawv qhia cua sov ntawm cov qauv metabolite ntau. Lub dendrogram sawv cev rau Ward's clustering ntawm Euclidean nrug ntawm cov qauv. (E) Kev tshuaj xyuas cov khoom tseem ceeb (PCA) ntawm daim ntawv qhia metabolite qauv, qhia peb daim ntawv theej ntawm txhua tus qauv, cov qauv los ntawm tib tus neeg mob tau txuas nrog los ntawm ib txoj kab. (F) PCA ntawm cov qauv metabolite ntawm cov qauv tau teeb tsa rau tus neeg mob (piv txwv li, siv ib feem ntawm kev rov ua dua); hom qauv raug txwv los ntawm lub cev convex. PC1, cov khoom tseem ceeb 1; PC2, cov khoom tseem ceeb 2.
Tom ntej no, peb tau siv txoj kev txhim kho no los tshuaj xyuas 99 cov metabolites hauv CD4 +, CD8 + thiab CD45-cell fractions hauv cov ascites thiab qog nqaij hlav thawj zaug ntawm rau tus neeg mob HGSC (Daim Duab 2C, Daim Duab S3A thiab Rooj S3 thiab S4). Cov pej xeem uas xav paub txog 2% txog 70% ntawm cov qauv loj thawj zaug ntawm cov hlwb muaj sia nyob, thiab qhov sib piv ntawm cov hlwb sib txawv ntawm cov neeg mob. Tom qab cais cov hlaws dai, cov feem pua ​​ntxiv ntawm kev txaus siab (CD4 +, CD8 + lossis CD45-) suav ntau dua 85% ntawm tag nrho cov hlwb muaj sia nyob hauv cov qauv nruab nrab. Txoj kev txhim kho no tso cai rau peb tshuaj xyuas cov pej xeem ntawm cov hlwb los ntawm cov metabolism ntawm cov ntaub so ntswg ntawm tib neeg, uas tsis yooj yim sua los ntawm cov qauv loj. Siv cov txheej txheem no, peb tau txiav txim siab tias l-kynurenine thiab adenosine, ob qho metabolites immunosuppressive zoo no tau nce siab hauv cov hlwb T lossis cov hlwb qog nqaij hlav (Daim Duab S3, B thiab C). Yog li ntawd, cov txiaj ntsig no qhia txog kev ua siab ncaj thiab lub peev xwm ntawm peb cov kev sib cais ntawm cov hlwb thiab cov txheej txheem spectrometry los nrhiav cov metabolites tseem ceeb hauv cov ntaub so ntswg ntawm tus neeg mob.
Peb qhov kev tshuaj xyuas kuj tau qhia txog kev sib cais ntawm cov hom cell hauv thiab ntawm cov neeg mob (Daim Duab 2D thiab Daim Duab S4A). Tshwj xeeb, piv rau lwm tus neeg mob, tus neeg mob 70 tau qhia txog cov yam ntxwv sib txawv ntawm cov metabolism (Daim Duab 2E thiab Daim Duab S4B), qhia tias tej zaum yuav muaj kev sib txawv ntawm cov metabolism ntawm cov neeg mob. Nws tsim nyog sau tseg tias piv rau lwm tus neeg mob (1.2 txog 2 litres; Rooj S1), tag nrho cov ascites sau hauv tus neeg mob 70 (80 ml) yog me dua. Kev tswj hwm ntawm kev sib txawv ntawm cov neeg mob thaum lub sijhawm tshuaj xyuas cov khoom tseem ceeb (piv txwv li, siv kev tshuaj xyuas ib nrab redundancy) qhia txog kev hloov pauv sib xws ntawm cov hom cell, thiab cov hom cell thiab / lossis microenvironment tau sib sau ua ke raws li cov ntaub ntawv metabolite (Daim Duab 2F). Kev tshuaj xyuas ntawm cov metabolites ib leeg tau hais txog cov teebmeem no thiab qhia txog qhov sib txawv tseem ceeb ntawm cov hom cell thiab microenvironment. Nws tsim nyog sau tseg tias qhov sib txawv tshaj plaws pom yog MNA, uas feem ntau yog nplua nuj hauv CD45- hlwb thiab hauv CD4 + thiab CD8 + hlwb uas nkag mus rau hauv qog (Daim Duab 3A). Rau cov hlwb CD4 +, qhov cuam tshuam no pom tseeb tshaj plaws, thiab MNA hauv CD8 + hlwb kuj zoo li raug cuam tshuam los ntawm ib puag ncig. Txawm li cas los xij, qhov no tsis tseem ceeb, vim tias tsuas yog peb ntawm rau tus neeg mob tuaj yeem soj ntsuam rau cov qhab nia CD8 + qog. Ntxiv rau MNA, hauv ntau hom hlwb hauv ascites thiab qog, lwm cov metabolites uas tsis zoo hauv TIL kuj nplua nuj sib txawv (Cov Duab S3 thiab S4). Yog li ntawd, cov ntaub ntawv no qhia txog cov txheej txheem zoo ntawm cov metabolites immunomodulatory rau kev tshawb fawb ntxiv.
(A) Cov ntsiab lus ntawm MNA hauv CD4 +, CD8 + thiab CD45- hlwb los ntawm ascites thiab qog. Daim duab qhia lub thawv qhia qhov nruab nrab (kab), interquartile ntau yam (ncej hinge) thiab ntau yam ntaub ntawv, txog li 1.5 npaug ntawm interquartile ntau yam (ncej whisker). Raws li tau piav qhia hauv Cov Khoom Siv thiab Txoj Kev rau Tus Neeg Mob, siv tus nqi limma ntawm tus neeg mob los txiav txim siab tus nqi P (*P <0.05 thiab **P <0.01). (B) Daim duab kos ntawm MNA metabolism (60). Metabolites: S-adenosyl-1-methionine; SAH, S-adenosine-1-homocysteine; NA, nicotinamide; MNA, 1-methylnicotinamide; 2-PY, 1-methyl- 2-pyridone-5-carboxamide; 4-PY, 1-methyl-4-pyridone-5-carboxamide; NR, nicotinamide ribose; NMN, nicotinamide mononucleotide. Cov Enzymes (ntsuab): NNMT, nicotinamide N-methyltransferase; SIRT, sirtuins; NAMPT, nicotinamide phosphoribosyl transferase; AOX1, aldehyde oxidase 1; NRK, nicotinamide riboside kinase; NMNAT, nicotinamide mono Nucleotide adenylate transferase; Pnp1, purine nucleoside phosphorylase. (C) t-SNE ntawm scRNA-seq ntawm ascites (grey) thiab qog (liab; n = 3 tus neeg mob). (D) NNMT kev qhia tawm hauv cov pej xeem sib txawv uas tau txheeb xyuas siv scRNA-seq. (E) Kev qhia tawm ntawm NNMT thiab AOX1 hauv SK-OV-3, tib neeg lub raum embryonic (HEK) 293T, T hlwb thiab MNA-kho T hlwb. Qhov kev qhia tawm uas tau muab tais tau pom piv rau SK-OV-3. Tus qauv kev qhia tawm nrog SEM tau pom (n = 6 tus neeg pub dawb noj qab haus huv). Cov nqi Ct ntau dua 35 raug suav hais tias tsis pom (UD). (F) Kev qhia tawm ntawm SLC22A1 thiab SLC22A2 hauv SK-OV-3, HEK293T, T hlwb thiab T hlwb kho nrog 8mM MNA. Qhov kev qhia tawm uas tau muab tais tau pom piv rau SK-OV-3. Tus qauv kev qhia tawm nrog SEM tau pom (n = 6 tus neeg pub dawb noj qab haus huv). Tus nqi Ct ntau dua 35 raug suav hais tias tsis pom (UD). (G) Cov ntsiab lus ntawm Cell MNA hauv cov hlwb pub dawb noj qab haus huv uas tau qhib tom qab 72 teev ntawm kev sib xyaw nrog MNA. Tus qauv kev qhia tawm nrog SEM tau pom (n = 4 tus neeg pub dawb noj qab haus huv).
MNA yog tsim los ntawm kev hloov cov pab pawg methyl los ntawm S-adenosyl-1-methionine (SAM) mus rau nicotinamide (NA) los ntawm nicotinamide N-methyltransferase (NNMT; Daim Duab 3B). NNMT yog overexpressed nyob rau hauv ntau yam mob qog noj ntshav tib neeg thiab yog txuam nrog kev loj hlob, kev nkag mus thiab metastasis (25-27). Txhawm rau kom nkag siab zoo dua qhov chaw ntawm MNA hauv T hlwb hauv TME, peb siv scRNA-seq los piav qhia txog kev qhia ntawm NNMT hla cov hom hlwb hauv ascites thiab qog ntawm peb tus neeg mob HGSC (Rooj S5). Kev tshuaj xyuas ntawm kwv yees li 6,500 hlwb tau qhia tias hauv ascites thiab qog ib puag ncig, NNMT kev qhia tau txwv rau cov pej xeem fibroblast thiab qog hlwb xav tias (Daim Duab 3, C thiab D). Nws yog ib qho tsim nyog sau tseg tias tsis muaj kev qhia NNMT pom tseeb hauv txhua tus pej xeem uas qhia PTPRC (CD45 +) (Daim Duab 3D thiab Daim Duab S5A), uas qhia tias MNA pom hauv cov metabolite spectrum tau nkag mus rau hauv T hlwb. Qhov kev qhia tawm ntawm aldehyde oxidase 1 (AOX1) hloov MNA mus rau hauv 1-methyl-2-pyridone-5-carboxamide (2-PYR) lossis 1-methyl-4-pyridone-5-carboxamide (4-PYR); Daim Duab 3B) kuj tseem txwv rau cov pej xeem ntawm fibroblasts qhia COL1A1 (Daim Duab S5A), uas ua ke qhia tias T cell tsis muaj peev xwm ntawm kev ua haujlwm ntawm MNA ib txwm muaj. Tus qauv kev qhia tawm ntawm cov noob caj noob ces MNA no tau txheeb xyuas siv cov ntaub ntawv cell thib ob ywj pheej los ntawm ascites los ntawm cov neeg mob HGSC (Daim Duab S5B; n = 6) (16). Tsis tas li ntawd, kev tshuaj xyuas cov tshuaj polymerase chain reaction (qPCR) ntawm cov cell pub dawb noj qab haus huv kho nrog MNA qhia tau hais tias piv nrog kev tswj hwm SK-OV-3 ovarian qog nqaij hlav cell, NNMT lossis AOX1 yuav luag tsis tau qhia tawm (Daim Duab 3E). Cov txiaj ntsig tsis tau xav txog no qhia tau hais tias MNA yuav raug tso tawm los ntawm fibroblasts lossis qog nqaij hlav rau hauv cov T cell sib ze hauv TME.
Txawm hais tias cov neeg sib tw suav nrog tsev neeg ntawm cov organic cation transporters 1 txog 3 (OCT1, OCT2 thiab OCT3) encoded los ntawm cov soluble carrier 22 (SLC22) tsev neeg (SLC22A1, SLC22A2 thiab SLC22A3), cov peev xwm transporters ntawm MNA tseem tsis tau txhais (28). QPCR ntawm mRNA los ntawm cov qe ntshav dawb noj qab haus huv tau qhia cov qib qis ntawm SLC22A1 tab sis tsis pom cov qib ntawm SLC22A2, uas lees paub tias nws tau tshaj tawm yav dhau los hauv cov ntaub ntawv (Daim Duab 3F) (29). Qhov sib piv, SK-OV-3 ovarian qog hlwb kab tau qhia cov qib siab ntawm ob qho tib si transporters (Daim Duab 3F).
Yuav kom sim seb cov T cell puas muaj peev xwm nqus tau MNA txawv teb chaws, cov T cell noj qab haus huv tau cog rau 72 teev nyob rau hauv qhov muaj ntau yam MNA sib txawv. Yog tsis muaj MNA sab nraud, cov ntsiab lus ntawm MNA hauv cell tsis tuaj yeem pom (Daim Duab 3G). Txawm li cas los xij, cov T cell uas tau qhib kho nrog MNA sab nraud tau qhia txog kev nce ntxiv ntawm cov ntsiab lus MNA hauv cov cell, txog li 6 mM MNA (Daim Duab 3G). Qhov tshwm sim no qhia tau tias txawm hais tias qib qis ntawm kev qhia tawm ntawm tus neeg thauj khoom thiab tsis muaj cov enzyme tseem ceeb uas ua rau muaj kev hloov pauv MNA hauv cell, TIL tseem tuaj yeem nqus tau MNA.
Cov tshuaj metabolites hauv cov neeg mob T hlwb thiab kev sim nqus MNA hauv vitro ua rau muaj peev xwm ntau ntxiv tias cov fibroblasts uas cuam tshuam nrog mob qog noj ntshav (CAF) tso MNA thiab cov hlwb qog nqaij hlav tuaj yeem tswj hwm cov phenotype thiab kev ua haujlwm ntawm TIL. Txhawm rau txiav txim siab qhov cuam tshuam ntawm MNA rau T hlwb, cov hlwb pub dawb T noj qab haus huv tau qhib hauv vitro thaum muaj lossis tsis muaj MNA, thiab lawv cov kev loj hlob thiab cytokine tsim tau raug soj ntsuam. Tom qab 7 hnub ntawm kev ntxiv MNA ntawm qhov ntau tshaj plaws, cov pej xeem ob npaug tau txo qis me ntsis, thaum lub zog tau khaws cia ntawm txhua koob tshuaj (Daim Duab 4A). Tsis tas li ntawd, kev kho mob ntawm exogenous MNA ua rau muaj kev nce ntxiv ntawm CD4 + thiab CD8 + T hlwb qhia txog tumor necrosis factor-α (TNFα; Daim Duab 4B). Qhov sib piv, kev tsim cov intracellular ntawm IFN-γ tau txo qis hauv CD4 + T hlwb, tab sis tsis yog hauv CD8 + T hlwb, thiab tsis muaj kev hloov pauv tseem ceeb hauv interleukin 2 (IL-2; Daim Duab 4, C thiab D). Yog li ntawd, enzyme-linked immunosorbent assay (ELISA) ntawm cov supernatants los ntawm cov kab lis kev cai T cell kho MNA no tau qhia txog kev nce ntxiv ntawm TNFα, kev txo qis ntawm IFN-γ, thiab tsis muaj kev hloov pauv ntawm IL-2 (Daim Duab 4, E rau G). . Kev txo qis ntawm IFN-γ qhia tau hais tias MNA yuav ua lub luag haujlwm hauv kev tiv thaiv kev ua haujlwm tiv thaiv qog ntawm T cell. Txhawm rau simulate qhov cuam tshuam ntawm MNA ntawm T cell-mediated cytotoxicity, chimeric antigen receptor T (FRα-CAR-T) cells targeting folate receptor α thiab CAR-T (GFP) tswj hwm los ntawm cov protein fluorescent ntsuab (GFP) -CAR-T) cells yog tsim los ntawm cov donor peripheral ntshav mononuclear cells (PBMC) noj qab haus huv. CAR-T cells tau cog qoob loo rau 24 teev nyob rau hauv lub xub ntiag ntawm MNA, thiab tom qab ntawd co-cultured nrog tib neeg SK-OV-3 ovarian qog hlwb qhia folate receptor α ntawm tus effector rau lub hom phiaj piv ntawm 10: 1. Kev kho mob MNA ua rau muaj kev txo qis ntawm kev tua cov qe FRα-CAR-T, uas zoo ib yam li cov qe FRα-CAR-T uas kho nrog adenosine (Daim Duab 4H).
(A) Tag nrho cov cell uas muaj sia nyob thiab cov pejxeem ob npaug (PD) ncaj qha los ntawm kev cog qoob loo rau hnub 7. Daim duab bar sawv cev rau qhov nruab nrab + SEM ntawm rau tus neeg pub dawb noj qab haus huv. Sawv cev rau cov ntaub ntawv los ntawm tsawg kawg n = 3 qhov kev sim ywj pheej. (B txog D) CD3/CD28 thiab IL-2 tau siv los ua kom T cell ua haujlwm ntawm lawv cov MNA concentration rau 7 hnub. Ua ntej kev tshuaj xyuas, cov cell tau raug txhawb nrog PMA/ionomycin nrog GolgiStop rau 4 teev. TNFα (B) kev qhia tawm hauv T cell. Piv txwv li duab (sab laug) thiab cov ntaub ntawv tabular (sab xis) ntawm TNFα kev qhia tawm hauv cov cell uas muaj sia nyob. IFN-γ (C) thiab IL-2 (D) kev qhia tawm hauv T cell. Kev qhia tawm ntawm cytokines tau ntsuas los ntawm kev ntws cytometry. Daim duab bar sawv cev rau qhov nruab nrab (n = 6 tus neeg pub dawb noj qab haus huv) + SEM. Siv kev tshuaj xyuas ib txoj kev ntawm qhov sib txawv thiab kev ntsuas rov ua dua (*P <0.05 thiab **P <0.01) los txiav txim siab tus nqi P. Sawv cev rau cov ntaub ntawv los ntawm tsawg kawg n = 3 qhov kev sim ywj pheej. (E txog G) CD3/CD28 thiab IL-2 tau siv los ua kom cov T hlwb ua haujlwm ntawm lawv cov MNA concentration rau 7 hnub. Cov khoom nruab nrab tau sau ua ntej thiab tom qab 4 teev ntawm PMA/ionomycin stimulation. Cov concentration ntawm TNFα (E), IFN-γ (F) thiab IL-2 (G) tau ntsuas los ntawm ELISA. Daim duab bar sawv cev rau qhov nruab nrab (n = 5 tus neeg pub dawb noj qab haus huv) + SEM. Tus nqi P txiav txim siab siv kev tshuaj xyuas ib txoj kev ntawm qhov sib txawv thiab kev ntsuas rov ua dua (*P<0.05). Cov kab dotted qhia txog qhov txwv ntawm kev kuaj pom. (H) Kev ntsuam xyuas Cell lysis. FRα-CAR-T lossis GFP-CAR-T hlwb tau kho nrog adenosine (250μM) lossis MNA (10 mM) rau 24 teev, lossis tsis kho (Ctrl). Feem pua ​​​​​​ntawm kev tua ntawm SK-OV-3 hlwb tau ntsuas. Tus nqi P txiav txim siab los ntawm Welch t xeem (*P<0.5 thiab **P<0.01).
Yuav kom nkag siab txog kev tswj hwm kev qhia tawm ntawm TNFα uas nyob ntawm MNA, cov kev hloov pauv hauv TNFα mRNA ntawm cov T hlwb uas kho nrog MNA tau raug soj ntsuam (Daim Duab 5A). Cov hlwb pub dawb noj qab haus huv uas kho nrog MNA tau qhia tias muaj kev nce ob npaug ntawm TNFα transcription theem, qhia tias MNA nyob ntawm kev tswj hwm TNFα transcriptional. Txhawm rau tshawb nrhiav qhov kev tswj hwm no, ob qho kev paub txog transcription factors uas tswj hwm TNFα, uas yog activated T cell nuclear factor (NFAT) thiab cov protein tshwj xeeb 1 (Sp1), tau raug soj ntsuam los teb rau MNA kev khi rau tus proximal TNFα promoter (30). Tus promoter TNFα muaj 6 qhov chaw khi NFAT thiab 2 qhov chaw khi Sp1, sib tshooj ntawm ib qho chaw [-55 base pairs (bp) los ntawm 5'cap] (30). Chromatin immunoprecipitation (ChIP) tau qhia tias thaum kho nrog MNA, kev khi ntawm Sp1 rau tus promoter TNFα tau nce peb npaug. Kev koom ua ke ntawm NFAT kuj nce ntxiv thiab mus txog qhov tseem ceeb (Daim Duab 5B). Cov ntaub ntawv no qhia tau tias MNA tswj kev qhia tawm ntawm TNFα los ntawm Sp1 transcription, thiab rau qhov tsawg dua qhov kev qhia tawm ntawm NFAT.
(A) Piv nrog cov T hlwb uas cog qoob loo tsis muaj MNA, qhov kev hloov pauv ntawm TNFα kev qhia tawm hauv T hlwb kho nrog MNA. Tus qauv kev qhia tawm nrog SEM tau pom (n = 5 tus neeg pub dawb noj qab haus huv). Sawv cev rau cov ntaub ntawv los ntawm tsawg kawg n = 3 qhov kev sim ywj pheej. (B) Tus txhawb nqa TNFα ntawm T hlwb kho nrog lossis tsis muaj 8 mM MNA tom qab NFAT thiab Sp1 tau ua ke nrog (Ctrl) thiab PMA / ionomycin stimulation rau 4 teev. Immunoglobulin G (IgG) thiab H3 tau siv ua qhov tsis zoo thiab zoo tswj rau immunoprecipitation, raws li. Kev ntsuas ntawm ChIP qhia tau hais tias qhov kev khi ntawm Sp1 thiab NFAT rau TNFα promoter hauv MNA-kho hlwb tau nce ntau zaus piv nrog kev tswj hwm. Sawv cev rau cov ntaub ntawv los ntawm tsawg kawg n = 3 qhov kev sim ywj pheej. Tus nqi P txiav txim siab los ntawm ntau qhov kev xeem t (*** P <0.01). (C) Piv rau ascites ntawm HGSC, T hlwb (tsis yog cytotoxic) tau qhia txog kev qhia tawm ntawm TNF hauv qog. Cov xim sawv cev rau cov neeg mob sib txawv. Cov hlwb uas tau tso tawm tau raug kuaj xyuas tsis raws cai mus txog 300 thiab jittered kom txwv tsis pub overdrawing (** Padj = 0.0076). (D) Tus qauv MNA rau mob qog nqaij hlav zes qe menyuam. MNA yog tsim tawm hauv cov hlwb qog thiab fibroblasts hauv TME thiab raug coj los ntawm T hlwb. MNA ua rau muaj kev sib txuas ntawm Sp1 rau TNFα promoter, ua rau muaj kev nce ntxiv ntawm TNFα transcription thiab TNFα cytokine ntau lawm. MNA kuj ua rau muaj kev txo qis hauv IFN-γ. Kev txwv ntawm T cell ua haujlwm ua rau muaj peev xwm tua neeg tsawg dua thiab ua kom cov qog loj hlob sai dua.
Raws li cov ntawv ceeb toom, TNFα muaj cov teebmeem tiv thaiv kab mob qog nqaij hlav thiab tiv thaiv kab mob qog nqaij hlav pem hauv ntej thiab nraub qaum, tab sis nws muaj lub luag haujlwm paub zoo hauv kev txhawb nqa kev loj hlob thiab metastasis ntawm mob qog nqaij hlav zes qe menyuam (31-33). Raws li cov ntawv ceeb toom, qhov concentration ntawm TNFα hauv ascites thiab cov nqaij hlav hauv cov neeg mob uas muaj mob qog nqaij hlav zes qe menyuam siab dua li ntawm cov nqaij hlav benign (34-36). Hais txog lub tshuab, TNFα tuaj yeem tswj hwm kev ua haujlwm, kev ua haujlwm thiab kev loj hlob ntawm cov qe ntshav dawb, thiab hloov cov phenotype ntawm cov qe ntshav qog nqaij hlav (37, 38). Sib xws nrog cov kev tshawb pom no, kev tshuaj xyuas kev qhia tawm ntawm cov noob caj noob ces qhia tau tias TNF tau nce siab hauv T hlwb hauv cov nqaij hlav piv rau ascites (Daim Duab 5C). Kev nce hauv TNF kev qhia tawm tsuas yog pom tseeb hauv cov pej xeem T hlwb nrog cov phenotype tsis-cytotoxic (Daim Duab S5A). Hauv kev xaus, cov ntaub ntawv no txhawb nqa qhov kev xav tias MNA muaj ob qho kev tiv thaiv kab mob thiab kev txhawb nqa qog nqaij hlav hauv HGSC.
Kev siv fluorescent labeling raws li flow cytometry tau dhau los ua txoj kev tseem ceeb rau kev kawm txog TIL metabolism. Cov kev tshawb fawb no tau qhia tias piv nrog cov ntshav peripheral lymphocytes lossis T cell los ntawm cov kab mob lymphoid theem ob, murine thiab tib neeg TIL muaj qhov siab dua rau kev nqus glucose (4, 39) thiab qhov maj mam poob ntawm mitochondrial ua haujlwm (19, 40). Txawm hais tias peb tau pom cov txiaj ntsig zoo sib xws hauv kev tshawb fawb no, qhov kev txhim kho tseem ceeb yog los sib piv cov metabolism ntawm cov hlwb qog thiab TIL los ntawm cov ntaub so ntswg qog tib yam. Sib xws nrog qee cov ntawv ceeb toom dhau los no, cov hlwb qog (CD45-EpCAM +) los ntawm ascites thiab cov qog muaj kev nqus glucose ntau dua li CD8 + thiab CD4 + T cell, txhawb nqa tias kev nqus glucose ntau ntawm cov hlwb qog tuaj yeem piv nrog T cell. Lub tswv yim ntawm kev sib tw T cell. TME. Txawm li cas los xij, kev ua haujlwm mitochondrial ntawm cov hlwb qog yog siab dua li ntawm CD8 + T cell, tab sis kev ua haujlwm mitochondrial zoo ib yam li ntawm CD4 + T cell. Cov txiaj ntsig no txhawb nqa lub ntsiab lus tshiab uas oxidative metabolism yog qhov tseem ceeb rau cov hlwb qog (41, 42). Lawv kuj qhia tias CD8 + T hlwb tej zaum yuav muaj kev cuam tshuam ntau dua rau kev ua haujlwm tsis zoo ntawm oxidative dua li CD4 + T hlwb, lossis CD4 + T hlwb tej zaum yuav siv cov pa roj carbon uas tsis yog glucose los tswj kev ua haujlwm mitochondrial (43, 44). Nws yuav tsum tau sau tseg tias peb tsis pom qhov sib txawv ntawm kev nqus glucose lossis kev ua haujlwm mitochondrial ntawm CD4 + T effectors, T effector nco thiab T central nco hlwb hauv ascites. Ib yam li ntawd, qhov sib txawv ntawm CD8 + T hlwb hauv qog tsis muaj dab tsi ua rau kev hloov pauv ntawm kev nqus glucose, qhia txog qhov sib txawv tseem ceeb ntawm T hlwb cog qoob loo hauv vitro thiab tib neeg TIL hauv vivo (22). Cov kev soj ntsuam no kuj tau lees paub los ntawm kev siv cov pej xeem cell tsis muaj kev ntxub ntxaug, uas qhia ntxiv tias CD45 + / CD3- / CD4 + / CD45RO + hlwb nrog kev nqus glucose ntau dua thiab kev ua haujlwm mitochondrial dua li cov hlwb qog muaj ntau tab sis muaj cov pej xeem Metabolic nquag. Cov pej xeem no yuav sawv cev rau cov subpopulation ntawm myeloid suppressor hlwb lossis plasmacytoid dendritic hlwb uas tau txheeb xyuas hauv scRNA-seq kev tshuaj xyuas. Txawm hais tias ob qho no tau raug tshaj tawm hauv tib neeg cov qog nqaij hlav zes qe menyuam [45], lawv tseem xav tau kev ua haujlwm ntxiv yog los piav qhia txog cov myeloid subpopulation no.
Txawm hais tias cov txheej txheem raws li kev ntws cytometry tuaj yeem ua kom meej meej qhov sib txawv ntawm cov piam thaj thiab oxidative metabolism ntawm cov hom cell, cov metabolites tseeb tsim los ntawm glucose lossis lwm cov pa roj carbon rau mitochondrial metabolism hauv TME tseem tsis tau txiav txim siab. Kev muab qhov muaj lossis tsis muaj cov metabolites rau ib pawg TIL xav tau kev ntxuav cov pej xeem cell los ntawm cov ntaub so ntswg excised. Yog li ntawd, peb txoj kev txhim kho cell ua ke nrog cov spectrometry loj tuaj yeem muab kev nkag siab rau cov metabolites uas muaj ntau yam sib txawv hauv T cell thiab cov pej xeem qog nqaij hlav hauv cov qauv neeg mob sib phim. Txawm hais tias txoj kev no muaj qhov zoo dua li fluorescence-activated cell sorting, qee cov tsev qiv ntawv metabolite yuav raug cuam tshuam vim muaj kev ruaj khov thiab / lossis tus nqi hloov pauv sai (22). Txawm li cas los xij, peb txoj kev tau txheeb xyuas ob qho kev lees paub immunosuppressive metabolites, adenosine thiab kynurenine, vim tias lawv sib txawv heev ntawm cov hom qauv.
Peb qhov kev tshuaj xyuas metabonomic ntawm cov qog thiab TIL subtypes muab kev nkag siab ntau ntxiv rau lub luag haujlwm ntawm cov metabolites hauv ovarian TME. Ua ntej, siv cov flow cytometry, peb tau txiav txim siab tias tsis muaj qhov sib txawv hauv mitochondrial kev ua haujlwm ntawm cov qog thiab CD4 + T hlwb. Txawm li cas los xij, LC-MS / MS kev tshuaj xyuas tau qhia txog kev hloov pauv tseem ceeb hauv qhov muaj ntau ntawm cov metabolites ntawm cov pej xeem no, qhia tias cov lus xaus txog TIL metabolism thiab nws cov haujlwm metabolic tag nrho yuav tsum tau txhais lus zoo. Qhov thib ob, MNA yog cov metabolite nrog qhov sib txawv loj tshaj plaws ntawm CD45-cells thiab T hlwb hauv ascites, tsis yog qog. Yog li ntawd, kev faib ua feem thiab qhov chaw ntawm qog yuav muaj cov teebmeem sib txawv ntawm TIL metabolism, uas qhia txog qhov ua tau heterogeneity hauv ib qho chaw me me. Qhov thib peb, kev qhia tawm ntawm MNA-tsim enzyme NNMT feem ntau txwv rau CAF, uas yog cov qog rau qhov tsawg dua, tab sis cov qib MNA pom tau pom hauv cov qog-los ntawm T hlwb. Kev overexpression ntawm NNMT hauv ovarian CAF muaj cov nyhuv txhawb nqa mob qog noj ntshav, ib feem vim yog kev txhawb nqa CAF metabolism, qog nkag mus thiab metastasis (27). Txawm hais tias qib tag nrho ntawm TIL yog qhov nruab nrab, qhov kev qhia tawm ntawm NNMT hauv CAF muaj feem cuam tshuam nrog Cancer Genome Atlas (TCGA) mesenchymal subtype, uas cuam tshuam nrog kev kwv yees tsis zoo (27, 46, 47). Thaum kawg, qhov kev qhia tawm ntawm cov enzyme AOX1 uas ua rau MNA puas tsuaj kuj txwv rau cov pej xeem CAF, uas qhia tau tias T cell tsis muaj peev xwm metabolize MNA. Cov txiaj ntsig no txhawb nqa lub tswv yim tias txawm hais tias xav tau kev ua haujlwm ntxiv los txheeb xyuas qhov kev tshawb pom no, cov qib siab ntawm MNA hauv T cell yuav qhia tau tias muaj ib puag ncig tiv thaiv kab mob CAF microenvironment.
Vim tias cov MNA transporters tsis tshua muaj kev qhia tawm thiab cov protein tseem ceeb uas koom nrog MNA metabolism tsis pom, qhov muaj MNA hauv T cell yog qhov tsis tau xav txog. Tsis yog NNMT lossis AOX1 tuaj yeem pom los ntawm kev tshuaj xyuas scRNA-seq thiab targeted qPCR ntawm ob pawg neeg ywj pheej. Cov txiaj ntsig no qhia tau tias MNA tsis yog synthesized los ntawm T cell, tab sis nqus los ntawm TME nyob ib puag ncig. Kev sim hauv vitro qhia tau tias T cell feem ntau khaws cov MNA sab nraud.
Peb cov kev tshawb fawb hauv vitro tau qhia tias MNA sab nraud ua rau muaj kev qhia tawm ntawm TNFα hauv T hlwb thiab ua rau Sp1 txuas rau TNFα promoter zoo dua. Txawm hais tias TNFα muaj ob qho tib si tiv thaiv qog thiab tiv thaiv qog, hauv mob qog zes qe menyuam, TNFα tuaj yeem txhawb kev loj hlob ntawm mob qog zes qe menyuam (31-33). Kev tsis ua kom TNFα tsis zoo hauv kev cog qoob loo ntawm cov qog zes qe menyuam lossis kev tshem tawm ntawm TNFα teeb liab hauv cov qauv nas tuaj yeem txhim kho TNFα-mediated inflammatory cytokine ntau lawm thiab tiv thaiv kev loj hlob ntawm qog (32, 35). Yog li ntawd, hauv qhov no, TME-derived MNA tuaj yeem ua haujlwm ua pro-inflammatory metabolite los ntawm TNFα-dependent mechanism los ntawm autocrine loop, yog li txhawb kev tshwm sim thiab kis ntawm mob qog zes qe menyuam (31). Raws li qhov ua tau no, TNFα blockade tab tom kawm ua tus neeg sawv cev kho mob rau mob qog zes qe menyuam (37, 48, 49). Tsis tas li ntawd, MNA ua rau cytotoxicity ntawm CAR-T hlwb rau cov qog zes qe menyuam, muab pov thawj ntxiv rau MNA-mediated immune suppression. Ua ke, cov txiaj ntsig no qhia txog tus qauv uas cov qog nqaij hlav thiab CAF hlwb tso MNA rau hauv extracellular TME. Los ntawm (i) TNF-induced ovarian cancer loj hlob stimulation thiab (ii) MNA-induced T cell cytotoxic activity inhibition, qhov no yuav muaj ob qho nyhuv qog nqaij hlav (Daim Duab 5D).
Xaus lus, los ntawm kev siv kev sib xyaw ua ke ntawm kev nplua nuj ntawm cov hlwb sai, kev txheeb xyuas cov hlwb ib leeg thiab kev txheeb xyuas cov metabolism, txoj kev tshawb fawb no tau qhia txog qhov sib txawv loj ntawm cov kab mob qog nqaij hlav thiab cov hlwb ascites hauv cov neeg mob HGSC. Qhov kev tshuaj xyuas no tau qhia tias muaj qhov sib txawv ntawm kev nqus cov piam thaj thiab kev ua haujlwm mitochondrial ntawm T hlwb, thiab tau txheeb xyuas MNA ua cov tshuaj metabolite tswj kev tiv thaiv kab mob uas tsis yog hlwb. Cov ntaub ntawv no muaj kev cuam tshuam rau TME cuam tshuam li cas rau T hlwb metabolism hauv tib neeg mob qog nqaij hlav. Txawm hais tias kev sib tw ncaj qha rau cov as-ham ntawm T hlwb thiab cov hlwb mob qog nqaij hlav tau tshaj tawm, cov metabolites kuj tseem tuaj yeem ua haujlwm ua cov neeg tswj hwm tsis ncaj qha los txhawb kev loj hlob ntawm qog nqaij hlav thiab tej zaum yuav txwv tsis pub muaj kev tiv thaiv kab mob endogenous. Kev piav qhia ntxiv txog lub luag haujlwm ntawm cov metabolites tswj hwm no yuav qhib lwm txoj hauv kev los txhim kho kev tiv thaiv kab mob qog nqaij hlav.
Cov qauv ntawm tus neeg mob thiab cov ntaub ntawv kho mob tau txais los ntawm BC mob qog nqaij hlav qhov chaw khaws cov ntaub so ntswg uas tau lees paub los ntawm Canadian Tissue Repository Network. Raws li cov txheej txheem tau pom zoo los ntawm BC Cancer Research Ethics Committee thiab University of British Columbia (H07-00463), txhua tus neeg mob cov qauv thiab cov ntaub ntawv kho mob tau txais kev pom zoo sau ntawv lossis zam lawv txoj kev pom zoo. Cov qauv tau khaws cia rau hauv BioBank uas tau lees paub (BRC-00290). Cov yam ntxwv ntawm tus neeg mob kom ntxaws tau qhia hauv Cov Lus Qhia S1 thiab S5. Rau kev khaws cia khov kho, siv rab riam phais kom rhuav tshem tus neeg mob cov qauv qog nqaij hlav thiab tom qab ntawd thawb nws los ntawm lub lim dej 100-micron kom tau txais ib lub cell suspension. Tus neeg mob lub ascites tau centrifuged ntawm 1500 rpm rau 10 feeb ntawm 4 ° C kom pellet cov cell thiab tshem tawm cov supernatant. Cov hlwb tau los ntawm qog thiab ascites tau raug khaws cia rau hauv 50% cov ntshav AB uas tsis ua haujlwm los ntawm cua sov (Sigma-Aldrich), 40% RPMI-1640 (Thermo Fisher Scientific) thiab 10% dimethyl sulfoxide. Cov hlwb ib leeg uas khaws cia no tau yaj thiab siv rau kev txiav txim siab txog metabolic thiab metabolite raws li tau piav qhia hauv qab no.
Cov khoom siv tag nrho muaj 0.22 μm lim 50:50 ntxiv RPMI 1640: AimV. RPMI 1640 + 2.05 mM l-glutamine (Thermo Fisher Scientific) ntxiv nrog 10% cov tshuaj AB tib neeg uas tsis ua haujlwm kub (Sigma-Aldrich), 12.5 mM Hepes (Thermo Fisher Scientific), 2 mM l-glutamine (Thermo Fisher Scientific) Fisher Scientific), 1 x Penicillin Streptomycin (PenStrep) kua (Thermo Fisher Scientific) thiab 50 μMB-mercaptoethanol. AimV (Invitrogen) ntxiv nrog 20 mM Hepes (Thermo Fisher Scientific) thiab 2 mM l-glutamine (Thermo Fisher Scientific). Tus flow cytometer staining buffer muaj 0.22μm filtered phosphate buffered saline (PBS; Invitrogen) ntxiv nrog 3% heat-inactivated AB human serum (Sigma). Tus cell enrichment buffer yog muaj 0.22μm filtered PBS thiab ntxiv nrog 0.5% heat-inactivated human AB serum (Sigma-Aldrich).
Hauv 37°C tag nrho cov khoom nruab nrab, cov cell tau pleev xim nrog 10 nM MT DR thiab 100 μM 2-NBDG rau 30 feeb. Tom ntej no, cov cell tau pleev xim nrog cov xim muaj sia nyob eF506 ntawm 4°C rau 15 feeb. Rov ua kom cov cell rov qab zoo li qub hauv FC Block (eBioscience) thiab Brilliant Stain Buffer (BD Biosciences), dilute hauv flow cytometer staining buffer (raws li cov lus qhia ntawm cov chaw tsim khoom), thiab incubate rau 10 feeb ntawm chav tsev kub. Pleev xim rau cov cell nrog ib pawg ntawm cov tshuaj tiv thaiv (Rooj S2) hauv flow cytometry staining buffer ntawm 4°C rau 20 feeb. Rov ua kom cov cell rov qab zoo li qub hauv flow cytometry staining buffer (Cytek Aurora; 3L-16V-14B-8R configuration) ua ntej kev tshuaj xyuas. Siv SpectroFlo thiab FlowJo V10 los tshuaj xyuas cov ntaub ntawv suav cell, thiab siv GraphPad Prism 8 los tsim cov ntaub ntawv. Qhov nruab nrab ntawm qhov ci ntsa iab (MFI) ntawm 2-NBDG thiab MT DR tau raug hloov kho kom zoo, thiab tom qab ntawd kev xeem t sib koom ua ke tau siv rau kev tshuaj xyuas suav lej los suav cov neeg mob sib phim. Tshem tawm tag nrho cov pej xeem uas muaj tsawg dua 40 qhov xwm txheej los ntawm kev tshuaj xyuas; nkag mus rau tus nqi MFI ntawm 1 rau txhua tus nqi tsis zoo ua ntej ua qhov kev tshuaj xyuas suav lej thiab kev pom cov ntaub ntawv.
Yuav kom ntxiv rau txoj kev npaj gating ntawm tes ntawm cov txheej txheem saum toj no, peb tau siv cov lus piav qhia tag nrho los ntawm tsob ntoo txwv duab (FAUST) (21) kom muab cov hlwb rau cov pej xeem tom qab tshem tawm cov hlwb tuag hauv FlowJo. Peb tswj hwm cov zis los sib koom ua ke cov pej xeem uas zoo li tau muab faib tsis raug (ua ke PD1 + nrog PD1-qog hlwb) thiab cov pej xeem khaws cia. Txhua tus qauv muaj qhov nruab nrab ntau dua 2% hlwb, rau tag nrho 11 cov pej xeem.
Siv Ficoll gradient density centrifugation los cais PBMC ntawm cov khoom sib cais leukocyte (STEMCELL Technologies). CD8 + T cell tau raug rho tawm ntawm PBMC siv CD8 MicroBeads (Miltenyi) thiab nthuav dav hauv cov khoom siv tag nrho siv TransAct (Miltenyi) rau 2 lub lis piam raws li cov lus qhia ntawm cov chaw tsim khoom. Cov cell tau tso cai rau sawv rau 5 hnub hauv cov khoom siv tag nrho uas muaj IL-7 (10 ng/ml; PeproTech), thiab tom qab ntawd rov ua kom muaj zog nrog TransAct. Nyob rau hnub 7, raws li cov lus qhia ntawm cov chaw tsim khoom, tib neeg CD45 MicroBeads (Miltenyi) tau siv los nplua nuj cov cell hauv peb qhov sib law liag. Cov cell tau raug faib rau kev tshuaj xyuas flow cytometry (raws li tau piav qhia saum toj no), thiab ib lab lub cell tau raug faib peb zaug rau kev tshuaj xyuas LC-MS/MS. Cov qauv tau ua tiav los ntawm LC-MS/MS raws li tau piav qhia hauv qab no. Peb kwv yees tus nqi metabolite uas ploj lawm nrog tus lej ion ntawm 1,000. Txhua tus qauv raug normalized los ntawm tag nrho cov ion number (TIC), hloov pauv logarithmically thiab cia li normalized hauv MetaboAnalystR ua ntej kev tshuaj xyuas.
Cov kua nplaum ib lub cell ntawm txhua tus neeg mob tau yaj thiab lim los ntawm lub lim dej 40 μm mus rau hauv cov khoom siv tag nrho (raws li tau piav qhia saum toj no). Raws li cov txheej txheem ntawm cov chaw tsim khoom, peb qhov kev xaiv zoo sib law liag los ntawm kev sib cais cov hlaws dai sib nqus siv MicroBeads (Miltenyi) tau siv los ua kom cov qauv rau CD8 +, CD4 + thiab CD45- hlwb (ntawm dej khov). Hauv ntej, cov hlwb tau rov ua dua hauv cov cell enrichment buffer (raws li tau piav qhia saum toj no) thiab suav. Cov hlwb tau incubated nrog tib neeg CD8 hlaws dai, tib neeg CD4 hlaws dai lossis tib neeg CD45 hlaws dai (Miltenyi) ntawm 4 ° C rau 15 feeb, thiab tom qab ntawd ntxuav nrog cell enrichment buffer. Cov qauv raug dhau los ntawm LS kem (Miltenyi), thiab cov feem pua ​​​​​​zoo thiab tsis zoo raug sau. Txhawm rau kom txo lub sijhawm thiab ua kom cov kauj ruam rov qab los ntawm lub cell, CD8-fraction ces siv rau qhov thib ob ntawm CD4 + enrichment, thiab CD4-fraction siv rau CD45-enrichment tom qab. Khaws cov tshuaj rau ntawm dej khov thoob plaws hauv cov txheej txheem sib cais.
Yuav npaj cov qauv rau kev tshuaj xyuas metabolite, cov cell tau ntxuav ib zaug nrog cov dej khov-txias ntsev, thiab 1 ml ntawm 80% methanol tau ntxiv rau txhua tus qauv, tom qab ntawd vortexed thiab snap khov hauv cov kua nitrogen. Cov qauv tau raug rau peb lub voj voog khov-thaw thiab centrifuged ntawm 14,000 rpm rau 15 feeb ntawm 4 ° C. Cov supernatant uas muaj cov metabolites tau evaporated kom txog thaum qhuav. Cov metabolites tau rov yaj hauv 50 μl ntawm 0.03% formic acid, vortexed kom sib tov, thiab tom qab ntawd centrifuged kom tshem tawm cov khib nyiab.
Tshem tawm cov metabolites raws li tau piav qhia saum toj no. Hloov cov supernatant mus rau lub raj mis kua chromatography ua haujlwm siab rau kev tshawb fawb metabolomics. Siv cov txheej txheem kho mob random los kho txhua tus qauv nrog cov lej zoo sib xws ntawm cov hlwb kom tiv thaiv cov teebmeem batch. Peb tau ua qhov kev ntsuam xyuas zoo ntawm cov metabolites thoob ntiaj teb uas tau tshaj tawm ua ntej ntawm AB SCIEX QTRAP 5500 Triple Quadrupole Mass Spectrometer (50). Kev tshuaj xyuas Chromatographic thiab kev koom ua ke ntawm thaj chaw siab tshaj plaws tau ua tiav siv MultiQuant version 2.1 software (Applied Biosystems SCIEX).
Ib qho suav ion ntawm 1000 tau siv los kwv yees tus nqi metabolite uas ploj lawm, thiab TIC ntawm txhua tus qauv tau siv los xam qhov chaw siab tshaj plaws ntawm txhua tus metabolite uas tau kuaj pom kom kho rau cov kev hloov pauv uas tau qhia los ntawm kev tshuaj xyuas cov cuab yeej los ntawm kev ua cov qauv. Tom qab TIC raug kho kom zoo, MetaboAnalystR (51) (tus qauv tsis siv neeg) yog siv rau kev hloov pauv logarithmic thiab kev ntsuas kab tsis siv neeg. Peb siv PCA nrog vegan R pob los ua qhov kev tshuaj xyuas tshawb nrhiav ntawm qhov sib txawv ntawm cov qauv metabolome, thiab siv kev tshuaj xyuas ib nrab redundancy los tshuaj xyuas cov neeg mob. Siv txoj kev Ward los tsim daim ntawv qhia cua sov dendrogram los sib sau ua ke qhov deb Euclidean ntawm cov qauv. Peb siv limma (52) ntawm cov qauv metabolite nplua nuj los txheeb xyuas cov metabolites nplua nuj sib txawv thoob plaws tag nrho cov hom cell thiab microenvironment. Txhawm rau kom yooj yim rau kev piav qhia, peb siv cov qauv nruab nrab ntawm pawg los teev tus qauv, thiab xav txog cov hom cell hauv microenvironment ua txhua pawg (n = 6 pawg); rau qhov kev xeem tseem ceeb, peb tau ua peb qhov kev ntsuas rov ua dua rau txhua lub metabolite Txhawm rau kom tsis txhob muaj kev rov ua dua cuav, tus neeg mob tau suav nrog ua qhov teeb meem hauv kev tsim limma. Txhawm rau txheeb xyuas qhov sib txawv ntawm cov metabolites ntawm cov neeg mob sib txawv, peb tau kho cov qauv limma suav nrog cov neeg mob hauv txoj kev ruaj khov. Peb tshaj tawm qhov tseem ceeb ntawm qhov sib txawv ua ntej ntawm hom cell thiab microenvironment ntawm Padj <0.05 (Benjamini-Hochberg kho).
Tom qab siv Miltenyi Dead Cell Removal Kit (>80% viability), kev txheeb xyuas cov noob caj noob ces ib leeg tau ua tiav rau tag nrho cov noob caj noob ces khov thiab cov qauv qog nqaij hlav siv 10x 5′gene expression protocol. Tsib rooj plaub nrog cov qog nqaij hlav thiab ascites sib phim tau raug tshuaj xyuas, txawm hais tias qhov tsis muaj zog los ntawm ib qho qauv qog nqaij hlav tiv thaiv nws suav nrog. Txhawm rau kom ua tiav ntau qhov kev xaiv ntawm cov neeg mob, peb tau muab cov qauv ntawm txhua tus neeg mob ua ke hauv txoj kab ntawm 10x chromium controller, thiab tshuaj xyuas cov noob caj noob ces thiab cov chaw qog nqaij hlav sib cais. Tom qab txheeb xyuas [Illumina HiSeq 4000 28 × 98 bp paired end (PE), Quebec genome; qhov nruab nrab ntawm 73,488 thiab 41,378 nyeem ib lub cell rau qog thiab ascites feem]], peb siv CellSNP thiab Vireo (53) (raws li CellSNP raws li Tus neeg SNP (VCF) uas muab los ntawm GRCh38 tau muab tus kheej pub dawb. Peb siv SNPRelate los xav txog tus kheej ze tshaj plaws (IBS) ntawm tus neeg mob genotype xwm txheej (IBS), tsis suav nrog cov hlwb tsis tau muab faib thiab cov hlwb txheeb xyuas ua duplexes thiab cov neeg pub dawb sib phim ntawm ascites thiab qog qauv (54). Raws li txoj haujlwm no, peb khaws peb rooj plaub nrog cov sawv cev ntawm cov hlwb ntau hauv qog thiab ascites rau kev tshuaj xyuas downstream. Tom qab ua tiav cov kauj ruam lim dej loj hauv lub scater (55) thiab scran (56) BioConductor ntim, qhov no tau tsim 6975 lub hlwb (2792 thiab 4183 lub hlwb los ntawm qog thiab ascites, feem) rau kev tshuaj xyuas. Peb siv igraph's (57) Louvain clustering ntawm cov neeg nyob ze tshaj plaws network (SNN) raws li Jaccard qhov deb rau cov hlwb sib sau ua ke los ntawm kev qhia tawm. Cov pawg tau sau ua ke rau cov hom hlwb uas muaj peev xwm raws li cov cim qhia txog kev qhia tawm thiab pom nrog t-SNE. Cov hlwb Cytotoxic T tau txhais los ntawm kev qhia tawm ntawm CD8A thiab GZMA, tsis suav nrog cov pawg me me uas muaj cov protein qhia tawm ribosomal tsawg. Peb tau nkag mus rau cov ntaub ntawv luam tawm ntawm Izar et al. (16), suav nrog lawv cov t-SNE embedding, tuaj yeem tswj kev qhia tawm sib tshooj ntawm cov cim qhia txog cov hlwb tiv thaiv kab mob thiab kev qhia tawm NNMT.
PBMC raug cais tawm ntawm cov khoom sib cais leukocyte (STEMCELL Technologies) los ntawm Ficoll gradient density centrifugation. Cov hlwb CD3 + raug cais tawm ntawm PBMC siv cov hlaws CD3 (Miltenyi). Yog tias muaj lossis tsis muaj MNA, cov hlwb CD3 + raug qhib nrog plate-bound CD3 (5μg/ml), soluble CD28 (3μg/ml) thiab IL-2 (300 U/ml; Proleukin). Nyob rau hnub kawg ntawm kev nthuav dav, qhov muaj sia nyob (Fixable Viability Dye eFluor450, eBioscience) thiab kev loj hlob (123count eBeads, Thermo Fisher Scientific) raug soj ntsuam los ntawm flow cytometry. Soj ntsuam kev ua haujlwm ntawm cov teebmeem los ntawm kev txhawb cov hlwb nrog PMA (20 ng/ml) thiab ionomycin (1μg/ml) nrog GolgiStop rau 4 teev, thiab saib xyuas CD8-PerCP (RPA-T8, BioLegend), CD4-AF700 (RPA-T4), BioLegend) thiab TNFα-fluorescein isothiocyanate (FITC) (MAb11, BD). Txhawb cov hlwb qPCR thiab ChIP nrog PMA (20 ng/ml) thiab ionomycin (1μg/ml) rau 4 teev. Cov ELISA supernatant tau sau ua ntej thiab tom qab kev txhawb nrog PMA (20 ng/ml) thiab ionomycin (1 μg/ml) rau 4 teev.
Ua raws li cov txheej txheem ntawm cov chaw tsim khoom kom cais RNA siv RNeasy Plus Mini Kit (QIAGEN). Siv QIAshredder (QIAGEN) los ua kom cov qauv sib xyaw. Siv cov khoom siv RNA rau cDNA muaj peev xwm siab (Thermo Fisher Scientific) los tsim cov DNA sib txuas (cDNA). Siv TaqMan Rapid Advanced Master Mix (Thermo Fisher Scientific) los ntsuas qhov kev qhia tawm ntawm cov noob caj noob ces (raws li cov txheej txheem ntawm cov chaw tsim khoom) nrog cov probes hauv qab no: Hs00196287_m1 (NNMT), Hs00154079_m1 (AOX1), Hs00427552_m1 (SLC22A1), Hs02786624_g1 [glyceraldehyde-3-phosphate off Hydrogen (GAPDH)] thiab Hs01010726_m1 (SLC22A2). Cov qauv kuaj tau khiav ntawm StepOnePlus real-time PCR system (Applied Biosystems) (Applied Biosystems) hauv MicroAmp fast optical 96-well reaction plate (Applied Biosystems) nrog MicroAmp optical film. Txhua tus nqi Ct uas tshaj 35 suav tias yog siab dua qhov ntsuas pom thiab raug cim tias tsis pom.
Ua ChIP raws li tau piav qhia ua ntej (58). Hauv ntej, cov hlwb tau kho nrog formaldehyde (qhov kawg concentration 1.42%) thiab incubated ntawm chav tsev kub rau 10 feeb. Siv cov tshuaj ntxiv o (25 mM Hepes, 1.5 mM MgCl2, 10 mM KCl thiab 0.1% NP-40) ntawm dej khov rau 10 feeb, tom qab ntawd rov ua dua hauv immunoprecipitation buffer raws li tau piav qhia (58). Cov qauv tau sonicated nrog cov voj voog hauv qab no: 10 voj voog (20 1-ob vib nas this pulses) thiab lub sijhawm static ntawm 40 vib nas this. Incubate ChIP-qib immunoglobulin G (Cell Signaling Technology; 1μl), histone H3 (Cell Signaling Technology; 3μl), NFAT (Invitrogen; 3μl) thiab SP1 (Cell Signaling Technology; 3μl) antibodies nrog cov qauv ntawm 4 ° CC co ib hmos. Muab cov protein A beads (Thermo Fisher Scientific) tso rau hauv cov qauv ntawm 4°C nrog kev co maj mam rau 1 teev, tom qab ntawd siv cov chelex beads (Bio-Rad) los nplua nuj DNA, thiab siv proteinase K (Thermo Fisher) rau kev zom protein. TNFα promoter tau kuaj pom los ntawm PCR: forward, GGG TAT CCT TGA TGC TTG TGT; ntawm qhov tsis sib xws, GTG CCA ACA ACT GCC TTT ATA TG (207-bp khoom). Cov duab tau tsim los ntawm Image Lab (Bio-Rad) thiab ntsuas siv ImageJ software.
Cov kua nplaum kab mob hauv lub cell tau sau raws li tau piav qhia saum toj no. Kev txiav txim siab tau ua raws li cov txheej txheem ntawm cov chaw tsim khoom ntawm tib neeg TNFα ELISA kit (Invitrogen), tib neeg IL-2 ELISA kit (Invitrogen) thiab tib neeg IFN-γ ELISA kit (Abcam). Raws li cov txheej txheem ntawm cov chaw tsim khoom, cov kua nplaum kab mob tau diluted 1: 100 los ntes TNFα thiab IL-2, thiab 1: 3 los ntes IFN-γ. Siv EnVision 2104 Multilabel Reader (PerkinElmer) los ntsuas qhov nqus ntawm 450 nm.
PBMC raug cais tawm ntawm cov khoom sib cais leukocyte (STEMCELL Technologies) los ntawm Ficoll gradient density centrifugation. Cov hlwb CD3 + raug cais tawm ntawm PBMC siv cov hlaws CD3 (Miltenyi). Yog tias muaj lossis tsis muaj MNA, cov hlwb CD3+ raug qhib nrog plate-bound CD3 (5μg/ml), soluble CD28 (3μg/ml) thiab IL-2 (300 U/ml; Proleukin) rau 3 hnub. Tom qab 3 hnub, cov hlwb raug sau thiab ntxuav nrog 0.9% saline, thiab cov pellet raug khov. Kev suav cov hlwb tau ua los ntawm flow cytometry (Cytek Aurora; 3L-16V-14B-8R configuration) siv 123count eBeads.
Cov tshuaj rho tawm raws li tau piav qhia saum toj no. Cov tshuaj qhuav tau rov ua dua ntawm qhov concentration ntawm 4000 cell equivalents / μl. Tshuaj xyuas cov qauv los ntawm reversed-phase chromatography (1290 Infinity II, Agilent Technologies, Santa Clara, CA) thiab CORTECS T3 kem (2.1 × 150 mm, qhov loj me ntawm cov khoom me me 1.6-μm, qhov loj me ntawm qhov pore 120-Å; #186008500, Waters). Polar mass spectrometer (6470, Agilent), uas electrospray ionization ua haujlwm hauv hom zoo. Mobile theem A yog 0.1% formic acid (hauv H2O), mobile theem B yog 90% acetonitrile, 0.1% formic acid. Qhov LC gradient yog 0 txog 2 feeb rau 100% A, 2 txog 7.1 feeb rau 99% B, thiab 7.1 txog 8 feeb rau 99% B. Tom qab ntawd rov sib npaug lub kem nrog theem txav tau A ntawm tus nqi ntws ntawm 0.6 ml / feeb rau 3 feeb. . Tus nqi ntws yog 0.4 ml / feeb, thiab lub chamber kem raug cua sov rau 50 ° C. Siv MNA tus qauv tshuaj ntshiab (M320995, Toronto Research Chemical Company, North York, Ontario, Canada) los tsim lub sijhawm khaws cia (RT) thiab kev hloov pauv (RT = 0.882 feeb, kev hloov pauv 1 = 137 → 94.1, kev hloov pauv 2 = 137 → 92, Kev Hloov Pauv 3 = 137 → 78). Thaum tag nrho peb qhov kev hloov pauv tshwm sim ntawm lub sijhawm khaws cia kom raug, kev hloov pauv 1 yog siv rau kev ntsuas kom ntseeg tau tias muaj qhov tshwj xeeb. Tus qauv nkhaus ntawm MNA (Toronto Research Chemical Company) tau tsim los ntawm rau qhov kev sib xyaw ua ke ntawm cov kua tshuaj (1 mg/ml) kom tau txais cov qauv ntawm 0.1, 1.0, 10 thiab 100 ng/ml thiab 1.0 thiab 10μg/ml feem kua. Qhov txwv ntawm kev ntes yog 1 ng/ml, thiab cov lus teb linear yog ntawm 10 ng/ml thiab 10μg/ml. Txhua qhov kev txhaj tshuaj ntawm ob microliters ntawm cov qauv thiab tus qauv yog siv rau kev tshuaj xyuas LC/MS, thiab cov qauv tswj kev zoo sib xyaw ua ke raug khiav txhua yim qhov kev txhaj tshuaj kom ntseeg tau tias lub platform tshuaj xyuas ruaj khov. Cov lus teb MNA ntawm txhua cov qauv cell kho MNA yog nyob rau hauv qhov ntau ntawm cov kev sim. Kev tshuaj xyuas cov ntaub ntawv tau ua tiav siv MassHunter quantitative analysis software (v9.0, Agilent).
Lub cim αFR-CAR tiam thib ob tau coj los ntawm Song et al. (59). Hauv ntej, lub cim muaj cov ntsiab lus hauv qab no: CD8a tus thawj coj kab ke, tib neeg αFR-tshwj xeeb ib-saw hloov pauv fragment, CD8a hinge thiab transmembrane cheeb tsam, CD27 intracellular domain thiab CD3z intracellular domain. Tag nrho CAR kab ke tau synthesized los ntawm GenScript, thiab tom qab ntawd cloned rau hauv tiam thib ob lentiviral expression vector upstream ntawm GFP expression cassette siv los ntsuas qhov ua tau zoo ntawm transduction.
Lentivirus yog tsim los ntawm kev hloov pauv ntawm HEK293T hlwb [American Type Culture Collection (ATCC); loj hlob hauv Dulbecco's modified Eagle medium uas muaj 10% fetal bovine serum (FBS) thiab 1% PenStrep, thiab siv CAR-GFP vector thiab Cov plasmids ntim (psPAX2 thiab pMD2.G, Addgene) siv lipofection amine (Sigma-Aldrich). Cov supernatant uas muaj tus kab mob tau sau 48 thiab 72 teev tom qab transfection, lim, thiab concentrated los ntawm ultracentrifugation. Khaws cov concentrated viral supernatant ntawm -80 ° C kom txog thaum transduction.
PBMC raug cais tawm ntawm cov khoom siv sib cais leukocyte uas pub dawb noj qab haus huv (STEMCELL Technologies) los ntawm Ficoll gradient density centrifugation. Siv cov positive selection CD8 microbeads (Miltenyi) los cais cov hlwb CD8+ ntawm PBMC. Txhawb cov hlwb T nrog TransAct (Miltenyi) thiab hauv TexMACS medium [Miltenyi; ntxiv nrog 3% heat-inactivated human serum, 1% PenStrep thiab IL-2 (300 U/ml)]. Nees nkaum plaub teev tom qab txhawb, cov hlwb T tau hloov pauv nrog lentivirus (10 μl concentrated virus supernatant rau 106 lub hlwb). 1 txog 3 hnub tom qab hloov pauv ntawm Cytek Aurora (ntawm FSC (Forward Scatter)/SSC (Side Scatter), Singlet, GFP+), ntsuas qhov kev qhia GFP ntawm cov hlwb kom pom qhov ua tau zoo ntawm kev hloov pauv tsawg kawg 30%.
Cov hlwb CAR-T tau cog qoob loo rau 24 teev hauv Immunocult (STEMCELL Technologies; ntxiv nrog 1% PenStrep) nyob rau hauv cov xwm txheej hauv qab no: tsis kho, kho nrog 250 μM adenosine lossis 10 mM MNA. Tom qab kev kho mob ua ntej, cov hlwb CAR-T tau ntxuav nrog PBS thiab ua ke nrog 20,000 SK-OV-3 hlwb [ATCC; hauv McCoy 5A nruab nrab (Sigma-Aldrich) ntxiv nrog 10% FBS thiab 1% PenStrep ntawm 10: Tus effector rau lub hom phiaj piv ntawm 1 tau amplified hauv triplicate hauv cov nruab nrab Immunocult ntxiv. SK-OV-3 hlwb thiab SK-OV-3 hlwb lysed nrog digitalis saponin (0.5mg / ml; Sigma-Aldrich) tau siv ua cov kev tswj tsis zoo thiab zoo, raws li. Tom qab 24 teev ntawm kev cog ua ke, cov supernatant tau sau thiab cov lactate dehydrogenase (LDH) tau ntsuas raws li cov lus qhia ntawm cov chaw tsim khoom (LDH Glo Cytotoxicity Assay Kit, Promega). Cov LDH supernatant tau diluted 1:50 hauv LDH buffer. Feem pua ​​ntawm kev tua tau ntsuas siv cov mis hauv qab no: feem pua ​​ntawm kev tua = feem pua ​​ntawm kev kho / qhov siab tshaj plaws tua tus nqi x 100%, qhov twg feem pua ​​ntawm kev kho = co-culture-T cell xwb, thiab qhov siab tshaj plaws tua tus nqi = kev tswj hwm zoo-kev tswj hwm tsis zoo.
Raws li tau piav qhia hauv cov ntawv nyeem lossis cov ntaub ntawv thiab cov txheej txheem, siv GraphPad Prism 8, Microsoft Excel lossis R v3.6.0 rau kev tshuaj xyuas lej. Yog tias ntau cov qauv raug sau los ntawm tib tus neeg mob (xws li ascites thiab qog), peb siv kev xeem t khub lossis suav nrog tus neeg mob ua qhov cuam tshuam random hauv tus qauv linear lossis dav dav raws li qhov tsim nyog. Rau kev tshuaj xyuas metabolomics, qhov kev xeem tseem ceeb yog ua tiav hauv triplicate.
Yog xav paub ntxiv txog tsab xov xwm no, thov mus saib http://advances.sciencemag.org/cgi/content/full/7/4/eabe1174/DC1
Qhov no yog ib tsab xov xwm qhib rau kev nkag mus tau faib raws li cov nqe lus ntawm Creative Commons Attribution-Non-Commercial License, uas tso cai rau kev siv, kev faib tawm thiab kev tsim dua tshiab hauv txhua qhov chaw, tsuav yog qhov kev siv zaum kawg tsis yog rau kev lag luam thiab lub hauv paus yog tias cov haujlwm thawj yog qhov tseeb. Kev siv.
Lus Cim: Peb tsuas thov kom koj muab koj qhov chaw nyob email kom tus neeg uas koj pom zoo rau nplooj ntawv paub tias koj xav kom lawv pom email thiab nws tsis yog spam. Peb yuav tsis ntes cov chaw nyob email twg li.
Lo lus nug no yog siv los sim seb koj puas yog tus qhua thiab tiv thaiv kev xa tawm spam tsis siv neeg.
Marisa K. Kilgour (Marisa K. Kilgour), Sarah MacPherson (Sarah MacPherson), Lauren G. Zacharias (Lauren G. Zacharias), Abigail Eli Aris G. Watson (H. Watson), John Stagg (John Stagg), Brad H. Nelson (Brad H. Nelson), Ralph J.R. Dellini (J.R. G. Jones (Russell G. Jones), Phineas T. Hamilton (Phineas T.
MNA pab txhawb rau kev tiv thaiv kab mob ntawm cov qe ntshav T thiab sawv cev rau lub hom phiaj kho mob rau kev kho mob qog noj ntshav hauv tib neeg.
Marisa K. Kilgour (Marisa K. Kilgour), Sarah MacPherson (Sarah MacPherson), Lauren G. Zacharias (Lauren G. Zacharias), Abigail Eli Aris G. Watson (H. Watson), John Stagg (John Stagg), Brad H. Nelson (Brad H. Nelson), Ralph J.R. Dellini (J.R. G. Jones (Russell G. Jones), Phineas T. Hamilton (Phineas T.
MNA pab txhawb rau kev tiv thaiv kab mob ntawm cov qe ntshav T thiab sawv cev rau lub hom phiaj kho mob rau kev kho mob qog noj ntshav hauv tib neeg.
© 2021 American Association rau Advancement of Science. tag nrho cov cai. AAAS yog tus khub ntawm HINARI, AGORA, OARE, CHORUS, CLOCKSS, CrossRef thiab COUNTER. ScienceAdvances ISSN 2375-2548.