Deng immunomodulatory metabolites metung lang susi keng tumor microenvironment (TME), pero kareng mapilan a exceptions, ing karelang pangatau e pa balu. keni, sinuri mi la reng tumor ampo T cells ibat kareng tumor ampo ascites da reng pasyenti na ating high-grade serous carcinoma (HGSC) para abalu mi ing metabolome da reng miyayaliwang TME compartments. Deng ascites ampo reng tumor cells atin lang maragul a pamiyaliwa keng metabolite. Nung ikumpara la kareng ascites, deng tumor-infiltrating T cells mas dakal la keng 1-methylnicotinamide (MNA). Agyang matas ya ing level ning MNA kareng T cells, ing pamagpahayag ning nicotinamide N-methyltransferase (metung a enzyme a mag-catalyze king pamaglipat da reng methyl groups ibat king S-adenosylmethionine papunta king nicotinamide) limitadu ya kareng fibroblasts ampong tumor cells. keng gagawan na, ing MNA mag-induce yang T cells para mag-secrete yang tumor-promoting cytokine tumor necrosis factor alpha. Uli na niti, ing TME-derived MNA makaambag ya keng immune regulation da reng T cells ampo magrepresenta yang potensyal a target ning immunotherapy para keng pamanulu keng cancer ning tau.
Deng tumor-derived metabolites atin lang malalam a inhibitory effect keng anti-tumor immunity, at dakal la reng ebidensya a papakit na malyari la namang magsilbing susi para keng pamagprogresu ning sakit (1). Dagdag pa keng Warburg effect, ing bayung obra megumpisa yang mag characterize keng metabolic state da reng tumor cells ampo ing kayang relasyun keng immune state ning tumor microenvironment (TME). Deng pamagaral kareng modelung mouse ampo kareng T cells ning tau pepakit da na ing glutamine metabolism (2), oxidative metabolism (3) ampo ing glucose metabolism (4) malyari lang kumilos independenti kareng miyayaliwang immune cell subgroups. Deng mapilan a metabolites kareng pathways a reni makapigil la keng anti-tumor function da reng T cells. Mepatunayan na ing pamag-blockade ning coenzyme tetrahydrobiopterin (BH4) malyari yang makasira keng pamagdakal da reng T cells, at ing pamagdakal ning BH4 keng katawan malyari yang magpasanting keng anti-tumor immune response a mediated ning CD4 ampo CD8. Dagdag pa, ing immunosuppressive effect ning kynurenine malyari yang iligtas kapamilatan ning pamamie BH4 (5). Ing isocitrate dehydrogenase (IDH) mutant glioblastoma, ing pamaglual ning enantiometabolic (R)-2-hydroxyglutarate (R-2-HG) makapigil ya keng T cell activation, proliferation ampo cytolysis Activity (6). Bayu-bayu pa mu, meyakit na ing methylglyoxal, metung a by-product ning glycolysis, gagawan de reng suppressor cells ning myeloid origin, at ing T cell transfer ning methylglyoxal malyari yang makapigil keng effector T cell function. Ing pamanulu, ing pamag-neutralize ning methylglyoxal malyari yang lampasan ing aktibidad da reng myeloid-derived suppressor cells (MDSC) at synergistically a pasikanan ing checkpoint blockade therapy kareng modelung mouse (7). Deng pamagaral a reni parehu lang didinan pansin ing susi da reng TME-derived metabolites keng pamagregulate kareng T cell function ampo activity.
Ing T cell dysfunction maralas yang mibalita keng ovarian cancer (8). Iti metung yang bage uli ning metabolic characteristics a atyu keng hypoxia ampo abnormal a tumor vasculature (9), na magresulta keng pamagbayu ning glucose ampo tryptophan keng by-products antimo ing lactic acid ampo kynurenine. Ing sobrang extracellular lactate babawasan na ing pamaglalang ning interferon-γ (IFN-γ) at mag drive ya keng pamiyaliwa da reng myelosuppressive subgroups (10, 11). Ing pamangan ning tryptophan direktang makapigil keng T cell proliferation ampo makapigil keng T cell receptor signaling (12-14). Agyang makanyan, dakal a obra ing makapadurut keng immune metabolism ing megawa keng in vitro T cell culture gamit ing optimized media, o limitadu kareng homologous mouse models in vivo, ala metung man kareti ing lubus a magpakit keng heterogeneity da reng cancer ning tau ampo ing Physiological macro ampo micro a kapaligiran.
Ing karaniwan a katangian ning ovarian cancer yapin ing peritoneal spread ampo ing itsura ning ascites. Ing pamitipun ning cell fluid keng ascites makasuglung ya keng advanced a sakit ampo e masanting a prognosis (15). Agpang kareng ulat, ining metung a compartment hypoxic ya, atin yang matas a lebel ning vascular endothelial growth factor (VEGF) ampo indoleamine 2,3-dioxygenase (IDO), ampo makalub ya kareng T regulatory cells ampo myeloid inhibitory cells (15-18). Ing metabolic a kapaligiran ning ascites malyari yang miyayaliwa keng tumor mismu, inya e malino ing pamag-reprogram kareng T cells keng peritoneal space. Dagdag pa, ing susi a pamiyaliwa ampo ing heterogeneity pilatan da reng ascites ampo reng metabolites a atyu keng kapaligiran ning tumor malyari yang maka-abala keng pamaglub da reng immune cells ampo ing karelang gagawan kareng tumor, ampo kailangan ing karagdagang pamanyaliksik.
Para asolve la deng problemang deni, dinisenyu mi ing metung a sensitive cell separation ampo liquid chromatography tandem mass spectrometry (LC-MS/MS) a paralan para abalu ing miyayaliwang uri ning cell (kayabe na ing CD4 + ampo CD8 + T cells) ampo ing kilub ampo pilatan da reng tumor Deng kayang metabolites makayabe la kareng cells keng pareung ascites ampo tumor a kapaligiran ning pasyenti. Gagamitan mi ya ining paralan kayabe ning high-dimensional flow cytometry ampo ing single-cell RNA sequencing (scRNA-seq) para mamye matas a resolution a larawan ning metabolic status da reng susi a populasyun a reni. Ing paralan ayni pepakit na ing maragul a pamagtas ning level ning 1-methylnicotinamide (MNA) kareng tumor T cells, at ing in vitro experiments pepakit na ing immunomodulatory effect ning MNA keng T cell function e ya pa balu kanita. Keraklan, ing paralan ayni papakit na ing mutual metabolic interactions pilatan da reng tumor ampo reng immune cells, ampo mamye yang makatagulaling a panaun kareng immune regulation metabolites, na malyaring magamit para keng pamanulu keng T cell-based ovarian cancer immunotherapy Treatment opportunities.
Ginamit mi ing high-dimensional flow cytometry para a-quantify ing glucose uptake [2-(N-(7-nitrophenyl-2-oxa-1,3-diaza-4-yl)amino)-2-deoxyglucose (2-NBDG) ampo ing mitochondrial activity [MitoTracker Deep Red (MT DR) (MT DR, 2019, side side) tipikal a marker a makayaliwa kareng immune cells ampo kareng populasyun da reng tumor cell (Table S2 ampo Figure S1A). Ing pamagsuri ayni pepakit na nung ikumpara la kareng T cells, deng ascites ampo tumor cells atin lang mas matas a glucose uptake levels, pero mas malati la pamiyaliwa keng mitochondrial activity. Ing average a glucose uptake da reng tumor cells [CD45-EpCAM (EpCAM)+] atlu anggang apat a besis ya kesa kareng T cells, at ing average a glucose uptake da reng CD4 + T cells 1.2 besis ya kesa kareng CD8 + T cells, a magpakit na ing Tumor infiltrating lymphocytes (TIL) atin lang miyayaliwang metabolic requirements agyang parehu la keng figure TAME (Figure 1). Ing pamiyaliwa, ing mitochondrial activity kareng tumor cells kalupa ne ning CD4 + T cells, at ing mitochondrial activity da reng adwang uri ning cell mas matas ya kesa kareng CD8 + T cells (Figure 1B). Keraklan, deng resulta a reni papakit da ing metabolic level. Ing metabolic activity da reng tumor cells mas matas ya kesa kareng CD4 + T cells, ampo ing metabolic activity da reng CD4 + T cells mas matas ya kesa kareng CD8 + T cells. Agyang makanyan la reng epektu kareng uri da reng cell, alang pare-pareung pamiyaliwa keng metabolic status da reng CD4 + ampo CD8 + T cells o ing karelang relatibung proporsiun keng ascites kumpara kareng tumor (Figure 1C). Ing pamiyaliwa, keng CD45-cell fraction, ing proporsiun da reng EpCAM+ cells keng tumor meragdagan ya kumpara keng ascites (Figure 1D). Atin mu namang ikit a malino pamiyaliwa ning metabolic pilatan da reng EpCAM+ ampo EpCAM- cell components. Deng EpCAM+ (tumor) cells atin lang mas matas a glucose uptake ampo mitochondrial activity kesa kareng EpCAM- cells, na mas matas kesa keng metabolic activity da reng fibroblasts kareng tumor cells keng TME (Figure 1, E ampo F).
(A ampo B) Median fluorescence intensity (MFI) ning glucose uptake (2-NBDG) (A) ampo mitochondrial activity da reng CD4 + T cells (MitoTracker dark red) (B) Representative graphs (kaili) ampo tabulated data (Wan), CD8 + T cells ampo EpCAM + CD45-tumor cells ampo tumor cells ibat kareng tumor. (C) Ing ratio da reng CD4 + ampo CD8 + cells (da reng CD3 + T cells) keng ascites ampo tumor. (D) Proportion ning EpCAM + tumor cells keng ascites ampo tumor (CD45−). (E ampo F) EpCAM + CD45-tumor ampo EpCAM-CD45-matrix glucose uptake (2-NBDG) (E) ampo mitochondrial activity (MitoTracker dark red) (F) representative graphs (kayli) ampo tabulated data (Wan) Ascites ampo tumor cells. (G) Representative graphs ning CD25, CD137 ampo PD1 expression kapamilatan ning flow cytometry. (H ampo I) CD25, CD137 ampo PD1 expression keng CD4 + T cells (H) ampo CD8 + T cells (I). (J ampo K) Naive, central memory (Tcm), effector (Teff) ampo effector memory (Tem) phenotypes base keng expression ning CCR7 ampo CD45RO. Representative images (kaili) ampo tabular data (wanan) da reng CD4 + T cells (J) ampo CD8 + T cells (K) keng ascites ampo tumor. P values ​​fidetermined by paired t-test (*P<0.05, **P<0.01 and ***P<0.001). Ing linya mangabaldugan yang makatuglung a pasyenti (n = 6). FMO, fluorescence minus metung; MFI, median fluorescence intensity.
Ing karagdagang pamagsuri pepakit na ing aliwang makabaldugan a pamiyaliwa king pilatan ning matas a resolution ning T cell phenotypic status. Ing activated (Figure 1, G to I) ampo ing effector memory (Figure 1, J ampo K) kareng tumor mas maralas la kesa keng ascites (proportion ning CD3 + T cells). makanyan mu naman, ing pamagsuri keng phenotype kapamilatan ning pamagpahayag da reng activation markers (CD25 ampo CD137) ampo reng depletion markers [programmed cell death protein 1 (PD1)] pepakit na agyang miyayaliwa la reng metabolic characteristics da reng populasyun a reni (Figure S1, B to E), pero alang makabaldugan a metabolic a pamiyaliwa ing pare-pareung mayayakit pilatan da reng naive, o subset a epektu ning memorya (Figure S1, S1 YAKU). Deting resulta mekumpirma la kapamilatan ning pamangamit kareng paralan ning pamagaral ning makina para automatic lang i-assign deng cell phenotypes (21), a lalu pang pepakit ing presensya ning maragul a bilang da reng bone marrow cells (CD45 + / CD3- / CD4 + / CD45RO +) king ascites ning pasyenti (Figure S2A ). Kareng eganaganang mekilalang cell type, ining myeloid cell population pepakit na ing pekamatas a glucose uptake ampo mitochondrial activity (Figure S2, B to G). Deng resulta a reni papakit da ing masikan a pamiyaliwa da reng miyayaliwang uri da reng cell a mayayakit kareng ascites ampo kareng tumor kareng pasyenti ning HGSC.
Ing peka-hamon keng pamanintindi kareng metabonomic a katangian ning TIL yapin ing kailangang i-isolate la reng T cell samples a sapat a kalinisan, kalidad ampo dagul ibat kareng tumor. Deng bayung pamagaral pepakit da na ing pamag-sort ampo pamag-enrich ning bead base keng flow cytometry malyari yang magdalang pamagbayu keng cellular metabolite profiles (22-24). Para alampasan ing problemang ayni, pepa-optimize mi ing bead enrichment method para i-isolate at i-isolate ing TIL ibat keng surgically resected a human ovarian cancer bayu ing pamag-analisa kapamilatan ning LC-MS/MS (lawen ya ing Materials and Methods; Figure 2A). Para abalu ing kabilugan a epekto na niting protocol kareng pamagbayu ning metabolite, ikumpara mi la reng metabolite profiles da reng T cells a me-activate kareng malusug a donors kaybat ning bead separation step kareng cells a e me-bead separated pero menatili la keng yelo. Ing quality control analysis ayni menakit yang matas a correlation kareng adwang kundisyun (r = 0.77), at ing technical repeatability ning grupu da reng 86 metabolites atin yang matas a repeatability (Figure 2B). Uli na niti, deng paralan a reni malyari lang gawang ustu metabolite analysis kareng cells a daralan keng cell type enrichment, inya sa’t mamye yang mumunang high-resolution platform para akilala la reng specific metabolites keng HGSC, inya sa’t malyari lang mika mas malalam a pamanintindi deng tau keng cell specificity Sexual metabolism program.
(A) Schematic diagram ning magnetic bead enrichment. Bayu ing pamagsuri kapamilatan ning LC-MS/MS, ding cell dumalan lang atlung sunod-sunod a magnetic bead enrichment o manatili la king yelo. (B) Ing epektu ning enrichment type keng karakal da reng metabolites. Ing average da reng atlung sukad para keng balang uri ning pamagpayaman ± SE. Ing grey a linya mangabaldugan yang 1:1 a relasyun. Ing intra-class correlation (ICC) da reng paulit-ulit a sukad a mayayakit keng axis label. NAD, nicotinamide adenine dinucleotide. (C) Schematic diagram ning workflow ning pasyenti metabolite analysis. Deng ascites o tumor kukunan da la kareng pasyenti at i-cryopreserve la. Ing malating dake ning balang sample me-analisa ya kapamilatan ning flow cytometry, kabang ding mitagan a sample dinalan lang atlung round ning enrichment para kareng CD4+, CD8+ ampo CD45- cells. Deting cell fractions me-analisa la gamit ing LC-MS/MS. (D) Heat map ning standardized metabolite abundance. Ing dendrogram magrepresenta yang Ward's clustering da reng Euclidean a distansya pilatan da reng sample. (E) Principal component analysis (PCA) ning sample metabolite map, papakit na ing atlung replicate ning balang sample, deng sample ibat keng parehung pasyenti makasuglung la keng metung a linya. (F) Ing PCA ning metabolite profile ning sample a makakondisyon keng pasyenti (ie, gamit ing partial redundancy); ing sample type limitadu ya keng convex hull. PC1, main component 1; PC2, main component 2.
Kaybat, ginamit mi ining paralan ning pamagpayaman para asuri ing 99 a metabolites keng CD4 +, CD8 + ampo CD45-cell fractions keng mumunang ascites ampo tumor da reng anam a pasyenti ning HGSC (Figure 2C, Figure S3A ampo Table S3 ampo S4). Ing populasyun a interesadu yapin ing 2% anggang 70% ning orihinal a maragul a sample da reng mabie cell, at ing proporsiun da reng cell miyayaliwa yang maragul kareng pasyenti. Kaybat dang mikawani deng beads, ing enriched fraction of interest (CD4+, CD8+ o CD45-) mag-account ya keng maigit 85% da reng eganaganang mabibye cells keng sample keng average. Ing paralan ning pamagpayaman paintulutan na kaming mag-analisa kareng populasyun da reng cell ibat keng human tumor tissue metabolism, na imposibli gawan ibat kareng mangaragul a sample. Gamit ining protocol, abalu mi na ing l-kynurenine ampo ing adenosine, deng adwang well-characterized immunosuppressive metabolites mitas la keng tumor T cells o tumor cells (Figure S3, B ampo C). Uli na niti, deng resulta a reni papakit da ing katapatan ampo ing abilidad ning kekatamung cell separation ampo mass spectrometry technology para akit la reng biologically important metabolites kareng tissue da reng pasyenti.
Ing kekaming pamagsuri pepakit na ing masikan a metabolic separation da reng cell types kilub ampo pilatan da reng pasyenti (Figure 2D ampo Figure S4A). Partikular, nung ikumpara la kareng aliwang pasyenti, ing pasyenti 70 pepakit na ing miyayaliwang metabolic characteristics (Figure 2E ampo Figure S4B), a magpakit na atin yang maragul a metabolic heterogeneity kareng pasyenti. Dapat papansinan na nung ikumpara la kareng aliwang pasyenti (1.2 anggang 2 litru; Table S1), ing kabilugan a dagul ning ascites a tinipun da reng pasyenti 70 (80 ml) mas malati ya. Ing pamagkontrol king inter-patient heterogeneity kabang mag-analisa kareng principal component (alimbawa, gamit ing partial redundancy analysis) papakit na ing pare-pareung pamagbayu pilatan da reng uri da reng cell, at deng uri da reng cell ampo/o microenvironment malino lang mipagkalub agpang king profile ning metabolite (Figure 2F ). Ing pamagsuri kareng metung a metabolites dininan nong pansin deng epektu a reni at pepakit na ing maragul a pamiyaliwa da reng uri da reng cell ampo ing microenvironment. Dapat papansinan na ing peka-extreme a pamiyaliwa a mayayakit yapin ing MNA, na keraklan mayaman ya keng CD45- cells ampo keng CD4+ ampo CD8+ cells na lulub keng tumor (Figure 3A). Para kareng CD4 + cells, ing epektu ayni malino ya, at ing MNA kareng CD8 + cells balamu masikan ya apektadu keng kapaligiran. Makanyan man, ali ya importanti iti, uling atlu lamu kareng anam a pasyenti deng malyaring evaluate para kareng tumor CD8+ scores. Dagdag pa keng MNA, kareng miyayaliwang uri da reng cell keng ascites ampo tumor, deng aliwang metabolites na e masyadung makakilala keng TIL miyayaliwa la naman (Figures S3 ampo S4). Uli na niti, deng data a reni papakit da ing metung a promising set da reng immunomodulatory metabolites para keng karagdagang pamanyaliksik.
(A) Normalized a laman ning MNA keng CD4+, CD8+ ampo CD45- cells ibat keng ascites ampo tumor. Ing box plot papakit ne ing median (line), interquartile range (frame hinge) ampo ing data range, anggang 1.5 besis ing interquartile range (frame whisker). Antimo ing makasulat keng Patient Materials and Methods, gamitan me ing limma value ning pasyenti para abalu ing P value (*P<0.05 ampo **P<0.01). (B) Schematic diagram ning 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 (berdi): 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 ning scRNA-seq ning ascites (grey) ampo tumor (pula; n = 3 pasyenti). (D) NNMT expression kareng miyayaliwang cell populations a mekilala gamit ing scRNA-seq. (E) Expression ning NNMT ampo AOX1 keng SK-OV-3, human embryonic kidney (HEK) 293T, T cells ampo MNA-treated T cells. Ing folded expression makayagpang ya keng SK-OV-3. Ing expression pattern keng SEM mipalabas ya (n = 6 a malusog a donors). Deng Ct a maygit 35 tuturing dong e akit (UD). (F) Expression ning SLC22A1 ampo SLC22A2 keng SK-OV-3, HEK293T, T cells ampo T cells a me-treat keng 8mM MNA. Ing folded expression makayagpang ya keng SK-OV-3. Ing expression pattern keng SEM mipalabas ya (n = 6 a malusog a donors). Deng Ct a maygit 35 tuturing dong e akit (UD). (G) Cell MNA content keng activated healthy donor T cells kaybat ning 72 oras a pamag-incubate keng MNA. Ing expression pattern keng SEM mipalabas ya (n = 4 a malusog a donors).
Ing MNA gagawan de kapamilatan ning pamaglipat ning methyl group ibat king S-adenosyl-1-methionine (SAM) papunta king nicotinamide (NA) kapamilatan ning nicotinamide N-methyltransferase (NNMT; Figure 3B). Ing NNMT overexpressed ya kareng miyayaliwang cancer ning tau at makasuglung ya keng proliferation, invasion ampo metastasis (25-27). Para mas aintindian ing panibatan ning MNA kareng T cells keng TME, ginamit mi ing scRNA-seq para abalu mi ing expression ning NNMT kareng cell types kareng ascites ampo tumors da reng atlung pasyenti ning HGSC (Table S5). Ing pamagsuri kareng 6,500 a cell pepakit na na keng ascites ampo tumor a kapaligiran, ing NNMT expression makabawal ya keng presumed fibroblast ampo tumor cell populations (Figure 3, C ampo D). Dapat papansinan na alang malino NNMT expression keng nanu mang populasyun na mag express PTPRC (CD45 +) (Figure 3D ampo Figure S5A), na magpakit na ing MNA a akit keng metabolite spectrum mipakilala ya keng T cells. Ing pamagpalwal ning aldehyde oxidase 1 (AOX1) mag convert yang MNA keng 1-methyl-2-pyridone-5-carboxamide (2-PYR) o 1-methyl-4-pyridone-5-carboxamide (4- PYR); Ing Figure 3B) makabawal ya keng populasyun da reng fibroblasts a mag-express COL1A1 (Figure S5A), na nung nukarin papakit da na deng T cells ala lang abilidad keng conventional MNA metabolism. Ing expression pattern da reng MNA-related genes me-verify ya gamit ing kaduang independent cell data set ibat kareng ascites ibat kareng pasyenti ning HGSC (Figure S5B; n = 6) (16). Dagdag pa, ing quantitative polymerase chain reaction (qPCR) analysis da reng malusog a donor T cells a me-treat king MNA pepakit na nung ikumpara la kareng control SK-OV-3 ovarian tumor cells, ing NNMT o AOX1 halus e ya mipalabas (Figure 3E). Deting e asahan a resulta papakit da na ing MNA malyari yang mipalabas ibat kareng fibroblasts o tumor papunta kareng makalapit a T cells keng TME.
Agyang deng kandidatu kayabe la reng pamilya da reng organic cation transporters 1 anggang 3 (OCT1, OCT2 ampo OCT3) a maka-encode keng soluble carrier 22 (SLC22) a pamilya (SLC22A1, SLC22A2 ampo SLC22A3), deng potensyal a transporters ning MNA e la pa malino (28) . Ing QPCR ning mRNA ibat kareng malusog a donor T cells pepakit na ing mababang expression levels ning SLC22A1 pero e akit ing SLC22A2, na mekumpirma na minunang mibalita ya keng literatura (Figure 3F) (29). Ing pamiyaliwa, ing SK-OV-3 ovarian tumor cell line pepakit na ing matas a lebel da reng adwang transporters (Figure 3F).
Para asuri ing posibilidad na deng T cells atin lang abilidad a manyipsip kareng dayu a MNA, deng malusug a donor T cells me-culture la kilub ning 72 oras king presensya da reng miyayaliwang konsentrasyun ning MNA. Nung ala yang exogenous MNA, e malyaring akit ing cellular content ning MNA (Figure 3G). Makanyan man, deng activated T cells a me-treat keng exogenous MNA pepakit da ing dose-dependent a pamagtas ning MNA content kareng cells, anggang 6 mM MNA (Figure 3G). Ing resulta ayni papakit na agyang mababa ya ing transporter expression ampo ing kakulangan ning main enzyme a maki pakibatan keng intracellular MNA metabolism, ing TIL malyari ya pa muring tanggapan ing MNA.
Ing spectrum da reng metabolites kareng T cells da reng pasyenti ampo reng in vitro MNA absorption experiments daragdagan da ing posibilidad na deng cancer-associated fibroblasts (CAF) magsecrete lang MNA ampo deng tumor cells malyari lang magregulate keng phenotype ampo ing function ning TIL. Para abalu ing epektu ning MNA kareng T cells, deng malusog a donor T cells me-activate la in vitro keng presensya o ala nang MNA, at ing karelang proliferation ampo cytokine production me-evaluate la. Kaybat ning 7 aldo ning pamagdagdag MNA keng pekamatas a dosis, ing bilang ning pamagdoble ning populasyun metung yang katamtaman a pamagbawas, kabang ing sikanan menatili ya kareng sablang dosis (Figure 4A). Dagdag pa, ing pamanulu keng exogenous MNA meging sangkan ning pamagtas ning proporsiun da reng CD4 + ampo CD8 + T cells a magpalwal tumor necrosis factor-α (TNFα; Figure 4B). Ing pamiyaliwa, ing intracellular a pamaglalang ning IFN-γ makabaldugan yang mebawas kareng CD4 + T cells, pero ali kareng CD8 + T cells, at alang makabaldugan a pamagbayu keng interleukin 2 (IL-2; Figure 4, C and D). Uli na niti, ing enzyme-linked immunosorbent assay (ELISA) da reng supernatants ibat kareng MNA-treated T cell cultures pepakit na ing maragul a pamagtas ning TNFα, pamagbaba ning IFN-γ, at alang pamagbayu king IL-2 (Figure 4, E to G). . Ing pamagbaba ning IFN-γ papakit na ing MNA malyari yang mika papel keng pamag-inhibit keng anti-tumor activity da reng T cells. Para abalu ing epektu ning MNA keng T cell-mediated cytotoxicity, deng chimeric antigen receptor T (FRα-CAR-T) cells a magtarget keng folate receptor α ampo CAR-T (GFP) a reguladu ning green fluorescent protein (GFP) -CAR-T) cells gagawan da la reng malusog a donor peripheral blood mononuclear cells (PBMC). Deng CAR-T cells me-culture la kilub ning 24 oras keng presensya ning MNA, at kaybat me-co-culture la kareng human SK-OV-3 ovarian tumor cells a mag-express folate receptor α keng effector to target ratio a 10:1. Ing MNA treatment meging sangkan ning maragul a pamagbawas keng pamakamate kareng FRα-CAR-T cells, na kalupa da reng FRα-CAR-T cells a metreat keng adenosine (Figure 4H).
(A) Total viable cell count ampo population doubling (PD) diretsu ibat keng culture keng aldo 7. Ing bar graph magrepresenta ya keng mean + SEM da reng anam a malusog a donors. Magrepresenta yang data ibat keng n = 3 a independenting experiment. (B to D) CD3/CD28 ampo IL-2 ginamit la para i-activate la reng T cells keng karelang MNA concentrations kilub ning 7 aldo. Bayu ing pamag-analisa, ding cells me-stimulate la keng PMA/ionomycin keng GolgiStop kilub ning 4 oras. TNFα (B) expression keng T cells. Alimbawa king larawan (kayli) ampo tabular data (wanan) ning TNFα expression kareng mabibye cells. IFN-γ (C) ampo IL-2 (D) expression kareng T cells. Ing expression da reng cytokines mesukad ya kapamilatan ning flow cytometry. Ing bar graph magrepresenta yang mean (n = 6 healthy donors) + SEM. Gamitan ing one-way analysis ning variance ampo paulit-ulit a sukad (*P<0.05 ampo **P<0.01) para abalu ing P value. Magrepresenta yang data ibat keng n = 3 a independenting experiment. (E to G) CD3/CD28 ampo IL-2 ginamit la para i-activate la reng T cells keng karelang MNA concentrations kilub ning 7 aldo. Ing medium mekolekta ya bayu at kaybat ning 4 oras ning PMA/ionomycin stimulation. Ing konsentrasyun ning TNFα (E), IFN-γ (F) ampo IL-2 (G) mesukad ya kapamilatan ning ELISA. Ing bar graph magrepresenta yang mean (n = 5 healthy donors) + SEM. Ing P value meyakit ya gamit ing one-way analysis ning variance ampo paulit-ulit a sukad (*P<0.05). Ing makatulduk a gulis papakit na ing limitasyun ning pamagtuklas. (H) Cell lysis assay. Deng FRα-CAR-T o GFP-CAR-T cells me-adjust la keng adenosine (250μM) o MNA (10 mM) kilub ning 24 oras, o me-adjust la (Ctrl). Ing porsyentu ning pamakamate da reng SK-OV-3 cells mesukad ya. P value a meyakit keng Welch t test (*P<0.5 ampo **P<0.01).
Para mika mechanistic a pamanintindi keng MNA-dependent TNFα expression regulation, ing pamagbayu keng TNFα mRNA da reng MNA-treated T cells me evaluate la (Figure 5A). Deng malusog a donor T cells a me-treat keng MNA pepakit da ing adwang besis a pamagtas keng TNFα transcription levels, a magpakit na ing MNA makadependi ya keng TNFα transcriptional regulation. Para abalu ing posibling regulatory mechanism, adwa lang balu transcription factors na magregulate keng TNFα, ilapin deng activated T cell nuclear factor (NFAT) ampo ing specific protein 1 (Sp1), me evaluate la bilang pakibat keng MNA binding keng proximal TNFα promoter ( 30). Ing TNFα promoter atin yang 6 a mekilalang NFAT binding sites ampo 2 Sp1 binding sites, a mipapalibut keng metung a site [-55 base pairs (bp) ibat keng 5'cap] (30). Ing chromatin immunoprecipitation (ChIP) pepakit na nung gamitan me ing MNA, ing pamagtali ning Sp1 keng TNFα promoter miragdagan yang atlung besis. Ing pamaglub ning NFAT meragdagan ya at linapit ya keng importansya (Figure 5B). Deng data a reni papakit da na ing MNA magregulate ya keng expression ning TNFα kapamilatan ning Sp1 transcription, ampo keng mas ditak a paralan ing expression ning NFAT.
(A) Nung ikumpara la kareng T cells a me-culture a alang MNA, ing fold change ning TNFα expression kareng T cells a me-treat keng MNA. Ing expression pattern keng SEM mipalabas ya (n = 5 healthy donors). Magrepresenta yang data ibat keng n = 3 a independenting experiment. (B) Ing TNFα promoter da reng T cells a me-treat o alang 8 mM MNA kaybat ning NFAT ampo Sp1 metung la kareng (Ctrl) ampo PMA/ionomycin stimulation kilub ning 4 oras. Ing immunoglobulin G (IgG) ampo ing H3 ginamit la bilang negatibo ampo positibong kontrol para keng immunoprecipitation, agpang kareti. Ing quantification ning ChIP pepakit na ing pamag-bind ning Sp1 ampo NFAT keng TNFα promoter kareng MNA-treated cells meragdagan yang marakal a besis kumpara keng control. Magrepresenta yang data ibat keng n = 3 a independenting experiment. P value a meyakit kareng dakal a t-tests (*** P <0.01). (C) Nung ikumpara la kareng ascites ning HGSC, deng T cells (e cytotoxic) pepakit da ing mas maragul a expression ning TNF keng tumor. Deng kule mangabaldugan la kareng miyayaliwang pasyenti. Deng mipalabas a cell randomly lang me-sample keng 300 ampo me-jitter la para limitahan ing overdrawing (** Padj = 0.0076). (D) Ing mipanukalang model ning MNA para keng ovarian cancer. Ing MNA gagawan de reng tumor cells ampo fibroblasts keng TME ampo kukunan de reng T cells. Ing MNA daragdagan na ing pamagtali ning Sp1 keng TNFα promoter, a magdalang pamagtas ning TNFα transcription ampo ing TNFα cytokine production. Ing MNA magdalang pamagbaba keng IFN-γ. Ing pamag-inhibite keng T cell function magdalang mabawas a abilidad a makamate ampo mas mabilis a pamandagul ning tumor.
Agpang kareng ulat, ing TNFα atin yang front ampo back-dependent anti-tumor ampo anti-tumor effects, pero atin yang balu a papil keng pamag promote keng pamandagul ampo metastasis ning ovarian cancer (31-33). Agpang kareng ulat, ing konsentrasyun ning TNFα kareng ascites ampo tumor tissues kareng pasyenti a ating ovarian cancer mas matas ya kesa kareng benign tissues (34-36). keng mekanismo, ing TNFα malyari yang magregulate keng activation, function ampo proliferation da reng white blood cells, ampo alilan ne ing phenotype da reng cancer cells (37, 38). Agpang kareng akit a reni, ing differential gene expression analysis pepakit na ing TNF makabaldugan yang up-regulated kareng T cells kareng tumor tissues kumpara kareng ascites (Figure 5C). Ing pamagtas ning TNF expression mayayakit yamu kareng T cell populations a alang cytotoxic a phenotype (Figure S5A). keng pamamutut, deng data a reni suportan de ing panaun na ing MNA atin yang dual immunosuppressive ampo tumor promoting effects keng HGSC.
Ing fluorescent labeling base keng flow cytometry meging yang peka paralan para abalu ing TIL metabolism. Deng pamagaral a reni pepakit da na nung ikumpara la kareng peripheral blood lymphocytes o T cells ibat kareng secondary lymphoid organs, ing murine ampo human TIL atin lang mas matas a tendency keng pamag-uptake glucose (4, 39) ampo ing gradual a pangawala ning mitochondrial function (19, 40). Agyang atin kaming ikit a parehung resulta keng pamagaral ayni, ing susi a panyulung yapin ing pamagkumpara keng metabolism da reng tumor cells ampo ing TIL ibat keng parehung me-resect a tumor tissue. Agpang kareng mapilan kareng minunang ulat, deng tumor (CD45-EpCAM +) cells ibat keng ascites ampo tumor atin lang mas matas a glucose uptake kesa kareng CD8 + ampo CD4 + T cells, a susuporta na ing matas a glucose uptake da reng tumor cells malyari yang ikumpara kareng T cells. Ing konsepto ning T cell competition. TME. Nanupata, mas matas ya ing mitochondrial activity da reng tumor cells kesa kareng CD8 + T cells, dapot ing mitochondrial activity kalupa ne ning CD4 + T cells. Deting resulta pepatibe da ing lulual a tema na ing oxidative metabolism importanti ya para kareng tumor cells (41, 42). Ila ring magsuggest na deng CD8 + T cells malyari lang mas susceptible keng oxidative dysfunction kesa kareng CD4 + T cells, o deng CD4 + T cells malyari lang gumamit carbon sources bukud keng glucose para manatili ing mitochondrial activity (43, 44). Dapat papansinan na ala kaming ikit a pamiyaliwa keng glucose uptake o mitochondrial activity pilatan da reng CD4 + T effectors, T effector memory ampo T central memory cells keng ascites. makanyan mu naman, ing differentiation state da reng CD8 + T cells kareng tumor ala yang kaugnayan keng pamagbayu keng glucose uptake, papakit na ing makabaldugan a pamiyaliwa da reng T cells a me-culture in vitro ampo ing human TIL in vivo (22). Deting obserbasyun mekumpirma la naman kapamilatan ning pamangamit king unbiased automatic cell population allocation, a lalu pang pepakit na ing CD45 + / CD3- / CD4 + / CD45RO + cells a maki mas matas a glucose uptake ampo mitochondrial activity kesa kareng tumor cells ila ring keraklan dapot atin lang Metabolic active cell population. Ing populasyun ayni malyari yang magrepresenta keng putative subpopulation da reng myeloid suppressor cells o plasmacytoid dendritic cells a mekilala keng scRNA-seq analysis. Agyang parehu lang mireport deti kareng human ovarian tumors [45], kailangan da pa mu rin ing karagdagang obra yapin ing pamaglarawan kaniting myeloid subpopulation.
Agyang deng paralan a makabasi keng flow cytometry malyari lang malino deng pangkabilugan a pamiyaliwa ning glucose ampo oxidative metabolism kareng uri da reng cell, e la pa meyakit deng eksaktung metabolites a gagawan ning glucose o aliwang carbon sources para keng mitochondrial metabolism keng TME. Ing pamag-assign keng presensya o alang metabolites keng metung a TIL subset mangailangan yang pamaglinis keng cell population ibat keng me-excised a tissue. Uli na niti, ing kekaming cell enrichment method a kayabe ning mass spectrometry malyari yang mamye insights kareng metabolites na miyayaliwang enrichment kareng T cells ampo tumor cell populations keng matching pasyenti samples. Agyang atin yang advantage ining paralan keng fluorescence-activated cell sorting, deng mapilan a metabolite libraries malyari lang maapektuan pauli ning inherent stability ampo/o mabilis a turnover rate (22). Makanyan man, ing kekaming paralan mekakilala yang adwang kikilalanan a immunosuppressive metabolites, adenosine ampo kynurenine, uling miyayaliwa la kareng sample types.
Ing kekaming metabonomic analysis kareng tumor ampo TIL subtypes mamye yang mas marakal a insights keng papil da reng metabolites keng ovarian TME. Mumuna, gamit ing flow cytometry, abalu mi na ala yang pamiyaliwa ing mitochondrial activity kareng tumor ampo CD4 + T cells. Makanyan man, ing LC-MS/MS analysis pepakit na ing maragul a pamagbayu keng karakal da reng metabolites kareng populasyun a reni, a magpakit na ing konklusyun tungkul keng TIL metabolism ampo ing kayang kabilugan a metabolic activity mangailangan yang maingat a interpretasyun. Kadwa, ing MNA ya ing metabolite na atin pekamaragul a pamiyaliwa kareng CD45-cells ampo T cells keng ascites, ali kareng tumor. Uli na niti, ing compartmentalization ampo ing tumor location malyari lang mika miyayaliwang epektu keng TIL metabolism, na magpakit keng posibling heterogeneity keng metung a microenvironment. Katlu, ing pamagpakilala ning MNA-producing enzyme NNMT keraklan limitadu ya keng CAF, na tumor cells keng mas ditak a sukad, pero deng detectable MNA levels mayayakit la kareng tumor-derived T cells. Ing overexpression ning NNMT keng ovarian CAF atin yang balu a cancer-promoting effect, metung a bage uli ning pamagpromote ning CAF metabolism, tumor invasion ampo metastasis (27). Agyang ing kabilugan a antas ning TIL katamtaman ya, ing pamagpakilala ning NNMT king CAF malapit yang makaugne king Cancer Genome Atlas (TCGA) mesenchymal subtype, a maki kaugnayan king e masanting a prognosis (27, 46, 47). Katataulian, ing pamagpakilala ning enzyme AOX1 a maki pakibatan king pangasira ning MNA limitadu ya mu rin king populasyun ning CAF, a magpakit a deng T cells ala lang abilidad a mag-metabolize king MNA. Deting resulta suportan de ing ideya na agyang kailangan ing karagdagang obra para abalu ing akit a ini, ing matas a antas ning MNA kareng T cells malyari yang magpakit king presensya ning immunosuppressive CAF microenvironment.
Uling mababa ing expression level da reng MNA transporters ampo ing e mayayakit a level da reng key proteins a maki kaugnayan keng MNA metabolism, ing presensya ning MNA kareng T cells e ya asahan. E la malyaring akit deng NNMT o AOX1 kapamilatan ning scRNA-seq analysis ampo ing targeted qPCR da reng adwang independenting cohort. Deng resulta papakit da na ing MNA ali ya gagawan da reng T cells, pero masipsip ya ibat keng makapadurut a TME. Deng in vitro experiment papakit da na deng T cells atin lang ugaling magtipun exogenous MNA.
Ing kekaming in vitro a pamagaral pepakit na ing exogenous MNA mag-induce yang expression ning TNFα kareng T cells ampo papaslag ne ing pamag-bind ning Sp1 keng TNFα promoter. Agyang ing TNFα atin yang anti-tumor ampo anti-tumor a gagawan, keng ovarian cancer, ing TNFα malyari yang magpromote keng pamandagul ning ovarian cancer (31-33). Ing neutralization ning TNFα keng ovarian tumor cell culture o ing pamaglako ning TNFα signal keng mouse models malyari yang magpasanting keng TNFα-mediated inflammatory cytokine production ampo makapigil keng pamandagul ning tumor (32, 35). Uli na niti, keng kasu ayni, ing TME-derived MNA malyari yang maging pro-inflammatory metabolite kapamilatan ning TNFα-dependent a mekanismo kapamilatan ning autocrine loop, at makanyan yang magpromote king pangapalyari at pangakalat ning ovarian cancer (31). Base keng posibilidad ayni, ing TNFα blockade me-aral ya bilang metung a potensyal a therapeutic agent para keng ovarian cancer (37, 48, 49). Dagdag pa, ing MNA makasira ya keng cytotoxicity da reng CAR-T cells kareng ovarian tumor cells, mamye yang karagdagang ebidensya para keng MNA-mediated immune suppression. Ing kabilugan, deng resulta a reni magsuggest lang model nung nukarin deng tumor ampo reng CAF cells magsecrete lang MNA keng extracellular TME. Kapamilatan ning (i) TNF-induced ovarian cancer growth stimulation ampo (ii) MNA-induced T cell cytotoxic activity inhibition, malyari yang mika dual tumor effect (Figure 5D).
keng pangwakas, kapamilatan ning pamag apply keng kombinasyun ning mabilis a cell enrichment, single-cell sequencing ampo metabolic profiling, ining pamagaral pepakit na ing maragul a pamiyaliwa ning immunometabolomic pilatan da reng tumor ampo ascites cells kareng pasyenti ning HGSC. Iting komprehensibung pamagsuri pepakit na atin pamiyaliwa keng glucose uptake ampo ing mitochondrial activity kareng T cells, ampo mekilala ya ing MNA bilang metung a non-cell autonomous immune regulatory metabolite. Deng data a reni atin lang epektu nung makananu ya makaapektu ing TME keng T cell metabolism kareng cancer da reng tau. Agyang ing direktang kompetisyon para kareng sustansya pilatan da reng T cells ampo reng cancer cells mereport na, deng metabolites malyari la namang maging e direktang regulator para isulong ing tumor progression ampo posibling pigilan ing endogenous immune responses. Ing karagdagang pamaglarawan keng functional role da reng regulatory metabolites malyari yang magbukas alternatibong diskarti para keng pamagpabuti keng anti-tumor immune response.
Deng specimen da reng pasyenti ampo reng clinical data mekwa la kapamilatan ning BC cancer tumor tissue repository a sertipikadu ning Canadian Tissue Repository Network. Agpang keng protocol a me-aprubahan ning BC Cancer Research Ethics Committee ampo ing University of British Columbia (H07-00463), ding sablang specimen da reng pasyenti ampo reng clinical data mekwa lang informed written consent o pormal lang me-waive keng karelang consent. Deng sample makalage la keng certified BioBank (BRC-00290). Deng detalyadung katangian da reng pasyenti mayayakit la keng Tables S1 ampo S5. Para keng cryopreservation, ing scalpel magagamit ya para mechanically decompose ing tumor sample ning pasyenti at kaybat itulak ne keng 100-micron filter para mika metung a cell suspension. Ing ascites ning pasyenti me-centrifuge ya keng 1500 rpm kilub ning 10 minutu keng 4°C para i-pellet la reng cells ampo ilako ing supernatant. Deng cell a ibat keng tumor ampo ascites me-cryopreserve la keng 50% heat-inactivated human AB serum (Sigma-Aldrich), 40% RPMI-1640 (Thermo Fisher Scientific) ampo 10% dimethyl sulfoxide. Deting me-preserba a single cell suspensions me-thaw la at ginamit la para keng metabolomics ampo metabolite determination a makalarawan lalam.
Ing kumpletung medium atin yang 0.22 μm a me-filter 50:50 supplemented RPMI 1640: AimV. RPMI 1640 + 2.05 mM l-glutamine (Thermo Fisher Scientific) a midagdag keng 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) ampo 50 μMB-mercaptoethanol. Ing AimV (Invitrogen) metung yang 20 mM Hepes (Thermo Fisher Scientific) ampo 2 mM l-glutamine (Thermo Fisher Scientific). Ing flow cytometer staining buffer atin yang 0.22μm a filtered phosphate buffered saline (PBS; Invitrogen) a mika 3% heat-inactivated AB human serum (Sigma). Ing cell enrichment buffer metung yang 0.22μm a filtered PBS ampo atin yang 0.5% heat-inactivated human AB serum (Sigma-Aldrich).
keng 37°C kumpletung medium, ding cell me-stain la keng 10 nM MT DR ampo 100 μM 2-NBDG kilub ning 30 minutu. Kaybat, ding cells me-stain la keng viability dye eF506 keng 4°C kilub ning 15 minutus. I-resuspend la reng cell keng FC Block (eBioscience) ampo ing Brilliant Stain Buffer (BD Biosciences), i-dilute keng flow cytometer staining buffer (agpang keng instruksiun ning manufacturer), ampo i-incubate kilub ning 10 minutu keng room temperature. Stain la reng cells gamit ing set da reng antibodies (Table S2) keng flow cytometry staining buffer keng 4°C kilub ning 20 minutus. I-resuspend la reng cell keng flow cytometry staining buffer (Cytek Aurora; 3L-16V-14B-8R configuration) bayu ing pamag-analisa. Gamitan me ing SpectroFlo ampo FlowJo V10 para abalu me ing cell count data, ampo gamitan me ing GraphPad Prism 8 para gawan me ing data. Ing median fluorescence intensity (MFI) ning 2-NBDG ampo MT DR me-log-normalized ya, at kaybat ing paired t test ginamit ya para keng statistical analysis para akit la reng pasyenti. Ilako la ngan deng populasyun a ating mas ditak a 40 a events keng pamagsuri; isulud me ing MFI value a 1 para kareng nanu mang negatibong ulaga bayu ka mag gawa keng statistical analysis ampo data visualization.
Para dagdagan ing manual gating strategy ning process panel king babo, ginamit mi ing ganap a annotation ning shape restriction tree (FAUST) (21) para automatic lang i-assign deng cells keng populasyun kaybat dang leko deng mete cells keng FlowJo. Manual mi yang a-manage ing output para a-merge la reng populasyun na balamu e la misallocated (sasamut ing PD1+ kareng PD1-tumor cells) ampo reng retained a populasyun. Balang sample atin yang average a maigit 2% cells, para keng kabilugan a 11 a populasyun.
Ing Ficoll gradient density centrifugation ginamit ya para ilako ing PBMC kareng leukocyte separation products (STEMCELL Technologies). Deng CD8 + T cells me-isolate la keng PBMC gamit ing CD8 MicroBeads (Miltenyi) ampo mipalapad la keng kumpletung medium gamit ing TransAct (Miltenyi) kilub ning 2 duminggu agpang kareng instruksiun ning manufacturer. Deng cells pepaintulutan dong makatalakad kilub ning 5 aldo keng kumpletung medium a maki IL-7 (10 ng/ml; PeproTech), at kaybat mibalik la keng TransAct. keng aldo 7, agpang kareng instruksyun ning manufacturer, human CD45 MicroBeads (Miltenyi) ing ginamit para pasikanan la reng cells keng atlung sunod-sunod a round. Deng cells me-aliquot la para keng flow cytometry analysis (antimo ing makasulat babo), ampo metung a milyung cells ing me-aliquot atlung besis para keng LC-MS/MS analysis. Deng sample meprosesu la kapamilatan ning LC-MS/MS antimo ing makasulat lalam. Tinaya mi ing kulang a ulaga ning metabolite a maki ion a 1,000. Balang sample me-normalize ya keng total ion number (TIC), me-convert ya logarithmically ampo automatic yang me-normalize keng MetaboAnalystR bayu ya me-analisa.
Ing metung a cell suspension ning balang pasyenti me-thaw ya at me-filter ya kapamilatan ning 40 μm filter papunta king kumpletung medium (antimo ing makasulat babo). Agpang keng protocol ning manufacturer, atlung sunod-sunod a round ning positibong pamili kapamilatan ning magnetic bead separation gamit ing MicroBeads (Miltenyi) ing ginamit para pasikanan la reng sample para kareng CD8+, CD4+ ampo CD45- cells (keng yelo). king makuyad a salita, deng cells mibalik la keng cell enrichment buffer (antimo ing makasulat babo) ampo mibilang la. Deng cells me incubate la kareng human CD8 beads, human CD4 beads o human CD45 beads (Miltenyi) keng 4°C kilub ning 15 minutus, at kaybat me hugas la keng cell enrichment buffer. Ing sample mipalabas ya keng LS column (Miltenyi), at ding positibo ampo negatibong fractions tinipun la. Para mabawas ing panaun ampo mas maragul ing cell recovery step, ing CD8-fraction gamitan ya para keng kaduang round ning CD4+ enrichment, ampo ing CD4-fraction gamitan ya para keng tutuking CD45-enrichment. Itabi me ing solusyun keng yelo keng mabilug a prosesu ning pamikawani.
Para makasadya lang sample para keng metabolite analysis, ding cells me hugas lang misan keng ice-cold salt solution, ampo 1 ml ning 80% methanol ing miyabe keng balang sample, kaybat mi vortex ya ampo me snap frozen ya keng liquid nitrogen. Deng sample mipailalam la keng atlung freeze-thaw cycles ampo mi centrifuge la keng 14,000 rpm kilub ning 15 minutus keng 4°C. Ing supernatant a maki metabolites mipapalibut ya anggang matuling ya. Deng metabolites me-dissolve lang pasibayu keng 50 μl ning 0.03% formic acid, me-vortex la para mi-mix, at kaybat me-centrifuge la para milako la reng debris.
I-extract la reng metabolites antimo ing makasulat babo. Ilipat me ing supernatant keng high performance liquid chromatography bottle para keng metabolomics research. Gamitan me ing random a protocol ning pamanulu para a-treat ing balang sample kareng parehung bilang da reng cells para aiwasan ing batch effects. Ginawa kaming qualitative assessment kareng global metabolites na minunang mibulalag keng AB SCIEX QTRAP 5500 Triple Quadrupole Mass Spectrometer (50). Ing chromatographic analysis ampo ing peak area integration megawa la gamit ing MultiQuant version 2.1 software (Applied Biosystems SCIEX).
Ing ion count a 1000 ginamit ya para abalu ing kulang a ulaga ning metabolite, at ing TIC ning balang sample ginamit ya para makalkula ing normalized peak area ning balang meyakit a metabolite para i-correct ing pamagbayu a pepalub ning instrumental analysis ibat keng sample processing. Kaybat nang me-normalize ing TIC, ing MetaboAnalystR(51) (default parameter) ing gagamitan para keng logarithmic conversion ampo ing automatic norm line scaling. Ginamit mi ing PCA a maki vegan R package para gawan ing exploratory analysis da reng pamiyaliwa da reng metabolome kareng sample types, ampo ginamit mi ing partial redundancy analysis para abalu mi la reng pasyenti. Gamitan ing paralan ning Ward para gawan ing heat map dendrogram para i-cluster ing Euclidean a distansya pilatan da reng sample. Ginamit mi ing limma (52) keng standardized metabolite abundance para akilala mi la reng miyayaliwang metabolites keng mabilug a cell type ampo microenvironment. Para maging simpli ing paliwanag, gamitan mi ing group mean parameter para abalu mi ing model, ampo isipan mi la reng cell types keng microenvironment bilang balang grupu (n = 6 groups); para keng significance test, ginawa kaming atlung paulit-ulit a sukad para keng balang metabolite Para aiwasan ing maling replication, ing pasyenti metung yang sagabal keng limma design. Para akit ing pamiyaliwa da reng metabolites kareng miyayaliwang pasyenti, inayus mi ing limma model kayabe la reng pasyenti king metung a fixed a paralan. Ireport mi ing kabaldugan ning pre-specified a pamiyaliwa ning cell type ampo ing microenvironment ning Padj <0.05 (Benjamini-Hochberg correction).
Kaybat ning vigor enrichment gamit ing Miltenyi Dead Cell Removal Kit (>80% viability), ing single-cell transcriptome sequencing megawa ya keng kabilugan a live frozen ascites ampo tumor samples gamit ing 10x 5′gene expression protocol. Limang kasu a maki matching a tumor ampo ascites ing me-analisa, agyang ing mababang viability ibat keng metung a sample ning tumor me-prevent ya keng pamaglub na. Para akit mi la reng dakal a pasyenti, pisamut mi la reng sample da reng balang pasyenti kareng lane ning 10x chromium controller, at inanalisa mi la reng ascites ampo reng tumor sites. Kaybat ning pamag-sequence [Illumina HiSeq 4000 28×98 bp paired end (PE), Quebec genome; ing average a 73,488 ampo 41,378 a pamamasa balang cell para keng tumor ampo ascites]], ginamit mi ing CellSNP ampo Vireo (53) (base keng CellSNP bilang Ing karaniwan a human SNP (VCF) a binie ning GRCh38 metung yang donor identity. Gagamitan mi ing SNPRelate para abalu ing pekamalapit a identity (IBS) ning genotype da reng pasyenti (IBS) e la kayabe deng e me-assign a cells ampo reng cells a mekilala bilang duplexes ampo deng matching donors pilatan da reng ascites ampo reng tumor samples (54). (2792 ampo 4183 cells ibat keng tumor ampo ascites, respectively) para keng pamagsuri. deng cells maka define la keng expression ning CD8A ampo GZMA, ali la kayabe deng subclusters na ating mababang ribosomal protein expression.
Deng PBMC mekawani la kareng leukocyte separation products (STEMCELL Technologies) kapamilatan ning Ficoll gradient density centrifugation. CD3 + cells me-isolate la keng PBMC gamit ing CD3 beads (Miltenyi). keng presensya o ala ning MNA, deng CD3+ cells me-activate la keng plate-bound CD3 (5μg/ml), soluble CD28 (3μg/ml) ampo IL-2 (300 U/ml; Proleukin). keng tauling aldo ning pamagpalawak, ing viability (Fixable Viability Dye eFluor450, eBioscience) ampo ing proliferation (123count eBeads, Thermo Fisher Scientific) me evaluate la kapamilatan ning flow cytometry. I-evaluate ing effector function kapamilatan ning pamag-stimulate kareng cells gamit ing PMA (20 ng/ml) ampo ionomycin (1μg/ml) gamit ing GolgiStop kilub ning 4 oras, ampo i-monitor ing CD8-PerCP (RPA-T8, BioLegend), CD4-AF700 (RPA-T4) , BioLegend) ampo TNFα-fluoresce isothianate (TCb1) BD). I-stimulate ing qPCR ampo reng ChIP cells gamit ing PMA (20 ng/ml) ampo ionomycin (1μg/ml) kilub ning 4 oras. Ing ELISA supernatant mekolekta ya bayu at kaybat ning stimulation gamit ing PMA (20 ng/ml) ampo ionomycin (1 μg/ml) kilub ning 4 oras.
Tukian me ing protocol ning manufacturer para maka isolate kang RNA gamit ing RNeasy Plus Mini Kit (QIAGEN). Gamitan me ing QIAshredder (QIAGEN) para i-homogenize ing sample. Gamitan ing high-capacity RNA to cDNA kit (Thermo Fisher Scientific) para gawan ing complementary DNA (cDNA). Gamitan me ing TaqMan Rapid Advanced Master Mix (Thermo Fisher Scientific) para abalu me ing gene expression (agpang keng protocol ning manufacturer) kareng makatuking probe: Hs00196287_m1 (NNMT), Hs00154079_m1 (AOX1), Hs004275555S, 2012 Hs02786624_g1 [glyceraldehyde-3-phosphate off Hydrogen (GAPDH)] ampo Hs01010726_m1 (SLC22A2). Deng sample me-run la keng StepOnePlus real-time PCR system (Applied Biosystems) (Applied Biosystems) keng MicroAmp fast optical 96-well reaction plate (Applied Biosystems) gamit ing MicroAmp optical film. Nanu mang Ct value na lalagpus keng 35 tuturing deng babo ning detection threshold at makamarka ya bilang undetectable.
Gawa kang ChIP antimo ing minunang misulat (58). king makuyad a salita, ding cells me-treat la keng formaldehyde (final concentration 1.42%) ampo me-incubate la keng room temperature kilub ning 10 minutus. Gamitan me ing supplemented swelling buffer (25 mM Hepes, 1.5 mM MgCl2, 10 mM KCl ampo 0.1% NP-40) keng yelo kilub ning 10 minutu, kaybat i-resuspend me keng immunoprecipitation buffer antimo ing makasulat (58 ). Ing sample me sonicate ya kareng makatuking siklo: 10 siklo (20 1-second pulses) ampo ing static a oras a 40 segundo. I-incubate ing ChIP-grade immunoglobulin G (Cell Signaling Technology; 1μl), histone H3 (Cell Signaling Technology; 3μl), NFAT (Invitrogen; 3μl) ampo SP1 (Cell Signaling Technology; 3μl) antibodies keng sample keng 4°CC shake magdamag. I-incubate ing protein A beads (Thermo Fisher Scientific) keng sample keng 4°C a maki malumanay a pamagyugse kilub ning 1 oras, kaybat gamitan me ing chelex beads (Bio-Rad) para pasikanan ing DNA, ampo gamitan me ing proteinase K (Thermo Fisher) para keng pamaglaso ning protina. TNFα promoter meyakit ya kapamilatan ning PCR: forward, GGG TAT CCT TGA TGC TTG TGT; ing kabaligtaran, GTG CCA ACA ACT GCC TTT ATA TG (207-bp product). Deng larawan megawa la keng Image Lab (Bio-Rad) ampo me-quantify la gamit ing ImageJ software.
Ing cell culture supernatant mekolekta ya antimo ing makasulat babo. Ing pamagdesisyun megawa ya agpang keng pamamaralan ning manufacturer ning human TNFα ELISA kit (Invitrogen), human IL-2 ELISA kit (Invitrogen) ampo human IFN-γ ELISA kit (Abcam). Agpang keng protocol ning manufacturer, ing supernatant me dilute yang 1:100 para akit ing TNFα ampo IL-2, ampo 1:3 para akit ing IFN-γ. Gamitan me ing EnVision 2104 Multilabel Reader (PerkinElmer) para sukatan ing absorbance keng 450 nm.
Deng PBMC mekawani la kareng leukocyte separation products (STEMCELL Technologies) kapamilatan ning Ficoll gradient density centrifugation. CD3 + cells me-isolate la keng PBMC gamit ing CD3 beads (Miltenyi). keng presensya o ala ning MNA, deng CD3+ cells me-activate la keng plate-bound CD3 (5μg/ml), soluble CD28 (3μg/ml) ampo IL-2 (300 U/ml; Proleukin) kilub ning 3 aldo. Kaybat ning atlung aldo, tinipun la reng cells at mehugas la keng 0.9% saline, ampo ing pellet me-snap frozen ya. Ing bilang da reng cell megawa ya kapamilatan ning flow cytometry (Cytek Aurora; 3L-16V-14B-8R configuration) gamit ing 123count eBeads.
I-extract la reng metabolites antimo ing makasulat babo. Ing metuling a extract mibalik ya keng konsentrasyun a 4000 cell equivalents/μl. Analisahan ing sample kapamilatan ning reversed-phase chromatography (1290 Infinity II, Agilent Technologies, Santa Clara, CA) ampo CORTECS T3 column (2.1×150 mm, particle size 1.6-μm, pore size 120-Å; #186008500, Waters). Ing polar mass spectrometer (6470, Agilent), nung nukarin ing electrospray ionization mag-operate ya keng positibong paralan. Ing mobile phase A 0.1% ya formic acid (keng H2O), ing mobile phase B 90% ya acetonitrile, 0.1% ya formic acid. Ing LC gradient 0 anggang 2 minutu para keng 100% A, 2 anggang 7.1 minutu para keng 99% B, ampo 7.1 anggang 8 minutu para keng 99% B. Kaybat, i-re-equilibrate me ing column keng mobile phase A keng flow rate a 0.6 ml/min kilub ning 3 minutu. . Ing flow rate 0.4ml/min ya, at ing column chamber mipainit ya anggang 50°C. Gamitan me ing MNA's pure chemical standard (M320995, Toronto Research Chemical Company, North York, Ontario, Canada) para mitatag ing retention time (RT) ampo ing pamagbayu (RT = 0.882 minutu, pamagbayu 1 = 137→94.1, pamagbayu 2 = 137→92 , Conversion = 13→77). Nung deng atlung pamagbayu malilyari la keng ustu a oras ning pamag-ingat, ing pamagbayu 1 magagamit ya para keng pamag-quantify para makasiguradung specificity. Ing standard curve ning MNA (Toronto Research Chemical Company) megawa ya kapamilatan ning anam a serial dilutions ning stock solution (1 mg/ml) para mika standards a 0.1, 1.0, 10 ampo 100 ng/ml ampo 1.0 ampo 10μg/ml agpang keng likidu. Ing limitasyun ning pamanalbe 1 ng/ml ya, at ing linear a pakibat pilatan ning 10 ng/ml ampong 10μg/ml. Balang injection ning adwang microliters ning sample ampo standard magagamit ya para keng LC/MS analysis, ampo ing metung a mixed quality control sample gagawan de balang walung injection para siguraduan ing katatagan ning analysis platform. Ing MNA a pakibat da reng eganaganang MNA-treated cell samples atyu la keng linear range ning assay. Ing pamagsuri keng data megawa ya gamit ing MassHunter quantitative analysis software (v9.0, Agilent).
Ing kaduang henerasyun a αFR-CAR construct mekwa ya kang Song et al. (59). king makuyad a salita, ing construct atin yang laman: CD8a leader sequence, human αFR-specific single-chain variable fragment, CD8a hinge ampo transmembrane region, CD27 intracellular domain ampo CD3z intracellular domain. Ing kumpletung CAR sequence me-synthesize ya kapamilatan ning GenScript, at kaybat me-clone ya king second-generation lentiviral expression vector king babo ning GFP expression cassette a ginamit para abalu ing transduction efficiency.
Ing lentivirus gagawan de kapamilatan ning pamag-transfection kareng HEK293T cells [American Type Culture Collection (ATCC); meragul ya keng Dulbecco's modified Eagle medium a maki 10% fetal bovine serum (FBS) ampo 1% PenStrep, ampo ginamit yang CAR-GFP vector ampo ing packaging plasmids (psPAX2 ampo pMD2.G, Addgene) gagamit yang lipofection amine (Sigma-Aldrich). Ing virus-containing supernatant mekolekta ya 48 ampo 72 oras kaybat ning transfection, me-filter ya, ampo me-concentrate ya kapamilatan ning ultracentrifugation. Ibili me ing concentrated viral supernatant keng -80°C anggang mag transduction.
Deng PBMC makabukud la kareng malusog a donor leukocyte separation products (STEMCELL Technologies) kapamilatan ning Ficoll gradient density centrifugation. Gamitan ing positive selection CD8 microbeads (Miltenyi) para i-isolate la reng CD8+ cells ibat keng PBMC. Pasiknangan la reng T cells gamit ing TransAct (Miltenyi) ampo ing TexMACS medium [Miltenyi; mika 3% heat-inactivated human serum, 1% PenStrep ampo IL-2 (300 U/ml)]. Aduang pulu't apat a oras kaybat ning stimulation, deng T cells mi-transduce la keng lentivirus (10 μl concentrated virus supernatant balang 106 cells). 1 anggang 3 aldo kaybat ning transduction keng Cytek Aurora (keng FSC (Forward Scatter)/SSC (Side Scatter), Singlet, GFP+), evaluate ing GFP expression da reng cells para ipakit ing transduction efficiency a at least 30% .
Deng CAR-T cells me-culture la kilub ning 24 oras keng Immunocult (STEMCELL Technologies; midagdagan lang 1% PenStrep) lalam da reng makatuking kundisyun: e me-treat, me-treat keng 250 μM adenosine o 10 mM MNA. Kaybat ning pretreatment, ding CAR-T cells mehugas la keng PBS ampo miyabe la kareng 20,000 SK-OV-3 cells [ATCC; keng McCoy 5A medium (Sigma-Aldrich) a midagdag 10% FBS ampo 1% PenStrep keng 10: Ing effector to target ratio ning 1 me amplify ya keng triplicate keng midagdag a Immunocult medium. SK-OV-3 cells ampo SK-OV-3 cells a me-lysed keng digitalis saponin (0.5mg/ml; Sigma-Aldrich) ginamit la bilang negatibo ampo positibong kontrol, agpang kareti. Kaybat ning 24 oras a co-cultivation, ing supernatant mekolekta ya at ing lactate dehydrogenase (LDH) mesukad ya agpang kareng instruksiun ning manufacturer (LDH Glo Cytotoxicity Assay Kit, Promega). Ing LDH supernatant me dilute yang 1:50 keng LDH buffer. Ing porsyentu ning pamakamate mesukad ya gamit ing makatuking formula: porsyentu ning pamakamate = porsyentu ning pamag-correct / maximum killing rate x 100%, nung nukarin porsyentu ning pamag-correct = co-culture-T cells mu, at maximum killing rate = positive control-negative control.
Antimo ing makasulat keng text o materyales ampo paralan, gamitan me ing GraphPad Prism 8, Microsoft Excel o R v3.6.0 para keng statistical analysis. nung dakal a sample ing mekolekta ibat keng metung a pasyenti (antimo ing ascites ampo ing tumor), gamitan mi ing paired t test o iyabe mi ya ing pasyenti bilang random a epektu keng linear o generalized model nung kailangan. Para keng metabolomics analysis, ing importance test gagawan de keng atlung replicate.
Para kareng karagdagang materyales para kening artikulu, lawan me ing 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.
Ing MNA makaambag ya keng immune suppression da reng T cells ampo magrepresenta yang potensyal a target ning immunotherapy para keng pamanulu keng cancer ning tau.
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.
Ing MNA makaambag ya keng immune suppression da reng T cells ampo magrepresenta yang potensyal a target ning immunotherapy para keng pamanulu keng cancer ning tau.
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