Immunomodulatory metabolites hi tumor microenvironment (TME)-a thil pawimawh tak a ni a, mahse tlemte tih loh chu an nihna hi a tam zawkin hriat a la ni lo. Hetah hian high-grade serous carcinoma (HGSC) vei damlote tumor leh ascites atanga tumor leh T cells te chu kan zirchiang a, heng TME compartment hrang hrangte metabolome kan hmuchhuak a ni. Ascites leh tumor cells te hian metabolite danglamna nasa tak an nei a. Ascites nena khaikhin chuan tumor-infiltrating T cells te hi 1-methylnicotinamide (MNA) ah nasa takin an hausa a ni. T cell-a MNA level sang tak ni mah se, nicotinamide N-methyltransferase (S-adenosylmethionine atanga nicotinamide-a methyl group transfer tichaktu enzyme) expression hi fibroblast leh tumor cell-ah chauh a awm a ni. A hnathawh danah chuan MNA hian T cells te chu tumor tichaktu cytokine tumor necrosis factor alpha secrete turin a tichak a ni. Chuvangin, TME atanga chhuak MNA hian T cells immune regulation-ah a pui a, mihring cancer enkawlna atana immunotherapy target awm thei a ni.
Tumor-derived metabolites hian anti-tumor immunity-ah inhibitory effect thuk tak a nei thei a, evidence tam zawk chuan natna zual zelna tur key driving force a ni thei tih a tarlang bawk (1). Warburg effect bakah hian tun hnaiah tumor cells metabolic state leh tumor microenvironment (TME) immune state nena a inzawmna te hriat chian hna thawh tan a ni. Mouse model leh mihring T cells chungchanga zirchianna an neihah chuan glutamine metabolism (2), oxidative metabolism (3) leh glucose metabolism (4) te hian immune cell subgroup hrang hrangah independent takin hna an thawk thei tih hmuhchhuah a ni. Heng kawng hrang hranga metabolite engemaw zat hian T cells te anti-tumor function chu a titawp a ni. Coenzyme tetrahydrobiopterin (BH4) blockade hian T cells proliferation a tichhe thei tih finfiah a ni tawh a, taksaa BH4 tihpun hian CD4 leh CD8 mediated anti-tumor immune response a tichak thei tih finfiah a ni. Tin, kynurenine hian immunosuppressive effect a neih chu BH4 pek hian a chhanchhuak thei bawk (5). Isocitrate dehydrogenase (IDH) mutant glioblastoma-ah chuan enantiometabolic (R)-2-hydroxyglutarate (R-2-HG) secretion hian T cell activation, proliferation leh cytolysis Activity a titawp a ni (6). Tun hnaiah glycolysis by-product methylglyoxal hi myeloid origin atanga suppressor cells atanga siam a ni tih hmuhchhuah a ni a, methylglyoxal T cell transfer hian effector T cell function a titawp thei tih hmuhchhuah a ni. Treatment-ah chuan methylglyoxal neutralization hian myeloid-derived suppressor cells (MDSC) activity a hneh thei a, mouse model-a checkpoint blockade therapy chu synergistically-in a tichak thei bawk (7). Heng zirchiannate hian an vaiin TME-derived metabolites te hian T cell function leh activity tihregulate-na atana an chanvo pawimawh tak chu an sawi uar hle.
Ovarian cancer-ah hian T cell dysfunction a awm tih an sawi nasa hle (8). Hei hi hypoxia leh tumor vasculature pangngai lo (9)-a awm metabolic characteristics vang a ni a, chu chuan glucose leh tryptophan chu by-product lactic acid leh kynurenine-ah a chantir a ni. Extracellular lactate tam lutuk hian interferon-γ (IFN-γ) siam chhuah a ti tlem a, myelosuppressive subgroup hrang hrangte inthliarna a tichak bawk (10, 11). Tryptophan ei hian T cell proliferation direct-in a titawp a, T cell receptor signaling a titawp bawk (12-14). Heng thil hmuhchhuah te hi awm mahse, immune metabolism chhehvel hna tam tak chu in vitro T cell culture-ah optimized media hmangin an thawk a, a nih loh leh homologous mouse models in vivo-ah chauh tihkhawtlai a ni a, chung zinga pakhat mah hian mihring cancer leh Physiological macro leh micro environment heterogeneity chu a lantir kim lo.
Ovarian cancer natna langsar tak chu peritoneal spread leh ascites lo lang hi a ni. Ascites-a cell fluid awmkhawm hi natna zual leh prognosis tha lo nen a inzawm a ni (15). Report-a a lan dan chuan he compartment danglam tak hi hypoxic a ni a, vascular endothelial growth factor (VEGF) leh indoleamine 2,3-dioxygenase (IDO) a tam hle a, T regulatory cells leh myeloid inhibitory cells te hian an infiltrate a ni (15-18). Ascites metabolic environment hi tumor ngei nen a danglam thei a, chuvangin peritoneal space-a T cells te reprogramming hi a chiang lo hle. Chu bakah, tumor environment-a ascites leh metabolites awmte danglamna leh heterogeneity pawimawh tak takte hian immune cells infiltration leh tumor-a an hnathawh dan a tikhawlo thei a, zirchianna neih belh a ngai a ni.
Heng harsatnate sutkian nan hian sensitive cell separation leh liquid chromatography tandem mass spectrometry (LC-MS/MS) method kan design a, chu chuan cell chi hrang hrang (CD4 + leh CD8 + T cells te pawh tel) bakah tumor chhung leh inkara zirchianna a nei a Its metabolites span cells in the same ascites and tumor environment of the patient. He method hi high-dimensional flow cytometry leh single-cell RNA sequencing (scRNA-seq) te nen kan hmang a, heng key population te metabolic status hi highly resolved portrait kan pe thei a ni. Hetiang method hmang hian tumor T cell-a 1-methylnicotinamide (MNA) level a sang hle tih hmuhchhuah a ni a, in vitro experiment-ah chuan MNA hian T cell function-a immunomodulatory effect a neih dan hi a hmain hriat a la ni lo. A tlangpuiin, he method hian tumor leh immune cells inkara mutual metabolic interactions a pholang a, immune regulation metabolites chungchangah pawh hriatna danglam tak a pe a, hei hi T cell-based ovarian cancer immunotherapy Treatment opportunities enkawlna atan a tangkai thei ang.
High-dimensional flow cytometry hmangin a rualin glucose lakluh zat kan teh a [2-(N-(7-nitrophenyl-2-oxa-1,3-diaza-4-yl)amino)-2-deoxyglucose (2-NBDG) leh mitochondrial activity [MitoTracker Deep Red (MT DR)] (7, 19, 20) te hi side by side typical an ni immune cell leh tumor cell population thliar hrangtu marker (Table S2 leh Figure S1A). He analysis hian T cells nena khaikhin chuan ascites leh tumor cells te hian glucose uptake level an nei sang zawk a, mahse mitochondrial activity-ah erawh danglamna tlem zawk an nei tih a tarlang. Tumor cells [CD45-EpCAM (EpCAM)+] glucose uptake average chu T cells aiin a let thum atanga a let li vel a ni a, CD4 + T cells te glucose uptake average chu CD8 + T cells te aiin a let 1.2 in a tam a, hei hian Tumor infiltrating lymphocytes (TIL) te hian TME khatah pawh metabolic requirement hrang hrang an nei tih a tilang a ni (Figure 1A). Chumi danglamna chu tumor cell-a mitochondrial activity hi CD4 + T cells nen a inang a, cell chi hnih mitochondrial activity hi CD8 + T cells aiin a sang zawk a ni (Figure 1B). A tlangpuiin heng result te hian metabolic level a pholang thin. Tumor cells metabolic activity hi CD4 + T cells aiin a sang zawk a, CD4 + T cells te metabolic activity hi CD8 + T cells aiin a sang zawk bawk. Cell chi hrang hrangah heng nghawngte hi awm mahse, tumor nena khaikhin chuan CD4 + leh CD8 + T cells metabolic status emaw, ascites-a an relative proportion-ah emaw danglamna a awm lo (Figure 1C). Chumi danglamna chu CD45-cell fraction-ah chuan tumor-a EpCAM+ cell awm zat chu ascites nena khaikhin chuan a pung a ni (Figure 1D). EpCAM+ leh EpCAM- cell components te metabolic difference chiang tak kan hmu bawk. EpCAM+ (tumor) cells hian EpCAM- cells aiin glucose uptake leh mitochondrial activity a nei sang zawk a, hei hi TME-a tumor cells-a fibroblasts te metabolic activity aiin a sang zawk hle (Figure 1, E leh F).
(A leh B) CD4 + T cells (MitoTracker dark red) te glucose lakluhna (2-NBDG) (A) leh mitochondrial activity median fluorescence intensity (MFI) (B) Representative graphs (khawi lamah nge) leh tabulated data (Right), CD8 + T cells leh EpCAM + CD45-tumor cells te chu ascites leh tumor atanga lak chhuah a ni. (C) Ascites leh tumor-a CD4 + leh CD8 + cells (CD3 + T cells) ratio. (D) Ascites leh tumor (CD45−)-a EpCAM + tumor cell awm zat. (E leh F) EpCAM + CD45-tumor leh EpCAM-CD45-matrix glucose lakluh (2-NBDG) (E) leh mitochondrial activity (MitoTracker dark red) (F) aiawhtu graph (khawi lamah nge) leh tabulated data ( Dinglam) Ascites leh tumor cells te. (G) Flow cytometry hmanga CD25, CD137 leh PD1 expression tarlanna graph. (H leh I) CD4 + T cells (H) leh CD8 + T cells (I)-a CD25, CD137 leh PD1 expression. (J leh K) CCR7 leh CD45RO expression atanga chhut naive, central memory (Tcm), effector (Teff) leh effector memory (Tem) phenotype te. Ascites leh tumor-a CD4 + T cells (J) leh CD8 + T cells (K) te aiawhtu image (khawi lamah nge) leh tabular data (dinglamah). P value te chu paired t-test hmanga teh a ni (*P<0.05, **P<0.01 leh ***P<0.001). Line hian matched patients (n = 6) a entir a ni. FMO, fluorescence atanga pakhat paih chhuah; MFI, median fluorescence chakna a ni.
Analysis dang an neih chuan highly resolved T cell phenotypic status inkarah hian danglamna langsar dang a awm tih hmuhchhuah a ni. Tumor-a activated (Figure 1, G to I) leh effector memory (Figure 1, J leh K) te hi ascites (CD3 + T cells proportion) aiin a tam zawk hle. Chutiang bawkin, activation marker (CD25 leh CD137) leh depletion marker [programmed cell death protein 1 (PD1)] expression hmanga phenotype an zirchiannaah chuan heng population-te metabolic characteristic hi inang lo mah se (Figure S1, B to E) , Mahse naive, effector emaw memory subset-te inkarah hian metabolic danglamna lian tham hmuh tur a awm lo (Figure S1, F to I). Heng result te hi machine learning method hmanga cell phenotypes (21) automatic-a assign hmanga finfiah a ni a, chu chuan damlo ascites-ah bone marrow cells (CD45 + / CD3- / CD4 + / CD45RO +) tam tak a awm tih a tilang lehzual a ni (Figure S2A ). Cell chi hrang hrang hriat chhuah zawng zawng zingah hian he myeloid cell population hian glucose uptake leh mitochondrial activity a nei sang ber a ni (Figure S2, B to G). Heng result te hian HGSC vei te ascites leh tumor-a cell chi hrang hrang hmuhchhuah te inkara metabolic danglamna nasa tak a tarlang a ni.
TIL metabolic characteristic hriatthiamna kawnga harsatna lian ber chu tumor atanga T cell sample thianghlim, quality leh quantity tling isolate a ngai a ni. Tun hnaia zirchianna hrang hrangah chuan flow cytometry hmanga sorting leh bead enrichment hman dan hian cellular metabolite profile a tidanglam thei tih hmuhchhuah a ni (22-24). He harsatna hi sutkian nan hian bead enrichment method chu LC-MS/MS hmanga zirchianna hmain surgically resected human ovarian cancer atanga TIL isolate leh isolate turin kan optimize a (Materials and Methods; Figure 2A en rawh). He protocol hian metabolite inthlak danglamna a nghawng dan tlangpui tehna atan a chunga bead separation step hnua donor hrisel tak takte activate T cells te metabolite profile te chu bead separated ni lo mahse ice-a awm reng cell te nen kan khaikhin a. He quality control analysis hian heng condition pahnih inkarah hian inzawmna sang tak a awm tih hmuhchhuah a ni (r = 0.77), metabolite 86 awmna group-a technical repeatability hian repeatability sang tak a nei tih hmuhchhuah a ni (Figure 2B). Chuvangin, heng hmanrua te hian cell type enrichment nei cell-ah metabolite analysis dik tak a ti thei a, chu chuan HGSC-a metabolite bik hriat theihna tur high-resolution platform hmasa ber a siam thei a, chu chuan miten cell specificity chungchang hriatthiamna thuk zawk a neih theih phah a ni Sexual metabolism program.
(A) Magnetic bead tihpun dan schematic diagram. LC-MS/MS hmanga an zirchian hmain cell te hi magnetic bead enrichment vawi thum a zawnin an paltlang ang a, ice-ah an awm reng ang. (B) Enrichment type hian metabolite tamna a nghawng dan. Enrichment type tin atana tehna pathum awm zat average ± SE. Gray line hian 1:1 inzawmna a entir a ni. Axis label-a tehna repeated te intra-class correlation (ICC) a ni. NAD, nicotinamide adenine dinucleotide hmanga siam a ni. (C) Damlo metabolite thlirletna hnathawh dan schematic diagram. Ascites emaw tumor emaw chu damlo atanga lakkhawm niin cryopreserved a ni. Sample tin atanga a tlem ber chu flow cytometry hmangin an zirchiang a, sample dangte chu CD4+, CD8+ leh CD45- cells te tan enrichment round thum an nei bawk. Heng cell fraction te hi LC-MS/MS hmangin an zirchiang a. (D) Standardized metabolite tam dan heat map. Dendrogram hian Ward-a’n sample inkar Euclidean distance a clustering a entir a ni. (E) Sample metabolite map-a principal component analysis (PCA), sample tin replicate pathum tarlanna, damlo pakhat atanga sample lakte chu line hmanga inzawmkhawm a ni. (F) Damlo chunga sample conditioned (chu chu, partial redundancy hmanga) metabolite profile PCA; sample type chu convex hull vang a ni. PC1, a bulpui ber 1; PC2, a bulpui ber 2.
A dawtah chuan he enrichment method hmang hian HGSC vei paruk primary ascites leh tumor-a CD4 +, CD8 + leh CD45-cell fraction-a metabolite 99 kan zirchiang a (Figure 2C, Figure S3A leh Table S3 leh S4). Population of interest hi original large sample of living cells atanga 2% atanga 70% vel a ni a, cell proportion hi damlo hrang hrangah a inang lo hle. Beads te hi an inthen hnuah enriched fraction of interest (CD4+, CD8+ emaw CD45-) hian sample chhunga cell nung zawng zawng zingah a vaiin 85% aia tam a luah a ni. He enrichment method hmang hian mihring tumor tissue metabolism atanga cell population te kan zirchiang thei a, hei hi sample lian tak tak atanga tih theih loh a ni. He protocol hmang hian l-kynurenine leh adenosine, heng immunosuppressive metabolite chiang tak pahnih te hi tumor T cell emaw tumor cell emaw ah an sang tih kan hmu chhuak a (Figure S3, B leh C). Chuvangin, heng result te hian kan cell separation leh mass spectrometry technology hmanga damlo tissue-a biologically important metabolites hmuhchhuah theihna fidelity leh theihna a lantir a ni.
Kan zirchiannaah hian damlo chhung leh damlo inkara cell chi hrang hrangte metabolic separation chak tak a awm tih kan hmu bawk (Figure 2D leh Figure S4A). A bik takin, damlo dangte nena khaikhin chuan damlo 70 hian metabolic characteristic hrang hrang a nei a (Figure 2E leh Figure S4B), hei hian damlote inkarah metabolic heterogeneity nasa tak a awm thei tih a tilang. Hriat tur chu damlo dang (liter 1.2 atanga 2; Table S1) nena khaikhin chuan damlo 70 (80 ml)-a ascites lakkhawm zawng zawng chu a tlem zawk a ni. Principal component analysis neih chhunga inter-patient heterogeneity control (entir nan, partial redundancy analysis hmanga) hian cell chi hrang hrang inkara inthlak danglamna awm reng a lantir a, cell chi hrang hrang leh/ emaw microenvironment te chu metabolite profile angin chiang takin aggregate a ni (Figure 2F ). Single metabolites an zirchiannaah hian heng nghawngte hi a ngaih pawimawh hle a, cell chi hrang hrang leh microenvironment inkarah danglamna nasa tak a awm tih hmuhchhuah a ni. Hriat tur chu a danglamna nasa ber hmuh chhuah chu MNA a ni a, hei hi CD45- cells leh tumor chhunga lut CD4+ leh CD8+ cells-ah te a tam tlangpui (Figure 3A). CD4 + cells tan chuan he effect hi a langsar ber a, CD8 + cells-a MNA pawh hi environment-in nasa takin a nghawng niin a lang. Mahse, hei hi a pawimawh lo, a chhan chu damlo paruk zinga pathum chauh hi tumor CD8+ score endik theih a ni. MNA bakah hian ascites leh tumor-a cell chi hrang hrangah hian TIL-a characterized tha lo metabolite dangte pawh hi differentially rich an ni (Figure S3 leh S4). Chuvangin, heng data te hian zirchianna dang neih belh zelna tur immunomodulatory metabolites set beisei awm tak a rawn pholang a ni.
(A) Ascites leh tumor atanga CD4+, CD8+ leh CD45- cells a MNA awm zat normalized. Box plot-ah hian median (line), interquartile range (frame hinge) leh data range te tarlan a ni a, interquartile range (frame whisker) aiin a let 1.5 thleng a ni. Patient Materials and Methods-a tarlan angin damlo limma value hmangin P value (*P<0.05 leh **P<0.01) teh rawh. (B) MNA metabolism schematic diagram (60) te chu a hnuaia mi ang hian a ni. Thil chi hrang hrang: S-adenosyl-1-methionine; SAH, S-adenosine-1-homocysteine ​​te chu a awm a; NA, nicotinamide tih a ni a; MNA, 1-methylnicotinamide hmanga siam a ni a; 2-PY, 1-methyl- 2-pyridone-5-carboxamide hmanga siam a ni a; 4-PY, 1-methyl-4-pyridone-5-carboxamide hmanga siam a ni a; NR, nicotinamide ribose tih chu a ni a; NMN, nicotinamide mononucleotide hmanga siam a ni. Enzyme (green): NNMT, nicotinamide N-methyltransferase te chu a awm a; SIRT, sirtuins tih te; NAMPT, nicotinamide phosphoribosyl transferase hmanga siam a ni a; AOX1, aldehyde oxidase 1 te chu a awm a; NRK, nicotinamide riboside kinase tih chu a ni a; NMNAT, nicotinamide mono Nucleotide adenylate transferase hmanga siam a ni a; Pnp1, purine nucleoside phosphorylase hmanga siam a ni. (C) ascites (grey) leh tumor (red; n = damlo 3) scRNA-seq-a t-SNE. (D) scRNA-seq hmanga cell population hrang hranga NNMT expression hmuhchhuah. (E) SK-OV-3, human embryonic kidney (HEK) 293T, T cells leh MNA hmanga enkawl T cells-a NNMT leh AOX1 expression. Folded expression chu SK-OV-3 nena khaikhin chuan a lantir a ni. SEM hmanga expression pattern tarlan a ni (n = 6 healthy donors). Ct value 35 aia tam chu undetectable (UD) anga ngaih a ni. (F) SK-OV-3, HEK293T, T cells leh T cells 8mM MNA hmanga enkawl ah SLC22A1 leh SLC22A2 te expression. Folded expression chu SK-OV-3 nena khaikhin chuan a lantir a ni. SEM hmanga expression pattern tarlan a ni (n = 6 healthy donors). Ct value 35 aia tam chu undetectable (UD) anga ngaih a ni. (G) MNA nena darkar 72 chhung incubation hnua activated healthy donor T cells-a cell MNA awm zat. SEM hmanga expression pattern tarlan a ni (n = 4 healthy donors).
MNA hi nicotinamide N-methyltransferase (NNMT; Figure 3B) hmanga S-adenosyl-1-methionine (SAM) atanga nicotinamide (NA)-a methyl group transfer atanga siam a ni. NNMT hi mihring cancer chi hrang hrangah a overexpress a, proliferation, invasion leh metastasis nen a inzawm a ni (25-27). TME-a T cell-a MNA awmna hriat chian zawk nan scRNA-seq hmangin HGSC damlo pathum ascites leh tumor-a cell chi hrang hranga NNMT expression characterize kan hmang a (Table S5). Cell 6,500 vel an zirchiannaah chuan ascites leh tumor environment-ah chuan NNMT expression hi fibroblast leh tumor cell population nia ngaihte chauh a ni tih hmuhchhuah a ni (Figure 3, C leh D). Hriat tur pawimawh tak chu PTPRC (CD45 +) (CD45 +) (Figure 3D leh Figure S5A) express tu population-ah NNMT expression langsar tak a awm lo a, hei hian metabolite spectrum-a MNA hmuhchhuah chu T cell-ah a lut tih a tilang a ni. Aldehyde oxidase 1 (AOX1) expression hian MNA chu 1-methyl-2-pyridone-5-carboxamide (2-PYR) emaw 1-methyl-4-pyridone-5-carboxamide (4- PYR) emaw ah a chantir a Figure 3B) hi COL1A1 (Figure S5A) expressing fibroblast population-ah chauh tihkhawtlai a ni bawk a, chungte chu an inzawm khawm chuan T cells te hian MNA metabolism pangngaia theihna an nei lo tih a tilang a ni. Heng MNA nena inzawm genes te expression pattern hi HGSC vei atanga ascites atanga independent cell data set pahnihna hmangin an finfiah a (Figure S5B; n = 6) (16). Tin, MNA hmanga enkawl donor T cell hrisel tak takte quantitative polymerase chain reaction (qPCR) analysis hmangin control SK-OV-3 ovarian tumor cells nena khaikhin chuan NNMT emaw AOX1 emaw chu a lang lo tluk a ni (Figure 3E). Heng beisei loh result te hian MNA hi fibroblast emaw tumor emaw atang hian TME-a T cell inhnaih tak takah a chhuak thei tih a tarlang.
Candidate zingah hian soluble carrier 22 (SLC22) chhungkaw (SLC22A1, SLC22A2 leh SLC22A3)-in a encode organic cation transporter 1 atanga 3 (OCT1, OCT2 leh OCT3) chhungkua te pawh tel ve mah se, MNA-a transporter awm thei tur chu hriat chian a la ni lo (28) Donor T cell hrisel tak tak atanga mRNA QPCR hmangin SLC22A1 expression level hniam mahse SLC22A2 chu hmuh theih loh level a awm tih hmuhchhuah a ni a, hei hian a hmain literature-ah report a nih thu a nemnghet a ni (Figure 3F) (29). Chumi danglamna chu SK-OV-3 ovarian tumor cell line hian transporter pahnih level sang tak a lantir a (Figure 3F).
T cells te hian ramdang MNA an lakluh theihna an neih leh neih loh enfiah nan donor T cell hrisel te chu MNA concentration hrang hrang awmnaah darkar 72 chhung culture an ni. Exogenous MNA awm lohnaah chuan MNA cellular content hi hriat theih a ni lo (Figure 3G). Mahse, activated T cells exogenous MNA hmanga enkawl te chuan dose-dependent in cells chhunga MNA awm zat a pung a, 6 mM MNA thlengin a pung (Figure 3G). He result hian transporter expression level hniam tak leh intracellular MNA metabolism mawhphurtu enzyme awm lo mahse TIL hian MNA a la la thei tih a tilang.
Damlote T cell-a metabolite spectrum leh in vitro MNA absorption experiment-ah hian cancer-associated fibroblasts (CAF)-in MNA a chhuah theihna a tipung a, tumor cell-te chuan TIL phenotype leh hnathawh a tidanglam thei bawk. T cells-a MNA-in nghawng a neih dan hriat nan donor T cell hrisel tak takte chu MNA awm leh awm lohah in vitro-ah activate a ni a, an proliferation leh cytokine production te chu an zirchiang a ni. Dose sang berah MNA dah belh ni 7 hnuah population doubling number chu a tlahniam tawk a, dose zawng zawngah vigor chu a awm reng thung (Figure 4A). Tin, exogenous MNA enkawlnaah hian CD4 + leh CD8 + T cells tumor necrosis factor-α expressing proportion a pung bawk (TNFα; Figure 4B). Chumi danglamna chu CD4 + T cell-ah chuan intracellular production of IFN-γ chu nasa takin a tlahniam a, CD8 + T cell-ah erawh a tlahniam lo va, interleukin 2-ah pawh danglamna lian tham a awm lo (IL-2; Figure 4, C leh D). Chuvangin, heng MNA hmanga enkawl T cell culture atanga supernatants te enzyme-linked immunosorbent assay (ELISA) hmangin TNFα a pung nasa hle a, IFN-γ a tlahniam a, IL-2 ah pawh danglamna a awm lo (Figure 4, E to G). . IFN-γ tlahniam hian MNA hian T cells anti-tumor activity tihtawp kawngah chanvo a nei thei tih a tilang. T cell-mediated cytotoxicity-a MNA-in nghawng a neih dan simulate turin, green fluorescent protein (GFP) -CAR-T) cells-in a enkawl folate receptor α leh CAR-T (GFP) target-tu chimeric antigen receptor T (FRα-CAR-T) cells te chu healthy donor peripheral blood mononuclear cells (PBMC) hmangin an siam chhuak a ni. CAR-T cells chu MNA awmnaah darkar 24 chhung culture a ni a, chutah chuan folate receptor α expressing human SK-OV-3 ovarian tumor cells nen effector to target ratio 10:1-ah co-culture a ni. MNA hmanga enkawlnaah hian FRα-CAR-T cells killing activity a tlahniam nasa hle a, hei hi adenosine hmanga enkawl FRα-CAR-T cells nen a inang hle (Figure 4H).
(A) Total viable cell count leh population doubling (PD) ni 7-naah culture atanga direct a ni.Bar graph hian donor hrisel paruk te mean + SEM a tarlang a ni. Independent experiment n = 3 tal atanga data a entir. (B to D) CD3/CD28 leh IL-2 hmangin T cells te chu an MNA concentration hrang hrangah ni 7 chhung activate an ni. Analysis hmain cell te chu PMA/ionomycin hmangin GolgiStop hmangin darkar 4 chhung an stimulate a. T cells ah TNFα (B) expression a awm. Cell nung a TNFα expression entirnan image (khawi lamah nge) leh tabular data (dinglamah). T cell-a IFN-γ (C) leh IL-2 (D) te hi a langsar hle. Cytokines expression chu flow cytometry hmangin an teh a. Bar graph hian mean (n = 6 healthy donors) + SEM a entir a ni. P value hriat nan one-way analysis of variance leh repeated measures (*P<0.05 leh **P<0.01) hmangin. Independent experiment n = 3 tal atanga data a entir. (E to G) CD3/CD28 leh IL-2 hmangin T cells te chu an MNA concentration hrang hrangah ni 7 chhung activate an ni. Medium hi PMA/ionomycin stimulation darkar 4 hma leh a hnuah lakkhawm a ni. TNFα (E), IFN-γ (F) leh IL-2 (G) te concentration chu ELISA hmangin an teh a. Bar graph hian mean (n = 5 healthy donors) + SEM a entir a ni. P value chu one-way analysis of variance leh repeated measurement hmanga teh a ni (*P<0.05). Dotted line hian detection a detection limit a tarlang a. (H) Cell lysis enfiahna. FRα-CAR-T emaw GFP-CAR-T cell te chu adenosine (250μM) emaw MNA (10 mM) hmangin darkar 24 chhung an siamrem a, enkawl lohvin an dah (Ctrl). SK-OV-3 cells te percentage killing chu an teh a ni. P value chu Welch t test hmanga teh a ni (*P<0.5 leh **P<0.01).
MNA-dependent TNFα expression regulation chungchanga mechanistic understanding neih theih nan MNA hmanga enkawl T cells TNFα mRNA inthlak danglamna chu an zirchiang a (Figure 5A). MNA hmanga enkawl donor T cell hrisel tak takte chuan TNFα transcription level chu a let hnih zetin a pung tih an hmuchhuak a, hei hian MNA hi TNFα transcriptional regulation-ah a innghat tih a tilang a ni. He regulatory mechanism awm thei hi chhui chian nan hian TNFα regulate tu transcription factor hriat lar pahnih, activated T cell nuclear factor (NFAT) leh specific protein 1 (Sp1) te chu MNA binding to the proximal TNFα promoter chhanna atan an evaluate a ( 30 ). TNFα promoter hian NFAT binding site hriatchhuah 6 leh Sp1 binding site 2 a nei a, hmun khatah a inzawm khawm a [5’cap atanga base pairs (bp) 55] (30). Chromatin immunoprecipitation (ChIP) hmangin MNA hmanga enkawl a nih chuan Sp1 leh TNFα promoter inzawmna chu a let thum zetin a pung tih hmuhchhuah a ni. NFAT telh pawh a pung a, a pawimawhna a hnaih hle bawk (Figure 5B). Heng data te hian MNA hian Sp1 transcription hmangin TNFα expression a tidanglam tih a tilang a, a tlem berah chuan NFAT expression a tidanglam bawk.
(A) MNA tel lova cultured T cells nena khaikhin chuan MNA hmanga enkawl T cells-a TNFα expression fold change a ni. SEM hmanga expression pattern tarlan a ni (n = 5 healthy donors). Independent experiment n = 3 tal atanga data a entir. (B) NFAT leh Sp1 hnua 8 mM MNA hmanga enkawl emaw, tel lo emawa enkawl T cells TNFα promoter chu (Ctrl) leh PMA/ionomycin stimulation nen darkar 4 chhung an inzawm khawm a. Immunoprecipitation atana negative leh positive control atan immunoglobulin G (IgG) leh H3 te hman a ni. ChIP quantification-ah chuan MNA hmanga enkawl cell-a Sp1 leh NFAT te TNFα promoter nena inzawmna chu control nen khaikhin chuan vawi tam tak a pung tih hmuhchhuah a ni. Independent experiment n = 3 tal atanga data a entir. P value chu t-test tam tak hmanga teh a ni (*** P <0.01). (C) HGSC ascites nen khaikhin chuan T cells (non-cytotoxic) te hian tumor-ah TNF expression a tipung tih an hmuchhuak. A rawng te hian damlo hrang hrang a entir a ni. Displayed cells te chu randomly sample 300 ah lak a ni a, overdrawing tihtlem nan jittered a ni (** Padj = 0.0076). (D) Ovarian cancer atana MNA model ruahman. MNA hi tumor cell-ah leh TME-ah fibroblast-ah siam a ni a, T cell-in a la a ni. MNA hian Sp1 chu TNFα promoter nena inzawmna a tipung a, chu chuan TNFα transcription leh TNFα cytokine siam chhuahna a tipung a ni. MNA hian IFN-γ a tihhniam bawk. T cell hnathawh tihkhawtlai hian thah theihna a ti tlem a, tumor a tipung chak zawk bawk.
Report-a a lan dan chuan TNFα hian hmalam leh hnung lamah anti-tumor leh anti-tumor effects a nei a, mahse ovarian cancer thanna leh metastasis tichaktuah hian chanvo hriat hlawh tak a nei a ni (31-33). Report-a a lan dan chuan ovarian cancer vei damlote zingah ascites leh tumor tissue-a TNFα concentration hi benign tissue aiin a tam zawk a ni (34-36). Mechanism lamah chuan TNFα hian thisen varte activation, function leh proliferation a tidanglam thei a, cancer cells phenotype pawh a thlak danglam thei bawk (37, 38). Heng thil hmuhchhuah te nen hian a inmil hle a, differential gene expression analysis hmangin tumor tissue-a T cells-ah TNF chu ascites nena khaikhin chuan nasa takin a up-regulated tih hmuhchhuah a ni (Figure 5C). TNF expression tihpun hi non-cytotoxic phenotype nei T cell population-ah chauh a lang (Figure S5A). A tawi zawngin, heng data te hian MNA hian HGSC ah hian dual immunosuppressive leh tumor promoting effects a nei tih ngaihdan hi a thlawp a ni.
Flow cytometry hmanga fluorescent labeling hman hi TIL metabolism zir chianna atana hmanraw pawimawh ber a ni ta a ni. Heng zirchiannate hian secondary lymphoid organ atanga peripheral blood lymphocytes emaw T cells emaw nena khaikhin chuan murine leh mihring TIL te hian glucose lakluh duhna an nei sang zawk tih an hmuchhuak a ( 4 , 39 ) a, mitochondrial function pawh a hloh zauh zauh bawk ( 19 , 40 ). He zirchiannaah hian result inang chiah kan hmu a, mahse, a hmasawnna ber chu tumor cell leh TIL resected tumor tissue inang atanga metabolism tehkhin hi a ni. Heng report hmasa thenkhat nen hian a inmil hle a, ascites leh tumor atanga tumor (CD45-EpCAM +) cells te hian CD8 + leh CD4 + T cells te aiin glucose uptake an nei sang zawk a, hei hian tumor cells te glucose uptake sang tak hi T cells nen tehkhin theih a nih thu a thlawp a ni. T cell inelna tih concept. TME a ni. Mahse, tumor cells mitochondrial activity hi CD8 + T cells aiin a sang zawk a, mahse mitochondrial activity erawh CD4 + T cells te nen a inang thung. Heng results te hian tumor cells tan oxidative metabolism a pawimawh tih thupui lo chhuak chu a tichak lehzual a ni (41, 42). Tin, CD4 + T cells te hi CD4 + T cells aiin oxidative dysfunction an nei awlsam zawk thei tih an sawi bawk a, a nih loh leh CD4 + T cells te hian mitochondrial activity vawng reng turin glucose tih loh carbon source dang an hmang thei tih an sawi bawk (43, 44). Hriat tur chu ascites-a CD4 + T effector, T effector memory leh T central memory cell-te zingah glucose uptake emaw mitochondrial activity-ah danglamna kan hmu lo. Chutiang bawkin, tumor-a CD8 + T cells differentiation state hian glucose uptake inthlak danglamna nen hian inzawmna a nei lo a, hei hian in vitro-a cultured T cells leh human TIL in vivo-a danglamna nasa tak a tarlang a ni (22). Heng thil hmuhchhuah te hi unbiased automatic cell population allocation hman hmangin a nemnghet bawk a, hei hian tumor cell aiin glucose uptake sang zawk leh mitochondrial activity nei CD45 + / CD3- / CD4 + / CD45RO + cells te chu an tam zawk a, mahse Metabolic active cell population an nei tih a tilang lehzual a ni. He population hian scRNA-seq analysis-a myeloid suppressor cells emaw plasmacytoid dendritic cells emaw hmuhchhuah subpopulation putative a entir thei a ni. Heng pahnih hi mihring ovarian tumor-ah report ni mahse [45], an la mamawh Further work is to describe this myeloid subpopulation.
Flow cytometry hmanga siam dan hian cell chi hrang hrang glucose leh oxidative metabolism inthlauhna tlangpui chu tichiang thei mah se, TME-a mitochondrial metabolism atana glucose emaw carbon source dang emaw atanga siam metabolite dik tak chu hriat chian a la ni lo. TIL subset pek tawh pakhata metabolite awm leh awm loh assign tur chuan excised tissue atanga cell population tihthianghlim a ngai a ni. Chuvangin, kan cell enrichment method hi mass spectrometry nena inzawm hian damlo sample matching-a T cells leh tumor cell population-a differentially enriched metabolites te chungchang hriatna a pe thei a ni. Hetiang method hian fluorescence-activated cell sorting aiin thatna nei mahse, metabolite library thenkhat chu inherent stability leh/ emaw rapid turnover rate vangin a nghawng thei a ni (22). Chuti chung pawh chuan kan method hian immunosuppressive metabolite hriat chhuah pahnih adenosine leh kynurenine te chu a hmuchhuak thei a, a chhan chu sample chi hrang hrangah an inang lo hle a ni.
Tumor leh TIL subtype te kan metabolite analysis hian ovarian TME-a metabolites te chanvo chungchang hi a hriat chian zawk a ni. Pakhatnaah chuan flow cytometry hmangin tumor leh CD4 + T cells te mitochondrial activity ah danglamna a awm lo tih kan hmuchhuak a. Mahse, LC-MS/MS analysis-ah hian heng population zingah hian metabolite tamnaah danglamna nasa tak a awm tih hmuhchhuah a ni a, hei hian TIL metabolism leh a metabolic activity zawng zawng chungchanga thutlukna siamte chu uluk taka hrilhfiah a ngai tih a tilang. Pahnihnaah chuan MNA hi ascites-a CD45-cells leh T cells inkara danglamna lian ber nei metabolite a ni a, tumor-ah a ni lo. Chuvangin, compartmentalization leh tumor awmna hmun hian TIL metabolism-ah nghawng hrang hrang a nei thei a, hei hian microenvironment pakhata heterogeneity awm thei chu a tarlang a ni. Pathumnaah chuan MNA siamtu enzyme NNMT expression hi CAF-ah chauh a innghat ber a, chu chu tumor cells a ni tlem zawk a, mahse tumor atanga chhuak T cells-ah chuan detectable MNA level hmuh tur a awm thung. Ovarian CAF-a NNMT overexpression hian cancer tichaktu thiltihtheihna a nei tih hriat a ni a, a chhan pakhat chu CAF metabolism, tumor invasion leh metastasis tihhmasawn vang a ni (27). TIL level pumpui hi a hniam lam ni mah se, CAF-a NNMT expression hi Cancer Genome Atlas (TCGA) mesenchymal subtype nen a inzawm tlat a, hei hi prognosis tha lo nen a inzawm a ni (27, 46, 47). A tawp berah chuan MNA tihchhiatna atana mawhphurtu enzyme AOX1 expression pawh CAF population-ah a innghat a, hei hian T cells te hian MNA metabolize theihna an tlachham tih a tilang a ni. Heng results te hian he thil hmuhchhuah hi finfiah turin hna thawh belh a ngai a, mahse T cells-a MNA level sang chuan immunosuppressive CAF microenvironment a awm tih a tilang thei tih ngaihdan hi a thlawp a ni.
MNA transporter expression level hniam leh MNA metabolism-a inrawlh key proteins level hriat theih loh te ngaihtuah chuan T cells-a MNA awm hi beisei loh tak a ni. Independent cohort pahnih scRNA-seq analysis leh targeted qPCR hmangin NNMT emaw AOX1 emaw pawh hmuhchhuah theih a ni lo. Heng results te hian MNA hi T cells ten an siam lo va, a chhehvel TME atanga absorbed a ni tih a tarlang. In vitro experiment-ah chuan T cells hian exogenous MNA an khawlkhawm duh hle tih hmuhchhuah a ni.
Kan in vitro study atanga kan hmuh dan chuan exogenous MNA hian T cells ah TNFα expression a tichhuak a, Sp1 chu TNFα promoter nena inzawmna a tichak bawk. TNFα hian anti-tumor leh anti-tumor function pahnih nei mahse, ovarian cancer-ah chuan TNFα hian ovarian cancer a tipung thei a ni (31-33). Ovarian tumor cell culture-a TNFα neutralization emaw, mouse model-a TNFα signal tihbo emaw hian TNFα-mediated inflammatory cytokine production a tichak thei a, tumor thanna a titawp thei bawk (32, 35). Chuvangin, hetiang a nih chuan TME atanga chhuak MNA hian autocrine loop kaltlangin TNFα-dependent mechanism hmangin pro-inflammatory metabolite angin hna a thawk thei a, chu chuan ovarian cancer lo awm leh darh zelna a tichak thei a ni (31). Hetiang thil awm theihna atang hian TNFα blockade hi ovarian cancer enkawlna atana hman theih tur anga zirchian mek a ni (37, 48, 49). Hei bakah hian MNA hian CAR-T cells te ovarian tumor cells te cytotoxicity a tichhe a, hei hian MNA-mediated immune suppression a awm theihna tur evidence dang a pe belh bawk. Heng result zawng zawng hi a vaiin, tumor leh CAF cells te hian MNA chu extracellular TME ah an secrete dan model a ni tih a tilang a ni. (i) TNF-induced ovarian cancer growth stimulation leh (ii) MNA-induced T cell cytotoxic activity inhibition hmangin hei hian dual tumor effect a nei thei a ni (Figure 5D).
Thutawp atan chuan, rapid cell enrichment, single-cell sequencing leh metabolic profiling te inzawmkhawm hmangin, he zirchianna hian HGSC vei te tumor leh ascites cell te immunometabolomic danglamna lian tak a hmuchhuak a ni. He zirchianna kimchang tak hian T cells te zingah glucose uptake leh mitochondrial activity ah danglamna a awm tih a tarlang a, MNA chu non-cell autonomous immune regulatory metabolite a nih thu a tarlang bawk. Heng data te hian TME hian mihring cancer-a T cell metabolism a nghawng danah nghawng a nei a ni. T cells leh cancer cells te inkara nutrients inelna direct a awm tih report ni mahse, metabolites te hian tumor progression tichak turin indirect regulator angin hna an thawk thei bawk a, endogenous immune response te pawh a titawp thei bawk. Heng regulatory metabolites te hnathawh dan sawifiah belh hian anti-tumor immune response tihchakna tur strategy dang a hawng thei a ni.
Damlo specimen leh clinical data te hi Canadian Tissue Repository Network-in a certified BC cancer tumor tissue repository kaltlangin an la a ni. BC Cancer Research Ethics Committee leh University of British Columbia (H07-00463)-in an pawmpui protocol angin, damlo specimen leh clinical data zawng zawngte chu informed written consent emaw formally waived emaw an ni. Sample te hi Certified BioBank (BRC-00290) ah dah a ni. Damlo mizia chipchiar tak chu Table S1 leh S5-ah hian tarlan a ni. Cryopreservation atan chuan scalpel hmangin damlo tumor sample chu mechanically decompose a ni a, chu chu 100-micron filter hmangin nawr chhuakin cell suspension pakhat an hmu thei a ni. Damlo ascites chu 1500 rpm-ah minute 10 chhung 4°C-ah centrifuge-in cell-te chu pellet-in supernatant chu lakchhuah a ni. Tumor leh ascites atanga cell lakchhuah te chu 50% heat-inactivated human AB serum (Sigma-Aldrich), 40% RPMI-1640 (Thermo Fisher Scientific) leh 10% dimethyl sulfoxide-ah cryopreserved a ni. Heng preserved single cell suspensions te hi thawed niin a hnuaia tarlan ang hian metabolomics leh metabolite determination atan hman a ni.
Medium kimchang chu 0.22 μm filtered 50:50 supplemented RPMI 1640: AimV a ni. RPMI 1640 + 2.05 mM l-glutamine (Thermo Fisher Scientific) chu 10% heat-inactivated human AB serum (Sigma-Aldrich), 12.5 mM Hepes (Thermo Fisher Scientific), 2 mM l-glutamine (Thermo Fisher Scientific) Fisher Scientific), 1 x Penicillin Streptomycin (PenStrep) solution (Thermo Fisher Scientific) leh 50 μMB-mercaptoethanol hmanga siam a ni. AimV (Invitrogen) hi Hepes 20 mM (Thermo Fisher Scientific) leh l-glutamine 2 mM (Thermo Fisher Scientific) te nen a tihpun a ni. Flow cytometer staining buffer hi 0.22μm filtered phosphate buffered saline (PBS; Invitrogen) a ni a, 3% heat-inactivated AB human serum (Sigma) nen a inzawm a ni. Cell enrichment buffer hi 0.22μm filtered PBS atanga siam niin 0.5% heat-inactivated human AB serum (Sigma-Aldrich) hmanga tihpun a ni.
37°C complete medium-ah cell te chu 10 nM MT DR leh 100 μM 2-NBDG hmangin minute 30 chhung stain a ni. A dawtah chuan cell te chu viability dye eF506 hmangin 4°C ah minute 15 chhung an stain a. Cell te chu FC Block (eBioscience) leh Brilliant Stain Buffer (BD Biosciences) ah te resuspend la, flow cytometer staining buffer ah dilute la (a siamtu thupek angin), room temperature ah minute 10 chhung incubate rawh. Cell te chu antibodies set (Table S2) hmangin flow cytometry staining buffer ah 4°C ah minute 20 chhung stain rawh. Cell te chu flow cytometry staining buffer (Cytek Aurora; 3L-16V-14B-8R configuration) ah resuspend leh la, chu chu analysis hmain dah leh rawh. Cell count data thlirletna atan SpectroFlo leh FlowJo V10 hmang la, GraphPad Prism 8 hmangin data siam rawh. 2-NBDG leh MT DR-a median fluorescence intensity (MFI) chu log-normalized a ni a, chutah chuan paired t test hmangin statistical analysis hmangin matched patients te account a ni. Analysis atanga thil thleng 40 aia tlem nei population zawng zawng paih vek; statistical analysis leh data visualization tih hmain negative value eng pawh tan MFI value 1 dah hmasa phawt ang che.
A chunga process panel-a manual gating strategy tihpun nan hian FlowJo-a cell thite tihbo hnuah cells chu population-ah automatic-a assign turin shape restriction tree (FAUST) (21) hmanga full annotation kan hmang a. Misallocated ang maia lang (PD1+ leh PD1-tumor cells inzawmkhawm) leh retained population te inzawmkhawm turin output chu manual-in kan enkawl a ni. Sample khatah hian a vaiin cell 2% aia tam a awm a, a vaiin population 11 a awm a ni.
Ficoll gradient density centrifugation hmangin PBMC chu leukocyte separation products (STEMCELL Technologies) atanga thliar hran a ni. CD8 + T cells chu PBMC atangin CD8 MicroBeads (Miltenyi) hmangin lakchhuah a ni a, siamtu thupek angin TransAct (Miltenyi) hmangin complete medium-ah kar 2 chhung expand a ni. Cell te chu IL-7 (10 ng/ml; PeproTech) awmna complete medium-ah ni 5 chhung dah a ni a, chutah chuan TransAct hmangin re-stimulate leh a ni. Ni 7-naah chuan siamtu thupek angin mihring CD45 MicroBeads (Miltenyi) hmangin cell te chu round thum a zawnin an tihausa a. Cell te chu flow cytometry analysis atan (a chunga kan sawi tawh ang khan) aliquoted a ni a, LC-MS/MS analysis atan cell maktaduai khat chu vawi thum aliquoted a ni bawk. Sample te hi a hnuaia tarlan ang hian LC-MS/MS hmangin an process a ni. Ion number 1,000 hmangin metabolite value hloh chu kan chhut a. Sample tin hi total ion number (TIC) hmangin normalized a ni a, logarithmically convert a ni a, analysis hmain MetaboAnalystR-ah automatic-in normalized a ni.
Damlo pakhat zel single cell suspension chu thaw in 40 μm filter hmangin complete medium (a chunga kan sawi tawh ang khan) ah filter a ni. Manufacturer protocol ang chuan MicroBeads (Miltenyi) hmanga magnetic bead separation hmanga positive selection round thum a zawnin CD8+, CD4+ leh CD45- cells (ice-a) sample enrich turin hman a ni. A tawi zawngin, cell te chu cell enrichment buffer-ah (a chunga kan sawi tawh ang khan) resuspended an ni a, chhiar an ni. Cell te chu mihring CD8 beads, mihring CD4 beads emaw mihring CD45 beads (Miltenyi) emaw hmangin 4°C-ah minute 15 chhung an incubate a, chutah chuan cell enrichment buffer hmangin an silfai a ni. Sample chu LS column (Miltenyi) kaltlangin, positive leh negative fraction te chu lakkhawm a ni. Duration tihtlem leh cell recovery step tihpun nan CD8-fraction chu CD4+ enrichment round hnihna atan hman a ni a, chu CD4-fraction chu a hnua CD45-enrichment atan hman a ni. Separation process chhung zawng hian solution chu ice-ah dah tur a ni.
Metabolite analysis atana sample buatsaih nan cell te chu ice-cold salt solution hmangin vawi khat silfai a ni a, sample tinah 80% methanol 1 ml dah a ni a, chutah chuan vortexed niin liquid nitrogen-ah snap frozen a ni. Sample te hi freeze-thaw cycle pathum hmangin 14,000 rpm-ah minute 15 chhung 4°C-ah centrifuged an ni. Metabolite awmna supernatant chu a vawt thlengin a vawt a. Metabolite te chu 0.03% formic acid 50 μl-ah an hmin leh a, vortex-in an inhmeh tir a, chutah chuan centrifuged-in bawlhhlawh paih chhuah an ni.
A chunga kan sawi tawh ang khan metabolites te chu extract rawh. Supernatant chu metabolomics research atan high performance liquid chromatography bottle-ah dah la. Random treatment protocol hmangin sample tinte chu cell zat inang chiah hmanga enkawl la, batch effects a awm loh nan. A hmaa AB SCIEX QTRAP 5500 Triple Quadrupole Mass Spectrometer (50)-a global metabolites tihchhuah tawhte chu qualitative assessment kan nei a. Chromatographic analysis leh peak area integration te chu MultiQuant version 2.1 software (Applied Biosystems SCIEX) hmangin an ti a.
Ion count 1000 hmangin metabolite value awm lo chu chhut a ni a, sample tinte TIC hmangin sample processing atanga instrumental analysis atanga inthlak danglamna lo awmte siamthat nan metabolite hmuhchhuah tinte normalized peak area chhut a ni bawk. TIC chu normalized a nih hnuah MetaboAnalystR(51) (default parameter) chu logarithmic conversion leh automatic norm line scaling atan hman a ni. Sample chi hrang hrang metabolome danglamna exploratory analysis neih nan vegan R package nei PCA kan hmang a, damlote zirchianna atan partial redundancy analysis kan hmang bawk. Ward method hmangin sample inkar Euclidean distance cluster turin heat map dendrogram siam rawh. Cell type leh microenvironment pumpui huapa metabolite tam dan inang lo tak takte hriatchhuah nan standardized metabolite abundance-a limma (52) kan hmang a. Thuhmahruai awlsam zawk nan group mean parameter hmangin model kan tarlang a, microenvironment-a cell chi hrang hrangte chu group tin angin kan ngaihtuah a (n = group 6) for the significance test, we performed three repeated measurement for each metabolite False replication awm loh nan damlo chu limma design-ah daltu atan kan dah tel a ni. Damlo hrang hrangte metabolite danglamna enfiah nan damlo te pawh huamin limma model chu fixed takin kan siamrem a. Padj <0.05 (Benjamini-Hochberg correction) cell type leh microenvironment inkara pre-specified contrast awmzia kan report a ni.
Miltenyi Dead Cell Removal Kit (>80% viability) hmanga vigor enrichment hnuah 10x 5′gene expression protocol hmangin total live frozen ascites leh tumor sample-ah single-cell transcriptome sequencing an ti a. Tumor leh ascites inmil case panga an zirchiang a, mahse tumor sample pakhat atanga viability hniam vangin a tel theih loh phah a ni. Damlo thlan chhuah tam tak neih theih nan 10x chromium controller lanes-a damlo pakhat zel sample kan dah khawm a, ascites leh tumor hmun hrang hrang kan zirchiang a. Sequencing hnuah [Illumina HiSeq 4000 28×98 bp paired end (PE), Quebec genome; an average of 73,488 and 41,378 reads per cell for tumor and ascites respectively]], kan hmang a, CellSNP leh Vireo (53) (based on CellSNP as The common human SNP (VCF) provided by GRCh38 is assigned a donor identity. SNPRelate hmangin damlo genotype status (IBS) closest identity (IBS) infer nan kan hmang a, excluding unassigned cells and cells identified as duplexes and matching donors between ascites and tumor samples (54) He hna a\ang hian tumor leh ascites-a cell representation tam tak nei case pathum chu downstream analysis atan kan vawng reng a, scater (55) leh scran (56) BioConductor packaging-a mass filtration step kan tih hnuah, hei hian cell 697 a pe chhuak a ni (2792 leh 4183 cells from tumor and ascites, respectively) for analysis Kan hmang a, igraph’s (57) Louvain clustering of shared nearest neighbor network (SNN) chu Jaccard distance to cluster cells by expression hmanga siam a ni defined by the expression of CD8A and GZMA, excluding subclusters with low ribosomal protein expression Kan t-SNE embedding telin, immune cell marker leh NNMT expression inkara expression overlap control thei.
PBMC te hi leukocyte separation products (STEMCELL Technologies) atanga Ficoll gradient density centrifugation hmangin an thliar hrang a. PBMC atang hian CD3 beads (Miltenyi) hmangin CD3 + cells te chu lakchhuah a ni. MNA awm leh awm lohah CD3+ cell te chu plate-bound CD3 (5μg/ml), soluble CD28 (3μg/ml) leh IL-2 (300 U/ml; Proleukin) hmangin an activate a. Expansion ni hnuhnung berah chuan flow cytometry hmangin a viability (Fixable Viability Dye eFluor450, eBioscience) leh proliferation (123count eBeads, Thermo Fisher Scientific) te chu an zirchiang a. Effector hnathawh dan chu PMA (20 ng/ml) leh ionomycin (1μg/ml) hmanga cell te GolgiStop hmanga darkar 4 chhung tihchak hmangin endik la, CD8-PerCP (RPA-T8, BioLegend), CD4-AF700 (RPA-T4) , BioLegend) leh TNFα-fluorescein isothiocyanate (FITC) (MAb11, BD) te enfiah bawk ang che. qPCR leh ChIP cell te chu PMA (20 ng/ml) leh ionomycin (1μg/ml) hmangin darkar 4 chhung stimulate tur a ni. ELISA supernatant chu PMA (20 ng/ml) leh ionomycin (1 μg/ml) hmangin darkar 4 chhung stimulation hma leh hnuah lakkhawm a ni.
RNeasy Plus Mini Kit (QIAGEN) hmanga RNA isolate turin siamtute protocol zawm rawh. Sample chu homogenize turin QIAshredder (QIAGEN) hmang ang che. Complementary DNA (cDNA) siam nan hian high-capacity RNA to cDNA kit (Thermo Fisher Scientific) hmang rawh. TaqMan Rapid Advanced Master Mix (Thermo Fisher Scientific) hmangin a hnuaia probes te hmang hian gene expression (a siamtu protocol angin) quantified rawh: Hs00196287_m1 (NNMT), Hs00154079_m1 (AOX1), Hs00427552_m1 (SLC22A1), Hs02786624_g1 [glyceraldehyde-3-phosphate off Hydrogen (GAPDH)] leh Hs01010726_m1 (SLC22A2) te a awm bawk. Sample te hi StepOnePlus real-time PCR system (Applied Biosystems) (Applied Biosystems) hmangin MicroAmp fast optical 96-well reaction plate (Applied Biosystems)-ah MicroAmp optical film hmanga kalpui a ni. Ct value 35 aia tam chu detection threshold aia sang anga ngaih a ni a, undetectable anga chhinchhiah a ni.
A hmaa kan sawi tawh angin ChIP ti rawh (58). A tawi zawngin, cell te chu formaldehyde (final concentration 1.42%) hmangin an treat a, room temperature-ah minute 10 chhung an incubate a. ice-ah minute 10 chhung supplemented swelling buffer (25 mM Hepes, 1.5 mM MgCl2, 10 mM KCl leh 0.1% NP-40) hmang la, chutah chuan a sawi angin immunoprecipitation buffer-ah resuspend leh rawh (58 ). Chumi hnuah sample chu a hnuaia cycle hrang hrang hmangin sonicated a ni: cycle 10 (20 1-second pulses) leh static time second 40. ChIP-grade immunoglobulin G (Cell Signaling Technology; 1μl), histone H3 (Cell Signaling Technology; 3μl), NFAT (Invitrogen; 3μl) leh SP1 (Cell Signaling Technology; 3μl) antibodies te chu sample nen 4°CC shake-ah zan khat chhung incubate rawh. Protein A beads (Thermo Fisher Scientific) chu sample nen 4°C-ah darkar 1 chhung zawi zawiin incubate la, chutah chuan chelex beads (Bio-Rad) hmangin DNA chu tihausa la, proteinase K (Thermo Fisher) hmangin protein digestion atan hmang bawk ang che. TNFα promoter chu PCR hmangin an hmuchhuak a: hmalam, GGG TAT CCT TGA TGC TTG TGT; chu ai chuan GTG CCA ACA ACT GCC TTT ATA TG (207-bp product) a ni thung. Thlalak te hi Image Lab (Bio-Rad) siam niin, ImageJ software hmangin quantified a ni.
Cell culture supernatant chu a chunga kan sawi tawh ang khan an la khawm a. He determination hi siamtute tih dan angin human TNFα ELISA kit (Invitrogen), human IL-2 ELISA kit (Invitrogen) leh human IFN-γ ELISA kit (Abcam) te tih dan angin an ti a ni. Manufacturer protocol angin supernatant chu 1:100-a diluted-in TNFα leh IL-2 hmuhchhuah a ni a, 1:3-a diluted-in IFN-γ hmuhchhuah a ni bawk. EnVision 2104 Multilabel Reader (PerkinElmer) hmangin 450 nm-a absorbance teh theih a ni.
PBMC te hi leukocyte separation products (STEMCELL Technologies) atanga Ficoll gradient density centrifugation hmangin an thliar hrang a. PBMC atang hian CD3 beads (Miltenyi) hmangin CD3 + cells te chu lakchhuah a ni. MNA awm leh awm lohah CD3+ cells chu plate-bound CD3 (5μg/ml), soluble CD28 (3μg/ml) leh IL-2 (300 U/ml; Proleukin) hmangin ni 3 chhung activate a ni. Ni 3 hnuah cell te chu an la khawm a, 0.9% saline hmangin an silfai a, pellet chu snap frozen a ni. Cell chhiarna chu flow cytometry (Cytek Aurora; 3L-16V-14B-8R configuration) hmangin 123count eBeads hmangin an ti a.
A chunga kan sawi tawh ang khan metabolites te chu extract rawh. Dred extract chu 4000 cell equivalents/μl concentration-ah reconstituted a ni. Sample chu reversed-phase chromatography (1290 Infinity II, Agilent Technologies, Santa Clara, CA) leh CORTECS T3 column (2.1×150 mm, particle size 1.6-μm, pore size 120-Å; #186008500, Waters) hmangin enfiah rawh. Polar mass spectrometer (6470, Agilent), chutah chuan electrospray ionization chu positive mode-in a thawk a ni. Mobile phase A chu 0.1% formic acid (H2O-ah) a ni a, mobile phase B chu 90% acetonitrile, 0.1% formic acid a ni. LC gradient chu 100% A tan minute 0 atanga 2, 99% B tan minute 2 atanga 7.1, leh 99% B tan minute 7.1 atanga 8 a ni a, chutah chuan mobile phase A nen column chu flow rate 0.6 ml/min-ah minute 3 chhung equilibrate leh rawh. . Flow rate chu 0.4ml/min a ni a, column chamber chu 50°C thleng a lum a ni. MNA-a pure chemical standard (M320995, Toronto Research Chemical Company, North York, Ontario, Canada) hmangin retention time (RT) leh transformation (RT = 0.882 minutes, transformation 1 = 137→94.1, transformation 2 = 137→92 , Conversion 3 = 137→78) siam rawh. Transition pathumte hi retention time dik takah a awm vek chuan transition 1 hi specificity hriat chian nan quantification atan hman a ni. MNA (Toronto Research Chemical Company) standard curve chu stock solution (1 mg/ml) serial dilution paruk hmanga siam niin, standard 0.1, 1.0, 10 leh 100 ng/ml leh 1.0 leh 10μg/ml liquid a ni. Detection limit chu 1 ng/ml a ni a, linear response chu 10 ng/ml leh 10μg/ml inkar a ni. Sample leh standard microliter hnih injection khatah LC/MS analysis atan hman a ni a, analysis platform stability a awm theih nan injection vawi riat danah mixed quality control sample pakhat run a ni bawk. MNA hmanga enkawl cell sample zawng zawng MNA response chu assay linear range chhungah a awm a ni. Data thlirletna hi MassHunter quantitative analysis software (v9.0, Agilent) hmanga enfiah a ni.
Second generation αFR-CAR construct chu Song et al. (59) a ni. A tawi zawngin, construct-ah hian a hnuaia thu awmte hi a awm a: CD8a leader sequence, mihring αFR-specific single-chain variable fragment, CD8a hinge leh transmembrane region, CD27 intracellular domain leh CD3z intracellular domain. CAR sequence kimchang chu GenScript hmangin an siam a, chu chu transduction efficiency tehna atana hman GFP expression cassette upstream-a second-generation lentiviral expression vector-ah clone a ni.
Lentivirus hi HEK293T cells transfection hmanga siam a ni a [American Type Culture Collection (ATCC); Dulbecco-a modified Eagle medium-a 10% fetal bovine serum (FBS) leh 1% PenStrep awmnaah chin a ni a, CAR-GFP vector leh The packaging plasmids (psPAX2 leh pMD2.G, Addgene) hmangin lipofection amine (Sigma-Aldrich) hman a ni. Virus awmna supernatant chu transfection atanga darkar 48 leh 72 hnuah lakkhawm a ni a, filter a ni a, ultracentrifugation hmangin concentrate a ni. Concentrated viral supernatant chu -80°C ah transduction thlengin dah la.
PBMC hi donor leukocyte separation product hrisel (STEMCELL Technologies) atanga Ficoll gradient density centrifugation hmangin an thliar hrang a ni. PBMC atanga CD8+ cells te chu isolate nan positive selection CD8 microbeads (Miltenyi) hmang thin ang che. T cells te chu TransAct (Miltenyi) hmangin leh TexMACS medium [Miltenyi; heat-inactivated human serum 3%, PenStrep 1% leh IL-2 (300 U/ml) hmanga tihpun a ni]. Stimulation hnu darkar 24 hnuah T cells te chu lentivirus (cell 106 ah 10 μl concentrated virus supernatant) hmangin an transduce a. Cytek Aurora (on FSC (Forward Scatter)/SSC (Side Scatter), Singlet, GFP+) hmanga transduction hnu ni 1 atanga ni 3 hnuah, cells te GFP expression chu evaluate la, transduction efficiency 30% tal a lantir theih nan.
CAR-T cells te chu Immunocult (STEMCELL Technologies; supplemented with 1% PenStrep) ah darkar 24 chhung cultured a ni a, a hnuaia condition te hi: enkawl loh, 250 μM adenosine emaw 10 mM MNA hmanga enkawl. Pretreatment hnuah CAR-T cells chu PBS hmangin silfai a ni a, SK-OV-3 cell 20,000 nen an inzawm khawm a ni [ATCC; in McCoy 5A medium (Sigma-Aldrich) supplemented with 10% FBS and 1% PenStrep at 10: Effector to target ratio 1 chu supplemented Immunocult medium-ah vawi thum amplified a ni. SK-OV-3 cells leh SK-OV-3 cells digitalis saponin (0.5mg/ml; Sigma-Aldrich) hmanga lysed te chu negative leh positive control atan hman a ni. Darkar 24 chhung co-cultivation hnuah supernatant chu lakkhawm a ni a, siamtu thupek (LDH Glo Cytotoxicity Assay Kit, Promega) angin lactate dehydrogenase (LDH) chu teh a ni. LDH supernatant chu LDH buffer-ah 1:50 in an diluted a. Thih zat percentage chu a hnuaia formula hmang hian teh a ni: percentage of killing = correction percentage / maximum killing rate x 100%, chutah chuan correction percentage = co-culture-T cells chauh, leh maximum killing rate = positive control-negative control.
Text emaw materials leh methods-a tarlan angin statistical analysis atan GraphPad Prism 8, Microsoft Excel emaw R v3.6.0 emaw hmang ang che. Damlo pakhat (ascites leh tumor ang chi) atanga sample tam tak lakkhawm a nih chuan paired t test kan hmang emaw, a remchan dan angin linear emaw generalized model-ah random effect angin damlo chu kan dah tel bawk. Metabolomics analysis atan chuan importance test hi vawi thum tih a ni.
He thuziak atana supplementary materials hriat duh chuan http://advances.sciencemag.org/cgi/content/full/7/4/eabe1174/DC1 ah hian en theih a ni
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A rilru a hah lutuk chuan a rilru a buai em em a, a rilru a hah lutuk chuan a rilru a buai em em bawk a G. Jones), Phineas T. Hamilton (Phineas T.-a) te chuan an ziak a ni.
MNA hian T cells immune suppression-ah a pui a, mihring cancer enkawlna atana immunotherapy target awm thei a ni.
A rilru a hah lutuk chuan a rilru a buai em em a, a rilru a hah lutuk chuan a rilru a buai em em bawk a G. Jones), Phineas T. Hamilton (Phineas T.-a) te chuan an ziak a ni.
MNA hian T cells immune suppression-ah a pui a, mihring cancer enkawlna atana immunotherapy target awm thei a ni.
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