Immunomodulatory metabolites ubedo piny mapire tek pa tumor microenvironment (TME), ento ku moko manok, rwom migi podi ungeyere ngo. Kae, wa ngiyo two man T cells kud i two man ascites pa ju ma twoyo pa serous carcinoma (HGSC) pi nyutho metabolome pa TME compartments matung tung eni. Ascites ku tumor cells gitiye ku tung tung madit mi metabolite. Ka juporo ku ascites, tumor-infiltrating T cells utiye ku 1-methylnicotinamide (MNA) madwong. Kadi bende kadhiri pa MNA in T cells utiye malo, nyuthu pa nicotinamide N-methyltransferase (enzyme ma ketho methyl groups kud i S-adenosylmethionine ni nicotinamide) utiye kende kende i fibroblasts man tumor cells. I tic, MNA ketho T cells ni wodhu cytokine tumor necrosis factor alpha. Pieno, TME-derived MNA konyo i yub pa T cells man nyuthu yore mi immunotherapy pi thieth pa two kanca pa dhanu.
Jam piny ma wok kud i two copo bedu ku tic ma tek ikum gwoko kum ikum two, man lembe madwong nyutho nia gicopo bedu piny ma pire tek pi dongo pa two (1). Mi medo ikum adwogi pa Warburg, tic ma kawoni ucaku ni nyuthu kite ma dul kom ma remo timo kudu tic man kite ma dul kum dhanu timo kudu tic (TME). Somo ikum oyo man T cells pa dhanu unyutho nia glutamine metabolism (2), oxidative metabolism (3) man glucose metabolism (4) copo timo tic giri ikum immune cell subgroups matung tung. Jam piny mapol ma timo tic i yore eni juku tic pa T cells. Junyutho nia juku coenzyme tetrahydrobiopterin (BH4) ecopu nyayu peko i dongo pa T cells, man mediri pa BH4 i kum ecopu medo kero pa kum ma gwoko kum ma CD4 ku CD8 timo. Mi medo, tic pa kynurenine mi jwigo two copo bedu maber ku miyo BH4 (5). I isocitrate dehydrogenase (IDH) mutant glioblastoma, wok pa enantiometabolic (R)-2-hydroxyglutarate (R-2-HG) juku T cell activation, proliferation man cytolysis Activity (6). I kare ma ukadhu, junyutho nia methylglyoxal, piny ma wok kud i glycolysis, juyubu ku suppressor cells mi myeloid origin, man T cell transfer pa methylglyoxal copo juku tic pa effector T cells. I yore mi yath, dwoko piny pa methylglyoxal copo loyo tic pa myeloid-derived suppressor cells (MDSC) man konyowa ni medo kero pa checkpoint blockade therapy i mouse models (7). Somo eni uketho cwiny ikum tic ma pire tek pa TME-derived metabolites i yub pa tic pa T cell.
T cell dysfunction ungiyure lee i two mi ic (8). Eni utiye pi kit ma piny timo kudu tic ma nwangre i hypoxia man abnormal vasculature pa tumor (9), ma kelo loko pa glucose man tryptophan ni piny ma wok kud i iye calu lactic acid man kynurenine. Lactate madwong ma woko dwoko piny tic pa interferon-γ (IFN-γ) man eketho tung tung pa myelosuppressive subgroups (10, 11). Camu tryptophan juku T cell proliferation man juku T cell receptor signaling (12-14). Kadi bende lembe maeni nen, tic madwong ikum kit ma kum dhanu timo kudu tic i in vitro T cell culture ku tic ku jam piny maber, nyo kende kende i homologous mouse models in vivo, acel de mbe ma nyutho tung tung pa two kanca pa dhanu man Physiological macro man micro environment.
Lembe ma rwinyo pa two kanca mi ic utiye nyai pa ic man nen pa ascites. Coko pa pii pa cell i ascites utiye ku winjiri ku two ma ukadhu man timo marac (15). Ni lubre ku lapor, kabedo eni utiye ku hypoxic, utiye ku rwom ma malu pa vascular endothelial growth factor (VEGF) man indoleamine 2,3-dioxygenase (IDO), man utiye ku T regulatory cells man myeloid inhibitory cells (15-18). Kabedo mi tic pa ascites copo bedu tung tung ku pa two ne gire, pi meno dwoko rwom pa T cells i kabedo mi peritoneal ungeyere ngo. Mi medo, tung tung man apoka poka ma nuti ikind ascites ku metabolites ma nwangre i kabedu pa two copo juku dongo pa cell mi kum man tic migi ikum two, man lembe mukende mitere.
Pi ni daru peko eni, wayubu yore mi poko cell man liquid chromatography tandem mass spectrometry (LC-MS/MS) pi ponjo kit cell matung tung (ma ikind gi CD4 + man CD8 + T cells) man ikind man ikind tumor. Metabolites pare ucungo ikum cell i ascites man tumor environment acel pa jaratwo. Watiyo ku yore eni karacelu ku high-dimensional flow cytometry man single-cell RNA sequencing (scRNA-seq) mi mio cal maber pa kit ma dul pa dhanu eni gikwo kudu. Yore maeni unyutho mediri madit pa 1-methylnicotinamide (MNA) i tumor T cells, man in vitro experiments unyutho nia immunomodulatory effect pa MNA ikum tic pa T cells ubedu ungeyere ungo. I ayi ma rwinyo, yore eni nyutho kit ma two timo ku dul kom ma gwoko kum, man emiyo ngec ma ukoc ikum dul kom ma gwoko kum, ma copo bedo ber pi thieth pa T cell-based ovarian cancer immunotherapy Treatment opportunities.
Watiyo ku high-dimensional flow cytometry pi pimo glucose uptake [2-(N-(7-nitrophenyl-2-oxa-1,3-diaza-4-yl)amino)-2-deoxyglucose (2-NBDG) man mitochondrial activity [MitoTracker Deep Red (MT DR) (MT DR, 2017, 2019) i thenge acel lanyuth ma poko kin dul kom ma gwoko kum ku dul kom ma mako two (Table S2 man cal S1A). Lembe maeni nyutho nia ka juporo ku T cells, ascites man tumor cells gitiye ku glucose ma malu, ento gitiye ku tung tung manok i tic pa mitochondrial. Glucose ma romo pa tumor cells [CD45-EpCAM (EpCAM)+] utiye wang adek onyo angwen pa T cells, man rwom pa glucose ma CD4 + T cells maku utiye wang 1.2 pa CD8 + T cells, ma nyutho nia Tumor infiltrating lymphocytes (TIL) utiye ku yeny mi metabolic matung tung kadi i TAME marom (Figure 1). I ayi pa tung tung, tic pa mitochondrial in tumor cells urombo ku pa CD4 + T cells, man tic pa mitochondrial pa kit cells ario eno utiye malo uloyu pa CD8 + T cells (lanyut 1B). I ayi ma rwinyo, adwogi eni nyutho rwom pa metabolic. Tic mi metabolic pa cell mi two utiye malo uloyu pa CD4 + T cells, man tic mi metabolic pa CD4 + T cells utiye malo uloyu pa CD8 + T cells. Kadi bedu ku adwogi eni ikind kit cell, epe tung tung moku ikum kit ma CD4 + man CD8 + T cells timo kudu tic nyo wel migi i ascites ka juporo ku tumors (lanyut 1C). I ayi pa tung tung, i CD45-cell fraction, wel pa EpCAM+ cells i tumor umedire ka juporo ku ascites (lanyut 1D). Waneno bende tung tung ma nen kamaleng ikind EpCAM+ ku EpCAM- cell components. EpCAM+ (tumor) cells utiye ku glucose uptake ma malu man tic pa mitochondrial ma sagu EpCAM- cells, ma utiye malo uloyu tic pa fibroblasts in tumor cells in TME (Figure 1, E man F).
(A ku B) Median fluorescence intensity (MFI) mi glucose uptake (2-NBDG) (A) man mitochondrial activity pa CD4 + T cells (MitoTracker dark red) (B) Representative graphs (kor cing) man tabulated data (Right), CD8 + T cells man EpCAM + CD45-tumor cells calu tumor cells man tumor. (C) Ratio pa CD4 + man CD8 + cells (mi CD3 + T cells) in ascites man tumor. (D) Wel pa EpCAM + tumor cells in ascites man tumor (CD45−). (E ku F) EpCAM + CD45-tumor man EpCAM-CD45-matrix glucose uptake (2-NBDG) (E) man mitochondrial activity (MitoTracker dark red) (F) representative graphs (left) and tabulated data (Right) Ascites and tumor cells. (G) Graph ma nyutho CD25, CD137 man PD1 ku flow cytometry. (H ku I) CD25, CD137 man PD1 nyuthu ikum CD4 + T cells (H) man CD8 + T cells (I). (J ku K) Naive, central memory (Tcm), effector (Teff) man effector memory (Tem) phenotypes based on expression of CCR7 and CD45RO. Cal ma nyutho (kor cing) man lembe ma jugoro (kor cing) pa CD4 + T cells (J) man CD8 + T cells (K) i ascites man tumors. P values ​​ ma juporo ku paired t-test (*P<0.05, **P<0.01 man ***P<0.001). Laini nyuthu ju ma two (n = 6). FMO, fluorescence minus one; MFI, rwom pa ler pa ceng.
Lembe mukende ma jutimo unyutho tung tung mange ma pire tek ikind kit ma T cell nen kudu. Lembe ma jutiyo kudu (lanyut 1, G ku I) man ma jutiyo kudu (lanyut 1, J ku K) i two ma nyai utiye pol kare uloyu ascites (wel pa CD3 + T cells). Kumeno bende, nyang ikum kit ma nen pa dhanu ku nyuthu pa lanyuth pa tic (CD25 man CD137) man lanyuth pa tho [programmed cell death protein 1 (PD1)] unyutho nia kadi bende kit pa dhanu eni gi tung tung (lanyut S1, B nicidho E), ento tung tung moku ma pire tek ungo ikind adwogi pa naive, nyo poyo wic (lanyut S1, S1) AN). Adwogi eni juyike ku tic ku yore mi ponji pa cuma mi poko kit pa cell (21), ma unyutho bedo pa cell mapol pa cogo (CD45 + / CD3- / CD4 + / CD45RO +) i ascites pa jaratwo (Figure S2A ). Ikind kit cell ma junwango, dul pa cell ma myeloid eni unyutho rwom ma malu pa glucose man tic pa mitochondrial (lanyut S2, B ku G). Adwogi eni nyutho tung tung madit mi metabolic ikind kit cell mapol ma nwangre i ascites man tumors i ju ma two HGSC.
Peko madit mi nyang ikum kit pa TIL utiye mitu mi poko T cell ma leng, maber man madwong kud i kum two. Somo ma kawoni unyutho nia poko piny man yore mi medo piny ma dongo dongo ma lubu flow cytometry copo kelo aloka loka i kit ma piny timo kudu piny (22-24). Mi daru peko eni, wa medo yore mi medo rwom pa bead ni poko man poko TIL kud i two mi ic pa dhanu ma jutimo igi operation ma podi jutimo ngo sayusac ku LC-MS/MS (nen jam tic man yore; cal 2A). Pi ni ngiyo adwogi pa protocol eni ikum aloka loka pa metabolite, waporo metabolite profiles pa T cells ma jutimo ku ju ma kum gi yot ingey poko pa bead ku cells ma jupoko ngo ku bead ento udong iwi pee. Lembe maeni mi gwoko ber pa piny unwangu niya lembe ario eni utiye ku winjiri madit (r = 0.77), man dwoko wang lembe pa dul pa 86 metabolites utiye ku dwoko wang lembe madit (lanyut 2B). Pieno, yore eni copo timo sayusac maber pa metabolite i cell ma ubekadhu kud i cell type enrichment, ma eketho wabedo ku rwom ma malu mi ngiyo metabolites moko i HGSC, man ekonyo dhanu ni nwangu nyang ma thuc pa cell specificity Sexual metabolism program.
(A) Cal pa magnetic bead enrichment. Mapodi jutimo sayusac ku LC-MS/MS, dul kom ne bi kadhu kud i rwom adek ma lubo ku magnetic bead enrichment nyo bibedo iwi pee. (B) Adwogi pa kit mi medo piny ikum wel pa metabolites. Ikind piny adek ma jupimo pi kit piny acel acel ± SE. Laini macol nyuthu bedo pa 1:1. Intra-class correlation (ICC) mi pimo ma judwoko wang mapol nyutho i label mi axis. NAD, nicotinamide adenine dinucleotide. (C) Cal ma nen pa tic pa jaratwo. Ascites nyo tumors ju kwalo kud ibang jurutwo man ju gwoko gi i cryopreserved. Thenge ma nok pa lanyuth acel acel jutimo sayusac iwie ku flow cytometry, ento lanyuth mukende jutimo i iye rwom adek mi medo rwom pa CD4+, CD8+ man CD45- cells. Jutimo sayusac ikum dul pa cell eni ku kony pa LC-MS/MS. (D) Heat map pa rwom pa metabolite ma juporo. Dendrogram nyuthu dul pa Ward pa bor pa Euclidean ikind lanyuth. (E) Principal component analysis (PCA) pa lanyuth pa metabolite map, nyutho rwom adek pa lanyuth acel acel, lanyuth pa jaratwo acel juporo ku laini. (F) PCA pa metabolite profile pa sample ma juketho ikum jaratwo (niwacu, tic ku partial redundancy); kit lanyuth utiye ma nok ku convex hull. PC1, piny madit mir acel; PC2, piny madit mir 2.
I ngeye, watiyo ku yore mi medo piny eni ni ngiyo 99 metabolites in CD4 +, CD8 + man CD45-cell fractions in primary ascites man tumors pa ju HGSC abusiel (lanyut 2C, cal S3A man Table S3 ku S4). Wel pa dhanu ma juyenyo ubedo 2% ku 70% pa wel pa dhanu ma kwo, man wel pa dhanu ubedo tung tung ikind jurutwo. I ngei poko dul, dul ma ju medo (CD4+, CD8+ nyo CD45-) ubedo 85% pa dul ma kwo i lanyuth. Yore mi medo piny eni konyowa ni ngiyo wel pa cell kud i metabolism pa tumor tissue pa dhanu, ma epe ni timo kud i sample madwong. Ni tiyo ku protocol eni, wanwango ni l-kynurenine ku adenosine, piny aryo ma juyubu maber ma nyayu two ubedo malu in tumor T cells nyo tumor cells (Figure S3, B man C). Pieno, adwogi eni nyutho bedo mandha man kero pa poko pa cell man diro mi mass spectrometry ni nwangu metabolites ma pire tek i kum jurutwo.
Lembe ma wabeponjo de unyutho apoka poka madit pa kit cell ikind ju ma two (lanyut 2D man cal S4A). I andha, ka juporo ku jurutwo mukende, jurutwo 70 unyutho kit ma tung tung mi tic pa kum (lanyut 2E man cal S4B), ma nyutho nya copo bedu ku tung tung madit mi yot kum ikind jurutwo. Ber ni ngeyo ni ka juporo ku jurutwo mukende (1.2 ku 2 litres; Table S1), wel pa ascites ma jucoko i jurutwo 70 (80 ml) ubedo nok. Gwoko pa tung tung pa jurutwo i saa mi nyang ikum piny ma pire tek (lapor, nitio ku nyang ikum piny ma nok) nyutho aloka loka ma rwate ikind kit cell, man kit cell man/nyo microenvironment gicokiri maleng nimakere ku metabolite profile (Figure 2F ). Nyamu lembe pa metabolites acel acel unyutho adwogi eni man unyutho tung tung madit ikum kit cell ku microenvironment. Ber ni ngeyo ni tung tung madit uloyu ma nen utiye MNA, ma pol kare utiye ku CD45- cells man CD4+ man CD8+ cells ma dongo i tumor (lanyut 3A). Pi CD4 + cells, adwogi eni nenre kamaleng, man MNA in CD8 + cells de nen calu etiye ku adwogi marac ikum kabedu. Ento, eni piretek ungo, kum ju adek kende ikind ju abusiel ma jucopu pimo gi pi CD8+ scores. Mi medo ikum MNA, i kit cell matung tung i ascites man tumors, metabolites mange ma jukwanu ngo maber i TIL de gitiye ku lonyo matung tung (Figures S3 ku S4). Pieno, lembe eni nyutho dul maber pa immunomodulatory metabolites pi medo sayu lembe.
(A) Kite ma MNA utiye kudu in CD4+, CD8+ man CD45- cells kud i ascites man tumor. Box plot nyutho median (line), interquartile range (frame hinge) man data range, nitundo 1.5 times interquartile range (frame whisker). Calu ma jukoro i Patient Materials and Methods, ti ku limma value pa jaratwo ni nyang P value (*P<0.05 man **P<0.01). (B) Cal pa 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. Enzymes (macol): 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) man tumor (makwar; n = 3 jurutwo). (D) NNMT nyuthu in dul pa cell matung tung ma junwango ku tic pa scRNA-seq. (E) Nyuthu pa NNMT ku AOX1 in SK-OV-3, human embryonic kidney (HEK) 293T, T cells man MNA-treated T cells. Lembe ma jugoro nen ni rwate ku SK-OV-3. Kit ma SEM nyuthu kudu (n = 6 ju ma remo migi yot). Ct ma dit uloyu 35 jubinyuthe ni piny ma dhanu copo nwangu ngo (UD). (F) Nyuthu pa SLC22A1 man SLC22A2 in SK-OV-3, HEK293T, T cells man T cells ma jutimo ku 8mM MNA. Lembe ma jugoro nen ni rwate ku SK-OV-3. Kit ma SEM nyuthu kudu (n = 6 ju ma remo migi yot). Ct ma dit uloyu 35 jubinyuthe ni piny ma dhanu copo nwangu ngo (UD). (G) Cell MNA content in donor healthy T cells after 72 hours of incubation with MNA. Kit ma SEM nyuthu kudu (n = 4 ju ma gimio kony ma kum gi yot).
MNA juyubu ku loko methyl group kud i S-adenosyl-1-methionine (SAM) ni nicotinamide (NA) ku nicotinamide N-methyltransferase (NNMT; cal 3B). NNMT utiye ma juweco pire lee i kit two matung tung pa dhanu man etiye ku winjiri ku dongo, dongo man nyai (25-27). Mi nyang maber kama MNA uai i T cells in TME, watiyo ku scRNA-seq ni nyutho kite ma NNMT wok kudu i kind kit cells i ascites man tumors pa ju adek ma HGSC (Table S5). Nyamu lembe ikum 6,500 cells unyutho ni i ascites man tumor, NNMT expression ubedo kende kende i kum fibroblast man tumor cell populations (lanyut 3, C ku D). Ber ni ngeyo nya epe ku NNMT ma nyutho PTPRC (CD45 +) (lanyut 3D man cal S5A), ma nyutho nia MNA ma ju nwango i spectrum pa metabolite ju ketho i T cells. Nyuthu pa aldehyde oxidase 1 (AOX1) loko MNA ni 1-methyl-2-pyridone-5-carboxamide (2-PYR) nyo 1-methyl-4-pyridone-5-carboxamide (4-PYR); Cal 3B) de ucungo ikum wel pa fibroblasts ma nyutho COL1A1 (cal S5A), ma karacelu nyutho nia T cells gipe ku copo mi MNA metabolism macon. Kit ma MNA-related genes eni nyuthere kudu juyike ku tic ku lembe mir aryo ma jukwanu kud i ascites pa ju ma two HGSC (lanyut S5B; n = 6) (16). Mi medo, quantitative polymerase chain reaction (qPCR) analysis pa donor T cells ma kum gi yot ma jutiyo kudu ku MNA unyutho nia ka juporo ku control SK-OV-3 ovarian tumor cells, NNMT nyo AOX1 ubedu nya epe (lanyut 3E). Adwogi ma jukuro ngo nyutho nia MNA copo bedu ma juwodho kud i fibroblasts nyo tumors i T cells ma ceng i TME.
Kadi bende ju ma copo tio kudu uketho i iye thek pa organic cation transporters 1 ku 3 (OCT1, OCT2 man OCT3) ma jugoro ku soluble carrier 22 (SLC22) family (SLC22A1, SLC22A2 man SLC22A3), ju ma copo tero MNA podi ungiyu ngo (28) . QPCR pa mRNA kud i T cells ma kum gi yot unyutho rwom ma piny pa SLC22A1 ento rwom pa SLC22A2 ma nen ungo, ma nyutho nia jular jukoro pire con i giragora (lanyut 3F) (29). I ayi pa tung tung, SK-OV-3 ovarian tumor cell line unyutho rwom ma malu pa transporters ario eno (lanyut 3F).
Pi ni temo ka nyo T cells copo bedu ku copo mi mako MNA ma woko, ju gwoko T cells ma kum gi yot pi sawa 72 i bedo pa MNA matung tung. Ka MNA ma woko epe, piny ma nwang i MNA copo nwangire ngo (lanyut 3G). Ento, T cells ma jutiyo kudu ku exogenous MNA unyutho dongo pa MNA i cells, nitundo 6 mM MNA (lanyut 3G). Adwogi eni nyutho nia kadi nang rwom ma piny pa transporter expression man bedu pa enzyme madit ma timo tic mi intracellular MNA metabolism, TIL podi copo maku MNA.
Kite ma metabolites in T cells pa jurutwo man in vitro MNA absorption experiments medo kero pa cancer-associated fibroblasts (CAF) mi wodhu MNA man tumor cells copo doro kit man tic pa TIL. Mi nyang ikum adwogi pa MNA ikum T cells, T cells ma kum gi yot ju tiyo kudu in vitro i bedo tiye nyo epe pa MNA, man nyai migi man yik pa cytokine jutimo igi sayusac. I ngei nindu 7 mi medu MNA i rwom ma malu, wel pa dhanu ma umedire wang ario udoko piny, man tego ubedo i rwom zo (lanyut 4A). Mi medo, yath pa exogenous MNA ukelo dongo pa CD4 + man CD8 + T cells ma nyutho tumor necrosis factor-α (TNFα; cal 4B). I ayi pa tung tung, tic pa IFN-γ i kind dul kom ubedo nok in CD4 + T cells, ento ungo in CD8 + T cells, man aloka loka madit ope in interleukin 2 (IL-2; cal mir angwen, C ku D). Pieno, enzyme-linked immunosorbent assay (ELISA) pa supernatants kud i MNA-treated T cell cultures unyutho dongo madit pa TNFα, dwoko piny pa IFN-γ, man aloka loka moko ope pa IL-2 (lanyut 4, E ku G). . Dwoko piny pa IFN-γ nyutho nia MNA copo bedu ku tic moko i juku tic pa T cells. Pi niporo adwogi pa MNA ikum T cell-mediated cytotoxicity, chimeric antigen receptor T (FRα-CAR-T) cells ma targeting folate receptor α man CAR-T (GFP) regulated by green fluorescent protein (GFP) -CAR-T cells juyubu ku healthy donor peripheral blood mononuclear cells (PBMC). CAR-T cells jupidu pi sawa 24 i bedo pa MNA, man i ngeye jupidu karacelu ku human SK-OV-3 ovarian tumor cells ma nyuthu folate receptor α i effector ku target ratio pa 10:1. Tic pa MNA ukelo dwoko piny madit mi nek pa FRα-CAR-T cells, ma ubedo marom ku FRα-CAR-T cells ma jutimo ku adenosine (lanyut 4H).
(A) Wel pa cell ma kwo man wel pa dhanu (PD) ma wok kud i culture i ceng mir 7. Bar graph nyutho wel + SEM pa ju ma remo abusiel ma kum gi yot. Nyuthu lembe ma wok kud i n = 3 lembe ma jutimo giri. (B to D) CD3/CD28 man IL-2 jutiyo kudu pi ketho T cells tiyo i rwom migi mi MNA pi nindu 7. Mapodi jutimo ngo sayusac, jutimo tic ku PMA/ionomycin ku GolgiStop pi sawa angwen. TNFα (B) nyutho in T cells. Lapor pa cal (kor cing) man lembe ma jugoro (kor cing) pa TNFα ma nyuthu in cell makwo. IFN-γ (C) man IL-2 (D) nyutho in T cells. Nyuthu pa cytokines jupimu ku flow cytometry. Bar graph nyuthu wel (n = 6 ju ma remo migi yot) + SEM. Ti ku yore acel mi nyang ikum tung tung man dwoko wang lembe (*P<0.05 man **P<0.01) mi nwangu wel pa P. Nyuthu lembe ma wok kud i n = 3 lembe ma jutimo giri. (E to G) CD3/CD28 man IL-2 jutiyu kudu pi ketho T cells timo tic i MNA migi pi nindu 7. Jam piny ma jutiyo kudu eca ju kwalo mapodi man ingey sawa 4 mi PMA/ionomycin stimulation. Kite ma TNFα (E), IFN-γ (F) man IL-2 (G) jupimu kudu ku ELISA. Bar graph nyuthu wel (n = 5 ju ma remo migi yot) + SEM. P value juyike ku tiyo ku one-way analysis of variance man repeated measurements (*P<0.05). Lain ma jugoro ku dot nyutho rwom mi nwangu piny. (H) Cell lysis assay. FRα-CAR-T nyo GFP-CAR-T cells juyike ku adenosine (250μM) nyo MNA (10 mM) pi sawa 24, nyo juweko gi timo ngo (Ctrl). Wel pa tho pa SK-OV-3 cells jupimu. P value ma juporo ku Welch t test (*P<0.5 man **P<0.01).
Pi ni nwangu nyang ikum MNA-dependent TNFα expression regulation, aloka loka mi TNFα mRNA pa MNA-treated T cells jupimu (lanyut 5A). T cells ma kum gi yot ma jutiyo kudu ku MNA unyutho dongo pa TNFα, ma nyutho nia MNA ucungo iwi TNFα transcription regulation. Pi lubo tok lembe maeni, piny aryo ma jung'eyo ma doro TNFα, ma julwongo activated T cell nuclear factor (NFAT) man specific protein 1 (Sp1), jutimo sayusac ikumgi ni dwoko wang ikum MNA ma mako TNFα promoter ( 30). TNFα promoter utiye ku kabedo 6 ma NFAT mako man kabedo 2 ma mako Sp1, ma ucungo iwi kabedo acel [-55 base pairs (bp) kud i 5'cap] (30). Chromatin immunoprecipitation (ChIP) unyutho nia ka jutiyo kudu ku MNA, mako pa Sp1 ku TNFα promoter umedire wang adek. Kethu NFAT i iye de umedire man udok piny ma pire tek (lanyut 5B). Lembe eni nyutho nia MNA doro nyuthu pa TNFα nikadhu kud i Sp1 transcription, man i kadhiri manok nyuthu pa NFAT.
(A) Ka juporo ku T cells ma jutimo ku MNA, aloka loka pa TNFα i T cells ma jutimo ku MNA. Kit ma SEM nyuthu kudu (n = 5 ju ma gimio kony ma kum gi yot). Nyuthu lembe ma wok kud i n = 3 lembe ma jutimo giri. (B) TNFα promoter pa T cells ma jutiyo kudu ku nyo manangu 8 mM MNA ingey NFAT ku Sp1 juporo ku (Ctrl) man PMA/ionomycin pi sawa angwen. Immunoglobulin G (IgG) man H3 jutiyu kudu calu piny ma rac man maber pi immunoprecipitation, kubang wadi. Kwan pa ChIP unyutho nia mako pa Sp1 ku NFAT ku TNFα promoter i MNA-treated cells umedire wang mapol ka juporo ku control. Nyuthu lembe ma wok kud i n = 3 lembe ma jutimo giri. P value ma juporo ku t-tests madwong (*** P <0.01). (C) Ka juporo ku ascites pa HGSC, T cells (non-cytotoxic) unyutho medo pa TNF i tumor. Rangi nyuthu jurutwo matung tung. Cell ma ju nyuthu ju kwanu gi 300 man ju jwigo gi pi jwigo overdrawing (** Padj = 0.0076). (D) Model ma juyiko pa MNA pi two mi ic. MNA juyike i cell pa two man fibroblasts i TME man jutingo i T cells. MNA medo mako pa Sp1 ku TNFα promoter, ma ukelo dongo pa TNFα transcription man TNFα cytokine production. MNA de nyayu dwoko piny pa IFN-γ. Gengo tic pa T cell nyayu dwoko piny kero mi neko dhanu man medo dongo pa akala.
Ni lubre ku lapor, TNFα utiye ku adwogi maber ikum two man ikum two, ento etiye ku tic ma ungeyere maber i dongo man nyai pa two kanser mi ic (31-33). Ni lubre ku lapor, kadhiri pa TNFα in ascites man tumor tissues i ju ma twoyo pa ovarian cancer utiye malo uloyu i benign tissues (34-36). I yore mi tic, TNFα copo doro tic, tic man dongo pa rimo makwar, man loko kit ma rimo makwar nen kudu (37, 38). Malube ku lembe ma junwango, poko pa gene expression unyutho ni TNF ubedo ma malu in T cells in tumor tissues ka juporo ku ascites (lanyut 5C). Medu pa TNF nenre kende i T cell populations ku non-cytotoxic phenotype (Figure S5A). I ayi ma kicoko, lembe eni cwaku paru niya MNA utiye ku adwogi ario mi juku two man nyayu two i HGSC.
Label ma ler pa flow cytometry udoko yore madit mi ponjo TIL metabolism. Somo eni unyutho nia ka juporo ku lymphocytes mi rimo nyo T cells kud i lymphoid organs, murine man human TIL utiye ku kero madit mi maku glucose (4, 39) man rweny pa tic pa mitochondrial (19, 40). Kadi bende waneno adwogi marom i lembe maeni, dongo ma pire tek utiye ni poro metabolism pa tumor cells ku TIL kud i tumor tissue marom. Malube ku lapor moko mukadho, tumor (CD45-EpCAM +) cells kud i ascites man tumors utiye ku glucose ma malu uloyu CD8 + man CD4 + T cells, ma nyutho nia glucose ma malu pa tumor cells juporo ku T cells. Lembe pa pyem pa T cell. TME. Ento, tic pa mitochondrial pa tumor cells utiye malo uloyu pa CD8 + T cells, ento tic pa mitochondrial utiye marom ku pa CD4 + T cells. Adwogi eni medo kero pa lembe manyen ma ewacu niya oxidative metabolism piretek pi tumor cells (41, 42). Giwacu bende niya CD8 + T cells copo bedu ku kero mi timo tic maber uloyu CD4 + T cells, nyo CD4 + T cells copo tiyo ku piny mange ma nyayu carbon ma epe ku glucose pi gwoko tic pa mitochondrial (43, 44). Jucopo ngeyo nia waneno ngo tung tung moku ikum glucose uptake nyo mitochondrial activity ikind CD4 + T effectors, T effector memory man T central memory cells in ascites. Kumeno bende, tung tung pa CD8 + T cells in tumors epe ku aloka loka mi glucose uptake, ma nyutho tung tung madit ikum T cells ma jucul in vitro ku human TIL in vivo (22). Lembe ma waneno eni de juyike ku tic ku unbiased automatic cell population allocation, ma unyutho nia CD45 + / CD3- / CD4 + / CD45RO + cells ku glucose uptake ma malu man mitochondrial activity ma malu uloyo tumor cells utiye madwong ento gitiye ku Metabolic active cell population. Dhanu eni copo bedu dul ma nok pa myeloid suppressor cells nyo plasmacytoid dendritic cells ma junwango i scRNA-seq analysis. Kadi bende lembe ario eni jular jukoro pigi i two ma mako ic pa dhanu [45], podi gimito tic mukende utiye ni gonyo dul pa myeloid eni.
Kadi bende yore mi flow cytometry copo nyutho tung tung mi glucose man oxidative metabolism ikind kit cell, metabolites ma nwangre i glucose nyo carbon sources mange pi mitochondrial metabolism i TME podi ungiyu ngo. Kethu bedo tiye nyo bedo pee pa metabolites ni TIL subset moko mito juyik cell population kud i tissue ma juwodho. Pieno, yore mwa mi medo rwom pa cell ma rwate ku mass spectrometry ecopu mio nyang ikum metabolites ma dongo tung tung i T cells man tumor cell populations i sample pa jurutwo. Kadi bende yore eni utiye ku bero ikum poko cell ma tiyo ku fluorescence, metabolite moko copo bedo ku peko pi bedo maber pa piny man/nyoke pi loko piny pio pio (22). Ento, yore mwa ubedo ku copo mi nwangu piny ario ma nyayu two, adenosine man kynurenine, kum gi tung tung ikind kit lanyuth.
Kwano lembe mwa mi metabolomic pa two man TIL subtypes mio ngec madwong ikum tic pa metabolites in ovarian TME. Mir acel, nitio ku flow cytometry, wanwango nya epe ku tung tung ikind tic pa mitochondrial ikind two man CD4 + T cells. Ento, LC-MS/MS analysis unyutho aloka loka madit ikum wel pa metabolites ikind ju eni, ma nyutho nia lembe ma jumoko ikum TIL metabolism man tic pare mi metabolic mito ni gony maber. Mir aryo, MNA ubedo piny ma nyayu tung tung madit ikind CD45-cells ku T cells in ascites, ungo tumors. Pieno, poko poko man kabedo pa akala copo bedu ku adwogi matung tung ikum TIL metabolism, ma nyutho tung tung ma copo bedu i kabedu moko. Mir adek, nyuthu pa MNA-producing enzyme NNMT utiye kende kende i CAF, ma en e tumor cells i kadhiri ma nok, ento rwom pa MNA ma juromo nwangu nenre i tumor-derived T cells. Nyuthu pa NNMT in ovarian CAF utiye ku adwogi ma ungeyere mi kelo two kanca, moko ikume pi kelo CAF metabolism, tumor invasion man metastasis (27). Kadi bende rwom pa TIL utiye ma rwinyo, nyuthu pa NNMT in CAF utiye ku winjiri ku Cancer Genome Atlas (TCGA) mesenchymal subtype, ma rwate ku prognosis marac (27, 46, 47). Mi thum, nyuthu pa enzyme AOX1 ma kelo nyoth pa MNA de utiye kende kende i dul pa CAF, ma nyutho nia T cells gipe ku copo mi loko MNA. Adwogi eni cwaku paru niya kadi tic mukende mitere pi ketho lembe eni ucung matek, kadhiri madit pa MNA in T cells copo nyutho bedo tiye pa immunosuppressive CAF microenvironment.
Ni lubre ku rwom ma piny pa MNA transporters man rwom ma piny pa proteins ma pire tek ma tiyo i MNA metabolism, bedo pa MNA i T cells utiye piny ma jukuro ngo. NNMT nyo AOX1 de jucopo nwangu ngo ku scRNA-seq man qPCR pa dul ario ma tung tung. Adwogi eni nyutho nia MNA juyubu ngo ku T cells, ento juwodho kud i TME ma ucungo kudu. I vitro, lembe ma jutimo nyutho nia T cells copo coko MNA ma woko.
Somo mwa mi in vitro unyutho nia exogenous MNA nyayu nyuthu pa TNFα in T cells man medo kero pa Sp1 ku TNFα promoter. Kadi bende TNFα utiye ku tic mi jwigo two man jwigo two, i two mi ic, TNFα copo ketho two mi ic dongo (31-33). Weko TNFα in ovarian tumor cell culture nyoke weko TNFα signal in mouse models copo medo TNFα-mediated inflammatory cytokine production man juku dongo pa tumor (32, 35). Pieno, i lembe maeni, TME-derived MNA copo timo tic calu pro-inflammatory metabolite nikadhu kud i TNFα-dependent mechanism nikadhu kud i autocrine loop, ma copo ketho two mi ic wok man nyai (31). Ni lubre ku lembe maeni, TNFα blockade jubeponjo calu yore mi cango two mi ic (37, 48, 49). Mi medo, MNA nyayu peko i kum CAR-T cells ni ovarian tumor cells, ma mio lanyuth mange pa MNA-mediated immune suppression. I acel, adwogi eni nyutho lanyuth ma tumors man CAF cells giwodho MNA i extracellular TME. Ikind (i) TNF-induced ovarian cancer growth stimulation man (ii) MNA-induced T cell cytotoxic activity inhibition, eni copo bedu ku adwogi pa two (lanyut 5D).
I thum pa lembe, ni tiyo ku dikiri pa rwom pa cell ma nyap, rwom pa cell acel acel man metabolic profiling, somu eni unyutho tung tung madit pa immunometabolomic ikind tumors ku ascites cells ikind ju ma two HGSC. Lembe maeni ma jutimo unyutho nya tung tung utiye ikind glucose uptake man mitochondrial activity ikind T cells, man unyutho MNA calu non-cell autonomous immune regulatory metabolite. Lembe eni utiye ku adwogi ikum kite ma TME loko kudu T cell metabolism i two kanca pa dhanu. Kadi bende pyem ma rwate pi cam ikind T cells ku cancer cells jular jukoro, metabolites copo bedu calu regulators ma rwinyo pi medo dongo pa tumor man copo juku dwokowang pa kum. Wec mukende ikum tic pa jami ma nyayu two copo yabo yore mukende mi medo kero pa kum ma gwoko kum.
Lanyuth pa jurutwo man lembe mi yot kum ju nwango kud i BC cancer tumor tissue repository ma juyiko ku Canadian Tissue Repository Network. Nimakere ku cik ma juyio ku BC Cancer Research Ethics Committee man University of British Columbia (H07-00463), lanyuth pa jurutwo zo man lembe mi yot kum gi nwango waraga mi yioyic nyo giweko yioyic migi. Lanyuth ju gwoko gi i BioBank (BRC-00290). Kite pa jurutwo nen i Tables S1 ku S5. Pi cryopreservation, scalpel jutiyo kudu pi poko lanyuth pa two pa jaratwo man jucwalo i 100-micron filter pi nwangu cell acel kende. Ascites pa jaratwo juyike i 1500 rpm pi dakika 10 i 4°C mi poko cells man mi kwanyu supernatant. Cells maju nwango kud i tumor man ascites ju gwoko gi i 50% heat-inactivated human AB serum (Sigma-Aldrich), 40% RPMI-1640 (Thermo Fisher Scientific) man 10% dimethyl sulfoxide. Piny ma jugwoko eni juyike man jutiyo kudu pi metabolomics man metabolite determination ma juloko piny eno.
Piny ma iye zo utiye ku 0.22 μm ma juyiko 50:50 ma ju medo RPMI 1640: AimV. RPMI 1640 + 2.05 mM l-glutamine (Thermo Fisher Scientific) ma ju medo ku 10% heat-inactivated human AB serum (Sigma-Aldrich), 12.5 mM Hepes (Thermo Fisher Scientific), 2 mM l-glutamine (Thermo Fisher Scientific) x1 Penicillin Streptomycin (PenStrep) solution (Thermo Fisher Scientific) man 50 μMB-mercaptoethanol. AimV (Invitrogen) ju medo ku 20 mM Hepes (Thermo Fisher Scientific) man 2 mM l-glutamine (Thermo Fisher Scientific). Flow cytometer staining buffer ubedo ku 0.22μm filtered phosphate buffered saline (PBS; Invitrogen) ma ju medo ku 3% heat-inactivated AB human serum (Sigma). Buffer mi dongo pa cell utiye ku 0.22μm PBS ma juyiko man ju medo ku 0.5% heat-inactivated human AB serum (Sigma-Aldrich).
I 37°C ma pong, juyike ku 10 nM MT DR man 100 μM 2-NBDG pi dakika 30. I ngeye, juwiro cell ku rangi mi kwo eF506 i 4°C pi dakika apar wiye abic. Keth cells i FC Block (eBioscience) man Brilliant Stain Buffer (BD Biosciences), dilute in flow cytometer staining buffer (nimakere ku cik pa ju ma yiku), man iket pi dakika 10 i ot ma ngic. Keth rangi ikum cell ku dul pa antibodies (Table S2) i flow cytometry staining buffer i 4°C pi dakika 20. Keth cells i flow cytometry staining buffer (Cytek Aurora; 3L-16V-14B-8R configuration) mapodi jutimo ngo sayusac. Ti ku SpectroFlo man FlowJo V10 pi nyang ikum wel pa cell, man ti ku GraphPad Prism 8 pi cweyo lembe. Teko pa ler (MFI) pa 2-NBDG ku MT DR ubedo log-normalized, man i ngeye jutiyo ku t test ma juporo pi kwan pa ju ma two. Woth dhanu ceke ma gitiye ku lembe ma kadhu 40 kud i lembe ma jutimo; kyewo wel pa MFI pa 1 pi wel moku ci ma rac mapodi itimo ngo sayusac man nen pa lembe.
Pi medo kero mi timo tic ku cing ma malu, watiyo ku lembe ma jugoro ku shape restriction tree (FAUST) (21) mi ketho cell ni dul pa dhanu ingey kwanyu cell ma tho i FlowJo. Watimo tic ku cing wa pi ketho dul ma nen ni jupoko marac (ku PD1+ ku PD1-tumor cells) man dul ma jugwoko. Lanyuth acel acel utiye ku wel ma kadhu 2% pa cells, pi wel pa 11 pa dhanu.
Ficoll gradient density centrifugation jutiyo kudu pi poko PBMC ku leukocyte separation products (STEMCELL Technologies). CD8 + T cells jupoko kud i PBMC ku tic ku CD8 MicroBeads (Miltenyi) man ju medo gi i medium ma pong ku tic ku TransAct (Miltenyi) pi yenga 2 nimakere ku cik pa ju ma yiku. Juweko dul kom ne ucung pi nindu abic i piny ma IL-7 (10 ng/ml; PeproTech), man ju dwoko gi ku TransAct. I ceng mir 7, nimakere ku cik pa ju ma yiku, CD45 MicroBeads (Miltenyi) pa dhanu jutiyo kudu pi medo kero pa dul kom i rwom adek ma ulubu. Jupoko cell ne pi flow cytometry analysis (calu ma jukoro malu), man cell million acel jupoko wang adek pi LC-MS/MS analysis. Sampol ne jutiyo kudu ku LC-MS/MS calu ma juloko piny eno. Wapimo wel pa metabolite ma rwinyo ku wel pa ion 1,000. Lanyuth acel acel juyike ku wel pa ion (TIC), juloko logarithmic man juyike kende kende i MetaboAnalystR mapodi jutimo ngo sayusac.
Cell acel acel pa jaratwo acel acel juyike man juyike kud i 40 μm filter i medium ma jik (calu ma jukoro malu). Ni lubre ku cik pa ju ma yiku piny, timo adek ma ulubu ku poko piny ma tiyo ku MicroBeads (Miltenyi) jutiyo kudu pi medo CD8+, CD4+ man CD45- cells (i wi pee). I lembe ma nok, ju dwoko cell ne i cell enrichment buffer (calu ma jukoro malu) man ju kwanu. Juyiko cell ne ku CD8 pa dhanu, CD4 pa dhanu nyo CD45 pa dhanu (Miltenyi) i 4°C pi dakika 15, man juyike ku cell enrichment buffer. Lanyuth ne ukadhu kud i LS column (Miltenyi), man ju coko dul ma ber ku ma rac. Pi jwigo kare man medo kero pa cell, CD8-fraction jutiyo kudu pi rwom mir aryo mi CD4+, man CD4-fraction jutiyo kudu pi CD45-enrichment ma lubo. Keth jamb tiyo kudu iwi pee i kare mi poko piny.
Mi yiko lanyuth pi nyang ikum metabolite, ju lwoko cell wang acel ku pii ma ngic ma ngic, man 1 ml pa 80% methanol ju medu i lanyuth acel acel, man ju loko man ju ketho gi i nitrogen ma pii. Jam moko ma jucaku kudu juyike i rwom adek mi ngico man juyike i 14,000 rpm pi dakika 15 i 4°C. Piny ma ukadhu ma tingo metabolites juyike nitundo edoko muthwo. Jam piny ma jucwalo ju dwoko gi i 50 μl pa 0.03% formic acid, ju loko gi kara gi rombu, man ju loko gi i centrifuge pi kwanyu lum.
Woth metabolites calu ma jukoro malu. Lok pii ma ukadhu i pii ma tiyo maber pi timo sayusac ikum metabolomics. Ti ku yore mi timo yath ma rwinyo pi timo yath acel acel ku wel pa cell marom rom pi juku adwogi pa batch. Watimo pima maber pa piny ma wok kud i ngom zoo ma juwodho con iwi AB SCIEX QTRAP 5500 Triple Quadrupole Mass Spectrometer (50). Chromatographic analysis man peak area integration jutimo ku MultiQuant version 2.1 software (Applied Biosystems SCIEX).
Wel pa ion pa 1000 jutiyo kudu pi pimo wel pa metabolite ma rwinyo, man TIC pa sample acel acel jutiyo kudu pi kwanu normalized peak area pa metabolite acel acel ma junwango pi yiko aloka loka ma wok kud i instrumental analysis kud i sample processing. I ngei ma TIC ubedo maber, MetaboAnalystR(51) (default parameter) jutiyo kudu pi loko logarithmic man automatic norm line scaling. Watiyo ku PCA ku vegan R package pi timo sayusac ikum tung tung pa metabolome ikind kit lanyuth, man watiyo ku partial redundancy analysis pi ngiyo jurutwo. Ti ku yore pa Ward mi yubu heat map dendrogram mi poko bor pa Euclidean ikind lanyuth. Watiyo ku limma (52) iwi wel pa metabolite ma juyiko pi ni nyang metabolites ma tung tung ikind kit cell zo man microenvironment. Pi ni dwoko lembe yot, watiyo ku group mean parameter ni nyutho model, man waparu kit pa cell i microenvironment calu gurup acel acel (n = gurup 6); pi pimo lembe ma pire tek, watimo pima adek wang mapol pi metabolite acel acel Pi nigwoko replication ma ndra, ju medo jaratwo calu gin ma nyayu peko i limma design. Pi ni neno tung tung pa metabolites ikind jurutwo matung tung, wa loko limma model ma uketho i iye jurutwo i yo ma rwinyo. Wanyuthu beru pa tung tung ma jukoro con ikind kit cell man microenvironment pa Padj <0.05 (Benjamini-Hochberg correction).
I ngei medo kero ku tic pa Miltenyi Dead Cell Removal Kit (>80% viability), single-cell transcriptome sequencing jutimo iwi ascites ma kwo man tumor ma jutiyo kudu ku 10x 5′gene expression protocol. Jutimo sayusac iwi dhanu abic ma gibedo ku two ma rom rom man ascites, kadi nang nya kwo ma nok pa two acel uketho juketho ngo i iye. Pi ni nwangu yero pa jurutwo madwong, wa ketho lanyuth pa jurutwo acel acel i yore pa 10x chromium controller, man wa ngiyo kabedo pa ascites man tumor acel acel. I ngei lubo [Illumina HiSeq 4000 28×98 bp paired end (PE), Quebec genome; wel pa 73,488 man 41,378 kwan pi cell acel acel pi tumor man ascites]], watiyo ku CellSNP man Vireo (53) (ma lubere ku CellSNP calu The common human SNP (VCF) provided by GRCh38 is assigned a donor identity. Watiyo ku SNPRelate pi ni nyang ikum rwom pa jaratwo (IBS) weko cell ma jupoko ngo man cell ma juporo ku ascites man tumor samples (54). (2792 man 4183 cells kud i tumor man ascites, kubang acel acel) pi nyang ikumgi Watiyo ku igraph's (57) Louvain clustering of shared nearest neighbor network (SNN) malube ku Jaccard distance to cluster cells by expression. cells juweco pigi ku nyuthu pa CD8A man GZMA, muweko subclusters ku nyuthu pa ribosomal protein ma nok. Wanwango lembe ma juwodho pa Izar et al. (16), ikinde t-SNE embedding migi, ecopu doro nyuthu pa immune cell markers ku nyuthu pa NNMT.
PBMC jupoko kud i leukocyte separation products (STEMCELL Technologies) ku Ficoll gradient density centrifugation. CD3 + cells jupoko kud i PBMC ku tic ku CD3 beads (Miltenyi). Ka MNA utiye nyo epe, CD3+ cells juyike ku plate-bound CD3 (5μg/ml), soluble CD28 (3μg/ml) man IL-2 (300 U/ml; Proleukin). I ceng mir ajiki mi dongo, kwo (Fixable Viability Dye eFluor450, eBioscience) man dongo (123count eBeads, Thermo Fisher Scientific) jutimo ku flow cytometry. Pim tic pa jamb tiyo ku PMA (20 ng/ml) man ionomycin (1μg/ml) ku GolgiStop pi sawa angwen, man nen CD8-PerCP (RPA-T8, BioLegend), CD4-AF700 (RPA-T4) , BioLegend) man TNFα-fluoresceinsothianate (TCb) BD). Keth qPCR man ChIP cells ku PMA (20 ng/ml) man ionomycin (1μg/ml) pi sawa angwen. ELISA supernatant ju kwalo ma podi jutimo ngo ku PMA (20 ng/ml) man ionomycin (1 μg/ml) pi sawa angwen.
Lub cik pa ju ma yiku piny ni poko RNA ku RNeasy Plus Mini Kit (QIAGEN). Ti ku QIAshredder (QIAGEN) mi ketho lanyuth ubed acel. Ti ku RNA ma tek ni cDNA kit (Thermo Fisher Scientific) pi yubu DNA (cDNA). Ti ku TaqMan Rapid Advanced Master Mix (Thermo Fisher Scientific) pi pimo gene expression (nimakere ku cik pa ju ma yiku) ku probes ma e: Hs00196287_m1 (NNMT), Hs00154079_m1 (AOX1), Hs00427555155S (AX1) Hs02786624_g1 [glyceraldehyde-3-phosphate off Hydrogen (GAPDH)] man Hs01010726_m1 (SLC22A2). Jam lanyuth ne jutiyo kudu iwi StepOnePlus real-time PCR system (Applied Biosystems) (Applied Biosystems) i MicroAmp fast optical 96-well reaction plate (Applied Biosystems) ku MicroAmp optical film. Ct moku ci ma kadhu 35 jubinyuto ni etiye malo pa rwom ma jubi nwangu man jubiketho ni piny ma jubi nwangu ngo.
Tim ChIP calu ma jukoro con (58). I lembe ma nok, juyike ku formaldehyde (ma romo 1.42%) man juyike i ot pi dakika apar. Ti ku buffer mi lyethu (25 mM Hepes, 1.5 mM MgCl2, 10 mM KCl man 0.1% NP-40) iwi pee pi dakika 10, man idok i immunoprecipitation buffer calu ma jukoro (58). I ngeye jutimo sonication ku rwom ma e: 10 cycles (20 1-second pulses) man kare ma 40 seconds. Keth ChIP-grade immunoglobulin G (Cell Signaling Technology; 1μl), histone H3 (Cell Signaling Technology; 3μl), NFAT (Invitrogen; 3μl) man SP1 (Cell Signaling Technology; 3μl) antibodies ku lanyuth ma 4°CC yeng dyewor. Keth protein A beads (Thermo Fisher Scientific) ku lanyuth i 4°C ku yengo moth pi sawa acel, man ti ku chelex beads (Bio-Rad) mi medo DNA, man ti ku proteinase K (Thermo Fisher) pi nyayu protein. TNFα promoter junwango ku PCR: anyim, GGG TAT CCT TGA TGC TTG TGT; ento, GTG CCA ACA ACT GCC TTT ATA TG (207-bp product). Cal ne juyike ku Image Lab (Bio-Rad) man juporo ku tic pa ImageJ software.
Piny ma ucungo iwi cell culture jucoko calu ma jukoro malu. Lembe ma jutimo jutimo nimakere ku cik pa ju ma yiku kit pa human TNFα ELISA kit (Invitrogen), human IL-2 ELISA kit (Invitrogen) man human IFN-γ ELISA kit (Abcam). Nimakere ku cik pa ju ma yiku, pii ma ucungo iwi pii juyike 1:100 pi nwangu TNFα man IL-2, man 1:3 pi nwangu IFN-γ. Ti ku EnVision 2104 Multilabel Reader (PerkinElmer) pi pimo absorbance i 450 nm.
PBMC jupoko kud i leukocyte separation products (STEMCELL Technologies) ku Ficoll gradient density centrifugation. CD3 + cells jupoko kud i PBMC ku tic ku CD3 beads (Miltenyi). Ka MNA utiye nyo epe, CD3+ cells jutiyo kudu ku plate-bound CD3 (5μg/ml), soluble CD28 (3μg/ml) man IL-2 (300 U/ml; Proleukin) pi nindu 3. I ngei nindu 3, ju kwalo dul kom ne man ju lwoko ku 0.9% saline, man ju ketho pellet ne i friji. Kwan pa cell jutimo ku flow cytometry (Cytek Aurora; 3L-16V-14B-8R configuration) ma jutiyo kudu ku 123count eBeads.
Woth metabolites calu ma jukoro malu. Piny ma jukuboth ne judwoke kendu i kadhiri pa 4000 cell equivalents/μl. Nen lanyuth ku reversed-phase chromatography (1290 Infinity II, Agilent Technologies, Santa Clara, CA) man CORTECS T3 column (2.1×150 mm, particle size 1.6-μm, pore size 120-Å; #186008500, Waters). Polar mass spectrometer (6470, Agilent), ma i iye electrospray ionization timo tic maber. Mobile phase A utiye 0.1% formic acid (i H2O), mobile phase B utiye 90% acetonitrile, 0.1% formic acid. LC gradient utiye 0 ku 2 dakika pi 100% A, 2 ku 7.1 dakika pi 99% B, man 7.1 ku 8 dakika pi 99% B. I ngeye dwoko rwom pa column ku mobile phase A i flow rate pa 0.6 ml/min pi dakika 3. . Kite ma pii mol kudu ubedo 0.4ml/min, man ot pa column juyike ni 50°C. Ti ku MNA's pure chemical standard (M320995, Toronto Research Chemical Company, North York, Ontario, Canada) ni ketho kare mi gwoko (RT) man loko (RT = 0.882 dakika, loko 1 = 137→94.1, loko 2 = 137→92 , loko = 33→77). Ka aloka loka adek eni zo utimere i kare ma mitire, aloka loka mir acel jutiyu kudu pi pimo pi ni neno nia piny moko tiye maber. Standard curve pa MNA (Toronto Research Chemical Company) juyike ku dilutions abusiel mi stock solution (1 mg/ml) mi nwangu standards pa 0.1, 1.0, 10 man 100 ng/ml man 1.0 man 10μg/ml ma pii. Kabedo mi nwangu utiye 1 ng/ml, man dwokowang utiye ikind 10 ng/ml ku 10μg/ml. Kubang acel acel mi microliters aryo pa sample man standard jutiyu kudu pi LC/MS analysis, man mixed quality control sample jutiyo kudu kubang injection abora mi neno ni platform mi analysis utiye maber. Adwogi pa MNA pa lanyuth pa cell ma jutiyo kudu ku MNA ubedu ikind rwom pa assay. Kwano lembe jutimo ku kony pa MassHunter quantitative analysis software (v9.0, Agilent).
αFR-CAR mi rundi mir aryo juwodho kud i bang Song et al. (59). I lembe ma nok, yub ne otie ku lembe ma e: CD8a leader sequence, human αFR-specific single-chain variable fragment, CD8a hinge man transmembrane region, CD27 intracellular domain man CD3z intracellular domain. CAR sequence ma pong juyubu ku GenScript, man juyike i lentiviral expression vector mi rundi mir aryo ma malu pa GFP expression cassette ma jutiyo kudu pi pimo tic pa transduction.
Lentivirus juyubu ku timo transfection pa HEK293T cells [American Type Culture Collection (ATCC); judongo i Dulbecco's modified Eagle medium ma tye ku 10% fetal bovine serum (FBS) man 1% PenStrep, man jutiyo ku CAR-GFP vector man The packaging plasmids (psPAX2 man pMD2.G, Addgene) gitiyo ku lipofection amine (Sigma-Aldrich). Jam piny ma ucungo iwi two ne jucoko ingey sawa 48 ku 72 ingey transfection, juyike, man juyike ku ultracentrifugation. Gwok pii ma ucungo iwi kwidi i -80°C nitundu ikum transduction.
PBMC jupoko kud i kum piny ma jupoko ku rimo (STEMCELL Technologies) ku Ficoll gradient density centrifugation. Ti ku CD8 microbeads (Miltenyi) mi poko CD8+ kud i PBMC. Medu T cells ku TransAct (Miltenyi) man i TexMACS medium [Miltenyi; ju medo ku 3% pa ​​rimo pa dhanu ma lyeth ungo, 1% pa PenStrep man IL-2 (300 U/ml)]. I ngei sawa pyer aryo wi angwen, T cells juyike ku lentivirus (10 μl concentrated virus supernatant per 106 cells). 1 ku 3 ceng ingey loko ikum Cytek Aurora (i FSC (Forward Scatter)/SSC (Side Scatter), Singlet, GFP+), pim GFP expression pa cells ni nyutho transduction efficiency ma romo 30%.
CAR-T cells juyiko pi sawa 24 i Immunocult (STEMCELL Technologies; ju medo ku 1% PenStrep) ithe lembe ma e: ma jutimo ngo, jutimo ku 250 μM adenosine nyo 10 mM MNA. I ngei timo yath, juyiko CAR-T cells ku PBS man juporo ku 20,000 SK-OV-3 cells [ATCC; i McCoy 5A medium (Sigma-Aldrich) ma ju medo ku 10% FBS man 1% PenStrep i 10: Ratio pa effector ku target pa 1 ju medo i adek i Immunocult medium ma ju medo. SK-OV-3 cells man SK-OV-3 cells ma jupoko ku digitalis saponin (0.5mg/ml; Sigma-Aldrich) jutiyo kudu calu negative man positive controls, kubang wadi. I ngei sawa 24 mi co-cultivation, ju kwalo piny ma ukadhu man ju pimu lactate dehydrogenase (LDH) nimakere ku cik pa ju ma yiku (LDH Glo Cytotoxicity Assay Kit, Promega). LDH supernatant juyike 1:50 in LDH buffer. Wel mi nek jupimu ku formula ma e: wel mi nek = wel mi dwoko piny / wel mi nek ma malu x 100%, kama wel mi dwoko piny = co-culture-T cells kende, man wel mi nek madit = wel mi gwoko maber-ma rac.
Calu ma jukoro i lembe nyo jam tic man yore, ti ku GraphPad Prism 8, Microsoft Excel nyo R v3.6.0 pi nyang ikum wel. Ka juwodho lanyuth madwong kud i kum jaratwo acel (macalu ascites man tumor), watiyo ku paired t test nyo wa medo jaratwo calu random effect i linear nyo generalized model calu ma mitire. Pi nyang ikum metabolomics, lembe ma pire tek jutimo wang adek.
Pi lembe mukende ma medo ikum lembe maeni, 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. Dephrini (R.R.R. Bellnis), Deraph. Jones (Russell G. Jones), Phineas T. Hamilton (Phineas T.
MNA konyo i jwigo T cells man etiye yore maber mi gwoko kum dhanu pi remo pa dhanu.
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. Dephrini (R.R.R. Bellnis), Deraph. Jones (Russell G. Jones), Phineas T. Hamilton (Phineas T.
MNA konyo i jwigo T cells man etiye yore maber mi gwoko kum dhanu pi remo pa dhanu.
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