Jami ma konyo kom me lweny i kom two obedo gin ma pire tek i kom two ma mako kom (TME), ento ki jami manok ma ki kwanyo, gin ma nyutu gi pud pe ki ngeyo. Kany, wa kwano two ki T cells ki i two ki ascites pa lutwo ma tye ki two ma ki lwongo ni high-grade serous carcinoma (HGSC) me nyutu metabolome pa TME ma pat pat ni. Ascites ki tumor cells tye ki apoka poka mapol i kom jami ma gi tiyo kwede. Ka kiporo ki ascites, tumor-infiltrating T cells ni tye ki 1-methylnicotinamide (MNA) madwong. Kadi bed ni rwom me MNA i T cells tye malo, nyutu pa nicotinamide N-methyltransferase (enzyme ma weko methyl groups ki i S-adenosylmethionine cito i nicotinamide) tye ma ki keto i fibroblasts ki tumor cells. I tic, MNA weko T cells me cwalo cytokine tumor necrosis factor alpha. Pi meno, TME-derived MNA konyo i gwoko teko pa T cells ki nyutu gin ma twero konyo me cango two kanca pa dano.
Jami ma aa ki i kom two ma mako kom dano twero bedo ki adwogi ma gengo two ma mako kom dano, ki jami mapol ma ki nongo nyutu ni gin bene twero bedo gin ma weko two mede anyim (1). Medo i kom adwogi pa Warburg, tic manyen ni oyabe me nyutu kit ma cell me two ni loko kwede ki kit ma kom dano tiyo kwede ki kit ma kom dano gwoko kwede two ni (TME). Kwan ma kitimo i kom ler ma ki lwongo ni mouse ki T cells pa dano onyutu ni glutamine metabolism (2), oxidative metabolism (3) ki glucose metabolism (4) twero tic labongo kony pa dul kom ma pat pat. Jami mapol ma tye i yoo eni gengo tic pa T cells me lweny i kom two. Ki moko ni gengo coenzyme tetrahydrobiopterin (BH4) twero balo dongo pa T cells, ki medo pa BH4 i kom twero medo teko pa kom me lweny i kom two ma ki lwongo ni CD4 ki CD8. Medo ikom meno, tic pa kynurenine me gengo two ki twero lare ki miyo BH4 (5). I isocitrate dehydrogenase (IDH) mutant glioblastoma, cwalo pa enantiometabolic (R)-2-hydroxyglutarate (R-2-HG) gengo T cell activation, proliferation ki cytolysis Activity (6). I kare ni, ki nyutu ni methylglyoxal, gin ma aa ki i kom glycolysis, ki yubu ki cell ma gengo tic pa myeloid, ki T cell transfer pa methylglyoxal twero gengo tic pa effector T cell. I yat, dwoko piny me methylglyoxal twero loyo tic pa myeloid-derived suppressor cells (MDSC) ki medo tic pa checkpoint blockade therapy i kom ler ma ki lwongo ni mouse models (7). Kwan magi kacel nyutu tic ma pire tek pa TME-derived metabolites me doro tic ki tic pa T cell.
T cell dysfunction dong ki waco ne i kom two kanca me ic (8). Man tye pi kit ma kom dano tiyo kwede ma tye i hypoxia ki two ma pe rwate (9), ma kelo loko glucose ki tryptophan me doko jami ma calo lactic acid ki kynurenine. Lactate ma okato ki i cell dwoko piny rwom me interferon-γ (IFN-γ) ki kelo apoka poka i kin dul ma ki lwongo ni myelosuppressive (10, 11). Camo tryptophan gengo dongo pa T cell ki gengo tic pa T cell receptor (12-14). Kadi bed ni jami ma ki neno ni tye, tic mapol ma mako kit ma kom dano gwoko kwede kom dano ki timo i kom T cell culture ma ki tiyo kwede i kom ler ma ki lwongo ni in vitro kun kitiyo ki jami ma ki yubu maber, onyo ki keto i kom ler ma ki lwongo ni homologous mouse models in vivo, ma pe nyutu kit ma two kanca pa dano tye kwede ki kit ma kwo pa dano tye kwede.
Gin ma pol ma nyutu two kanca me ic tye nya pa ic ki nen pa ascites. Medo pii pa cell i ascites tye ki wat ki two ma odongo ki anyim marac (15). Malube ki ripot, dul man ma pat ni tye ki yamo manok, tye ki rwom ma malo me vascular endothelial growth factor (VEGF) ki indoleamine 2,3-dioxygenase (IDO), ki tye ki T regulatory cells ki myeloid inhibitory cells (15-18). Kabedo me tic pa ascites twero bedo pat ki pa two ma ki lwongo ni tumor kikome, ki dong loko T cells i kabedo me ic pe tye atir. Medo ikom meno, apoka poka ma pire tek ki apoka poka ma tye i kin ascites ki metabolites ma tye i kabedo ma two ni twero gengo donyo pa cell ma gwoko kom ki tic gi i kom two ni, ki kwan mukene mitte.
Me cobo peko eni, wa yubu yoo me poko cell ma rwate ki liquid chromatography tandem mass spectrometry (LC-MS/MS) me kwan kit cell ma pat pat (ma tye iye CD4 + ki CD8 + T cells) kacel ki i kin ki i kin two ma ki lwongo ni tumors. Wa tiyo ki yoo man kacel ki high-dimensional flow cytometry ki single-cell RNA sequencing (scRNA-seq) me miyo cal maber pa kit ma kom pa dano magi tye kwede. Yoo me tic man onyutu ni rwom me 1-methylnicotinamide (MNA) i kom two T cells omede, ki i kom jami ma ki temo i kom yat onyutu ni tic pa MNA i kom tic pa T cells onongo pe ki ngeyo con. I kom lok, yoo man nyutu kit ma two ma mako kom ki cell ma gwoko kom gi rwate kwede, ki miyo ngec ma pat pat i kom jami ma gwoko kom, ma twero bedo me kony pi cango two kanca ma mako ic ma ki lwongo ni T cell.
Watiyo ki high-dimensional flow cytometry me kwano wel glucose ma ki kwanyo [2-(N-(7-nitrophenyl-2-oxa-1,3-diaza-4-yl)amino)-2-deoxyglucose (2-NBDG) ki tic pa mitochondrial [MitoTracker Deep Red (MT DR)] (207, 2019) tye tung cing lanyut ma poko kin dul kom ma gwoko kom ki dul kom ma kelo two (Table S2 ki Figure S1A). Kwedo man onyutu ni ka kiporo ki T cells, ascites ki tumor cells tye ki rwom me gamo cukari ma malo, ento tye ki apokapoka manok i tic pa mitochondrial. Kit ma dul kom ma ki lwongo ni tumor cells [CD45-EpCAM (EpCAM)+] gamo kwede tye tyen adek onyo angwen ma kato pa T cells, ki wel glucose ma CD4 + T cells gamo tye tyen 1.2 pa CD8 + T cells, ma nyutu ni Tumor infiltrating lymphocytes (TIL) tye ki miti ma pat pat kadi wa i TAME acel-lu (lanyut 1). Ka i poro, tic pa mitochondrial i kom cell ma tye ki two ni rom ki pa CD4 + T cells, ki tic pa mitochondrial pa cell aryo ni tye malo loyo pa CD8 + T cells (lanyut 1B). I kom lok, adwogi eni nyutu rwom me tic pa kom. Tic pa cell me two ma mako kom dano tye malo loyo pa CD4 + T cells, ki tic pa CD4 + T cells tye malo loyo pa CD8 + T cells. Kadi bed ni adwogi magi tye i kin dul cell, pe tye apokapoka mo ma rwate i kit ma CD4 + ki CD8 + T cells tiyo kwede onyo kit ma gi rwate kwede i ascites ka kiporo ki tumors (lanyut 1C). Ka i poro, i dul CD45-cell, wel EpCAM+ cells i kom two ni omede ka kiporo ki ascites (lanyut 1D). Wa bene waneno apokapoka ma nen maber i kin EpCAM+ ki EpCAM- cell components. EpCAM+ (tumor) cells tye ki glucose ma malo ki tic pa mitochondrial ma loyo EpCAM- cells, ma tye malo loyo tic pa fibroblasts i tumor cells i TME (lanyut 1, E ki F).
(A ki B) Kit ma rwom me ryeny (MFI) me gamo glucose (2-NBDG) (A) ki tic pa mitochondrial pa CD4 + T cells (MitoTracker makwar macol) (B) cal ma nyutu (tung lacuc) ki ngec ma ki coyo (tung lacuc), CD8 + T cells ki EpCAM + CD45-tumor cells ki tumor cells ki tumor. (C) Kit ma CD4 + ki CD8 + cells (me CD3 + T cells) tye kwede i ascites ki tumor. (D) Kit ma EpCAM + tumor cells tye kwede i ascites ki tumor (CD45−). (E ki F) EpCAM + CD45-tumor ki EpCAM-CD45-matrix glucose uptake (2-NBDG) (E) ki tic pa mitochondrial (MitoTracker makwar macol) (F) cal ma nyutu (tung lacuc) ki ngec ma ki coyo (tung lacuc) Ascites ki tumor cells. (G) cal ma nyutu CD25, CD137 ki PD1 ma ki nyutu ki flow cytometry. (H ki I) CD25, CD137 ki PD1 ma ki nyutu i kom CD4 + T cells (H) ki CD8 + T cells (I). (J ki K) Naive, central memory (Tcm), effector (Teff) ki effector memory (Tem) kit ma ki nyutu kwede CCR7 ki CD45RO. Cal ma nyutu (tung lacuc) ki ngec ma ki coyo (tung lacuc) pa CD4 + T cells (J) ki CD8 + T cells (K) i ascites ki tumors. P ma ki nongo ki ikom t-test (*P<0.05, **P<0.01 ki ***P<0.001). Lain ni nyutu lutwo ma ki rwate (n = 6). FMO, ler ma ryeny ki kwanyo acel; MFI, rwom me ryeny ma tye i kin piny.
Kwedo lok mukene onyutu apokapoka mukene ma pire tek i kin kit ma T cell nen kwede maber. Gin ma ki keto (lanyut 1, G onyo I) ki gin ma ki lwongo ni effector memory (lanyut 1, J ki K) i kom two ma ki lwongo ni tumors pol kare loyo ascites (dul pa CD3 + T cells). I yo acel-lu, nyamo kit ma kom dano nen kwede ki nyutu pa lanyut me tic (CD25 ki CD137) ki lanyut me rweny [programmed cell death protein 1 (PD1)] onyutu ni kadi bed ni kit me tic pa dul kom magi pat pat (lanyut S1, B me o i E) , ento pe ki neno apokapoka mo madit i kin jami ma ki ngeyo, nyo gin ma ki poyo wic (lanyut S1, F1) AN). Adwogi eni ki moko ne kun kitiyo ki yoo me pwonyo jami ma ki lwongo ni machine learning me poko kit ma cell nen kwede (21), ma bene onyutu ni tye cell mapol ma ki lwongo ni bone marrow cells (CD45 + / CD3- / CD4 + / CD45RO +) i ascites pa latwo (Figure S2A ). I kin dul cell ma ki ngeyo, dul cell ma ki lwongo ni myeloid cell ni onyutu rwom me cukari ma malo ki tic pa mitochondrial (lanyut S2, B onyo G). Adwogi eni nyutu apokapoka ma tek i kin dul kom ma pat pat ma ki nongo i ascites ki tumors i kom lutwo HGSC.
Peko madit ma tye i niang kit ma TIL tiyo kwede i kom dano tye ni omyera ki poko T cell ma tye maleng, maber ki wel ma oromo ki i kom two ma mako kom. Kwan ma ki tiyo cok cok ni onyutu ni poko ki medo jami ma ki lwongo ni bead enrichment ma jenge i kom flow cytometry twero kelo alokaloka i kit ma cell metabolite nen kwede (22-24). Me cobo peko eni, wa yubu yoo me medo jami ma ki lwongo ni bead enrichment me poko ki poko TIL ki i kom two kanca me ic ma ki kwanyo ki i kom dano ma peya ki temo ki LC-MS/MS (nen jami ma ki tiyo kwede ki yoo; cal 2A). Me neno adwogi pa yub man i kom alokaloka me metabolite, wa poro metabolite profiles pa T cells ma ki tiyo kwede ki lumiyo kony ma kom gi yot inge poko kin nyig cam ma malo ni ki cell ma pe ki poko kin nyig cam ento odong i kom pee. Kwedo rwom me tic man onongo ni tye wat ma malo i kin jami aryo eni (r = 0.77), ki kit ma ki dwoko kwede cen pa dul pa 86 metabolites ni tye ki dwoko cen ma malo (lanyut 2B). Pi meno, yoo eni twero timo kwedo ma opore i kom cell ma tye ka nongo cell type enrichment, ma miyo yoo maber me nongo metabolites ma pat pat i HGSC, ma miyo dano nongo ngec matut i kom cell specificity Sexual metabolism program.
(A) cal ma ki nyutu me dongo pa magnetic bead. Mapwod pi ki temo ki LC-MS/MS, cell ni obi kato ki i dul adek ma lubo me magnetic bead enrichment onyo obi bedo i kom pee. (B) Adwogi pa kit ma ki medo kwede i kom wel jami ma ki kwanyo ki i kom dano. Wel ma ki nongo ki i kin jami adek ma ki pimo pi kit me dongo acel acel ± SE. Lain ma ryeny ni nyutu wat ma tye i kin 1:1. Intra-class correlation (ICC) me rwom ma ki dwoko cen ma ki nyutu i lanyut me axis. NAD, nicotinamide adenine dinucleotide. (C) cal ma ki nyutu kit ma tic pa latwo tye ka nyamo kwede jami ma mako kom dano. Two ma ki lwongo ni Ascites onyo two ma ki lwongo ni tumors ki kwanyo ki bot lutwo ki ki gwoko gi i piny ma lyet. Dul matidi pa sampol acel acel ki kwano ki flow cytometry, kun sampol mukene ni ki temo tyen adek me medo CD4+, CD8+ ki CD45- cells. Ki kwano dul kom magi kun kitiyo ki LC-MS/MS. (D) Map me lyeto pa jami ma ki lwongo ni metabolite ma ki ngeyo. Dendrogram nyutu kit ma Ward oketo kwede bor pa Euclidean i kin jami ma ki nyutu. (E) Kwedo gin ma pire tek (PCA) me map me metabolite, ma nyutu jami adek ma ki dwoko cen, lanyut ma aa ki bot latwo acel ki kubu ki lain. (F) PCA pa jami ma ki kwanyo ki i kom latwo (ie, kun kitiyo ki dul ma pe rwate); kit ma ki nyutu kwede ni ki gengo ki dul ma ki lwongo ni convex hull. PC1, gin ma pire tek 1; PC2, gin ma pire tek me 2.
Ma lubo, wa tiyo ki yoo me dongo man me nyamo 99 metabolites i CD4 +, CD8 + ki CD45-cell fractions i primary ascites ki tumors pa lutwo abicel me HGSC (lanyut 2C, cal S3A ki Table S3 ki S4). Wel dano ma ki miito ni tye 2% onyo 70% pa dul kom makwo ma ki kwanyo, ki wel dul kom ni pat pat i kin lutwo. Iyonge poko dul ma ki lwongo ni beads, dul ma ki medo ma mite (CD4+, CD8+ onyo CD45-) kwako 85% pa cell makwo weny i kom ma ki kwanyo ni. Yoo me dongo man miyo wa twero me nyamo wel cell ma aa ki i kom dano ma tye ki two ma mako kom, ma pe twere me tiimo ki i kom jami mapol. Nono yoo man, wamoko ni l-kynurenine ki adenosine, jami aryo magi ma ki ngeyo maber ma gengo two ni onongo tye malo i tumor T cells onyo tumor cells (lanyut S3, B ki C). Pi meno, adwogi eni nyutu gen ki kero pa cell separation ki diro me mass spectrometry me nongo jami ma pire tek i kom dano.
Kwedo lok wa bene onyutu apoka poka ma tek i kin dul kom ma i kin lutwo (lanyut 2D ki cal S4A). I kom lok, ka kiporo ki lutwo mukene, lutwo 70 onyutu kit ma kom gi tiyo kwede ma pat pat (lanyut 2E ki cal S4B), ma nyutu ni twero bedo ni tye apokapoka madit i kin lutwo. Ber me poyo ni ka kiporo ki lutwo mukene (1.2 onyo 2 liters; Table S1), wel ascites ma ki nongo i kom lutwo 70 (80 ml) onwongo nok. Kit me gwoko apokapoka i kin lutwo i kare me kwedo jami ma pire tek (me labolle, tic ki kwedo jami ma pe rwate) nyutu alokaloka ma rwate i kin dul cell, ki dul cell ki/onyo kabedo ma gi tye iye ki keto kacel ma lubu kit ma metabolite nen kwede (lanyut 2F ). Kwedo jami ma ki lwongo ni metabolites acel acel omoko adwogi eni ki onyutu apokapoka madit i kin dul cell ki kabedo ma gikwo iye. Ber me poyo ni apoka poka madit ma ki neno tye MNA, ma pol kare tye ki CD45- cells ki CD4+ ki CD8+ cells ma gi donyo i tumor (lanyut 3A). Pi CD4 + cells, adwogi man nen maber, ki MNA ma tye i CD8 + cells bene nen calo tye ka nongo adwogi marac ki kabedo. Ento, man pe pire tek, pien dano adek keken i kin lutwo abicel aye ki twero pimo gi pi CD8+ me two. Medo i kom MNA, i dul cell ma pat pat i ascites ki tumors, jami mukene ma ki ngeyo maber i TIL bene tye ki jami mapol ma pat pat (lanyut S3 ki S4). Pi meno, ngec eni nyutu jami ma twero konyo kom me lweny i kom two pi medo kwedo ngec.
(A) Gin ma ki medo i kom MNA i CD4+, CD8+ ki CD45- cells ki i ascites ki tumor. Gin ma ki nyutu i bok ni nyutu rwom ma tye i kin piny (line), rwom ma tye i kin dul angwen (gin ma ki lwongo ni frame hinge) ki rwom ma tye i kin jami ma tye i kin piny (frame whisker). Kit ma ki waco kwede i jami ma ki tiyo kwede ki yoo me tic pa latwo, tii ki wel limma pa latwo me nongo wel P (*P<0.05 ki **P<0.01). (B) cal ma nyutu kit ma MNA tiyo kwede (60). Jami ma mako kom: S-adenosyl-1-methionine; SAH, S-adenosine-1-homocysteine; NA, nicotinamide; MNA, 1-methylnicotinamide; 2-PY, 1-methyl- 2-pyridone-5-carboxamide; 4-PY, 1-methyl-4-pyridone-5-carboxamide; NR, nicotinamide ribose; NMN, nicotinamide mononucleotide. Enzymes (ma lum): 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 pa scRNA-seq pa ascites (macol) ki two (makwar; n = lutwo 3). (D) NNMT ma ki nyutu i dul cell ma pat pat ma ki nongo kun kitiyo ki scRNA-seq. (E) Nyutu pa NNMT ki AOX1 i SK-OV-3, human embryonic kidney (HEK) 293T, T cells ki MNA-treated T cells. Gin ma ki lwongo ni folded expression ni ki nyutu ma rwate ki SK-OV-3. Kit ma ki nyutu kwede ki SEM ki nyutu (n = 6 lumiyo kony ma kom gi yot). Ct ma kato 35 ki ngeyo ni pe kitwero nongo ne (UD). (F) Nyutu pa SLC22A1 ki SLC22A2 i SK-OV-3, HEK293T, T cells ki T cells ma ki tiyo kwede ki 8mM MNA. Gin ma ki lwongo ni folded expression ni ki nyutu ma rwate ki SK-OV-3. Kit ma ki nyutu kwede ki SEM ki nyutu (n = 6 lumiyo kony ma kom gi yot). Ct ma kato 35 ki ngeyo ni pe kitwero nongo ne (UD). (G) Cell MNA ma tye i kom T cells ma kom gi yot ma ki keto i tic ingee cawa 72 me gwoko ki MNA. Kit ma ki nyutu kwede ki SEM ki nyutu (n = 4 lumiyo kony ma kom gi yot).
MNA ki yubu ne ki loko dul me methyl ki i S-adenosyl-1-methionine (SAM) me doko nicotinamide (NA) ki nicotinamide N-methyltransferase (NNMT; cal 3B). NNMT ki nyutu ne i kom two ma pat pat pa dano ki bene rwate ki nyaa, donyo ki nyaa (25-27). Me niang maber kama MNA oa ki iye i T cells i TME, watiyo ki scRNA-seq me nyutu kit ma NNMT nyute kwede i kin cell ma tye i ascites ki tumors pa lutwo adek ma tye ki HGSC (Table S5). Kwedo i kom cell ma romo 6,500 onyutu ni i kabedo ma tye ki ascites ki tumor, nyutu NNMT onongo ki gengo i kom fibroblast ki tumor cell ma ki tamo ni (lanyut 3, C ki D). Ber me ngeyo ni pe tye gin mo ma nyutu NNMT i kin dano ma nyutu PTPRC (CD45 +) (lanyut 3D ki cal S5A), ma nyutu ni MNA ma kinongo i dul kom ma ki lwongo ni metabolite ni ki keto i T cells. Nyutu pa aldehyde oxidase 1 (AOX1) loko MNA me doko 1-methyl-2-pyridone-5-carboxamide (2-PYR) onyo 1-methyl-4-pyridone-5-carboxamide (4- PYR); cal 3B) bene ki keto i kom wel pa fibroblasts ma nyutu COL1A1 (lanyut S5A), ma kacel nyutu ni T cells gi pe ki kero me tic ki MNA macon. Kit ma MNA-related genes ni nyutu kwede ki moko kun kitiyo ki cell me aryo ma pe jenge i kom ngat mo ki bot lutwo HGSC (lanyut S5B; n = 6) (16). Medo ikom meno, kwan ma ki lwongo ni quantitative polymerase chain reaction (qPCR) i kom T cells ma kom gi yot ma ki miyo igi MNA onyutu ni ka kiporo ki SK-OV-3 ovarian tumor cells, NNMT onyo AOX1 onongo pe ki nyutu (lanyut 3E). Adwogi ma pe ki tamo ni nyutu ni MNA twero bedo ni ki kwanyo ki i fibroblasts onyo tumors i T cells ma tye cok kwede i TME.
Kadi bed ni jami ma ki yero ni tye iye dul pa organic cation transporters 1 onyo 3 (OCT1, OCT2 ki OCT3) ma ki coyo ki soluble carrier 22 (SLC22) family (SLC22A1, SLC22A2 ki SLC22A3), dul ma twero tero MNA pud pe ki ngeyo (28) . QPCR pa mRNA ma aa ki bot dano ma kom gi yot onyutu rwom ma piny pa SLC22A1 ento rwom ma pe nen pa SLC22A2, ma moko ni onongo dong ki coyo con i kwan (lanyut 3F) (29). Ka i poro, SK-OV-3 ovarian tumor cell line onyutu rwom ma malo pa jami aryo ni weng (lanyut 3F).
Me temo ka ce T cells twero bedo ki kero me mako MNA ma pat, ki gwoko T cells ma kom gi yot pi cawa 72 i kare ma tye ki MNA ma pat pat. Ka pe tye MNA ma aa ki woko, gin ma tye i cell pa MNA pe kitwero nongo ne (lanyut 3G). Ento, T cell ma ki tiyo kwede ma ki tiyo kwede ki exogenous MNA onyutu medo pa MNA i cell ni, nio wa i 6 mM MNA (lanyut 3G). Adwogi man nyutu ni kede rwom ma lapiny me nyutu jami ma tero ki peke pa enzyme ma loyo tic pa MNA i cell, TIL pud twero kwanyo MNA.
Kit ma jami ma kwo i T cells pa lutwo ki in vitro MNA absorption experiments ni medo kero pa cancer-associated fibroblasts (CAF) me cwalo MNA ki tumor cells twero doro kit ma TIL nen kwede ki tic pa TIL. Me moko adwogi pa MNA i kom T cells, ki keto T cells ma kom gi yot ma ki miyo i tic i vitro i kare ma MNA tye onyo pe, ki kit ma gi nya kwede ki kit ma gi yubu kwede cytokine ki kwano. Iyonge nino 7 me medo MNA i rwom ma malo, wel dano ma omede tyen aryo odok piny, kun guvu obedo ka mede i rwom weny (lanyut 4A). Medo ikom meno, tic ki MNA ma aa ki woko okelo dongo pa CD4 + ki CD8 + T cells ma nyutu tumor necrosis factor-α (TNFα; cal 4B). Ka i poro, yubu IFN-γ i kin cell onongo odok piny tutwal i CD4 + T cells, ento pe i CD8 + T cells, ki onongo pe tye alokaloka mo madit i interleukin 2 (IL-2; cal 4, C ki D). Pi meno, enzyme-linked immunosorbent assay (ELISA) me supernatants ki ikom MNA-treated T cell cultures onyutu dongo ma pire tek i TNFα, dwoko piny i IFN-γ, ki pe tye alokaloka mo i IL-2 (lanyut 4, E to G). . Dwoko piny pa IFN-γ nyutu ni MNA twero bedo ki tic me gengo tic pa T cells ma gengo two. Me temo adwogi pa MNA i kom T cell-mediated cytotoxicity, chimeric antigen receptor T (FRα-CAR-T) cells ma tye ka tic ki folate receptor α ki CAR-T (GFP) ma ki doro ki green fluorescent protein (GFP) -CAR-T) cells ki yubu ki donor ma kome yot peripheral blood mononuclear cells (PBMC). CAR-T cells ki pito pi cawa 24 i kare ma MNA tye iye, ki dong ki pito kacel ki human SK-OV-3 ovarian tumor cells ma nyutu folate receptor α i rwom me effector ki target ratio me 10:1. Yat me MNA okelo dwoko piny ma pire tek i tic me neko FRα-CAR-T cells, ma onongo rom ki FRα-CAR-T cells ma ki miyo iye adenosine (lanyut 4H).
(A) Wel cell ma twero kwo ki wel cell ma tye ka mede (PD) ma aa ki i kom jami ma ki pito i nino me 7. Bar graph nyutu wel + SEM pa dano abicel ma kom gi yot. Nyutu ngec ma aa ki n = 3 ma ki temo ma pat pat. (B to D) CD3/CD28 ki IL-2 ki tiyo kwede me tic ki T cells i rwom me MNA gi pi nino 7. Mapwod pi ki temo, ki juku cell ki PMA/ionomycin ki GolgiStop pi cawa 4. TNFα (B) nyutu i T cells. Cal ma ki nyutu (tung lacuc) ki ngec ma ki coyo (tung lacuc) pa TNFα ma ki nyutu i cell makwo. IFN-γ (C) ki IL-2 (D) nyutu i T cells. Kit ma cytokines nyute kwede ki pimo ki flow cytometry. cal ma ki nyutu ni nyutu wel (n = 6 dano ma kom gi yot) + SEM. Tii ki yoo acel me nyamo apoka poka ki dwoko cen rwom (*P<0.05 ki **P<0.01) me nongo wel P. Nyutu ngec ma aa ki n = 3 ma ki temo ma pat pat. (E to G) CD3/CD28 ki IL-2 ki tiyo kwede me tic ki T cells i rwom me MNA gi pi nino 7. Gin ma ki tiyo kwede ni ki kwanyo ma peya ki ingee cawa 4 me tic ki PMA/ionomycin. Ki pimo dwong pa TNFα (E), IFN-γ (F) ki IL-2 (G) ki ELISA. cal ma ki nyutu ni nyutu wel (n = 5 dano ma kom gi yot) + SEM. wel P ma ki nongo kun kitiyo ki yoo acel me nyamo apoka poka ki dwoko cen rwom (*P<0.05). Lain ma ki coyo ni nyutu rwom me nongo gin ma ki nongo ni. (H) Cell lysis assay. FRα-CAR-T onyo GFP-CAR-T cells ki medo gi ki adenosine (250μM) onyo MNA (10 mM) pi cawa 24, onyo ki weko labongo yat (Ctrl). Ki pimo wel ma ki neko pa SK-OV-3 cells. wel P ma ki nongo ki Welch t test (*P<0.5 ki **P<0.01).
Me nongo niang matut ikom kit ma MNA-dependent TNFα nyutu kwede, alokaloka ma tye i TNFα mRNA pa MNA-treated T cells ki kwano (lanyut 5A). T cell ma kom gi yot ma ki miyo igi MNA onyutu ni rwom me coc pa TNFα omede tyen aryo, ma nyutu ni MNA jenge i kom rwom me coc pa TNFα. Me kwedo kit ma twero bedo kwede, jami aryo ma ki ngeyo ma loyo TNFα, ma ki lwongo ni activated T cell nuclear factor (NFAT) ki specific protein 1 (Sp1), ki kwano me gamo MNA ma mako TNFα promoter ( 30). Gin ma ki lwongo ni TNFα promoter tye ki 6 ma ki ngeyo ni NFAT binding sites ki 2 Sp1 binding sites, ma gi rwate i kabedo acel [-55 base pairs (bp) from the 5'cap] (30). Chromatin immunoprecipitation (ChIP) onyutu ni ka ki tiyo kwede ki MNA, rwate pa Sp1 ki TNFα promoter omede tyen adek. Medo NFAT bene omede ki oo i rwom ma lamal (lanyut 5B). Lok ma ki nongo ni nyutu ni MNA doro nyutu pa TNFα kun wok ki i coc pa Sp1, ki i rwom ma lapiny nyutu pa NFAT.
(A) Ka kiporo ki T cells ma ki pito labongo MNA, alokaloka me TNFα ma ki nyutu i T cells ma ki pito ki MNA. Kit ma ki nyutu kwede ki SEM ki nyutu (n = 5 lumiyo kony ma kom gi yot). Nyutu ngec ma aa ki n = 3 ma ki temo ma pat pat. (B) TNFα promoter pa T cells ma ki tiyo kwede ki onyo labongo 8 mM MNA inge NFAT ki Sp1 ki rubu ki (Ctrl) ki PMA/ionomycin pi cawa 4. Immunoglobulin G (IgG) ki H3 ki tiyo kwede ma calo gin ma pe ber ki maber pi immunoprecipitation, kit meno. Kwano wel pa ChIP onyutu ni rwate pa Sp1 ki NFAT i kom TNFα promoter i MNA-treated cells omede tyen mapol ka kiporo ki control. Nyutu ngec ma aa ki n = 3 ma ki temo ma pat pat. P value ma ki nongo ki t-tests mapol (*** P <0.01). (C) Ka kiporo ki ascites pa HGSC, T cells (ma pe kelo two) onyutu medo rwom me tic pa TNF i kom two. Kala ni nyutu lutwo ma pat pat. Cell ma ki nyutu ni ki kwanyo gi labongo rwom me 300 ki ki jitter me gengo overdrawing (** Padj = 0.0076). (D) Kit ma ki yubu kwede MNA pi two kanca me ic. MNA ki yubu i cell me two ki fibroblasts i TME ki ki kwanyo ki i cell me T. MNA medo rwate pa Sp1 ki TNFα promoter, ma weko dongo TNFα transcription ki TNFα cytokine production. MNA bene kelo dwoko piny pa IFN-γ. Gengo tic pa T cell kelo dwoko piny kero me neko ki dongo pa two oyoto.
Malube ki lok ma ki waco, TNFα tye ki adwogi ma cung i kom two ma mako ic ki two ma mako ic, ento tye ki tic ma ki ngeyo maber me kelo dongo ki nya pa two ma mako ic (31-33). Malube ki ripot, dwong pa TNFα i ascites ki tumor tissues i kom lutwo ma tye ki two kanca me ic tye malo loyo ma tye i kom ma pe kelo two (34-36). I kom kit ma tic kwede, TNFα twero doro tic, tic ki dongo pa cell me remo ma ler, ki loko kit ma cell me two kanca nen kwede (37, 38). Ma rwate ki gin ma kinongo ni, rwom me gene ma pat pat onyutu ni TNF onongo tye ka mede i T cells i kom ma tye ki two ka kiporo ki ascites (lanyut 5C). Medo pa TNF onongo nen i kom T cell ma pe ki keto i kom cell (lanyut S5A). I agiki ne, ngec eni miiyo kony i kom neno ni MNA tye ki jami ma gengo two ki kelo two i HGSC.
Coyo nying ma ki lwongo ni Fluorescent labeling ma jenge i kom flow cytometry odoko yoo ma pire tek me kwan kit ma TIL tiyo kwede. Kwan magi onyutu ni ka kiporo ki dul kom ma ki lwongo ni peripheral blood lymphocytes onyo T cells ma aa ki i dul kom ma ki lwongo ni secondary lymphoid organs, murine ki human TIL tye ki rwom ma malo me gamo glucose (4, 39) ki rwenyo tic pa mitochondrial mot mot (19, 40). Kadi bed ni waneno adwogi marom i kwan man, yub ma pire tek tye me poro kit ma cell me two ki TIL tiyo kwede ki i kom two acel ma ki kwanyo. Ma rwate ki lok mogo macon ni, two (CD45-EpCAM +) cells ma aa ki i ascites ki tumors tye ki glucose ma malo loyo CD8 + ki CD4 + T cells, ma nyutu ni glucose ma malo pa tumor cells ki twero poro ki T cells. Tam me pyem pa T cell. TME. Ento, tic pa mitochondrial pa cell me two tye malo loyo pa CD8 + T cells, ento tic pa mitochondrial rom ki pa CD4 + T cells. Adwogi eni jingo lok ma tye ka nen ni tic pa jami ma kwako kom dano pire tek pi cell me two (41, 42). Gin bene owaco ni CD8 + T cells twero bedo ki peko me oxidative dysfunction ma loyo CD4 + T cells, onyo ni CD4 + T cells twero tic ki jami ma kelo carbon ma pat ki glucose me gwoko tic pa mitochondrial (43, 44). Omyera ki nge ni wan pe waneno apoka poka mo i kom rwom me cukari onyo tic pa mitochondrial i kin CD4 + T effectors, T effector memory ki T central memory cells i ascites. I yo acel-lu, kit ma CD8 + T cells gipoke kwede i kom two pe tye ki lok mo i kom alokaloka i rwom me gamo glucose, ma nyutu apokapoka madit i kin T cells ma ki dongo i vitro ki human TIL in vivo (22). Gin ma ki neno ni bene ki moko ki tic ki rwom me cell ma pe ki apokapoka, ma bene onyutu ni CD45 + / CD3- / CD4 + / CD45RO + cells ma tye ki glucose ma malo ki tic pa mitochondrial ma loyo tumor cells tye mapol ento tye ki Metabolic active cell population. Wel dano ni twero nyutu wel dano ma ki tamo ni tye i kom myeloid suppressor cells onyo plasmacytoid dendritic cells ma ki nongo i scRNA-seq analysis. Kadi bed ni gin aryo ni ki coyo gi i kom two ma mako ic pa dano [45], gin pud mito tic mukene me tito dul kom ma ki lwongo ni myeloid.
Kadi bed ni yoo me flow cytometry twero gonyo apokapoka ma tye i kin glucose ki oxidative metabolism i kin dul cell, jami ma ki yubu ki glucose onyo jami mukene ma kelo carbon pi mitochondrial metabolism i TME pwod pe ki ngeyo. Miiyo bedo tye onyo pe pa jami ma ki lwongo ni metabolites bot dul TIL ma kimiyo mito ni ki kwany wel cell ki i kom ma ki kwanyo. Pi meno, yoo me dongo cell wa kacel ki mass spectrometry twero miyo ngec i kom jami ma dongo ma pat pat i T cells ki tumor cell populations i kom labol pa lutwo. Kadi bed ni yoo man tye ki adwogi maber i kom poko kin cell ma kitiyo kwede ki fluorescence, jami mogo ma ki lwongo ni metabolite libraries twero bedo ki peko pi bedo maber ki/onyo rwom me loko oyotoyot (22). Kadi bed kumeno, yoo wa ni otwero nongo jami aryo ma ki ngeyo ma gengo two, adenosine ki kynurenine, pien gi pat pat i kin dul ma ki kwanyo.
Kwedo lok ma mako two ma mako kom ki dul kom ma ki lwongo ni TIL miyo ngec mukene i kom tic pa jami ma kwako kom i TME me ic. Mukwongo, kun wa tiyo ki flow cytometry, wa nongo ni onongo pe tye apokapoka mo i tic pa mitochondrial i kin two ki CD4 + T cells. Ento, LC-MS/MS ma ki kwano onyutu alokaloka madit i wel jami ma ki lwongo ni metabolites i kin dano magi, ma nyutu ni lok ma mako TIL metabolism ki tic pa metabolic ne mito gonyo ne maber. Me ariiyo, MNA tye gin ma tye ki apoka poka madit i kin CD45-cells ki T cells i ascites, ento pi tumors. Pi meno, poko kin dul ki kabedo pa buru twero bedo ki adwogi ma pat pat i kom tic pa TIL, ma nyutu apokapoka ma twero bedo tye i kabedo ma ki miyo. Me adek, nyutu pa enzyme ma kelo MNA NNMT tye ma ki keto i CAF, ma obedo cell me two ma nok, ento rwom me MNA ma ki twero nongo ki neno i cell me T ma aa ki i two. Nyutu pa NNMT i ovarian CAF tye ki adwogi ma ki ngeyo me kelo two kanca, ma tye pi kelo dongo pa CAF, donyo pa two ki nya pa two (27). Kadi bed ni rwom me TIL tye ma rwate, nyutu pa NNMT i CAF tye macok ki Cancer Genome Atlas (TCGA) mesenchymal subtype, ma tye ki rwate ki rwom me anyim marac (27, 46, 47). Me agiki ne, nyutu pa enzyme AOX1 ma tye ki twero me balo MNA bene tye i kin lwak CAF, ma nyutu ni T cells pe ki kero me loko MNA. Adwogi eni miiyo kony i kom tam ni kadi bed ni tic mukene mitte me moko gin ma kinongo ni, rwom ma malo pa MNA i T cells twero nyutu ni tye kabedo ma gengo two CAF.
Ki miiyo rwom ma piny pa MNA transporters ki rwom ma pe nen pa jami ma pire tek ma tye i MNA metabolism, bedo pa MNA i T cells pe ki tamo. NNMT onyo AOX1 pe ki twero nongo ne ki scRNA-seq analysis ki qPCR ma ki keto i kin dul aryo ma pat pat. Adwogi eni nyutu ni MNA pe ki yubu ki T cells, ento ki kwanyo ki i TME ma orumowa. Temo ma kitimo i kom ler ma ki lwongo ni T cells gi maro coko MNA ma aa ki woko.
Kwano wa ma kitimo i kom ler ma ki lwongo ni in vitro onyutu ni exogenous MNA kelo nyutu pa TNFα i T cells ki medo rwate pa Sp1 ki TNFα promoter. Kadi bed ni TNFα tye ki tic me gengo two ki two, i two kanca me ic, TNFα twero kelo dongo pa two kanca me ic (31-33). Kwanyo TNFα i kom cell me ovarian tumor onyo kwanyo TNFα i kom ler ma ki lwongo ni mouse models twero medo TNFα-mediated inflammatory cytokine production ki gengo dongo pa tumor (32, 35). Pi meno, i lok man, TME-derived MNA twero tic calo pro-inflammatory metabolite kun tiyo ki TNFα-dependent mechanism kun wok ki i autocrine loop, ma weko dongo ki nya pa two cancer me ic (31). Malube ki gin ma twero time ni, TNFα blockade tye ka kwan calo gin ma twero konyo two kanca me ic (37, 48, 49). Medo ikom meno, MNA balo teko pa CAR-T cells i kom ovarian tumor cells, ma miyo caden mukene pi MNA-mediated immune suppression. Ka ki keto kacel, adwogi eni nyutu kit ma two ma ki lwongo ni tumors ki CAF cells gi cwalo kwede MNA i TME ma tye woko. Ki i (i) TNF-ma okelo dongo pa two ma mako ic ki (ii) MNA-ma okelo T cell cytotoxic activity inhibition, man twero bedo ki adwogi marac i kom two (lanyut 5D).
Me agiki ne, no kitiyo ki dul ma ki lwongo ni rapid cell enrichment, single-cell sequencing ki metabolic profiling, kwan man onyutu apokapoka madit i kin immunometabolomic i kin tumor ki ascites cells i kom lutwo HGSC. Kwedo lok man onyutu ni tye apoka poka i kom rwom me cukari ki tic pa mitochondrial i kin T cells, ki onyutu MNA calo gin ma pe tiyo kene me gwoko kom. Lok ma ki nongo ni tye ki adwogi i kom kit ma TME gudo kwede tic pa T cell i two kanca pa dano. Kadi bed ni pyem ma rwate pi cam ma tye i kin T cells ki cancer cells dong ki tito, jami ma ki lwongo ni metabolites bene twero tic calo jami ma doro dongo pa two ma mako kom ki twero gengo tic pa kom ma gwoko kom. Lok mukene ma ki waco i kom tic pa jami ma ki lwongo ni regulatory metabolites ni twero yabo yoo mukene me medo teko pa kom ma lweny i kom two ma mako kom dano.
Labol pa latwo ki ngec ma mako yot kom ki nongo ki i BC cancer tumor tissue repository ma ki moko ki Canadian Tissue Repository Network. Malube ki cik ma ki moko ki BC Cancer Research Ethics Committee ki University of British Columbia (H07-00463), jami ma ki kwanyo ki i kom lutwo weny ki ngec ma ki nongo i ot yat gunongo cwak ma ki coyo onyo ki kwero cwak gi. Kamu kene ni ki gwoko gi i BioBank ma ki moko (BRC-00290). Kit pa latwo ma ki nyutu maber ki nyutu i Table S1 ki S5. Pi gwoko jami ma ngiic, ki tiyo ki gin ma ki lwongo ni scalpel me balo kamu kene pa latwo ki dong ki keto i 100-micron filter me nongo cell acel ma ki keto iye. Ascites pa latwo ni ki keto i 1500 rpm pi dakika 10 i 4°C me poko cell ki kwanyo gin ma odong. Cell ma ki nongo ki i kom two ma ki lwongo ni tumor ki ascites ki gwoko gi i 50% heat-inactivated human AB serum (Sigma-Aldrich), 40% RPMI-1640 (Thermo Fisher Scientific) ki 10% dimethyl sulfoxide. Gin ma ki gwoko ni ki juku ki dong ki tiyo kwede me nongo metabolomics ki metabolite determination ma kiloko iye piny kany.
Gin ma ki keto ni tye ki 0.22 μm ma ki yubu 50:50 ma ki medo iye RPMI 1640: AimV. RPMI 1640 + 2.05 mM l-glutamine (Thermo Fisher Scientific) ma ki medo ki 10% heat-inactivated human AB serum (Sigma-Aldrich), 12.5 mM Hepes (Thermo Fisher Scientific), 2 mM l-glutamine (Thermo Fisher Scientific) x1 Gin ma ki lwongo ni Penicillin Streptomycin (PenStrep) ma ki lwongo ni (Thermo Fisher Scientific) ki 50 μMB-mercaptoethanol. AimV (Invitrogen) ki medo ki 20 mM Hepes (Thermo Fisher Scientific) ki 2 mM l-glutamine (Thermo Fisher Scientific). Gin ma ki lwongo ni flow cytometer staining buffer ni tye ki 0.22μm ma ki lwongo ni filtered phosphate buffered saline (PBS; Invitrogen) ma ki medo ki 3% heat-inactivated AB human serum (Sigma). Gin ma ki lwongo ni cell enrichment buffer ni tye ki 0.22μm ma ki lwongo ni filtered PBS ki ki medo ki 0.5% heat-inactivated human AB serum (Sigma-Aldrich).
I 37°C ma opong, ki keto rangi i kom cell ki 10 nM MT DR ki 100 μM 2-NBDG pi dakika 30. Ma lubo, ki keto rangi i kom cell ni ki rangi ma ki lwongo ni viability dye eF506 i 4°C pi dakika 15. Medo cell ni i FC Block (eBioscience) ki Brilliant Stain Buffer (BD Biosciences), dilute i flow cytometer staining buffer (ma lubu cik pa layub), ki i ket pi dakika 10 i rwom me lyeto me ot. Ket rangi i kom cell ni ki jami ma gwoko kom (Table S2) i flow cytometry staining buffer i 4°C pi dakika 20. Medo cell ni i flow cytometry staining buffer (Cytek Aurora; 3L-16V-14B-8R configuration) ma peya ki temo. Tii ki SpectroFlo ki FlowJo V10 me nyamo wel cell, ki tii ki GraphPad Prism 8 me yubu ngec ni. Kit ma rwom me ryeny (MFI) pa 2-NBDG ki MT DR ni ki dwoko piny, ki dong ki tiyo ki t test ma ki keto me kwano wel dano ma kom gi lit. Kwanyo wel dano weny ma tye ki jami ma otiime ma pe okato 40 ki i kin jami ma ki kwano; ket wel MFI me 1 pi wel mo keken ma pe ber ma peya itimo kwan me wel ki neno ngec.
Me medo i kom yoo me tic ki cing ma malo ni, watiyo ki coc ma ki coyo ki shape restriction tree (FAUST) (21) me keto cell bot dano mapol ingee kwanyo cell ma otoo ki i FlowJo. Wa tiyo ki cing wa me tic ki jami ma ki nongo me ribo wel dano ma nen calo ki poko gi marac (medo PD1+ ki PD1-tumor cells) ki wel dano ma ki gwoko. Kamu kene acel acel tye ki wel ma kato 2% pa cell, pi wel ma romo 11.
Ki tiyo ki Ficoll gradient density centrifugation me poko PBMC ki jami ma ki poko ki leukocyte (STEMCELL Technologies). CD8 + T cells ki kwanyo gi ki i PBMC kun kitiyo ki CD8 MicroBeads (Miltenyi) ki ki medo gi i kin piny ma otum kun kitiyo ki TransAct (Miltenyi) pi cabit 2 kit ma luyub owaco kwede. Ki weko cell ni ocung pi nino 5 i kin piny ma tye ki IL-7 (10 ng/ml; PeproTech), ki dong ki dwoko cen ki TransAct. I nino dwe 7, malube ki cik pa luyub, dano CD45 MicroBeads (Miltenyi) ki tiyo kwede me dongo cell i dul adek ma lubo. Gin ma ki lwongo ni cell ni ki poko ne pi flow cytometry analysis (kit ma ki waco kwede malo ni), ki cell million acel ki poko ne tyen adek pi LC-MS/MS analysis. Kamu kene ni ki tiyo kwede ki LC-MS/MS kit ma ki waco kwede piny kany. Wa byeko wel metabolite ma orweny ki wel ion ma tye 1,000. Kamu kene acel acel ki dwoko rwom ne piny ki wel ion (TIC), ki loko ne ki rwom ne kene i MetaboAnalystR ma peya ki temo.
Gin ma ki keto i cell acel acel pa latwo acel acel ki juku ki ki kwanyo ki i 40 μm filter me doko gin ma opong (kit ma ki waco kwede malo ni). Malube ki cik pa luyub, dul adek ma lubo me yero maber ki poko jami ma ki lwongo ni magnetic bead separation kun kitiyo ki MicroBeads (Miltenyi) ki tiyo kwede me medo rwom me CD8+, CD4+ ki CD45- cells (i kom ice). Ki lok macek, cell ni ki dwoko gi i cell enrichment buffer (kit ma ki waco kwede malo ni) ki kwano gi. Ki gwoko cell ni ki CD8 pa dano, CD4 pa dano onyo CD45 pa dano (Miltenyi) i 4°C pi dakika 15, ki dong ki lwoko ki cell enrichment buffer. Labol ni ki kato ki i LS column (Miltenyi), ki dul ma tye maber ki marac ki kwanyo. Me dwoko piny kare ki medo rwom me dwogo pa cell, CD8-dul ni ki tiyo kwede pi dul me aryo me medo CD4+, ki CD4-dul ni ki tiyo kwede pi medo CD45 ma lubo. Gwok gin ma ki lwongo ni ice i kare me poko ne.
Me yubu kamu kene pi nyamo jami ma mako kom, ki lwoko cell ni ki pii ma ngiic ma ngiic, ki 1 ml me 80% methanol ki medo i kom kamu acel acel, ki dong ki keto i pii ma ki lwongo ni liquid nitrogen. Kamu kene ni ki keto gi i rwom adek me ngiico ki lyeto ki ki keto gi i 14,000 rpm pi dakika 15 i 4°C. Gin ma tye i kom ma tye ki jami ma ki lwongo ni metabolites ni ki loko nio ka otwo. Gin ma ki lwongo ni metabolites ni ki dwoko gi i 50 μl me 0.03% formic acid, ki loko me rubu, ki dong ki loko me kwanyo jami ma obale.
Kwanyo jami ma ki lwongo ni metabolites kit ma ki waco kwede malo ni. Lok gin ma odong i kom gin ma ki lwongo ni high chromatography bottle pi kwedo metabolomics. Tii ki yoo me tic ma pe ki ngeyo me tic ki lanyut acel acel ki wel cell ma rom me gengo adwogi marac. Watimo kwan ma rwate ki jami ma ki lwongo ni metabolites ma ki coyo con i AB SCIEX QTRAP 5500 Triple Quadrupole Mass Spectrometer (50). Kwedo rangi del kom ki keto kabedo ma malo ni ki tiimo kun kitiyo ki jami ma ki lwongo ni MultiQuant version 2.1 (Applied Biosystems SCIEX).
Wel ion ma romo 1000 ki tiyo kwede me byeko wel metabolite ma orweny, ki TIC pa sampol acel acel ki tiyo kwede me kwano rwom ma malo pa metabolite acel acel ma kinongo me yubu alokaloka ma okelo ki i tic ki jami ma ki tiyo kwede. Inge TIC dong ki dwoko piny, MetaboAnalystR(51) (gin ma ki keto) ki tiyo kwede me loko logarithmic ki dwoko rwom me rwom ma opore. Watiyo ki PCA ki vegan R package me timo kwedo ma mako apoka poka me metabolome i kin dul ma ki kwanyo, ki watiyo ki partial redundancy analysis me kwedo lutwo. Tii ki yoo pa Ward me yubu heat map dendrogram me poko bor pa Euclidean i kin jami ma ki nyutu. Watiyo ki limma (52) i kom rwom me metabolite ma ki ngeyo me nongo metabolites ma pat pat i kom cell type ki microenvironment. Me weko lok man bedo yot, wa tiyo ki dul ma ki lwongo ni group mean parameter me nyutu labol, ki tero dul cell ma tye i microenvironment calo dul acel acel (n = dul 6); pi temo gin ma pire tek, watimo rwom adek ma ki dwoko cen pi gin acel acel ma ki lwongo ni metabolite Me gengo nyaa ma pi adaa, latwo ni ki medo calo gin ma gengo i yub me limma. Me neno apoka poka ma tye i kin jami ma kom gi lit i kin lutwo ma pat pat, wa loko kit ma ki lwongo ni limma model ma tye ki lutwo i yoo ma ki moko. Wa tito rwom me apoka poka ma ki moko con i kin dul cell ki kabedo ma pat pat pa Padj <0.05 (Benjamini-Hochberg correction).
Iyonge medo teko kun kitiyo ki Miltenyi Dead Cell Removal Kit (>80% viability), kit ma ki lwongo ni single-cell transcriptome sequencing ki otimo i kom ascites ki tumor ma ki kwanyo kun kitiyo ki 10x 5′gene expression protocol. Dano abic ma tye ki two ma rwate ki ascites ki kwano, kadi bed ni kwo ma nok ki i kom two acel ma ki kwanyo ogengo keto ne. Me nongo yero lutwo mapol, wa ribo kamu kene pa latwo acel acel i dul ma ki lwongo ni 10x chromium controller, ki wa kwano ascites ki tumor sites ma pat pat. Iyonge keto rwom [Illumina HiSeq 4000 28×98 bp paired end (PE), Quebec genome; wel ma romo 73,488 ki 41,378 ma ki kwano pi cell acel acel pi two ki ascites]], watiyo ki CellSNP ki Vireo (53) (ma jenge i kom CellSNP ma calo SNP pa dano (VCF) ma kimiyo ki GRCh38 kimiyo bot ngat ma miyo kony. Watiyo ki SNPRelate me moko ngat ma cok loyo (IBS) kit pa latwo (IBS), kwanyo cell ma pe ki poko ki cell ma ki ngeyo ni tye duplexes ki ma rwate i kin ascites ki tumor samples (54). (2792 ki 4183 cells ma aa ki i kom two ki ascites, kit meno) pi nyamo lok. cell ni ki ngeyo ki nyutu pa CD8A ki GZMA, ma pe kwako dul ma tye ki nyutu pa ribosomal protein manok. Wanongo ngec ma ki coyo pa Izar et al. (16), medo ki t-SNE embedding gi, twero juku nyutu ma rwate i kin lanyut me kom ki nyutu pa NNMT.
PBMC ki poko ki i kom jami ma ki poko ki i kom remo (STEMCELL Technologies) ki Ficoll gradient density centrifugation. CD3 + cells ki kwanyo ki i PBMC kun kitiyo ki CD3 beads (Miltenyi). I kare ma MNA tye onyo pe, CD3+ cells ki oyabo ki plate-bound CD3 (5μg/ml), soluble CD28 (3μg/ml) ki IL-2 (300 U/ml; Proleukin). I nino me agiki me medo ne, kit ma twero bedo kwede (Fixable Viability Dye eFluor450, eBioscience) ki dongo ne (123count eBeads, Thermo Fisher Scientific) ki kwano ki flow cytometry. Temo tic pa gin ma ki lwongo ni effector kun i juku cell ki PMA (20 ng/ml) ki ionomycin (1μg/ml) ki GolgiStop pi cawa 4, ki nen CD8-PerCP (RPA-T8, BioLegend), CD4-AF700 (RPA-T4) , BioLegend) ki TNFα-fluoresce isothianate (TCb1) BD). Medo qPCR ki ChIP cells ki PMA (20 ng/ml) ki ionomycin (1μg/ml) pi cawa 4. Gin ma ki lwongo ni ELISA supernatant ni ki kwanyo ma peya ki inge tic ki PMA (20 ng/ml) ki ionomycin (1 μg/ml) pi cawa 4.
Lub cik pa luyub me poko RNA kun itiyo ki RNeasy Plus Mini Kit (QIAGEN). Tii ki QIAshredder (QIAGEN) me yubu gin ma ki nyutu ni. Tii ki jami ma ki lwongo ni RNA to cDNA (Thermo Fisher Scientific) me yubu DNA ma rwate (cDNA). Tii ki TaqMan Rapid Advanced Master Mix (Thermo Fisher Scientific) me kwano kit ma gene nyute kwede (malube ki cik pa luyub) ki jami ma lubo ni: Hs00196287_m1 (NNMT), Hs00154079_m1 (AOX1), Hs0042755m1 (AOX1), Hs02786624_g1 [glyceraldehyde-3-phosphate off Hydrogen (GAPDH)] ki Hs01010726_m1 (SLC22A2). Kamu kene ni ki tiyo kwede i kom StepOnePlus real-time PCR system (Applied Biosystems) (Applied Biosystems) i MicroAmp fast optical 96-well reaction plate (Applied Biosystems) ki MicroAmp optical film. Ct mo keken ma okato 35 ki ngeyo ni tye malo ki rwom me nongo ne ki ki keto ni pe kitwero nongo ne.
Tim ChIP kit ma kiloko kwede con (58). Ki lok macek, ki tiyo ki jami ma ki lwongo ni formaldehyde (rwom me agiki 1.42%) ki ki gwoko gi i rwom me lyeto me ot pi dakika 10. Tii ki gin ma ki medo me gengo two (25 mM Hepes, 1.5 mM MgCl2, 10 mM KCl ki 0.1% NP-40) i kom ice pi dakika 10, ki dong i dwoko i kom gin ma ki lwongo ni immunoprecipitation buffer kit ma ki waco kwede (58 ). Gin ma ki nyutu ni ki tiyo kwede ki rwom ma lubo ni: rwom 10 (20 1-ceken pulses) ki kare ma pe loko me ceken 40. Ket ChIP-grade immunoglobulin G (Cell Signaling Technology; 1μl), histone H3 (Cell Signaling Technology; 3μl), NFAT (Invitrogen; 3μl) ki SP1 (Cell Signaling Technology; 3μl) antibodies ki gin ma ki kwanyo i 4°CC ma ki yengo dyewor. Ket jami ma ki lwongo ni protein A (Thermo Fisher Scientific) ki gin ma ki kwanyo ni i 4°C ki yenge mot pi cawa 1, ki dong tic ki chelex beads (Bio-Rad) me medo DNA, ki tic ki proteinase K (Thermo Fisher) me juku protein. TNFα promoter onongo ki nongo ki PCR: anyim, GGG TAT CCT TGA TGC TTG TGT; i kom lok adaa, GTG CCA ACA ACT GCC TTT ATA TG (207-bp product). Cal ni ki yubu ki Image Lab (Bio-Rad) ki ki kwano ki tic ki jami ma ki lwongo ni ImageJ.
Gin ma ki lwongo ni cell culture supernatant ni ki kwanyo kit ma ki waco kwede malo ni. Ki moko tam ni malube ki yoo pa luyub jami ma ki lwongo ni human TNFα ELISA kit (Invitrogen), human IL-2 ELISA kit (Invitrogen) ki human IFN-γ ELISA kit (Abcam). Malube ki cik pa luyub, gin ma ki lwongo ni supernatant ni ki dilute 1:100 me nongo TNFα ki IL-2, ki 1:3 me nongo IFN-γ. Tii ki EnVision 2104 Multilabel Reader (PerkinElmer) me pimo rwom me 450 nm.
PBMC ki poko ki i kom jami ma ki poko ki i kom remo (STEMCELL Technologies) ki Ficoll gradient density centrifugation. CD3 + cells ki kwanyo ki i PBMC kun kitiyo ki CD3 beads (Miltenyi). I kare ma MNA tye onyo pe, CD3+ cells ki tiyo kwede ki plate-bound CD3 (5μg/ml), soluble CD28 (3μg/ml) ki IL-2 (300 U/ml; Proleukin) pi nino 3. Iyonge nino 3, ki kwanyo cell ni ki lwoko ki 0.9% saline, ki pellet ni ki juku. Kwano cell ni kitimo ki flow cytometry (Cytek Aurora; 3L-16V-14B-8R configuration) kun kitiyo ki 123count eBeads.
Kwanyo jami ma ki lwongo ni metabolites kit ma ki waco kwede malo ni. Gin ma ki kwanyo ma otwo ni ki dwoko cen i rwom me 4000 cell equivalents/μl. Kwan gin ma ki nyutu ni ki jami ma ki lwongo ni reversed-phase chromatography (1290 Infinity II, Agilent Technologies, Santa Clara, CA) ki CORTECS T3 column (2.1×150 mm, particle size 1.6-μm, pore size 120-Å; #186008500, Waters). Polar mass spectrometer (6470, Agilent), ma iye electrospray ionization tiyo i kit maber. Gin ma ki lwongo ni Mobile phase A tye 0.1% formic acid (i H2O), mobile phase B tye 90% acetonitrile, 0.1% formic acid. LC gradient tye 0 onyo 2 dakika pi 100% A, 2 onyo 7.1 dakika pi 99% B, ki 7.1 onyo 8 dakika pi 99% B. Ki dong dwoko rwom me column ni ki mobile phase A i rwom me mol pa 0.6 ml/min pi dakika 3. . Kit ma mol kwede tye 0.4ml/min, ki kama ki keto iye jami ni ki lyeto nio wa i 50°C. Tii ki MNA's pure chemical standard (M320995, Toronto Research Chemical Company, North York, Ontario, Canada) me moko kare me gwoko (RT) ki loko (RT = 0.882 dakika, loko 1 = 137→94.1, loko 2 = 137→92 , loko = 13→77). Ka alokaloka adek ni weng otimme i kare me gwoko ma opore, alokaloka 1 ki tiyo kwede me kwano wel me neno ni tye kakare. Gin ma ki lwongo ni standard curve pa MNA (Toronto Research Chemical Company) ki yubu ki dilution abicel ma ki lwongo ni stock solution (1 mg/ml) me nongo rwom me 0.1, 1.0, 10 ki 100 ng/ml ki 1.0 ki 10μg/ml ma rwate ki pii. Gin ma ki nongo tye 1 ng/ml, ki lagam ma rwate tye i kin 10 ng/ml ki 10μg/ml. Kit ma ki keto kwede microliters aryo me lanyut ki ma ki ngeyo ni ki tiyo kwede me nyamo LC/MS, ki sampol ma ki rubu ma ki tiyo kwede me neno rwom maber ki tiyo kwede i kom keto jami aboro weng me neno ni kabedo me kwano ni tye maber. Lagam pa MNA pa jami ma ki kwanyo ki MNA onongo tye i kin rwom me rwom me temo. Kwano ngec ni kitimo kun kitiyo ki jami ma ki lwongo ni MassHunter quantitative analysis software (v9.0, Agilent).
Gin ma ki yubu me aryo αFR-CAR ki kwanyo ki bot Song et al. (59). Ki lok macek, gin ma ki yubu ni tye ki jami ma lubo ni: CD8a leader sequence, human αFR-specific single-chain variable fragment, CD8a hinge ki transmembrane region, CD27 intracellular domain ki CD3z intracellular domain. Gin ma ki lwongo ni CAR sequence ni ki yubu ki GenScript, ki dong ki keto i dul me aryo ma ki lwongo ni lentiviral expression vector ma tye malo i kom GFP expression cassette ma ki tiyo kwede me pimo tic pa transduction.
Lentivirus ki yubu ki keto HEK293T cells [American Type Culture Collection (ATCC); ki pito i Dulbecco's modified Eagle medium ma tye ki 10% fetal bovine serum (FBS) ki 1% PenStrep, ki tiyo ki CAR-GFP vector ki The packaging plasmids (psPAX2 ki pMD2.G, Addgene) tiyo ki lipofection amine (Sigma-Aldrich). Gin ma tye ki kwidi ni ki kwanyo i nge cawa 48 ki 72 inge keto ne, ki kwanyo ne, ki ki keto ne i tic ki ultracentrifugation. Gwok gin ma ki lwongo ni viral supernatant ni i -80°C nio ka ki loko ne.
PBMC ki poko ki ikom jami ma ki poko ikom dano ma kom gi yot (STEMCELL Technologies) ki Ficoll gradient density centrifugation. Tii ki jami ma ki lwongo ni positive selection CD8 microbeads (Miltenyi) me poko CD8+ cells ki i PBMC. Medo T cells ki TransAct (Miltenyi) ki i TexMACS medium [Miltenyi; ki medo ki 3% me remo pa dano ma ki lyeto, 1% PenStrep ki IL-2 (300 U/ml)]. I nge cawa pyera aryo ki angwen me tic, T cells ki keto lentivirus (10 μl concentrated virus supernatant per 106 cells). 1 onyo 3 nino ingee keto i Cytek Aurora (i kom FSC (Forward Scatter)/SSC (Side Scatter), Singlet, GFP+), pimo kit ma GFP nyutu kwede i kom cell me nyutu tic maber me loko ma romo 30%.
CAR-T cells ki gwoko pi cawa 24 i Immunocult (STEMCELL Technologies; ki medo ki 1% PenStrep) i te jami ma lubo ni: ma pe ki tiyo kwede, ki keto iye 250 μM adenosine onyo 10 mM MNA. Iyonge yat, CAR-T cells ki lwoko ki PBS ki ki rubu ki 20,000 SK-OV-3 cells [ATCC; i McCoy 5A medium (Sigma-Aldrich) ma ki medo ki 10% FBS ki 1% PenStrep i 10: Ratio me effector ki target me 1 ki medo i tyen adek i immunocult medium ma ki medo iye. SK-OV-3 cells ki SK-OV-3 cells ma ki neko ki digitalis saponin (0.5mg/ml; Sigma-Aldrich) ki tiyo kwede calo gin ma pe ber ki ma ber, kit meno. Iyonge cawa 24 me dongo kacel, ki kwanyo gin ma odong ki lactate dehydrogenase (LDH) ki pimo kit ma ki waco kwede (LDH Glo Cytotoxicity Assay Kit, Promega). Gin ma ki lwongo ni LDH supernatant ni ki dilute 1:50 i LDH buffer. Wel ma ki neko ki pimo kun kitiyo ki yoo ma lubo ni: wel ma ki neko = wel ma ki loko / wel ma malo me neko x 100%, kan ma wel ma ki loko = co-culture-T cells keken, ki wel ma malo ma ki neko = gin ma ki keto maber-gin ma ki neko marac.
Kit ma ki waco kwede i coc onyo jami ki yoo, tii ki GraphPad Prism 8, Microsoft Excel onyo R v3.6.0 pi kwano wel. Ka ki kwanyo sampol mapol ki bot latwo acel (ma calo ascites ki tumor), wa tiyo ki t test ma ki rwate onyo wa medo latwo ni calo adwogi ma pe ki ngeyo i kit ma opore. Pi nyamo jami ma mako kom, temo ma pire tek ni ki tiimo i tyen adek.
Pi jami ma ki medo i kom pwony man, nen http://advances.sciencemag.org/cgi/content/full/7/4/eabe1174/DC1
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Marisa K. Kilgour (Marisa K. Kilgour), Sarah MacPherson (Sarah MacPherson), Lauren G. Zacharias (Lauren G. Zacharias), Abigail Eli Aris G. Watson (H. Watson), John Stagg (John Stagg), Brad H. Nelson (Brad H. Nelson), Ralph J. Dephardini (R.R.R.Bellnis), Deraphnis. Jones (Russell G. Jones), Phineas T. Hamilton (Phineas T.
MNA konyo me juku teko pa T cells ki nyutu yoo me cango two kanca pa dano.
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. Dephardini (R.R.R.Bellnis), Deraphnis. Jones (Russell G. Jones), Phineas T. Hamilton (Phineas T.
MNA konyo me juku teko pa T cells ki nyutu yoo me cango two kanca pa dano.
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