Saray immunomodulatory metabolites et manunan kabiangan na tumor microenvironment (TME), balet diad pigaran eksepsion, ag ni kabat so pakabidbiran da. Dia, inaral mi ray tumor tan T cells manlapud saray tumor tan ascites na saray pasyente ya walay high-grade serous carcinoma (HGSC) pian nanengneng so metabolome na sarayan nanduruman TME compartment. Say ascites tan tumor cells et walaan na malaknab a pandumaan na metabolite. No ikompara ed ascites, saray tumor-infiltrating T cells et signipikantin mayaman ed 1-methylnicotinamide (MNA). Anggaman atagey so lebel na MNA ed T cells, say pangibalikas na nicotinamide N-methyltransferase (sakey ya enzyme ya mamapakiwas ed iyalis na methyl groups manlapud S-adenosylmethionine ya anggad nicotinamide) et limitado ed saray fibroblast tan tumor cells. Diad kimey, say MNA et mamapawala na tumor-promoting cytokine tumor necrosis factor alpha. Kanian, say TME-derived MNA et makatulong ed immune regulation na T cells tan mangirerepresenta na posiblin target na immunotherapy parad panagtambal na kanser na too.
Saray metabolites ya nanlapud tumor et nayarin walaan na baleg ya epekto ed anti-tumor immunity, tan ondarakel so ebidensya ya mangipapanengneng ya saratan met so manserbin manunan puersa na iyaaligwas na sakit (1). Nilikud ed Warburg effect, say agano nin kimey et ginmapo lan mangibiig ed metabolic state na tumor cells tan say siglaotan to ed immune state na tumor microenvironment (TME). Saray impanaral ed saray modelo na ayep tan T cells na too so angipatnag ya say glutamine metabolism (2), oxidative metabolism (3) tan glucose metabolism (4) et nayarin onkiwas ya independente ed nanduruman immune cell subgroups. Pigaran metabolites ed sarayan dalan so mangaamper ed anti-tumor function na T cells. Napaneknekan ya say pangibara ed coenzyme tetrahydrobiopterin (BH4) et makaderal ed idakel na T cells, tan say idarakel na BH4 ed laman et makapaaligwas ed anti-tumor immune response ya ipapaarap na CD4 tan CD8. Niarum ni, say immunosuppressive ya epekto na kynurenine et nayarin nisalba diad pangiter na BH4 (5). Diad isocitrate dehydrogenase (IDH) mutant glioblastoma, say panag-secrete na enantiometabolic (R)-2-hydroxyglutarate (R-2-HG) et mamatunda ed T cell activation, proliferation tan cytolysis Activity (6). Diad agano ni, nipanengneng ya say methylglyoxal, sakey a produkto na glycolysis, et napapawala na saray suppressor cells ya nanlapud myeloid, tan say T cell transfer na methylglyoxal et nayarin mangamper ed effector T cell function. Diad panagtambal, say neutralisasyon na methylglyoxal et makatalona ed aktibidad na myeloid-derived suppressor cells (MDSC) tan makapaaligwas na checkpoint blockade therapy ed saray modelo na mouse (7). Sarayan impanaral so mankakasakey ya mangipapabitar ed manunan betang na saray metabolites ya nanlapud TME ed panangontrol ed kimey tan aktibidad na T cell.
Say T cell dysfunction et malaknab ya nireport ed ovarian cancer (8). Saya so lapud saray katangian na metabolismo ya walad hypoxia tan abnormal ya tumor vasculature (9), ya manresulta ed panguman na glucose tan tryptophan ya magmaliw ya by-products a singa say lactic acid tan kynurenine. Say alablabas ya extracellular lactate et mamapaabeba ed produksyon na interferon-γ (IFN-γ) tan mamapaarap ed pandumaan na myelosuppressive subgroups (10, 11). Say panangan na tryptophan et direktan mamatunda ed idakel na T cell tan mamatunda ed signaling na T cell receptor (12-14). Anggaman ed sarayan obserbasyon, dakel a kimey nipaakar ed immune metabolism so ginawa ed in vitro T cell culture ya inusar so optimized media, odino limitado ed homologous mouse models in vivo, ya anggapo ed saratan so sigpot a mangipapatnag na heterogeneity na saray kanser na too tan say Physiological macro tan micro environment.
Say kaslakan ya pakapatnagan na kanser ed obaryo et say ikayat na peritoneal tan say itsura na ascites. Say idakel na cell fluid ed ascites et misiglaotan ed graben sakit tan makapuy ya prognosis (15). Unong ed saray report, sayan nikadkaduman kompartimento et hypoxic, walaan na atagey a lebel na vascular endothelial growth factor (VEGF) tan indoleamine 2,3-dioxygenase (IDO), tan onloloob ed saray T regulatory cells tan myeloid inhibitory cells (15-18). Say metabolikon kaliberliber na ascites et nayarin duma ed tumor a mismo, kanian say pangi-reprogram na T cells ed peritoneal space et agmalinew. Niarum ni, saray manunan pandumaan tan agpantatalosan na ascites tan metabolites ya walad kaliberliber na tumor et nayarin mangamper ed iloob na saray immune cells tan say kimey da ed saray tumor, tan kaukolan so kaaruman ya panagsukimat.
Pian nasolbar irayan problema, nanggawa kami na sensitive cell separation tan liquid chromatography tandem mass spectrometry (LC-MS/MS) ya paraan pian aralen so nanduruman klase na selula (kaiba lay CD4 + tan CD8 + T cells) ontan met ed loob tan baetan na saray tumor Saray metabolites to et manlalapud saray selula ed parehon ascites tan tumor ya kaliberliber na pasyente. Uusaren mi iyan paraan a kaiba so high-dimensional flow cytometry tan single-cell RNA sequencing (scRNA-seq) pian nawalaan na malinew a litrato na metabolic status na sarayan manunan populasyon. Sayan paraan so angipatnag na signipikantin indaak na lebel na 1-methylnicotinamide (MNA) ed tumor T cells, tan saray eksperimento ed vitro so angipatnag ya say immunomodulatory ya epekto na MNA ed T cell function et ag ni amta nensaman. Diad inkalapagan, sayan paraan so mangipapatnag na saray metabolikon pankakasakey na saray tumor tan immune cells, tan mangiiter na nikadkaduman pakatalos ed saray metabolites na immune regulation, ya nayarin mausar ed panagtambal na T cell-based ovarian cancer immunotherapy Treatment opportunities.
Inusar mi so high-dimensional flow cytometry pian nabilang so glucose uptake [2-(N-(7-nitrophenyl-2-oxa-1,3-diaza-4-yl)amino)-2-deoxyglucose (2-NBDG) tan mitochondrial activity [MitoTracker Deep Red (MT DR) (MT DR, 2017, 2019) ed abay na side saray tipikal a marka ya mangibiig ed saray selula na immune tan saray populasyon na selula na tumor (Table S2 tan Figure S1A). Impanengneng na sayan analysis ya no ikompara ed T cells, say ascites tan tumor cells et mas atagey so glucose uptake levels da, balet mas melag so pandumaan da ed mitochondrial activity. Say average ya glucose uptake na tumor cells [CD45-EpCAM (EpCAM)+] et talora anggad apatiran danay nen say T cells, tan say average ya glucose uptake na CD4 + T cells et 1.2 danay nen say CD8 + T cells, ya mangipapanengneng ya saray Tumor infiltrating lymphocytes (TIL) et walaan na nanduruman metabolikon pankaukolan anggan diad parehon pigura TAME (Figure 1). Diad pidumaan, say aktibidad na mitochondrial ed saray selula na tumor et mipara ed CD4 + T cells, tan say aktibidad na mitochondrial na parehon klase na selula et mas atagey nen say CD8 + T cells (Figure 1B). Diad inkalapagan, sarayan resulta so mangipapatnag na lebel na metabolismo. Say metabolic activity na tumor cells et mas atagey nen say CD4 + T cells, tan say metabolic activity na CD4 + T cells et mas atagey nen say CD8 + T cells. Anggaman ed sarayan epekto ed nanduruman klase na selula, anggapoy mantutunosan a pandumaan ed metabolic status na CD4 + tan CD8 + T cells odino say relatibon proporsyon da ed ascites no ikompara ed saray tumor (Figure 1C). Diad pidumaan, diad CD45-cell fraction, say proporsyon na EpCAM+ cells ed tumor et dinmaak no ikompara ed ascites (Figure 1D). Naobserbaan mi met so malinew ya pandumaan na metabolismo ed baetan na EpCAM+ tan EpCAM- cell components. Saray EpCAM+ (tumor) cells et walaan na mas atagey ya glucose uptake tan mitochondrial activity nen saray EpCAM- cells, ya mas atagey nen say metabolic activity na fibroblasts ed tumor cells ed TME (Figure 1, E tan F).
(A tan B) Median fluorescence intensity (MFI) na glucose uptake (2-NBDG) (A) tan mitochondrial activity na CD4 + T cells (MitoTracker dark red) (B) Representative graphs (kawanan) tan tabulated data (Kawanan), CD8 + T cells tan EpCAM + CD45-tumor cells bilang tumor tan tumor. (C) Say ratio na CD4 + tan CD8 + cells (na CD3 + T cells) ed ascites tan tumor. (D) Proporsyon na EpCAM + tumor cells ed ascites tan tumor (CD45−). (E tan F) EpCAM + CD45-tumor tan EpCAM-CD45-matrix glucose uptake (2-NBDG) (E) tan mitochondrial activity (MitoTracker dark red) (F) representative graphs (kawanan) tan tabulated data (Kawanan) Ascites tan tumor cells. (G) Saray representatibon graph na CD25, CD137 tan PD1 ya ekspresyon panamegley na flow cytometry. (H tan I) CD25, CD137 tan PD1 ya ekspresyon ed CD4 + T cells (H) tan CD8 + T cells (I). (J tan K) Naive, central memory (Tcm), effector (Teff) tan effector memory (Tem) ya phenotypes base ed panangibalikas na CCR7 tan CD45RO. Saray representative ya imahen (kawanan) tan tabular data (kawanan) na CD4 + T cells (J) tan CD8 + T cells (K) ed ascites tan tumor. Saray P values ​​et nadetermina panamegley na paired t-test (*P<0.05, **P<0.01 tan ***P<0.001). Say linya et mangirerepresenta ed saray pasyente ya mipara (n = 6). FMO, fluorescence minus one; MFI, median ya inka-fluorescence intensity.
Say kaaruman ya impanaral et angipatnag na arum niran makatantandan pandumaan ed baetan na atagey a naresolbe ya T cell phenotypic status. Say activated (Figure 1, G anggad I) tan effector memory (Figure 1, J tan K) ed saray tumor et mas mabetbet nen say ascites (proporsyon na CD3 + T cells). Ontan met, say impanaral ed phenotype panamegley na pangibalikas na saray activation markers (CD25 tan CD137) tan saray depletion markers [programmed cell death protein 1 (PD1)] et angipatnag ya anggaman mandumaan so metabolikon katangian na sarayan populasyon (Figure S1, B anggad E), Balet anggapoy signipikantin metabolikon pandumaan ya naobserbaan ed baetan na naive, odino memoryal ya epekto na subset (Figure S1, F1) SIAK). Sarayan resulta et akumpirma diad impangusar na machine learning methods pian automatikon mangiter na cell phenotypes (21), ya lalon angipatnag na kiwawala na baleg a bilang na bone marrow cells (CD45 + / CD3- / CD4 + / CD45RO +) ed ascites na pasyente (Figure S2A ). Diad amin ya abitlan klase na selula, sayan myeloid cell population so angipatnag na sankatageyan ya glucose uptake tan mitochondrial activity (Figure S2, B anggad G). Ipapabitar na sarayan resulta so mabiskeg a pandumaan na metabolismo ed baetan na nanduruman klase na selula ya naromog ed ascites tan tumor ed saray pasyente na HGSC.
Say manunan angat ed pakatalos ed saray metabolomic characteristics na TIL et say pankaukolan ya i-isolate iray sample na T cell ya walaan na magenap a kalinisan, kalidad tan karakel manlapud saray tumor. Impanengneng na saray agano nin impanaral ya saray paraan na panag-sort tan panag-enrich na bead base ed flow cytometry et nayarin manresulta ed pananguman ed saray profile na metabolite na selula (22-24). Pian natalonaan iyan problema, in-optimize mi so bead enrichment method pian nai-isolate tan ni-isolate so TIL manlapud surgical resected human ovarian cancer antis ya analisaen panamegley na LC-MS/MS (nengnengen so Materials and Methods; Figure 2A). Pian naseguro so epekto na sayan protocol ed saray pananguman na metabolite, inkompara mi so profile na metabolite na T cells ya in-activate na saray mabunigas a donor kayari na bead separation step ed tagey ed saray selula ya ag-insian na bead balet ta nansiansia ed yelo. Sayan analysis control control et walay atagey ya siglaotan na sarayan duaran kondisyon (r = 0.77), tan say teknikal ya paulit-ulit na grupo na 86 ya metabolites et walaan na atagey ya paulit-ulit (Figure 2B). Kanian, sarayan paraan et makapanggawa na suston metabolite analysis ed saray selula ya manpapayaman na cell type, kanian mangiter na sankaunaan ya high-resolution platform parad pakabidbiran na espisipikon metabolites ed HGSC, diad ontan et nawalaan na mas aralem a pakatalos iray totoo ed cell specificity Sexual metabolism program.
(A) Schematic diagram na magnetic bead enrichment. Antis ya analisaen panamegley na LC-MS/MS, saray selula et ondalan ed taloran nantutumbokan ya round na magnetic bead enrichment odino mansiansia ed yelo. (B) Say epekto na klase na panagpayaman ed karakel na metabolites. Say average na taloran sukat parad kada klase na panagpayaman ± SE. Say grey line et mangirerepresenta na 1:1 ya relasyon. Say intra-class correlation (ICC) na paulit-ulit ya sukat et nanengneng ed axis label. NAD, nicotinamide adenine dinucleotide. (C) Schematic diagram na workflow na patient metabolite analysis. Saray ascites o tumor et naala ed saray pasyente tan i-cryopreserve. Say melag a kabiangan na kada sample et inanalisa panamegley na flow cytometry, bangbalet saray nakekeraan ya sample et linmabas ed taloran round na enrichment parad CD4+, CD8+ tan CD45- cells. Sarayan piraso na selula et inaral ya inusar so LC-MS/MS. (D) Heat map na standardized ya karakel na metabolite. Say dendrogram et mangirerepresenta ed panagtipon nen Ward na distansya na Euclidean ed baetan na saray sample. (E) Principal component analysis (PCA) na sample metabolite map, ya mangipapanengneng na taloran replicate na kada sample, saray sample manlapud parehon pasyente et konektado ed sakey a linya. (F) Say PCA na metabolite profile na sample ya akakondisyon ed pasyente (salanti, uusaren so partial redundancy); say klase na sample et limitado lapud convex hull. PC1, manunan kabiangan 1; PC2, manunan kabiangan 2.
Insan, inyaplika mi iyan paraan na panagpayaman pian naanalisa so 99 a metabolites ed CD4 +, CD8 + tan CD45-cell fractions ed saray manunan ascites tan tumor na anemiran pasyente na HGSC (Figure 2C, Figure S3A tan Table S3 tan S4). Say populasyon ya interesado et mankantidad na 2% anggad 70% na orihinal ya baleg a sample na mabilay iran selula, tan say proporsyon na saray selula et manduruman tuloy ed baetan na saray pasyente. Kayari na pangibiig ed saray beads, say pinayaman a fraction na interes (CD4+, CD8+ o CD45-) et mankantidad na masulok ya 85% na amin ya mabilay a selula ed sample ed average. Sayan paraan na panagpayaman et mamaabuloy ed sikatayo ya analisaen iray populasyon na selula manlapud metabolismo na tumor tissue na too, ya imposiblin gawaen manlapud angkakabaleg iran sample. Diad impangusar ed sayan protocol, nadetermina mi ya say l-kynurenine tan adenosine, sarayan duaran maong ya immunosuppressive metabolites et atagey ed tumor T cells odino tumor cells (Figure S3, B tan C). Kanian, sarayan resulta so mangipapanengneng na katooran tan abilidad na teknolohiya tayon mangibiig na selula tan mass spectrometry pian naromog iray importantin metabolites ed biolohiya ed saray tisyu na pasyente.
Say impanaral mi et angipatnag met na mabiskeg ya metabolic separation na saray klase na selula ed loob tan baetan na saray pasyente (Figure 2D tan Figure S4A). Diad partikular, no ikompara ed arum ya pasyente, say pasyente 70 et angipatnag na nanduruman metabolikon katangian (Figure 2E tan Figure S4B), ya mangipapanengneng ya nayarin walay baleg ya metabolikon heterogeneity ed baetan na saray pasyente. Nepeg ya imanoen ya no ikompara ed arum ya pasyente (1.2 anggad 2 litro; Table S1), say dagup na ascites ya naala ed pasyente 70 (80 ml) et mas melag. Say panangontrol ed inter-patient heterogeneity legan na manunan component analysis (alimbawa, say pangusar na partial redundancy analysis) et mangipapanengneng na mantutunosan iran pananguman ed baetan na saray klase na selula, tan saray klase na selula tan/odino microenvironment et malinew ya natipon unong ed profile na metabolite (Figure 2F ). Say impanaral ed saray saksakey a metabolites so angipabitar ed sarayan epekto tan angipatnag na signipikantin pandumaan na saray klase na selula tan say microenvironment. Nepeg ya imanoen ya say sankagrabian ya pandumaan ya naobserbaan et say MNA, ya kaslakan et mayaman ed CD45- cells tan ed CD4+ tan CD8+ cells ya onloloob ed tumor (Figure 3A). Parad saray CD4 + cells, sayan epekto so sankapatnagan, tan say MNA ed CD8 + cells et singa met mabiskeg ya apektado na kaliberliber. Balet, aliwan importante iya, ta talora labat ed anemiran pasyente so nayarin na-evaluate parad tumor CD8+ scores. Nilikud ed MNA, diad nanduruman klase na selula ed ascites tan tumor, arum niran metabolites ya aliwan maong so pakabidbiran ed TIL et walaan met na nanduruman klase na selula (Figures S3 tan S4). Kanian, sarayan datos so mangipapatnag na makapanpromisan set na immunomodulatory metabolites parad kaaruman a panagsukimat.
(A) Normalized ya karga na MNA ed CD4+, CD8+ tan CD45- cells manlapud ascites tan tumor. Ipapanengneng na box plot so median (linya), interquartile range (frame hinge) tan data range, anggad 1.5 beses na interquartile range (frame whisker). Unong ed nideskribe ed Patient Materials and Methods, usaren so limma value na pasyente pian naamtaan so P value (*P<0.05 tan **P<0.01). (B) Schematic diagram na metabolismo na MNA (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 (berde): 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 na scRNA-seq na ascites (abuek) tan tumor (ambalanga; n = 3 pasyente). (D) NNMT expression ed nanduruman populasyon na selula ya abitla ya inusar so scRNA-seq. (E) Say pangibalikas na NNMT tan AOX1 ed SK-OV-3, human embryonic kidney (HEK) 293T, T cells tan MNA-treated T cells. Say folded expression et nipanengneng no ikompara ed SK-OV-3. Say padron na panangibalikas na SEM et nipanengneng (n = 6 ya mabunigas ya nandonasyon). Saray Ct ya mas atagey nen say 35 et ipapasen ya ag nanengneng (UD). (F) Say pangibalikas na SLC22A1 tan SLC22A2 ed SK-OV-3, HEK293T, T cells tan T cells ya inikdan na 8mM MNA. Say folded expression et nipanengneng no ikompara ed SK-OV-3. Say padron na panangibalikas na SEM et nipanengneng (n = 6 ya mabunigas ya nandonasyon). Saray Ct ya mas atagey nen say 35 et ipapasen ya ag nanengneng (UD). (G) Cell MNA content ed saray activated healthy donor T cells kayari na 72 oras ya inkubasyon ed MNA. Say padron na panangibalikas na SEM et nipanengneng (n = 4 ya mabunigas ya donor).
Say MNA et napapawala diad pangiyalis na methyl group manlapud S-adenosyl-1-methionine (SAM) ya anggad nicotinamide (NA) panamegley na nicotinamide N-methyltransferase (NNMT; Figure 3B). Say NNMT et sobran napatnagan ed nanduruman kanser na too tan walay koneksion to ed idakel, invasion tan metastasis (25-27). Pian mas natalosan so lapuan na MNA ed T cells ed TME, inusar mi so scRNA-seq pian nabidbir so ekspresyon na NNMT ed nanduruman klase na selula ed ascites tan tumor na taloran pasyente na HGSC (Table S5). Say impanaral ed ngalngalin 6,500 ya selula et angipatnag ya diad ascites tan tumor ya kaliberliber, say NNMT expression et limitado ed saray ipapasen ya populasyon na fibroblast tan tumor cell (Figure 3, C tan D). Nepeg ya imanoen ya anggapoy mapatnag ya NNMT expression ed anggan dinan ya populasyon ya mangipapanengneng na PTPRC (CD45 +) (Figure 3D tan Figure S5A), ya mangipapanengneng ya say MNA ya anengneng ed metabolite spectrum et inloob ed T cells. Say panangibalikas na aldehyde oxidase 1 (AOX1) et manguuman ed MNA ya magmaliw ya 1-methyl-2-pyridone-5-carboxamide (2-PYR) odino 1-methyl-4-pyridone-5-carboxamide (4-PYR); Say Figure 3B) et limitado met ed populasyon na fibroblasts ya mangipapatnag na COL1A1 (Figure S5A), ya saniban mangipapanengneng ya saray T cells et andian na abilidad na conventional MNA metabolism. Say expression pattern na sarayan MNA-related genes et na-verify ya inusar so komaduan independentin cell data set manlapud ascites manlapud HGSC patients (Figure S5B; n = 6) (16). Niarum ni, say quantitative polymerase chain reaction (qPCR) analysis na saray mabunigas ya donor T cells ya inikdan na MNA et angipatnag ya no ikompara ed control SK-OV-3 ovarian tumor cells, say NNMT o AOX1 et ngalngali ag-napatnagan (Figure 3E). Sarayan ag-iilaloan ya resulta so mangipapanengneng ya say MNA et nayarin nasekreto manlapud fibroblasts odino tumor ya onloob ed asingger iran T cells ed TME.
Anggaman saray kandidato et kaiba so pamilya na organic cation transporters 1 anggad 3 (OCT1, OCT2 tan OCT3) ya in-encode na soluble carrier 22 (SLC22) ya pamilya (SLC22A1, SLC22A2 tan SLC22A3), saray posiblin transporter na MNA et agni nadetermina (28) . Say QPCR na mRNA manlapud mabunigas a donor T cells so angipatnag na abeban lebel na SLC22A1 balet ag nanengneng so lebel na SLC22A2, ya angikumpirma ya akauna lan nireport ed literatura (Figure 3F) (29). Diad pidumaan, say SK-OV-3 ovarian tumor cell line et angipatnag na atagey a lebel na parehon transporter (Figure 3F).
Pian nasubok so posibilidad na saray T cells ya walaan na abilidad ya mangawat na dayo ya MNA, saray mabunigas ya donor T cells so inkultura ed loob na 72 oras diad kiwawala na nanduruman konsentrasyon na MNA. No anggapoy exogenous MNA, say cellular content na MNA et ag nanengneng (Figure 3G). Balet, saray activated T cells ya inikdan na exogenous MNA et angipatnag na dose-dependent ya indaak na MNA content ed saray selula, ya anggad 6 mM MNA (Figure 3G). Ipapanengneng na sayan resulta ya anggaman ed abeban lebel na transporter expression tan kakulangan na manunan enzyme ya responsable ed intracellular MNA metabolism, say TIL et sarag nin siansia ya mangala na MNA.
Say spectrum na metabolites ed saray T cells na pasyente tan saray eksperimento ed panag-absorb na MNA ed vitro et mamaaligwas ed posibilidad ya saray cancer-associated fibroblasts (CAF) et mangisekreto na MNA tan saray tumor cells et nayarin mangiregula ed phenotype tan function na TIL. Pian naamtaan so epekto na MNA ed saray T cells, saray mabunigas a donor T cells so in-activate ed vitro diad kiwawala o anggapo na MNA, tan inebalwar so idarakel tan produksyon na cytokine da. Kayari na 7 ya agew ya inyarum na MNA ed sankatageyan ya dosis, say bilang na populasyon ya doble et medyo abawasan, bangta say biskeg et nansiansia ed amin ya dosis (Figure 4A). Niarum ni, say panagtambal ed exogenous MNA et nanresulta ed idakel na CD4 + tan CD8 + T cells ya mangipapatnag na tumor necrosis factor-α (TNFα; Figure 4B). Diad pidumaan, say intracellular ya produksyon na IFN-γ et signipikantin abawasan ed CD4 + T cells, balet aliwan diad CD8 + T cells, tan anggapoy signipikantin pananguman ed interleukin 2 (IL-2; Figure 4, C tan D). Kanian, say enzyme-linked immunosorbent assay (ELISA) na saray supernatant manlapud sarayan MNA-treated T cell cultures et angipatnag na signipikantin indakel na TNFα, inmabeba na IFN-γ, tan anggapoy impanguman na IL-2 (Figure 4, E anggad G). . Say ibabawas na IFN-γ et mangipapanengneng ya say MNA et nayarin walay papel to ed pangisebel ed anti-tumor ya aktibidad na T cells. Pian na-simulate so epekto na MNA ed T cell-mediated cytotoxicity, saray chimeric antigen receptor T (FRα-CAR-T) cells ya man-target na folate receptor α tan CAR-T (GFP) ya ireregulate na green fluorescent protein (GFP) -CAR-T) cells et gagawaen na mabunigas ya donor peripheral blood mononuclear cells (PBMC). Saray CAR-T cells so inkultura ed loob na 24 oras diad kiwawala na MNA, insan inkultura ed human SK-OV-3 ovarian tumor cells ya mangipapatnag na folate receptor α diad effector to target ratio ya 10:1. Say panagtambal na MNA et nanresulta ed signipikantin ibawas na aktibidad na panagpatey na FRα-CAR-T cells, ya mipara ed FRα-CAR-T cells ya inikdan na adenosine (Figure 4H).
(A) Say kabuoan na bilang na viable cell tan say populasyon ya doble (PD) ya direkta manlapud kultura nen koma-7 ya agew. Say bar graph et mangirerepresenta ed mean + SEM na anemiran mabunigas ya donor. Irerepresenta toy datos manlapud ag-onkulang ed n = 3 ya independentin eksperimento. (B anggad D) Say CD3/CD28 tan IL-2 so inusar pian na-activate iray T cells ed nanduruman MNA concentrations da ed loob na 7 ya agew. Antis na panagsukimat, saray selula so pinakiwas na PMA/ionomycin ya walay GolgiStop ed loob na 4 oras. TNFα (B) ya ekspresyon ed T cells. Alimbawa ya imahen (kawanan) tan tabular data (kawanan) na TNFα expression ed mabilay iran selula. IFN-γ (C) tan IL-2 (D) ya ekspresyon ed T cells. Say panangibalikas na cytokines et sinukat panamegley na flow cytometry. Say bar graph et mangirerepresenta ed average (n = 6 ya mabunigas ya donasyon) + SEM. Mangusar na one-way analysis of variance tan paulit-ulit ya sukat (*P<0.05 tan **P<0.01) pian naamtaan so P value. Irerepresenta toy datos manlapud ag-onkulang ed n = 3 ya independentin eksperimento. (E anggad G) Say CD3/CD28 tan IL-2 so inusar pian na-activate iray T cells ed nanduruman MNA concentrations da ed loob na 7 ya agew. Say medium et naala antis tan kayari na 4 oras ya PMA/ionomycin stimulation. Say konsentrasyon na TNFα (E), IFN-γ (F) tan IL-2 (G) et sinukat panamegley na ELISA. Say bar graph et mangirerepresenta ed average (n = 5 ya mabunigas ya donasyon) + SEM. Say P value et nadetermina ya inusar so one-way analysis of variance tan paulit-ulit ya panagsukat (*P<0.05). Say tuldok-tuldok a linya so mangipapanengneng na limitasyon na pakadetektaan na pakadetektaan. (H) Cell lysis assay. Saray FRα-CAR-T odino GFP-CAR-T ya selula et in-adjust na adenosine (250μM) odino MNA (10 mM) ed loob na 24 oras, odino ag-in-treat (Ctrl). Say porsiento na impamatey na SK-OV-3 cells so sinukat. Say P value et nadetermina na Welch t test (*P<0.5 tan **P<0.01).
Pian nawalaan na mekanistikon pakatalos ed MNA-dependent TNFα expression regulation, saray pananguman ed TNFα mRNA na MNA-treated T cells so inebalwar (Figure 5A). Saray mabunigas ya donor T cells ya inikdan na MNA et walaan na duaran danay ya indaak na TNFα transcription levels, ya mangipapanengneng ya say MNA et mandedependi ed TNFα transcription regulation. Pian nausisa iyan posiblin mekanismo na regulasyon, duaran kabkabat a transcription factors ya mangireregula ed TNFα, salanti, say activated T cell nuclear factor (NFAT) tan specific protein 1 (Sp1), so inebalwar bilang ebat ed MNA binding ed proximal TNFα promoter ( 30). Say TNFα promoter et walaan na 6 ya abitlan NFAT binding sites tan 2 Sp1 binding sites, ya mansasalet ed sakey a site [-55 base pairs (bp) manlapud 5'cap] (30). Impanengneng na Chromatin immunoprecipitation (ChIP) ya no tambalen na MNA, say pangisiglaot na Sp1 ed TNFα promoter et dinmaak na mamitlo. Say pangilaktip ed NFAT et dinmaak tan inmasingger met ed kaimportantian (Figure 5B). Ipapanengneng na sarayan datos ya say MNA so mangireregula ed pangibalikas na TNFα panamegley na Sp1 transcription, tan diad mas abeban laknab et say pangibalikas na NFAT.
(A) No ikompara ed saray T cells ya inkultura ya anggapoy MNA, say fold change na TNFα expression ed saray T cells ya inikdan na MNA. Say padron na panangibalikas na SEM et nipanengneng (n = 5 ya mabunigas ya nan-donate). Irerepresenta toy datos manlapud ag-onkulang ed n = 3 ya independentin eksperimento. (B) Say TNFα promoter na T cells ya inikdan na 8 mM MNA odino anggapoy 8 mM MNA kayari na NFAT tan Sp1 et inlaok ed (Ctrl) tan PMA/ionomycin stimulation ed loob na 4 oras. Say Immunoglobulin G (IgG) tan H3 so inusar bilang negatibo tan positibon kontrol parad immunoprecipitation, unong ed inkauksoy da. Say bilang na ChIP et angipatnag ya say pangisiglaot na Sp1 tan NFAT ed TNFα promoter ed saray selula ya inikdan na MNA et dinmaak na pigaran danay no ikompara ed kontrol. Irerepresenta toy datos manlapud ag-onkulang ed n = 3 ya independentin eksperimento. Say P value et nadetermina na dakel ya t-tests (*** P <0.01). (C) No ikompara ed ascites na HGSC, saray T cells (ya aliwan cytotoxic) so angipatnag na indakel na TNF ed tumor. Saray kolor et mangirerepresenta ed nanduruman pasyente. Saray nipanengneng ya selula et random ya in-sample ed 300 tan in-jitter pian nalimitaan so overdrawing (** Padj = 0.0076). (D) Niproponin modelo na MNA parad kanser ed obaryo. Say MNA et napapawala ed saray tumor cells tan fibroblasts ed TME tan naala na saray T cells. Say MNA et mamaaligwas ed pangibiig na Sp1 ed TNFα promoter, ya mangitonton ed idakel na TNFα transcription tan TNFα cytokine production. Say MNA et pansengegan met na ibawas na IFN-γ. Say pangisebel ed T cell function et manresulta ed nabawasan ya pakayarin mamatey tan maples ya idakel na tumor.
Unong ed saray report, say TNFα et walaan na anti-tumor tan anti-tumor ya epekto ya akadepende ed arap tan beneg, balet ta walaan itan na kabkabat a betang ed pangisulong na idakel tan iyaaligwas na kanser ed obaryo (31-33). Unong ed saray report, say konsentrasyon na TNFα ed ascites tan tumor tissues ed saray pasyente ya walay kanser ed obaryo et mas atagey nen saray benign tissues (34-36). No nipaakar ed mekanismo, say TNFα et nayarin mangiregula ed aktibasyon, kimey tan idarakel na saray selula na amputin dala, tan manguman ed itsura na saray selula na kanser (37, 38). Mitunosan ed sarayan naromog, say differential gene expression analysis so angipatnag ya say TNF et signipikantin inmatagey ed T cells ed tumor tissues no ikompara ed ascites (Figure 5C). Say idarakel na TNF expression et napatnagan labat ed saray populasyon na T cell ya walaan na non-cytotoxic phenotype (Figure S5A). Diad sumaryo, susuportaan na sarayan datos so panmoria ya say MNA et walaan na duaran immunosuppressive tan tumor promoting ya epekto ed HGSC.
Say fluorescent labeling base ed flow cytometry so nagmaliw ya manunan paraan pian naaral so metabolismo na TIL. Impanengneng na sarayan impanaral ya no ikompara ed saray peripheral blood lymphocytes odino T cells manlapud segundaryon lymphoid organs, say murine tan human TIL et walaan na mas atagey a tendensia ya mangala na glucose (4, 39) tan say kalkalnan pakaandi na mitochondrial function (19, 40). Anggaman naobserbaan mi so mipadparan resulta ed sayan impanaral, say manunan iyaaligwas et say pangikompara ed metabolismo na saray selula na tumor tan TIL manlapud parehon inekal a tisyu na tumor. Mitunosan ed pigara ed sarayan akaunan report, saray tumor (CD45-EpCAM +) cells manlapud ascites tan tumor et walaan na mas atagey ya glucose uptake nen saray CD8 + tan CD4 + T cells, ya manusuporta ya say atagey ya glucose uptake na tumor cells et nayarin ikompara ed saray T cells. Say konsepto na kompetisyon na T cell. TME. Balet, say mitochondrial activity na tumor cells et mas atagey nen say CD8 + T cells, balet say mitochondrial activity et mipara ed CD4 + T cells. Sarayan resulta so mamabiskeg ed onlelesa ya tema ya say oxidative metabolism et importante parad saray selula na tumor (41, 42). Isusuheri da met ya saray CD8 + T cells et nayarin mas mainomay ya na-oxidative dysfunction nen saray CD4 + T cells, odino saray CD4 + T cells et nayarin mangusar na carbon sources nilikud ed glucose pian napansiansia so mitochondrial activity (43, 44). Nepeg ya imanoen ya anggapoy anengneng min pandumaan ed glucose uptake odino mitochondrial activity ed baetan na CD4 + T effectors, T effector memory tan T central memory cells ed ascites. Ontan met, say pandumaan na CD8 + T cells ed saray tumor et anggapoy pibabali to ed pananguman ed glucose uptake, ya mangipapabitar ed signipikantin pandumaan na T cells ya inkultura ed vitro tan human TIL ed vivo (22). Sarayan obserbasyon et akumpirma met ed impangusar na unbiased ya automatic cell population allocation, ya lalon angipatnag ya saray CD45 + / CD3- / CD4 + / CD45RO + cells ya walaan na mas atagey ya glucose uptake tan mitochondrial activity nen saray tumor cells so kasmak balet ta walaan na Metabolic active cell population. Sayan populasyon et nayarin mangirerepresenta ed ipapasen ya subpopulasyon na myeloid suppressor cells odino plasmacytoid dendritic cells ya abitla ed scRNA-seq analysis. Anggaman parehon nireport irayan tumor ed obaryo na too [45], kaukolan da nin siansia so kaaruman a kimey pian nideskribe iyan myeloid subpopulation.
Anggaman saray paraan ya akabase ed flow cytometry et makapalinew ed inkalapagan a pandumaan na glucose tan oxidative metabolism ed baetan na saray klase na selula, saray eksakton metabolites ya napapawala na glucose odino arum niran lapuan na carbon parad mitochondrial metabolism ed TME et agni nadetermina. Say pangiter na kiwawala o kiwawala na saray metabolite ed sakey a TIL subset et mankaukolan na panaglinis ed populasyon na selula manlapud pinuteran a tisyu. Kanian, say paraan na panagpayaman na selula mi ya inlaok ed mass spectrometry et makapangiter na pakatalos ed saray metabolites ya mandumaan so inkayaman na T cells tan populasyon na tumor cell diad saray mipara ya sample na pasyente. Anggaman sayan paraan et walaan na bentaha nen say fluorescence-activated cell sorting, wala ray metabolite libraries ya nayarin naapektoan lapud inherent stability tan/odino maples ya turnover rate (22). Anggaman ontan, say paraan mi et akapangibiig na duaran kabkabat ya immunosuppressive metabolites, say adenosine tan kynurenine, lapud manduruma iratan ed baetan na saray klase na sample.
Say metabonomic analysis mi ed saray tumor tan TIL subtypes et mangiter na mas dakel ya pakatalos ed papel na saray metabolites ed ovarian TME. Unona, diad impangusar na flow cytometry, adetermina mi ya anggapoy pandumaan na aktibidad na mitochondrial ed baetan na saray tumor tan CD4 + T cells. Balet, say LC-MS/MS analysis et angipatnag na signipikantin pananguman ed karakel na metabolites ed sarayan populasyon, ya mangipapanengneng ya saray konklusyon nipaakar ed metabolismo na TIL tan say inkalapagan ya metabolikon aktibidad to et mankaukolan na maalwar ya interpretasyon. Kumadwa, say MNA so metabolite ya walaan na sankabalgan a pandumaan na CD45-cells tan T cells ed ascites, aliwan saray tumor. Kanian, say compartmentalization tan say kawalaan na tumor et nayarin walaan na nanduruman epekto ed TIL metabolism, ya mangipapabitar ed posiblin heterogeneity ed sakey a microenvironment. Komatlo, say panangibalikas na MNA-producing enzyme NNMT et manunan limitado ed CAF, ya saray tumor cells ed mas abeban laknab, balet saray nanengneng ya MNA levels et naobserbaan ed tumor-derived T cells. Say sobran panangibalikas na NNMT ed ovarian CAF et walay kabkabat ya epekto to ya mamaaligwas na kanser, lapud say iyaaligwas na metabolismo na CAF, invasion na tumor tan metastasis (27). Anggaman say inkalapagan a lebel na TIL et katamtaman, say panangibalikas na NNMT ed CAF et maapit a misiglaotan ed Cancer Genome Atlas (TCGA) mesenchymal subtype, ya misiglaotan ed makapuy a prognosis (27, 46, 47). Kaunoran, say panangibalikas na enzyme AOX1 ya responsable ed pakaderal na MNA et limitado met ed populasyon na CAF, ya mangipapanengneng ya saray T cells et anggapoy abilidad dan man-metabolize na MNA. Susuportaan na sarayan resulta so ideyan anggaman nakaukolan so kaaruman a kimey pian napaneknekan iyan naromog, say atagey a lebel na MNA ed T cells so nayarin mangipatnag na kiwawala na immunosuppressive CAF microenvironment.
Lapud abeban lebel na ekspresyon na MNA transporters tan say ag nanengneng ya lebel na saray manunan protina ya nalalanor ed metabolismo na MNA, say kiwawala na MNA ed T cells et ag-iilaloan. Anggapoy nanengneng ya NNMT odino AOX1 panamegley na scRNA-seq analysis tan targeted qPCR na duaran independentin cohort. Ipapanengneng na sarayan resulta ya say MNA et ag-nagawa na T cells, no ag ingen nasesepsep ed kaliberliber na TME. Ipapanengneng na saray eksperimento ed vitro ya saray T cells et walay tendensia dan mantipon na exogenous MNA.
Impanengneng na saray in vitro ya impanaral mi ya say exogenous MNA et mamapawala na TNFα ed T cells tan mamaaligwas ed pangibiig na Sp1 ed TNFα promoter. Anggaman say TNFα et walaan na parehon anti-tumor tan anti-tumor ya kimey, diad kanser ed obaryo, say TNFα et makapangiter na idakel na kanser ed obaryo (31-33). Say neutralization na TNFα ed ovarian tumor cell culture odino say pangekal na TNFα signal ed saray modelo na mouse et makapaaligwas na TNFα-mediated inflammatory cytokine production tan makaamper ed idakel na tumor (32, 35). Kanian, diad sayan kaso, say TME-derived MNA et nayarin onkiwas bilang pro-inflammatory metabolite panamegley na TNFα-dependent ya mekanismo panamegley na autocrine loop, diad ontan et napaaligwas so kiwawala tan ikayat na kanser ed obaryo (31). Base ed sayan posibilidad, say TNFα blockade et aaralen bilang posiblin tambal parad kanser ed obaryo (37, 48, 49). Niarum ni, say MNA et mamakapuy ed cytotoxicity na CAR-T cells ed ovarian tumor cells, ya mangiiter na kaaruman ya ebidensya parad MNA-mediated immune suppression. Diad inkalapagan, sarayan resulta et mangisusuheri na sakey a modelo no iner saray tumor tan CAF cells so mangisekreto na MNA ed extracellular TME. Diad panamegley na (i) TNF-induced ovarian cancer growth stimulation tan (ii) MNA-induced T cell cytotoxic activity inhibition, saya so nayarin walaan na duaran epekto ed tumor (Figure 5D).
Diad konklusyon, diad pangiyaplika na kombinasyon na maples ya cell enrichment, single-cell sequencing tan metabolic profiling, sayan impanaral so angipatnag na baleg a pandumaan na immunometabolomic ed baetan na saray tumor tan ascites cells ed saray pasyente na HGSC. Sayan komprehensibon panagsukimat so angipatnag ya walaray pandumaan ed glucose uptake tan mitochondrial activity ed baetan na T cells, tan abitla so MNA bilang sakey ya non-cell autonomous immune regulatory metabolite. Sarayan datos et walay epekto to no panon ya apektaen na TME so metabolismo na T cell ed saray kanser na too. Anggaman nireport so direktan kompetisyon na sustansya ed baetan na T cells tan cancer cells, saray metabolites et nayarin onkiwas met bilang agdirektan regulator pian napaaligwas so tumor progression tan posiblin naprebentaan iray endogenous immune responses. Say kaaruman a deskripsion na functional role na sarayan regulatory metabolites et nayarin mangilukas na alternatibon estratehiya parad pamaaligwas ed anti-tumor immune response.
Saray specimen na pasyente tan saray datos ed klinika et naala panamegley na BC cancer tumor tissue repository ya sertipikado na Canadian Tissue Repository Network. Unong ed protocol ya inaprobaan na BC Cancer Research Ethics Committee tan say University of British Columbia (H07-00463), amin ya specimen tan clinical data na pasyente et akaawat na insulat ya abobon odino pormal ya angibaliwala ed abobon da. Saray sample et akaamot ed sertipikadon BioBank (BRC-00290). Saray detalyadon katangian na pasyente et nanengneng ed Tables S1 tan S5. Parad cryopreservation, say scalpel et uusaren pian nabulok so sample na tumor na pasyente insan itulak ed 100-micron filter pian nawalaan na sakey a cell suspension. Say ascites na pasyente et in-centrifuge ed 1500 rpm ed loob na 10 minutos ed 4°C pian na-pellet iray selula tan naekal so supernatant. Saray selula ya naala ed tumor tan ascites et in-cryopreserve ed 50% heat-inactivated human AB serum (Sigma-Aldrich), 40% RPMI-1640 (Thermo Fisher Scientific) tan 10% dimethyl sulfoxide. Sarayan napreserba ya single cell suspensions et tinunaw tan inusar parad metabolomics tan metabolite determination ya adeskribe ed leksab.
Say kompleton medium et tugyopen na 0.22 μm ya sinala 50:50 ya inaruman na RPMI 1640: AimV. RPMI 1640 + 2.05 mM l-glutamine (Thermo Fisher Scientific) ya indagdag na 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) ya solusyon (Thermo Fisher Scientific) tan 50 μMB-mercaptoethanol. Say AimV (Invitrogen) et walay 20 mM Hepes (Thermo Fisher Scientific) tan 2 mM l-glutamine (Thermo Fisher Scientific). Say flow cytometer staining buffer et tugyopen na 0.22μm ya filtered phosphate buffered saline (PBS; Invitrogen) ya inaruman na 3% heat-inactivated AB human serum (Sigma). Say cell enrichment buffer et tugyopen na 0.22μm ya sinala ya PBS tan inaruman na 0.5% ya heat-inactivated human AB serum (Sigma-Aldrich).
Diad 37°C ya kompleton medium, saray selula et tinina na 10 nM MT DR tan 100 μM 2-NBDG ed loob na 30 minutos. Insan, saray selula so tinina na viability dye eF506 ed 4°C ed loob na 15 minutos. I-resuspend iray selula ed FC Block (eBioscience) tan Brilliant Stain Buffer (BD Biosciences), i-dilute ed flow cytometer staining buffer (unong ed instruksion na manggagawa), insan i-incubate ed loob na 10 minuto ed temperatura na kuarto. Mantsa na saray selula na sakey a set na antibodies (Table S2) diad flow cytometry staining buffer ed 4°C ed loob na 20 minutos. I-resuspend iray selula ed flow cytometry staining buffer (Cytek Aurora; 3L-16V-14B-8R configuration) antis ya analisaen. Usaren so SpectroFlo tan FlowJo V10 pian naanalisa so bilang na selula, tan usaren so GraphPad Prism 8 pian nagawa so datos. Say median fluorescence intensity (MFI) na 2-NBDG tan MT DR et log-normalized, insan inusar so paired t test parad statistical analysis pian nabilang iray pasyente ya mipara. Ekalen so amin ya populasyon ya walaan na kulang ya 40 ya ebento ed panagsukimat; mangiloob na MFI value ya 1 parad antokaman ya negatibon balor antis ya gawaen so statistical analysis tan data visualization.
Pian nadagdag so manual gating strategy na proseso panel ed tagey, inusar mi so kompleton annotation na shape restriction tree (FAUST) (21) pian automatikon mangiter na saray selula ed populasyon kayari na pangekal ed saray inatey a selula ed FlowJo. Manual min i-manage so output pian natipon iray populasyon ya singa ag-inmabobon (pankasakey so PD1+ tan PD1-tumor cells) tan saray populasyon ya nansiansia. Kada sample et walaan na average ya masulok ya 2% ya selula, parad dagup ya 11 ya populasyon.
Say Ficoll gradient density centrifugation so inusar pian naapag so PBMC manlapud saray produkto na leukocyte separation (STEMCELL Technologies). Saray CD8 + T cells et insian ed PBMC ya inusar so CD8 MicroBeads (Miltenyi) tan pinalaknab ed kompleton medium ya inusar so TransAct (Miltenyi) ed loob na 2 simba unong ed instruksion na manggagawa. Saray selula et inabuloyan ya onalagey ed loob na 5 agew diad kompleton medium ya walay IL-7 (10 ng/ml; PeproTech), insan in-re-stimulate na TransAct. Diad koma-7 ya agew, unong ed instruksion na manggagawa, say human CD45 MicroBeads (Miltenyi) so inusar pian napaaligwas iray selula ed taloran nantutumbokan a round. Saray selula so in-aliquot parad flow cytometry analysis (unong ed adeskribe ed tagey), tan sakey milyon a selula so in-aliquot na mamitlo parad LC-MS/MS analysis. Saray sample et inproseso panamegley na LC-MS/MS unong ed nideskribe ed leksab. Inkalkula mi so kulang ya metabolite value ya walay ion number ya 1,000. Kada sample et na-normalize ed total ion number (TIC), na-convert ed logarithm tan automatikon na-normalize ed MetaboAnalystR antis ya analisaen.
Say single cell suspension na kada pasyente et tinunaw tan sinala ed 40 μm ya filter ya anggad kompleton medium (unong ed adeskribe ed tagey). Unong ed protocol na manggagawa, taloran nantutumbokan ya positibon panagpili panamegley na magnetic bead separation ya inusar so MicroBeads (Miltenyi) so inusar pian napaaligwas iray sample parad CD8+, CD4+ tan CD45- cells (diad yelo). Diad antikey, saray selula et ipawil ed cell enrichment buffer (unong ed adeskribe ed tagey) tan bilangen. Saray selula so in-incubate ed human CD8 beads, human CD4 beads odino human CD45 beads (Miltenyi) ed 4°C ed loob na 15 minutos, insan inurasan na cell enrichment buffer. Say sample et ipasa ed LS column (Miltenyi), tan saray positibo tan negatibon fraction et nakolekta. Pian nabawasan so karukey tan napalaknab so kundang na pakabawi na selula, say CD8-fraction et usaren parad komaduan round na CD4+ enrichment, tan say CD4-fraction et usaren parad ontumbok ya CD45-enrichment. Iyan so solusyon ed yelo legan na proseso na panagbiig.
Pian niparaan iray sample parad metabolite analysis, saray selula et inurasan na maminsan na ice-cold salt solution, tan 1 ml na 80% methanol so inyarum ed kada sample, insan in-vortex tan in-snap frozen ed liquid nitrogen. Saray sample et inyarum ed taloran freeze-thaw cycles tan in-centrifuge ed 14,000 rpm ed loob na 15 minutos ed 4°C. Say supernatant ya walaan na saray metabolites et onsingaw anggad mamagaan. Saray metabolites et tinunaw lamet ed 50 μl na 0.03% formic acid, in-vortex pian manlaok, insan in-centrifuge pian naekal iray debris.
Ekalen iray metabolites unong ed adeskribe ed tagey. Iyalis so supernatant ed sakey a high performance liquid chromatography bottle parad metabolomics research. Mangusar na random ya protocol na panagtambal pian natambal so kada sample na parehon bilang na selula pian naprebentaan so epekto na batch. Nanggawa kami na kalidad na panangusisa ed saray global metabolites ya akauna lan nipalapag ed AB SCIEX QTRAP 5500 Triple Quadrupole Mass Spectrometer (50). Say chromatographic analysis tan say peak area integration et ginawa ya inusar so MultiQuant version 2.1 software (Applied Biosystems SCIEX).
Say ion count ya 1000 so inusar pian nakalkula so kulang ya metabolite value, tan say TIC na kada sample so inusar pian nakalkula so normalized peak area na kada naimano ya metabolite pian ipetek iray pananguman ya inloob na instrumental analysis manlapud sample processing. Kayari na inka-normalize na TIC, say MetaboAnalystR(51) (default parameter) so nausar parad logarithmic conversion tan automatic norm line scaling. Inusar mi so PCA ya walay vegan R package pian nagawaan so exploratory analysis na pandumaan na metabolome ed baetan na saray klase na sample, tan inusar mi so partial redundancy analysis pian naanalisa iray pasyente. Usaren so Ward method pian makagawa na heat map dendrogram pian na-cluster so Euclidean distance ed baetan na saray sample. Inusar mi so limma (52) ed standardized ya karakel na metabolite pian nabirbir so nanduruman karakel na metabolite ed interon klase na selula tan microenvironment. Pian napalemew so eksplinasyon, usaren mi so group mean parameter pian nibaga so modelo, tan ikonsidera iray klase na selula ed microenvironment bilang kada grupo (n = 6 a grupo); parad significance test, nanggawa kami na taloran paulit-ulit ya panagsukat parad kada metabolite Pian napaliisan so palson panagkopya, say pasyente so inlaktip bilang babeng ed desinyo na limma. Pian nanengneng so pandumaan na metabolites ed baetan na nanduruman pasyente, inuman mi so limma model ya kaiba iray pasyente diad pihon paraan. Irereport mi so kabaliksan na pre-specified ya pandumaan na klase na selula tan say microenvironment na Padj <0.05 (Benjamini-Hochberg correction).
Kayari na vigor enrichment ya inusar so Miltenyi Dead Cell Removal Kit (>80% viability), ginawa so single-cell transcriptome sequencing ed amin ya mabilay ya frozen ascites tan tumor samples ya inusar so 10x 5′gene expression protocol. Limaran kaso ya walay mipara iran tumor tan ascites so inanalisa, anggaman say abeban viability manlapud sakey a sample na tumor so angamper ed inlaktip to. Pian napili so dakel a pasyente, intipon mi ray sample na kada pasyente diad saray lane na 10x chromium controller, tan inaral mi so ascites tan tumor sites ya manbibiigan. Kayari na panag-sequence [Illumina HiSeq 4000 28×98 bp paired end (PE), Quebec genome; say average ya 73,488 tan 41,378 ya babasaen kada selula parad tumor tan ascites]], inusar mi so CellSNP tan Vireo (53) (base ed CellSNP bilang say kaslakan ya human SNP (VCF) ya inter na GRCh38 et naitdan na donor identity. Inusar mi so SNPRelate pian naamtaan so sankaasinggeran ya identity (IBS) na genotype na pasyente (IBS) ag-ilaktip iray ag-aka-assign ya selula tan saray selula ya abitla bilang duplex tan saray mantutumbokan ya donor ed baetan na saray sample na ascites tan tumor (54). (2792 tan 4183 ya selula manlapud tumor tan ascites, unong ed inkauksoy da) parad panagsukimat et inusar mi so igraph's (57) Louvain clustering na shared nearest neighbor network (SNN) base ed distansya na Jaccard ed cluster cells base ed expression. saray selula et nadetermina na CD8A tan GZMA, ya ag kaiba iray subclusters ya abeba so ribosomal protein expression. Naala mi so nipalapag ya datos nen Izar et al.
Saray PBMC et insian ed saray produkto na leukocyte separation (STEMCELL Technologies) panamegley na Ficoll gradient density centrifugation. Saray CD3 + cells et insian ed PBMC ya inusar so CD3 beads (Miltenyi). No wala o anggapo so MNA, saray CD3+ cells et na-activate panamegley na plate-bound CD3 (5μg/ml), soluble CD28 (3μg/ml) tan IL-2 (300 U/ml; Proleukin). Diad unor ya agew na panagpalaknab, say viability (Fixable Viability Dye eFluor450, eBioscience) tan say idakel (123count eBeads, Thermo Fisher Scientific) et inebalwar panamegley na flow cytometry. I-evaluate so effector function diad pangi-stimulate ed saray selula ya walay PMA (20 ng/ml) tan ionomycin (1μg/ml) ya walay GolgiStop ed loob na 4 oras, tan bantayan so CD8-PerCP (RPA-T8, BioLegend), CD4-AF700 (RPA-T4), BioLegend) tan TNFα-fluoresce isothianate (TCFIMA-T4), BD). I-stimulate iray qPCR tan ChIP cells na PMA (20 ng/ml) tan ionomycin (1μg/ml) ed loob na 4 oras. Say ELISA supernatant et naala antis tan kayari na stimulation ya walay PMA (20 ng/ml) tan ionomycin (1 μg/ml) ed loob na 4 oras.
Tumboken so protocol na manggagawa pian nai-isolate so RNA ya uusaren so RNeasy Plus Mini Kit (QIAGEN). Usaren so QIAshredder (QIAGEN) pian na-homogenize so sample. Mangusar na high-capacity RNA to cDNA kit (Thermo Fisher Scientific) pian makagawa na complementary DNA (cDNA). Usaren so TaqMan Rapid Advanced Master Mix (Thermo Fisher Scientific) pian nabilang so gene expression (unong ed protocol na manggagawa) ya uusaren iray onggendan ya probe: Hs00196287_m1 (NNMT), Hs00154079_m1 (AOX1), Hs00427555m1, 2012 Hs02786624_g1 [glyceraldehyde-3-phosphate off Hydrogen (GAPDH)] tan Hs01010726_m1 (SLC22A2). Saray sample et inusar ed StepOnePlus real-time PCR system (Applied Biosystems) (Applied Biosystems) diad MicroAmp fast optical 96-well reaction plate (Applied Biosystems) ya walay MicroAmp optical film. Antokaman ya Ct value ya onlampas ed 35 et ipapasen ya mas atagey nen say detection threshold tan markaan ya ag nanengneng.
Gawaen so ChIP unong ed akaunan adeskribe (58). Diad antikey, saray selula et inikdan na formaldehyde (pinal ya konsentrasyon 1.42%) tan inkulongan ed temperatura na kuarto ed loob na 10 minutos. Mangusar na kaaruman ya swelling buffer (25 mM Hepes, 1.5 mM MgCl2, 10 mM KCl tan 0.1% NP-40) ed yelo ed loob na 10 minuto, insan i-resuspend ed immunoprecipitation buffer unong ed adeskribe (58 ). Say sample et in-sonicate ed sarayan siklo: 10 siklo (20 1-segundo iran pulso) tan sakey ya static time ya 40 segundo. I-incubate so ChIP-grade immunoglobulin G (Cell Signaling Technology; 1μl), histone H3 (Cell Signaling Technology; 3μl), NFAT (Invitrogen; 3μl) tan SP1 (Cell Signaling Technology; 3μl) ya antibodies ed sample ed 4°CC ya yugyogen ed sanlabi. I-incubate so protein A beads (Thermo Fisher Scientific) ed sample ed 4°C ya maalwar ya yugyogen ed loob na 1 oras, insan usaren so chelex beads (Bio-Rad) pian napaaligwas so DNA, tan usaren so proteinase K (Thermo Fisher) parad panagtunaw na protina. Say TNFα promoter et adiskobre panamegley na PCR: forward, GGG TAT CCT TGA TGC TTG TGT; diad kasunian, GTG CCA ACA ACT GCC TTT ATA TG (207-bp ya produkto). Saray imahen et ginawa na Image Lab (Bio-Rad) tan kinantipikar ya inusar so ImageJ software.
Say cell culture supernatant et naala unong ed adeskribe ed tagey. Say determinasyon et ginawa unong ed saray proseso na manggagawa na human TNFα ELISA kit (Invitrogen), human IL-2 ELISA kit (Invitrogen) tan human IFN-γ ELISA kit (Abcam). Unong ed protocol na manggagawa, say supernatant et inlaok na 1:100 pian nanengneng so TNFα tan IL-2, tan 1:3 pian nanengneng so IFN-γ. Usaren so EnVision 2104 Multilabel Reader (PerkinElmer) pian nasukat so absorbance ed 450 nm.
Saray PBMC et insian ed saray produkto na leukocyte separation (STEMCELL Technologies) panamegley na Ficoll gradient density centrifugation. Saray CD3 + cells et insian ed PBMC ya inusar so CD3 beads (Miltenyi). No wala o anggapo so MNA, saray CD3+ cells et na-activate panamegley na plate-bound CD3 (5μg/ml), soluble CD28 (3μg/ml) tan IL-2 (300 U/ml; Proleukin) ed loob na 3 agew. Kayari 3 agew, saray selula so inala tan inurasan na 0.9% ya asin, tan say pellet so in-snap frozen. Say bilang na selula et ginawa panamegley na flow cytometry (Cytek Aurora; 3L-16V-14B-8R configuration) ya inusar so 123count eBeads.
Ekalen iray metabolites unong ed adeskribe ed tagey. Say pinamaga ya extract et inuman ed konsentrasyon ya 4000 cell equivalents/μl. Analisaen so sample panamegley na reversed-phase chromatography (1290 Infinity II, Agilent Technologies, Santa Clara, CA) tan CORTECS T3 column (2.1×150 mm, particle size 1.6-μm, pore size 120-Å; #186008500, Waters). Polar mass spectrometer (6470, Agilent), ya say electrospray ionization et mankikimey ed positibon paraan. Say mobile phase A et 0.1% formic acid (diad H2O), say mobile phase B et 90% acetonitrile, 0.1% formic acid. Say LC gradient et 0 anggad 2 minuto parad 100% A, 2 anggad 7.1 minuto parad 99% B, tan 7.1 anggad 8 minuto parad 99% B. Insan i-re-equilibrate so column na mobile phase A diad flow rate ya 0.6 ml/min ed loob na 3 minuto. . Say flow rate et 0.4ml/min, tan say column chamber et napainit ed 50°C. Usaren so puron chemical standard na MNA (M320995, Toronto Research Chemical Company, North York, Ontario, Canada) pian naestablisa so retention time (RT) tan say transformation (RT = 0.882 minutos, transformation 1 = 137→94.1, transformation 2 = 137→92 , Conversion = 13→787). No amin ya taloran transisyon et nagawa ed suston panaon na panag-ala, say transisyon 1 et usaren parad panagkuantipika pian naseguro so inka-espesipiko. Say standard curve na MNA (Toronto Research Chemical Company) et ginawa panamegley na anemiran serial dilution na stock solution (1 mg/ml) pian naala iray estandarte ya 0.1, 1.0, 10 tan 100 ng/ml tan 1.0 tan 10μg/ml ya likido. Say limitasyon na pakadetektaan et 1 ng/ml, tan say linear ya ebat et manlapud 10 ng/ml tan 10μg/ml. Kada ineksion na duaran microliter na sample tan standard et uusaren parad LC/MS ya panagsukimat, tan say mixed quality control sample et uusaren kada waloran ineksion pian naseguro so inkapekder na plataporma na panagsukimat. Say MNA ya ebat na amin ya MNA-treated cell samples et walad loob na linear range na assay. Say panagsukimat na datos et ginawa ya inusar so MassHunter quantitative analysis software (v9.0, Agilent).
Say komaduan henerasyon na αFR-CAR construct et inala ed Song et al. (59). Diad antikey, say construct et walaan na sarayan lugan: CD8a leader sequence, human αFR-specific single-chain variable fragment, CD8a hinge tan transmembrane region, CD27 intracellular domain tan CD3z intracellular domain. Say kompleton CAR sequence et ginawa na GenScript, insan in-clone ed second-generation lentiviral expression vector ya walad tagey na GFP expression cassette ya inusar pian nasuri so transduction efficiency.
Say lentivirus et napapawala panamegley na transfection na HEK293T cells [American Type Culture Collection (ATCC); pinabaleg ed Dulbecco's modified Eagle medium ya walay 10% fetal bovine serum (FBS) tan 1% PenStrep, tan inusar so CAR-GFP vector tan saray packaging plasmids (psPAX2 tan pMD2.G, Addgene) et manguusar na lipofection amine (Sigma-Aldrich). Say supernatant ya walay virus to et naala 48 tan 72 oras kayari na transfection, sinala, tan inkonsentra na ultracentrifugation. Isimpen so konsentradon viral supernatant ed -80°C anggad na-transduce.
Saray PBMC et nibiig ed saray mabunigas ya donor leukocyte separation products (STEMCELL Technologies) panamegley na Ficoll gradient density centrifugation. Mangusar na positibon pinilin CD8 microbeads (Miltenyi) pian nibiig iray CD8+ cells manlapud PBMC. Pabiskegen iray T cells panamegley na TransAct (Miltenyi) tan diad TexMACS medium [Miltenyi; inaruman na 3% heat-inactivated human serum, 1% PenStrep tan IL-2 (300 U/ml)]. Duamplo tan apat ya oras kayari na stimulation, saray T cells et in-transduce na lentivirus (10 μl concentrated virus supernatant kada 106 cells). 1 anggad 3 agew kayari na transduction ed Cytek Aurora (diad FSC (Forward Scatter)/SSC (Side Scatter), Singlet, GFP+), usisaen so GFP expression na saray selula pian nipanengneng so transduction efficiency ya ag-onkulang ed 30%.
Saray CAR-T cells et inkultura ed loob na 24 oras diad Immunocult (STEMCELL Technologies; indagdag na 1% PenStrep) diad sarayan kondisyon: ag-atambal, atambalan na 250 μM adenosine odino 10 mM MNA. Kayari na pretreatment, saray CAR-T cells et inurasan na PBS tan inlaok ed 20,000 SK-OV-3 cells [ATCC; diad McCoy 5A medium (Sigma-Aldrich) ya inaruman na 10% FBS tan 1% PenStrep ed 10: Say effector to target ratio ya 1 et pinalaknab ed taloran beses ed inaruman ya Immunocult medium. Saray SK-OV-3 cells tan SK-OV-3 cells ya in-lysed na digitalis saponin (0.5mg/ml; Sigma-Aldrich) so inusar bilang negatibo tan positibon kontrol, unong ed inkauksoy da. Kayari na 24 oras ya pankakasakey na panagtanem, say supernatant so inala tan say lactate dehydrogenase (LDH) so sinukat unong ed instruksion na manggagawa (LDH Glo Cytotoxicity Assay Kit, Promega). Say LDH supernatant et inlaok na 1:50 ed LDH buffer. Say porsiento na impamatey et sinukat ya inusar so onggendan a pormula: porsiento na impamatey = porsiento na panangipetek / sankatageyan a bilang na impamatey x 100%, no iner porsiento na panangipetek = co-culture-T cells labat, tan sankatageyan a bilang na impamatey = positibon kontrol-negatibon kontrol.
Unong ed nideskribe ed teksto odino materyales tan paraan, usaren so GraphPad Prism 8, Microsoft Excel odino R v3.6.0 parad statistical analysis. No dakel so sample ya naala ed parehon pasyente (singa say ascites tan tumor), mangusar kami na paired t test odino ilaktip so pasyente bilang random ya epekto ed linear odino generalized model no matukoy. Parad metabolomics analysis, say importance test et gagawaen ya mamitlo.
Parad kaaruman iran materyales parad sayan artikulo, nengnengen so 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. Bellnis), Deraph. Jones (Russell G. Jones), Phineas T. Hamilton (Phineas T.
Say MNA et ontutulong ed immune suppression na T cells tan mangirerepresenta na posiblin target na immunotherapy parad panagtambal na kanser na too.
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. Bellnis), Deraph. Jones (Russell G. Jones), Phineas T. Hamilton (Phineas T.
Say MNA et ontutulong ed immune suppression na T cells tan mangirerepresenta na posiblin target na immunotherapy parad panagtambal na kanser na too.
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